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2024 Articles

Griffin PT, Kane AE, Trapp A, Li J, Arnold M, Poganik JR, Conway RJ, McNamara MS, Meer MV, Hoffman N, Amorim JA, Tian X, MacArthur MR, Mitchell SJ, Mueller AL, Carmody C, Vera DL, Kerepesi C, Ying K, Noren Hooten N, Mitchell JR, Evans MK, Gladyshev VN, Sinclair DA. TIME-seq reduces time and cost of DNA methylation measurement for epigenetic clock construction. Nat Aging. doi: 10.1038/s43587-023-00555-2.

Abstract Epigenetic ‘clocks’ based on DNA methylation have emerged as the most robust and widely used aging biomarkers, but conventional methods for applying them are expensive and laborious. Here we develop tagmentation-based indexing for methylation sequencing (TIME-seq), a highly multiplexed and scalable method for low-cost epigenetic clocks. Using TIME-seq, we applied multi-tissue and tissue-specific epigenetic clocks in over 1,800 mouse DNA samples from eight tissue and cell types. We show that TIME-seq clocks are accurate and robust, enriched for polycomb repressive complex 2-regulated loci, and benchmark favorably against conventional methods despite being up to 100-fold less expensive. Using dietary treatments and gene therapy, we find that TIME-seq clocks reflect diverse interventions in multiple tissues. Finally, we develop an economical human blood clock (R > 0.96, median error = 3.39 years) in 1,056 demographically representative individuals. These methods will enable more efficient epigenetic clock measurement in larger-scale human and animal studies. More Information

Moqri M, Herzog C, Poganik JR, Ying K, Justice JN, Belsky DW, Higgins-Chen AT, Chen BH, Cohen AA, Fuellen G, Hägg S, Marioni RE, Widschwendter M, Fortney K, Fedichev PO, Zhavoronkov A, Barzilai N, Lasky-Su J, Kiel DP, Kennedy BK, Cummings S, Slagboom PE, Verdin E, Maier AB, Sebastiano V, Snyder MP, Gladyshev VN, Horvath S, Ferrucci L. Validation of biomarkers of aging. Nat Med. doi: 10.1038/s41591-023-02784-9.

Abstract The search for biomarkers that quantify biological aging (particularly ‘omic’-based biomarkers) has intensified in recent years. Such biomarkers could predict aging-related outcomes and could serve as surrogate endpoints for the evaluation of interventions promoting healthy aging and longevity. However, no consensus exists on how biomarkers of aging should be validated before their translation to the clinic. Here, we review current efforts to evaluate the predictive validity of omic biomarkers of aging in population studies, discuss challenges in comparability and generalizability and provide recommendations to facilitate future validation of biomarkers of aging. Finally, we discuss how systematic validation can accelerate clinical translation of biomarkers of aging and their use in gerotherapeutic clinical trials. More Information

Ying K, Liu H, Tarkhov AE, Sadler MC, Lu AT, Moqri M, Horvath S, Kutalik Z, Shen X, Gladyshev VN. Causality-enriched epigenetic age uncouples damage and adaptation. Nat Aging. doi: 10.1038/s43587-023-00557-0.

Abstract Machine learning models based on DNA methylation data can predict biological age but often lack causal insights. By harnessing large-scale genetic data through epigenome-wide Mendelian randomization, we identified CpG sites potentially causal for aging-related traits. Neither the existing epigenetic clocks nor age-related differential DNA methylation are enriched in these sites. These CpGs include sites that contribute to aging and protect against it, yet their combined contribution negatively affects age-related traits. We established a new framework to introduce causal information into epigenetic clocks, resulting in DamAge and AdaptAge—clocks that track detrimental and adaptive methylation changes, respectively. DamAge correlates with adverse outcomes, including mortality, while AdaptAge is associated with beneficial adaptations. These causality-enriched clocks exhibit sensitivity to short-term interventions. Our findings provide a detailed landscape of CpG sites with putative causal links to lifespan and healthspan, facilitating the development of aging biomarkers, assessing interventions, and studying reversibility of age-associated changes. More Information

2023 Articles

Li CZ, Haghani A, Yan Q, Lu AT, Zhang J, Fei Z, Ernst J, Yang W, Gladyshev VN, Raj K, Seluanov A, Gorbunova V, Horvath S. Epigenetic predictors of species maximum lifespan and other life history traits in mammals. bioRxiv. doi: https://doi.org/10.1101/2023.01.09.523139

AbstractMaximum lifespan is an intrinsic characteristic of a biological species and is defined as the longest time an individual of a species has been reported to survive. By analyzing 15K samples derived from 348 mammalian species representing 25 taxonomic orders we previously identified CpG methylation sites associated with maximum lifespan. Here we present accurate DNA methylation-based (DNAm) predictors of maximum lifespan (r=0.89), average gestation time (r=0.96), and age at sexual maturity (r=0.85). Our DNAm maximum lifespan predictor indicates a potential innate longevity advantage for females over males in 17 mammalian species such as humans, red deer, and cattle. The DNAm maximum lifespan predictions do not vary significantly by caloric restriction and partial reprogramming. Genetic disruptions in the somatotropic axis, which includes growth hormone, IGF-1, and their related receptors, have an impact on DNAm maximum lifespan only in select mouse tissues. Cancer mortality rates in major mammalian orders show no correlation with our epigenetic estimates of life history traits. The DNAm maximum lifespan predictor does not detect variation in lifespan between individuals of the same species, such as between the breeds of dogs. We also present the first prototypes of accurate pan mammalian DNAm predictors of sex and tissue type. Collectively, our findings indicate that maximum lifespan is determined, at least in part, by an epigenetic signature that is an intrinsic property of each species and is distinct from the signatures that relate to individual lifespan, which is unaffected by interventions influencing the mortality risk of individuals. More Information

Liu W, Zhu P, Li M, Li Z, Yu Y, Liu G, Du J, Wang X, Yang J, Tian R, Seim I, Kaya A, Li M, Li M, Gladyshev VN, Zhou X. Evolutionary transcriptomics reveals longevity mostly driven by polygenic and indirect selection in mammals. bioRxiv. doi: https://doi.org/10.1101/2023.01.09.523139

AbstractThe maximum lifespan varies more than 100-fold in mammals. This experiment of nature may uncover of the evolutionary forces and molecular features that define longevity. To understand the relationship between gene expression variation and maximum lifespan, we carried out a comparative transcriptomics analysis of liver, kidney, and brain tissues of 106 mammalian species. We found that expression is largely conserved and very limited genes exhibit common expression patterns with longevity in all the three organs analyzed. However, many pathways, e.g., “Insulin signaling pathway”, and “FoxO signaling pathway”, show accumulated correlations with maximum lifespan across mammals. Analyses of selection features further reveal that methionine restriction related genes whose expressions associated with longevity, are under strong selection in long-lived mammals, suggesting that a common approach could be utilized by natural selection and artificial intervention to control lifespan. These results suggest that natural lifespan regulation via gene expression is likely to be driven through polygenic model and indirect selection. More Information

Barlit H, Romero AM, Gülhan A, Patnaik PK, Tyshkovskiy A, Martínez-Pastor MT, Gladyshev VN, Puig S, Labunskyy VM. Genome-wide ribosome profiling uncovers the role of iron in the control of protein translation. bioRxiv. doi: https://doi.org/10.1101/2022.09.22.509115

AbstractIron is an essential trace element that serves as a cofactor for enzymes involved in multiple metabolic pathways, including ribosome biogenesis, protein translation, DNA synthesis and repair, lipid metabolism, and mitochondrial oxidative phosphorylation. In eukaryotes, iron deficiency leads to global inhibition of protein synthesis and coordinated changes in gene expression to limit iron utilization. Although several steps of protein translation depend on iron-containing enzymes, the contribution of iron to the translation process is not understood at the molecular level. Here, we report a genome-wide analysis of protein translation in response to iron deficiency in yeast using ribosome profiling. We show that iron depletion affects global protein synthesis as well as leads to translational repression of several groups of genes involved in iron-related processes. We further demonstrate that the RNA-binding proteins Cth1 and Cth2 play a central role in controlling the changes in protein translation by repressing the activity of the iron-dependent Rli1 ribosome recycling factor, inhibiting mitochondrial translation, and affecting the translation of genes involved in heme biosynthesis. We also discovered a mechanism, whereby iron deficiency represses translation of MRS3 mRNA, encoding mitochondrial iron transporter, through increased expression of antisense long non-coding RNA. Together, our results reveal complex gene expression and protein synthesis remodeling in response to low iron showing how this important metal affects protein translation at multiple levels. More Information

Ying K, Liu H, Tarkhov AE, Sadler MC, Lu AT, Moqri M, Horvath S, Kutalik Z, Shen X, Gladyshev VN. Causality-Enriched Epigenetic Age Uncouples Damage and Adaptation. Nat Aging. doi: 10.1038/s43587-023-00557-0.

AbstractMachine learning models based on DNA methylation data can predict biological age but often lack causal insights. By harnessing large-scale genetic data through epigenome-wide Mendelian randomization, we identified CpG sites potentially causal for aging-related traits. Neither the existing epigenetic clocks nor age-related differential DNA methylation are enriched in these sites. These CpGs include sites that contribute to aging and protect against it, yet their combined contribution negatively affects age-related traits. We established a new framework to introduce causal information into epigenetic clocks, resulting in DamAge and AdaptAge—clocks that track detrimental and adaptive methylation changes, respectively. DamAge correlates with adverse outcomes, including mortality, while AdaptAge is associated with beneficial adaptations. These causality-enriched clocks exhibit sensitivity to short-term interventions. Our findings provide a detailed landscape of CpG sites with putative causal links to lifespan and healthspan, facilitating the development of aging biomarkers, assessing interventions, and studying reversibility of age-associated changes. More Information

Zhang Z, Tian X, Lu JY, Boit K, Ablaeva J, Zakusilo FT, Emmrich S, Firsanov D, Rydkina E, Biashad SA, Lu Q, Tyshkovskiy A, Gladyshev VN, Horvath S, Seluanov A, Gorbunova V. Increased hyaluronan by naked mole-rat Has2 improves healthspan in mice. Nature. doi: 10.1038/s41586-023-06463-0.

AbstractAbundant high-molecular-mass hyaluronic acid (HMM-HA) contributes to cancer resistance and possibly to the longevity of the longest-lived rodent—the naked mole-rat1,2. To study whether the benefits of HMM-HA could be transferred to other animal species, we generated a transgenic mouse overexpressing naked mole-rat hyaluronic acid synthase 2 gene (nmrHas2). nmrHas2 mice showed an increase in hyaluronan levels in several tissues, and a lower incidence of spontaneous and induced cancer, extended lifespan and improved healthspan. The transcriptome signature of nmrHas2 mice shifted towards that of longer-lived species. The most notable change observed in nmrHas2 mice was attenuated inflammation across multiple tissues. HMM-HA reduced inflammation through several pathways, including a direct immunoregulatory effect on immune cells, protection from oxidative stress and improved gut barrier function during ageing. These beneficial effects were conferred by HMM-HA and were not specific to the nmrHas2 gene. These findings demonstrate that the longevity mechanism that evolved in the naked mole-rat can be exported to other species, and open new paths for using HMM-HA to improve lifespan and healthspan. More Information

Kerepesi C, Gladyshev VN. Intersection clock reveals a rejuvenation event during human embryogenesis. Aging Cell. doi: 10.1111/acel.13922.

AbstractRecent research revealed a rejuvenation event during early development of mice. Here, by examining epigenetic age dynamics of human embryogenesis, we tested whether a similar event exists in humans. For this purpose, we developed an epigenetic clock method, the intersection clock, that utilizes bisulfite sequencing in a way that maximizes the use of informative CpG sites with no missing clock CpG sites in test samples and applied it to human embryo development data. We observed no changes in the predicted epigenetic age between cleavage stage and blastocyst stage embryos; however, a significant decrease was observed between blastocysts and cells representing the epiblast. Additionally, by applying the intersection clock to datasets spanning pre and postimplantation, we found no significant change in the epigenetic age during preimplantation stages; however, the epigenetic age of postimplantation samples was lower compared to the preimplantation stages. We further investigated the epigenetic age of primed (representing early postimplantation) and naïve (representing preimplantation) pluripotent stem cells and observed that in all cases the epigenetic age of primed cells was significantly lower than that of naïve cells. Together, our data suggest that human embryos are rejuvenated during early embryogenesis. Hence, the rejuvenation event is conserved between the mouse and human, and it occurs around the gastrulation stage in both species. Beyond this advance, the intersection clock opens the way for other epigenetic age studies based on human bisulfite sequencing datasets as opposed to methylation arrays. More Information

Tarkhov AE, Lindstrom-Vautrin T, Zhang S, Ying K, Moqri M, Zhang B, Vadim N. Gladyshev. Nature of epigenetic aging from a single-cell perspective. bioRxiv. doi: https://doi.org/10.1101/2022.09.26.509592

AbstractAge-related changes in DNA methylation (DNAm) form the basis for the development of most robust predictors of age, epigenetic clocks, but a clear mechanistic basis for what exactly they quantify is lacking. Here, to clarify the nature of epigenetic aging, we analyzed the aging dynamics of bulk-tissue and single-cell DNAm, together with single-cell DNAm changes during early development. We show that aging DNAm changes are widespread, but are relatively slow and small in amplitude, with DNAm levels trending towards intermediate values and showing increased heterogeneity with age. By considering dominant types of DNAm changes, we find that aging manifests in the exponential decay-like loss or gain of methylation with a universal rate, independent of the initial level of DNAm. We further show that aging is dominated by the stochastic component, yet co-regulated changes are also present during both development and adulthood. We support the finding of stochastic epigenetic aging by direct single-cell DNAm analyses and modeling of aging DNAm trajectories with a stochastic process akin to radiocarbon decay. Finally, we describe a single-cell algorithm for the identification of co-regulated CpG clusters that may provide new opportunities for targeting aging and evaluating longevity interventions. More Information

Chen Q, Dwaraka VB, Carreras-Gallo N, Mendez K, Chen Y, Begum S, Kachroo P, Prince N, Went H, Mendez T, Lin A, Turner L, Moqri M, Chu SH, Kelly RS, Weiss ST, Rattray NJW, Gladyshev VN, Karlson E, Wheelock C, Mathé EA, Dahlin A, McGeachie MJ, Smith R, Lasky-Su JA. OMICmAge: An integrative multi-omics approach to quantify biological age with electronic medical records. bioRxiv. DOI: 10.1101/2023.10.16.562114

AbstractBiological aging is a multifactorial process involving complex interactions of cellular and biochemical processes that is reflected in omic profiles. Using common clinical laboratory measures in ~30,000 individuals from the MGB-Biobank, we developed a robust, predictive biological aging phenotype, EMRAge, that balances clinical biomarkers with overall mortality risk and can be broadly recapitulated across EMRs. We then applied elastic-net regression to model EMRAge with DNA-methylation (DNAm) and multiple omics, generating DNAmEMRAge and OMICmAge, respectively. Both biomarkers demonstrated strong associations with chronic diseases and mortality that outperform current biomarkers across our discovery (MGB-ABC, n=3,451) and validation (TruDiagnostic, n=12,666) cohorts. Through the use of epigenetic biomarker proxies, OMICmAge has the unique advantage of expanding the predictive search space to include epigenomic, proteomic, metabolomic, and clinical data while distilling this in a measure with DNAm alone, providing opportunities to identify clinically-relevant interconnections central to the aging process. More Information

Aguado J, Amarilla AA, Taherian Fard A, Albornoz EA, Tyshkovskiy A, Schwabenland M, Chaggar HK, Modhiran N, Gómez-Inclán C, Javed I, Baradar AA, Liang B, Peng L, Dharmaratne M, Pietrogrande G, Padmanabhan P, Freney ME, Parry R, Sng JDJ, Isaacs A, Khromykh AA, Valenzuela Nieto G, Rojas-Fernandez A, Davis TP, Prinz M, Bengsch B, Gladyshev VN, Woodruff TM, Mar JC, Watterson D, Wolvetang EJ. Senolytic therapy alleviates physiological human brain aging and COVID-19 neuropathology. Nat Aging. doi: 10.1038/s43587-023-00519-6.

AbstractAging is a major risk factor for neurodegenerative diseases, and coronavirus disease 2019 (COVID-19) is linked to severe neurological manifestations. Senescent cells contribute to brain aging, but the impact of virus-induced senescence on neuropathologies is unknown. Here we show that senescent cells accumulate in aged human brain organoids and that senolytics reduce age-related inflammation and rejuvenate transcriptomic aging clocks. In postmortem brains of patients with severe COVID-19 we observed increased senescent cell accumulation compared with age-matched controls. Exposure of human brain organoids to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induced cellular senescence, and transcriptomic analysis revealed a unique SARS-CoV-2 inflammatory signature. Senolytic treatment of infected brain organoids blocked viral replication and prevented senescence in distinct neuronal populations. In human-ACE2-overexpressing mice, senolytics improved COVID-19 clinical outcomes, promoted dopaminergic neuron survival and alleviated viral and proinflammatory gene expression. Collectively our results demonstrate an important role for cellular senescence in driving brain aging and SARS-CoV-2-induced neuropathology, and a therapeutic benefit of senolytic treatments. More Information

Poganik JR, Gladyshev VN. We need to shift the focus of aging research to aging itself Proc Natl Acad Sci U S A. DOI: 10.1073/pnas.2307449120

AbstractThe field of aging is at a precipice. Attention and funding are increasingly focused on this area, and exciting, fundamentally important findings are being reported literally every day. As the great promise of targeting aging comes into sharper focus, we are rapidly approaching the point where we must face the elephant in the room: We lack any semblance of a consensus on the nature of aging or, more fundamentally, on the essence of this process. Taking steps to resolve these foundational issues in aging biology will enable us to advance this field to the next level. More Information

Fernando R, Shindyapina AV, Ost M, Santesmasses D, Hu Y, Tyshkovskiy A, Yim SH, Weiss J, Gladyshev VN, Grune T Castro JP. Downregulation of mitochondrial metabolism is a driver for fast skeletal muscle loss during mouse aging. Commun Biol. doi: 10.1038/s42003-023-05595-3.

AbstractSkeletal muscle aging is characterized by the loss of muscle mass, strength and function, mainly attributed to the atrophy of glycolytic fibers. Underlying mechanisms driving the skeletal muscle functional impairment are yet to be elucidated. To unbiasedly uncover its molecular mechanisms, we recurred to gene expression and metabolite profiling in a glycolytic muscle, Extensor digitorum longus (EDL), from young and aged C57BL/6JRj mice. Employing multi-omics approaches we found that the main age-related changes are connected to mitochondria, exhibiting a downregulation in mitochondrial processes. Consistent is the altered mitochondrial morphology. We further compared our mouse EDL aging signature with human data from the GTEx database, reinforcing the idea that our model may recapitulate muscle loss in humans. We are able to show that age-related mitochondrial downregulation is likely to be detrimental, as gene expression signatures from commonly used lifespan extending interventions displayed the opposite direction compared to our EDL aging signature. More Information

Moqri M, Herzog C, Poganik JR; Biomarkers of Aging Consortium; Justice J, Belsky DW, Higgins-Chen A, Moskalev A, Fuellen G, Cohen AA, Bautmans I, Widschwendter M, Ding J, Fleming A, Mannick J, Han JJ, Zhavoronkov A, Barzilai N, Kaeberlein M, Cummings S, Kennedy BK, Ferrucci L, Horvath S, Verdin E, Maier AB, Snyder MP, Sebastiano V, Gladyshev VN. Biomarkers of aging for the identification and evaluation of longevity interventions. Cell. doi: 10.1016/j.cell.2023.08.003.

AbstractWith the rapid expansion of aging biology research, the identification and evaluation of longevity interventions in humans have become key goals of this field. Biomarkers of aging are critically important tools in achieving these objectives over realistic time frames. However, the current lack of standards and consensus on the properties of a reliable aging biomarker hinders their further development and validation for clinical applications. Here, we advance a framework for the terminology and characterization of biomarkers of aging, including classification and potential clinical use cases. We discuss validation steps and highlight ongoing challenges as potential areas in need of future research. This framework sets the stage for the development of valid biomarkers of aging and their ultimate utilization in clinical trials and practice. More Information

Tyshkovskiy A, Ma S, Shindyapina AV, Tikhonov S, Lee SG, Bozaykut P, Castro JP, Seluanov A, Schork NJ, Gorbunova V, Dmitriev SE, Miller RA, Gladyshev VN. (2023). Distinct longevity mechanisms across and within species and their association with aging. Cell. doi: 10.1016/j.cell.2023.05.002.

AbstractLifespan varies within and across species, but the general principles of its control remain unclear. Here, we conducted multi-tissue RNA-seq analyses across 41 mammalian species, identifying longevity signatures and examining their relationship with transcriptomic biomarkers of aging and established lifespan-extending interventions. An integrative analysis uncovered shared longevity mechanisms within and across species, including downregulated Igf1 and upregulated mitochondrial translation genes, and unique features, such as distinct regulation of the innate immune response and cellular respiration. Signatures of long-lived species were positively correlated with age-related changes and enriched for evolutionarily ancient essential genes, involved in proteolysis and PI3K-Akt signaling. Conversely, lifespan-extending interventions counteracted aging patterns and affected younger, mutable genes enriched for energy metabolism. The identified biomarkers revealed longevity interventions, including KU0063794, which extended mouse lifespan and healthspan. Overall, this study uncovers universal and distinct strategies of lifespan regulation within and across species and provides tools for discovering longevity interventions. More Information

Liu W, Zhu P, Li M, Li Z, Yu Y, Liu G, Du J, Wang X, Yang J, Tian R, Seim I, Kaya A, Li M, Li M, Gladyshev VN, Zhou X.(2023). Large-scale across species transcriptomic analysis identifies genetic selection signatures associated with longevity in mammals EMBO J. doi: 10.15252/embj.2022112740.

AbstractLifespan varies significantly among mammals, with more than 100-fold difference between the shortest and longest living species. This natural difference may uncover the evolutionary forces and molecular features that define longevity. To understand the relationship between gene expression variation and longevity, we conducted a comparative transcriptomics analysis of liver, kidney, and brain tissues of 103 mammalian species. We found that few genes exhibit common expression patterns with longevity in the three organs analyzed. However, pathways related to translation fidelity, such as nonsense-mediated decay and eukaryotic translation elongation, correlated with longevity across mammals. Analyses of selection pressure found that selection intensity related to the direction of longevity-correlated genes is inconsistent across organs. Furthermore, expression of methionine restriction-related genes correlated with longevity and was under strong selection in long-lived mammals, suggesting that a common strategy is utilized by natural selection and artificial intervention to control lifespan. Our results indicate that lifespan regulation via gene expression is driven through polygenic and indirect natural selection. More Information

Ogrodnik M, Gladyshev VN.(2023). The meaning of adaptation in aging: insights from cellular senescence, epigenetic clocks and stem cell alterations. Nat Aging. doi: 10.1038/s43587-023-00447-5.

AbstractWith recent rapid progress in research on aging, there is increasing evidence that many features commonly considered to be mechanisms or drivers of aging in fact represent adaptations. Here, we examine several such features, including cellular senescence, epigenetic aging and stem cell alterations. We draw a distinction between the causes and consequences of aging and define short-term consequences as ‘responses’ and long-term ones as ‘adaptations’. We also discuss ‘damaging adaptations’, which despite having beneficial effects in the short term, lead to exacerbation of the initial insult and acceleration of aging. Features commonly recognized as ‘basic mechanisms of the aging process’ are critically examined for the possibility of their adaptation-driven emergence from processes such as cell competition and the wound-like features of the aging body. Finally, we speculate on the meaning of these interactions for the aging process and their relevance for the development of antiaging interventions. More Information

Yang JH, Petty CA, Dixon-McDougall T, Lopez MV, Tyshkovskiy A, Maybury-Lewis S, Tian X, Ibrahim N, Chen Z, Griffin PT, Arnold M, Li J, Martinez OA, Behn A, Rogers-Hammond R, Angeli S, Gladyshev VN, Sinclair DA. (2023). Chemically induced reprogramming to reverse cellular aging. Aging (Albany NY). doi: 10.18632/aging.204896.

AbstractA hallmark of eukaryotic aging is a loss of epigenetic information, a process that can be reversed. We have previously shown that the ectopic induction of the Yamanaka factors OCT4, SOX2, and KLF4 (OSK) in mammals can restore youthful DNA methylation patterns, transcript profiles, and tissue function, without erasing cellular identity, a process that requires active DNA demethylation. To screen for molecules that reverse cellular aging and rejuvenate human cells without altering the genome, we developed high-throughput cell-based assays that distinguish young from old and senescent cells, including transcription-based aging clocks and a real-time nucleocytoplasmic compartmentalization (NCC) assay. We identify six chemical cocktails, which, in less than a week and without compromising cellular identity, restore a youthful genome-wide transcript profile and reverse transcriptomic age. Thus, rejuvenation by age reversal can be achieved, not only by genetic, but also chemical means. More Information

Lu AT, Fei Z, Haghani A, Robeck TR, Zoller JA, Li CZ, Lowe R, Yan Q, Zhang J, Vu H, Ablaeva J, Acosta-Rodriguez VA, Adams DM, Almunia J, Aloysius A, Ardehali R, Arneson A, Baker CS, Banks G, Belov K, Bennett NC, Black P, Blumstein DT, Bors EK, Breeze CE, Brooke RT, Brown JL, Carter GG, Caulton A, Cavin JM, Chakrabarti L, Chatzistamou I, Chen H, Cheng K, Chiavellini P, Choi OW, Clarke SM, Cooper LN, Cossette ML, Day J, DeYoung J, DiRocco S, Dold C, Ehmke EE, Emmons CK, Emmrich S, Erbay E, Erlacher-Reid C, Faulkes CG, Ferguson SH, Finno CJ, Flower JE, Gaillard JM, Garde E, Gerber L, Gladyshev VN, Gorbunova V, Goya RG, Grant MJ, Green CB, Hales EN, Hanson MB, Hart DW, Haulena M, Herrick K, Hogan AN, Hogg CJ, Hore TA, Huang T, Izpisua Belmonte JC, Jasinska AJ, Jones G, Jourdain E, Kashpur O, Katcher H, Katsumata E, Kaza V, Kiaris H, Kobor MS, Kordowitzki P, Koski WR, Krützen M, Kwon SB, Larison B, Lee SG, Lehmann M, Lemaitre JF, Levine AJ, Li C, Li X, Lim AR, Lin DTS, Lindemann DM, Little TJ, Macoretta N, Maddox D, Matkin CO, Mattison JA, McClure M, Mergl J, Meudt JJ, Montano GA, Mozhui K, Munshi-South J, Naderi A, Nagy M, Narayan P, Nathanielsz PW, Nguyen NB, Niehrs C, O’Brien JK, O’Tierney Ginn P, Odom DT, Ophir AG, Osborn S, Ostrander EA, Parsons KM, Paul KC, Pellegrini M, Peters KJ, Pedersen AB, Petersen JL, Pietersen DW, Pinho GM, Plassais J, Poganik JR, Prado NA, Reddy P, Rey B, Ritz BR, Robbins J, Rodriguez M, Russell J, Rydkina E, Sailer LL, Salmon AB, Sanghavi A, Schachtschneider KM, Schmitt D, Schmitt T, Schomacher L, Schook LB, Sears KE, Seifert AW, Seluanov A, Shafer ABA, Shanmuganayagam D, Shindyapina AV, Simmons M, Singh K, Sinha I, Slone J, Snell RG, Soltanmaohammadi E, Spangler ML, Spriggs MC, Staggs L, Stedman N, Steinman KJ, Stewart DT, Sugrue VJ, Szladovits B, Takahashi JS, Takasugi M, Teeling EC, Thompson MJ, Van Bonn B, Vernes SC, Villar D, Vinters HV, Wallingford MC, Wang N, Wayne RK, Wilkinson GS, Williams CK, Williams RW, Yang XW, Yao M, Young BG, Zhang B, Zhang Z, Zhao P, Zhao Y, Zhou W, Zimmermann J, Ernst J, Raj K, Horvath S.(2023). Universal DNA methylation age across mammalian tissues. Nat Aging. doi: 10.1038/s43587-023-00462-6.

AbstractAging, often considered a result of random cellular damage, can be accurately estimated using DNA methylation profiles, the foundation of pan-tissue epigenetic clocks. Here, we demonstrate the development of universal pan-mammalian clocks, using 11,754 methylation arrays from our Mammalian Methylation Consortium, which encompass 59 tissue types across 185 mammalian species. These predictive models estimate mammalian tissue age with high accuracy (r > 0.96). Age deviations correlate with human mortality risk, mouse somatotropic axis mutations and caloric restriction. We identified specific cytosines with methylation levels that change with age across numerous species. These sites, highly enriched in polycomb repressive complex 2-binding locations, are near genes implicated in mammalian development, cancer, obesity and longevity. Our findings offer new evidence suggesting that aging is evolutionarily conserved and intertwined with developmental processes across all mammals. More Information

Tyshkovskiy A, Zhang S, Gladyshev VN. (2023). Accelerated transcriptional elongation during aging impairs longevity. Cell Res. doi: 10.1038/s41422-023-00829-9.

AbstractIncreased transcriptional errors in aged organisms have been demonstrated for multiple species; however, mechanistic details remained elusive. In a recent issue of Nature, Debès et al. reveal a novel form of aging-associated molecular damage, demonstrating that Pol II transcriptional elongation rate increases with age, presumably due to reduced nucleosomal density, whereas targeted reduction of Pol II speed can partially ameliorate this deleterious phenotype and extend lifespan in animals, such as roundworms and fruit flies. More Information

Haghani A, Li CZ, Robeck TR, Zhang J, Lu AT, Ablaeva J, Acosta-Rodríguez VA, Adams DM, Alagaili AN, Almunia J, Aloysius A, Amor NMS, Ardehali R, Arneson A, Baker CS, Banks G, Belov K, Bennett NC, Black P, Blumstein DT, Bors EK, Breeze CE, Brooke RT, Brown JL, Carter G, Caulton A, Cavin JM, Chakrabarti L, Chatzistamou I, Chavez AS, Chen H, Cheng K, Chiavellini P, Choi OW, Clarke S, Cook JA, Cooper LN, Cossette ML, Day J, DeYoung J, Dirocco S, Dold C, Dunnum JL, Ehmke EE, Emmons CK, Emmrich S, Erbay E, Erlacher-Reid C, Faulkes CG, Fei Z, Ferguson SH, Finno CJ, Flower JE, Gaillard JM, Garde E, Gerber L, Gladyshev VN, Goya RG, Grant MJ, Green CB, Hanson MB, Hart DW, Haulena M, Herrick K, Hogan AN, Hogg CJ, Hore TA, Huang T, Izpisua Belmonte JC, Jasinska AJ, Jones G, Jourdain E, Kashpur O, Katcher H, Katsumata E, Kaza V, Kiaris H, Kobor MS, Kordowitzki P, Koski WR, Krützen M, Kwon SB, Larison B, Lee SG, Lehmann M, Lemaître JF, Levine AJ, Li X, Li C, Lim AR, Lin DTS, Lindemann DM, Liphardt SW, Little TJ, Macoretta N, Maddox D, Matkin CO, Mattison JA, McClure M, Mergl J, Meudt JJ, Montano GA, Mozhui K, Munshi-South J, Murphy WJ, Naderi A, Nagy M, Narayan P, Nathanielsz PW, Nguyen NB, Niehrs C, Nyamsuren B, O’Brien JK, Ginn PO, Odom DT, Ophir AG, Osborn S, Ostrander EA, Parsons KM, Paul KC, Pedersen AB, Pellegrini M, Peters KJ, Petersen JL, Pietersen DW, Pinho GM, Plassais J, Poganik JR, Prado NA, Reddy P, Rey B, Ritz BR, Robbins J, Rodriguez M, Russell J, Rydkina E, Sailer LL, Salmon AB, Sanghavi A, Schachtschneider KM, Schmitt D, Schmitt T, Schomacher L, Schook LB, Sears KE, Seifert AW, Shafer ABA, Shindyapina AV, Simmons M, Singh K, Sinha I, Slone J, Snell RG, Soltanmohammadi E, Spangler ML, Spriggs M, Staggs L, Stedman N, Steinman KJ, Stewart DT, Sugrue VJ, Szladovits B, Takahashi JS, Takasugi M, Teeling EC, Thompson MJ, Van Bonn B, Vernes SC, Villar D, Vinters HV, Vu H, Wallingford MC, Wang N, Wilkinson GS, Williams RW, Yan Q, Yao M, Young BG, Zhang B, Zhang Z, Zhao Y, Zhao P, Zhou W, Zoller JA, Ernst J, Seluanov A, Gorbunova V, Yang XW, Raj K, Horvath S.(2023). DNA methylation networks underlying mammalian traits. Science. doi: 10.1126/science.abq5693.

AbstractUsing DNA methylation profiles (n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species. More Information

Vijg J, Schumacher B, Abakir A, Antonov M, Bradley C, Cagan A, Church G, Gladyshev VN, Gorbunova V, Maslov AY, Reik W, Sharifi S, Suh Y, Walsh K (2023). Mitigating age-related somatic mutation burden. Trends Mol Med doi: 10.1016/j.molmed.2023.04.002.

AbstractGenomes are inherently unstable and require constant DNA repair to maintain their genetic information. However, selective pressure has optimized repair mechanisms in somatic cells only to allow transmitting genetic information to the next generation, not to maximize sequence integrity long beyond the reproductive age. Recent studies have confirmed that somatic mutations, due to errors during genome repair and replication, accumulate in tissues and organs of humans and model organisms. Here, we describe recent advances in the quantitative analysis of somatic mutations in vivo. We also review evidence for or against a possible causal role of somatic mutations in aging. Finally, we discuss options to prevent, delay or eliminate de novo, random somatic mutations as a cause of aging. More Information

Mitchell W, Goeminne L, Tyshkovskiy A, Zhang S, Paulo J, Pierce K, Choy A, Clish C, Gygi S, Gladyshev VN (2023). Multi-omics characterization of partial chemical reprogramming reveals evidence of cell rejuvenation. bioRxiv doi: https://doi.org/10.1101/2023.06.30.546730

AbstractPartial reprogramming by cyclic short-term expression of Yamanaka factors holds promise for shifting cells to younger states and consequently delaying the onset of many diseases of aging. However, the delivery of transgenes and potential risk of teratoma formation present challenges for in vivo applications. Recent advances include the use of cocktails of compounds to reprogram somatic cells, but the characteristics and mechanisms of partial cellular reprogramming by chemicals remain unclear. Here, we report a multi-omics characterization of partial chemical reprogramming in fibroblasts from young and aged mice. We measured the effects of partial chemical reprogramming on the epigenome, transcriptome, proteome, phosphoproteome, and metabolome. At the transcriptome, proteome, and phosphoproteome levels, we saw widescale changes induced by this treatment, with the most notable signature being an upregulation of mitochondrial oxidative phosphorylation. Furthermore, at the metabolome level, we observed a reduction in the accumulation of aging-related metabolites. Using both transcriptomic and epigenetic clock-based analyses, we show that partial chemical reprogramming reduces the biological age of mouse fibroblasts. We demonstrate that these changes have functional impacts, as evidenced by changes in cellular respiration and mitochondrial membrane potential. Taken together, these results illuminate the potential for chemical reprogramming reagents to rejuvenate aged biological systems, and warrant further investigation into adapting these approaches for in vivo age reversal. More Information

Zhang B, Lee DE, Trapp A, Tyshkovskiy A, Lu AT, Bareja A, Kerepesi C, McKay LK, Shindyapina AV, Dmitriev SE, Baht GS, Horvath S, Gladyshev VN, White JP. (2023). Multi-omic rejuvenation and life span extension on exposure to youthful circulation. Nature Aging doi: 10.1038/s43587-023-00451-9.

AbstractHeterochronic parabiosis (HPB) is known for its functional rejuvenation effects across several mouse tissues. However, its impact on biological age and long-term health is unknown. Here we performed extended (3-month) HPB, followed by a 2-month detachment period of anastomosed pairs. Old detached mice exhibited improved physiological parameters and lived longer than control isochronic mice. HPB drastically reduced the epigenetic age of blood and liver based on several clock models using two independent platforms. Remarkably, this rejuvenation effect persisted even after 2 months of detachment. Transcriptomic and epigenomic profiles of anastomosed mice showed an intermediate phenotype between old and young, suggesting a global multi-omic rejuvenation effect. In addition, old HPB mice showed gene expression changes opposite to aging but akin to several life span-extending interventions. Altogether, we reveal that long-term HPB results in lasting epigenetic and transcriptome remodeling, culminating in the extension of life span and health span. More Information

Poganik JR, Zhang B, Baht GS, Tyshkovskiy A, Deik A, Kerepesi C, Yim SH, Lu AT, Haghani A, Gong T, Hedman AM, Andolf E, Pershagen G, Almqvist C, Clish CB, Horvath S, White JP, Gladyshev VN. (2023). Biological age is increased by stress and restored upon recovery. Cell Metabolism. doi: 10.1016/j.cmet.2023.03.015.

AbstractAging is classically conceptualized as an ever-increasing trajectory of damage accumulation and loss of function, leading to increases in morbidity and mortality. However, recent in vitro studies have raised the possibility of age reversal. Here, we report that biological age is fluid and exhibits rapid changes in both directions. At epigenetic, transcriptomic, and metabolomic levels, we find that the biological age of young mice is increased by heterochronic parabiosis and restored following surgical detachment. We also identify transient changes in biological age during major surgery, pregnancy, and severe COVID-19 in humans and/or mice. Together, these data show that biological age undergoes a rapid increase in response to diverse forms of stress, which is reversed following recovery from stress. Our study uncovers a new layer of aging dynamics that should be considered in future studies. The elevation of biological age by stress may be a quantifiable and actionable target for future interventions. More Information

Ying K, Tyshkovskiy A, Trapp A, Liu H, Moqri M, Kerepesi C, Gladyshev VN (2023). ClockBase: a comprehensive platform for biological age profiling in human and mouse. bioRxiv. doi: 10.1101/2023.02.28.530532

AbstractAging represents the greatest risk factor for chronic diseases and mortality, but to understand it, we need the ability to measure biological age. In recent years, many machine learning algorithms based on omics data, termed aging clocks, have been developed that can accurately predict the age of biological samples. However, there is currently no resource for systematic profiling of biological age. Here, we describe ClockBase, a platform that features biological age estimates based on multiple aging clock models applied to more than 2,000 DNA methylation datasets and nearly 200,000 samples. We further provide an online interface for statistical analyses and visualization of the data. To show how this resource could facilitate the discovery of biological age-modifying factors, we describe a novel anti-aging drug candidate, zebularine, which reduces the biological age estimates based on all aging clock models tested. We also show that pulmonary fibrosis accelerates epigenetic age. Together, ClockBase provides a resource for the scientific community to quantify and explore biological ages of samples, thus facilitating discovery of new longevity interventions and age-accelerating conditions. More Information

Yang JH, Hayano M, Griffin PT, Amorim JA, Bonkowski MS, Apostolides JK, Salfati EL, Blanchette M, Munding EM, Bhakta M, Chew YC, Guo W, Yang X, Maybury-Lewis S, Tian X, Ross JM, Coppotelli G, Meer MV, Rogers-Hammond R, Sinclair DA (2023). Loss of epigenetic information as a cause of mammalian aging. Cell. doi: 10.1016/j.cell.2022.12.027

AbstractAll living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called “ICE” (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging. More Information

Moldakozhayev A, & Gladyshev VN (2023). Metabolism, homeostasis, and aging. Trends Endocrinol Metab. doi: 10.1016/j.tem.2023.01.003

AbstractWe propose a two-mode (pursuit/maintenance) model of metabolism defined by usable resource availability. Pursuit, consisting of anabolism and catabolism, dominates when usable resources are plentiful and leads to the generation of metabolic waste. In turn, maintenance of a system is activated by elevated metabolic waste during resource depletion. Interaction with the environment results in pendulum-like swings between these metabolic states in thriveless attempts to maintain the least deleterious organismal state – ephemeral homeostasis. Imperfectness of biological processes during these attempts supports the accumulation of the deleteriome, driving organismal aging. We discuss how metabolic adjustment by the environment and resource stabilization may modulate healthspan and lifespan. More Information

Liberman N, Rothi MN, Gerashchenko MV, Zorbas C, Boulias K, MacWhinnie FG, Ying AK, Taylor AF, Haddad JA, Shibuya H, Roach L, Dong A, Dellacona S, Lafontaine DL, Gladyshev VN, Greer EL. 18S rRNA methyltransferases DIMT1 and BUD23 drive intergenerational hormesis. Molecular Cell. doi: https://doi.org/10.1016/j.molcel.2023.08.014

AbstractHeritable non-genetic information can regulate a variety of complex phenotypes. However, what specific non-genetic cues are transmitted from parents to their descendants are poorly understood. Here, we perform metabolic methyl-labeling experiments to track the heritable transmission of methylation from ancestors to their descendants in the nematode Caenorhabditis elegans (C. elegans). We find heritable methylation in DNA, RNA, proteins, and lipids. We find that parental starvation elicits reduced fertility, increased heat stress resistance, and extended longevity in fed, naïve progeny. This intergenerational hormesis is accompanied by a heritable increase in N6ʹ-dimethyl adenosine (m6,2A) on the 18S ribosomal RNA at adenosines 1735 and 1736. We identified DIMT-1/DIMT1 as the m6,2A and BUD-23/BUD23 as the m7G methyltransferases in C. elegans that are both required for intergenerational hormesis, while other rRNA methyltransferases are dispensable. This study labels and tracks heritable non-genetic material across generations and demonstrates the importance of rRNA methylation for regulating epigenetic inheritance. More Information

Ying K, Paulson S, Perez-Guevara M, Emamifar M, Martínez MC, Kwon D, Poganik JR, Moqri M, Gladyshev VN. Biolearn, an open-source library for biomarkers of aging. bioRxiv doi: https://doi.org/10.1101/2023.12.02.569722

AbstractIdentifying and validating biomarkers of aging is pivotal for understanding the aging process and testing longevity interventions. Despite the development of numerous aging biomarkers, their clinical validation remains elusive, largely due to the lack of cross-population validation, which is hampered by disparate biomarker designs and inconsistencies in dataset structures. To bridge this gap, we introduce Biolearn, an innovative open-source library dedicated to the implementation and application of aging biomarkers. Biolearn facilitates (1) harmonization of existing aging biomarkers, while presenting a structured framework for novel biomarkers in standardized formats; (2) unification of public datasets, ensuring coherent structuring and formatting, thus simplifying cross-population validation studies; and (3) provision of computational methodologies to assess any harmonized biomarker against unified datasets. By furnishing a community-driven platform, Biolearn significantly augments the development, assessment, and validation trajectories of aging biomarkers, paving the way toward more rigorous clinical validation and, ultimately, application in clinical trials targeting healthy longevity. More Information

Firsanov D, Zacher M, Tian X, Zhao Y, George JC, Sformo TL, Tombline G, Biashad SA, Gilman A, Hamilton N, Patel A, Straight M, Lee M, Lu YJ, Haseljic E, Williams A, Miller N, Gladyshev VN, Zhang Z, Vijg J, Seluanov A, Gorbunova V. DNA repair and anti-cancer mechanisms in the longest-living mammal: the bowhead whale. bioRxiv doi: https://doi.org/10.1101/2023.05.07.539748

AbstractAt over 200 years, the maximum lifespan of the bowhead whale exceeds that of all other mammals. The bowhead is also the second-largest animal on Earth, reaching over 80,000 kg1. In spite of its very large number of cells, the bowhead is not highly cancer-prone, an incongruity termed Peto’s Paradox2. This has been explained by the evolution of additional tumor suppressor genes in larger animals, which is supported by research on elephants demonstrating expansion of the p53 gene3–5. However, we show here that bowhead whale fibroblasts undergo oncogenic transformation after disruption of fewer tumor suppressors than required for human fibroblasts. Instead, analysis of DNA repair revealed that bowhead cells repair double-strand breaks with uniquely high efficiency and accuracy compared to other mammals. Further, we identified two proteins, CIRBP and RPA2, that are present at high levels in bowhead fibroblasts and increase the efficiency and fidelity of DNA repair in human cells. These results suggest that rather than possessing additional tumor suppressor genes as barriers to oncogenesis, the bowhead whale relies on more accurate and efficient DNA repair to preserve genome integrity. This strategy that does not eliminate cells but repairs them may be critical for the long and cancer-free lifespan of the bowhead whale. Our work demonstrates the value of studying long-lived organisms in identifying novel longevity mechanisms and their potential for translation to humans. More Information

Ying K, Hanna Liu H, Andrei E. Tarkhov AE, Sadler MC, Lu AT, Moqri M, Horvath S, Kutalik Z, Shen X, Gladyshev VN. Causality-Enriched Epigenetic Age Uncouples Damage and Adaptation. bioRxiv doi: https://doi.org/10.1101/2022.10.07.511382

AbstractMachine learning models based on DNA methylation data can predict biological age but often lack causal insights. By harnessing large-scale genetic data through epigenome-wide Mendelian Randomization, we identified CpG sites potentially causal for aging-related traits. Neither the existing epigenetic clocks nor age-related differential DNA methylation are enriched in these sites. These CpGs include sites that contribute to aging and protect against it, yet their combined contribution negatively affects age-related traits. We established a novel framework to introduce causal information into epigenetic clocks, resulting in DamAge and AdaptAge—clocks that track detrimental and adaptive methylation changes, respectively. DamAge correlates with adverse outcomes, including mortality, while AdaptAge is associated with beneficial adaptations. These causality-enriched clocks exhibit sensitivity to short-term interventions. Our findings provide a detailed land-scape of CpG sites with putative causal links to lifespan and healthspan, facilitating the development of aging biomarkers, assessing interventions, and studying reversibility of age-associated changes. More Information

Bennett DF, Goyala A, Statzer C, Beckett CW, Tyshkovskiy A, Gladyshev VN, Ewald CY, & de Magalhães JP (2023). Rilmenidine extends lifespan and healthspan in Caenorhabditis elegans via a nischarin I1-imidazoline receptor. Aging Cell. doi: 10.1111/acel.13774

AbstractRepurposing drugs capable of extending lifespan and health span has a huge untapped potential in translational geroscience. Here, we searched for known compounds that elicit a similar gene expression signature to caloric restriction and identified rilmenidine, an I1-imidazoline receptor agonist and prescription medication for the treatment of hypertension. We then show that treating Caenorhabditis elegans with rilmenidine at young and older ages increases lifespan. We also demonstrate that the stress-resilience, health span, and lifespan benefits of rilmenidine treatment in C. elegans are mediated by the I1-imidazoline receptor nish-1, implicating this receptor as a potential longevity target. Consistent with the shared caloric-restriction-mimicking gene signature, supplementing rilmenidine to calorically restricted C. elegans, genetic reduction of TORC1 function, or rapamycin treatment did not further increase lifespan. The rilmenidine-induced longevity required the transcription factors FOXO/DAF-16 and NRF1,2,3/SKN-1. Furthermore, we find that autophagy, but not AMPK signaling, was needed for rilmenidine-induced longevity. Moreover, transcriptional changes similar to caloric restriction were observed in liver and kidney tissues in mice treated with rilmenidine. Together, these results reveal a geroprotective and potential caloric restriction mimetic effect by rilmenidine that warrant fresh lines of inquiry into this compound. More Information

2022 Articles

Petra A Tsuji, Didac Santesmasses, Byeong J Lee, Vadim N Gladyshev, Dolph L Hatfield (2022). Historical Roles of Selenium and Selenoproteins in Health and Development: The Good, the Bad and the Ugly. Int J Mol Sci. doi: 10.3390/ijms23010005

AbstractSelenium is a fascinating element that has a long history, most of which documents it as a deleterious element to health. In more recent years, selenium has been found to be an essential element in the diet of humans, all other mammals, and many other life forms. It has many health benefits that include, for example, roles in preventing heart disease and certain forms of cancer, slowing AIDS progression in HIV patients, supporting male reproduction, inhibiting viral expression, and boosting the immune system, and it also plays essential roles in mammalian development. Elucidating the molecular biology of selenium over the past 40 years generated an entirely new field of science which encompassed the many novel features of selenium. These features were (1) how this element makes its way into protein as the 21st amino acid in the genetic code, selenocysteine (Sec); (2) the vast amount of machinery dedicated to synthesizing Sec uniquely on its tRNA; (3) the incorporation of Sec into protein; and (4) the roles of the resulting Sec-containing proteins (selenoproteins) in health and development. One of the research areas receiving the most attention regarding selenium in health has been its role in cancer prevention, but further research has also exposed the role of this element as a facilitator of various maladies, including cancer. More Information

Huafeng Wang, Qianhui Dou, Kyung Jo Jeong, Jungmin Choi, Vadim N Gladyshev, Jean-Ju Chung (2022). Redox regulation by TXNRD3 during epididymal maturation underlies capacitation-associated mitochondrial activity and sperm motility in mice. J Biol Chem. doi: 10.1016/j.jbc.2022.102077

AbstractDuring epididymal transit, redox remodeling protects mammalian spermatozoa, preparing them for survival in the subsequent journey to fertilization. However, molecular mechanisms of redox regulation in sperm development and maturation remain largely elusive. In this study, we report that thioredoxin-glutathione reductase (TXNRD3), a thioredoxin reductase family member particularly abundant in elongating spermatids at the site of mitochondrial sheath formation, regulates redox homeostasis to support male fertility. Using Txnrd3-/- mice, our biochemical, ultrastructural, and live cell imaging analyses revealed impairments in sperm morphology and motility under conditions of TXNRD3 deficiency. We find that mitochondria develop more defined cristae during capacitation in wildtype sperm. Furthermore, we show that absence of TXNRD3 alters thiol redox status in both the head and tail during sperm maturation and capacitation, resulting in defective mitochondrial ultrastructure and activity under capacitating conditions. These findings provide insights into molecular mechanisms of redox homeostasis and bioenergetics during sperm maturation, capacitation, and fertilization. More Information

Stephan Emmrich, Alexandre Trapp, Frances Tolibzoda Zakusilo, Maggie E Straight, Albert K Ying, Alexander Tyshkovskiy, Marco Mariotti, Spencer Gray, Zhihui Zhang, Michael G Drage, Masaki Takasugi, Jan-Henning Klusmann , Vadim N Gladyshev, Andrei Seluanov , Vera Gorbunova (2022). Characterization of naked mole-rat hematopoiesis reveals unique stem and progenitor cell patterns and neotenic traits. EMBO J doi: 10.15252/embj.2021109694

AbstractNaked mole rats (NMRs) are the longest-lived rodents yet their stem cell characteristics remain enigmatic. Here, we comprehensively mapped the NMR hematopoietic landscape and identified unique features likely contributing to longevity. Adult NMRs form red blood cells in spleen and marrow, which comprise a myeloid bias toward granulopoiesis together with decreased B-lymphopoiesis. Remarkably, youthful blood and marrow single-cell transcriptomes and cell compositions are largely maintained until at least middle age. Similar to primates, the primitive stem and progenitor cell (HSPC) compartment is marked by CD34 and THY1. Stem cell polarity is seen for Tubulin but not CDC42, and is not lost until 12 years of age. HSPC respiration rates are as low as in purified human stem cells, in concert with a strong expression signature for fatty acid metabolism. The pool of quiescent stem cells is higher than in mice, and the cell cycle of hematopoietic cells is prolonged. By characterizing the NMR hematopoietic landscape, we identified resilience phenotypes such as an increased quiescent HSPC compartment, absence of age-related decline, and neotenic traits likely geared toward longevity. More Information

Didac Santesmasses, Vadim N Gladyshev (2022). Selenocysteine Machinery Primarily Supports TXNRD1 and GPX4 Functions and Together They Are Functionally Linked with SCD and PRDX6. Biomolecules. Biomolecules doi: 10.3390/biom12081049

AbstractThe human genome has 25 genes coding for selenocysteine (Sec)-containing proteins, whose synthesis is supported by specialized Sec machinery proteins. Here, we carried out an analysis of the co-essentiality network to identify functional partners of selenoproteins and Sec machinery. One outstanding cluster included all seven known Sec machinery proteins and two critical selenoproteins, GPX4 and TXNRD1. Additionally, these nine genes were further positively associated with PRDX6 and negatively with SCD, linking the latter two genes to the essential role of selenium. We analyzed the essentiality scores of gene knockouts in this cluster across one thousand cancer cell lines and found that Sec metabolism genes are strongly selective for a subset of primary tissues, suggesting that certain cancer cell lineages are particularly dependent on selenium. A separate outstanding cluster included selenophosphate synthetase SEPHS1, which was linked to a group of transcription factors, whereas the remaining selenoproteins were linked neither to these clusters nor among themselves. The data suggest that key components of Sec machinery have already been identified and that their primary role is to support the functions of GPX4 and TXNRD1, with further functional links to PRDX6 and SCD. More Information

Qianhui Dou, Anton A Turanov, Marco Mariotti, Jae Yeon Hwang, Huafeng Wang, Sang-Goo Lee, Joao A Paulo, Sun Hee Yim, Stephen P Gygi, Jean-Ju Chung, Vadim N Gladyshev (2022). Selenoprotein TXNRD3 supports male fertility via the redox regulation of spermatogenesis. J Biol Chem doi: 10.1016/j.jbc.2022.102183

AbstractThioredoxin/glutathione reductase (TXNRD3) is a selenoprotein composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent thiol oxidoreductase evolved through gene duplication within the Txnrd family, is expressed in the testes, and can reduce both thioredoxin and glutathione in vitro; however, the function of this enzyme remains unknown. To characterize the function of TXNRD3 in vivo, we generated a strain of mice bearing deletion of Txnrd3 gene. We show that these Txnrd3 knockout mice are viable and without discernable gross phenotypes, and also that TXNRD3 deficiency leads to fertility impairment in male mice. We found that Txnrd3 knockout animals exhibited a lower fertilization rate in vitro, a sperm movement phenotype, and an altered thiol redox status in sperm cells. Proteomic analyses further revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of sperm quality control. Taken together, these results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis. More Information

Lionel Tarrago, Alaattin Kaya, Hwa-Young Kim, Bruno Manta, Byung-Cheon Lee , Vadim N Gladyshev(2022) The selenoprotein methionine sulfoxide reductase B1 (MSRB1). Free Radic Biol Med doi: 10.1016/j.freeradbiomed.2022.08.043

AbstractMethionine (Met) can be oxidized to methionine sulfoxide (MetO), which exist as R- and S-diastereomers. Present in all three domains of life, methionine sulfoxide reductases (MSR) are the enzymes that reduce MetO back to Met. Most characterized among them are MSRA and MSRB, which are strictly stereospecific for the S- and R-diastereomers of MetO, respectively. While the majority of MSRs use a catalytic Cys to reduce their substrates, some employ selenocysteine. This is the case of mammalian MSRB1, which was initially discovered as selenoprotein SELR or SELX and later was found to exhibit an MSRB activity. Genomic analyses demonstrated its occurrence in most animal lineages, and biochemical and structural analyses uncovered its catalytic mechanism. The use of transgenic mice and mammalian cell culture revealed its physiological importance in the protection against oxidative stress, maintenance of neuronal cells, cognition, cancer cell proliferation, and the immune response. Coincident with the discovery of Met oxidizing MICAL enzymes, recent findings of MSRB1 regulating the innate immunity response through reversible stereospecific Met-R-oxidation of cytoskeletal actin opened up new avenues for biological importance of MSRB1 and its role in disease. In this review, we discuss the current state of research on MSRB1, compare it with other animal Msrs, and offer a perspective on further understanding of biological functions of this selenoprotein. More Information

Mahdi Moqri, Andrea Cipriano, Daniel Nachun, Tara Murty, Guilherme de Sena Brandine, Sajede Rasouli, Andrei Tarkhov, Karolina A. Aberg, Edwin van den Oord, Wanding Zhou, Andrew Smith, Crystal Mackall, Vadim Gladyshev, Steve Horvath, Michael P. Snyder, Vittorio Sebastiano (2022) PRC2 clock: a universal epigenetic biomarker of aging and rejuvenation. bioRxiv doi: https://doi.org/10.1101/2022.06.03.49460

AbstractDNA methylation (DNAm) is one of the most reliable biomarkers for aging across many mammalian tissues. While the age-dependent global loss of DNAm has been well characterized, age-dependent DNAm gain is less specified. Multiple studies have demonstrated that polycomb repressive complex 2 (PRC2) targets are enriched among the CpG sites which gain methylation with age. However, a systematic whole-genome examination of all PRC2 targets in the context of aging methylome as well as whether these associations are pan-tissue or tissue-specific is lacking. Here, by analyzing DNAm data from different assays and from multiple young and old human and mouse tissues, we found that low-methylated regions (LMRs) which are highly bound by PRC2 in embryonic stem cells gain methylation with age in all examined somatic mitotic cells. We also estimated that this epigenetic change represents around 90% of the age-dependent DNAm gain genome-wide. Therefore, we propose the “PRC2 clock,” defined as the average DNAm in PRC2 LMRs, as a universal biomarker of cellular aging in somatic cells. In addition, we demonstrate the application of this biomarker in the evaluation of different anti-aging interventions, including dietary restriction and partial epigenetic reprogramming. More Information

Kejun Ying, Hanna Liu, Andrei E. Tarkhov, Ake T. Lu, Steve Horvath, Zoltán Kutalik, Xia Shen, Vadim N. Gladyshev (2022) Causal Epigenetic Age Uncouples Damage and Adaptation. bioRxiv doi: https://doi.org/10.1101/2022.10.07.511382.

AbstractMachine learning models based on DNA methylation can be used to predict the age of biological samples, but their interpretability is limited due to the lack of causal inferences. Here, we lever-aged large-scale genetic data and performed epigenome-wide Mendelian Randomization to identify CpG sites causal to aging-related traits. We show that neither the existing epigenetic clocks nor DNA methylation changes are enriched in causal CpG sites. Causal CpGs include similar numbers of sites that contribute to aging and protect against it, yet their combined contribution negatively affects age-related traits. We developed a framework for integrating causal knowledge into epigenetic clock models and constructed DamAge and AdaptAge that measure age-related damaging and adaptive changes, respectively. DamAge acceleration is associated with various adverse conditions (e.g., mortality risk), whereas AdaptAge acceleration is related to beneficial adaptations. Only DamAge is reversed upon cell reprogramming. Our results offer a comprehensive map of CpG sites causal to lifespan and healthspan, allowing to build causal biomarkers of aging and rejuvenation and assess longevity interventions, age reversal, and aging-accelerating events. More Information

Andrei E. Tarkhov, Thomas Lindstrom-Vautrin, Sirui Zhang, Kejun Ying, Mahdi Moqri, Bohan Zhang, Vadim N. Gladyshev (2022) Nature of epigenetic aging from a single-cell perspective. bioRxiv doi: https://doi.org/10.1101/2022.09.26.509592.

Jeyoung Bang, Donghyun Kang, Jisu Jung, Tack-Jin Yoo, Myoung Sup Shim, Vadim N Gladyshev, Petra A Tsuji, Dolph L Hatfield, Jin-Hong Kim, Byeong Jae Lee (2022) SEPHS1: Its evolution, function and roles in development and diseases. Arch Biochem Biophys doi: 10.1016/j.abb.2022.109426.

AbstractSelenophosphate synthetase (SEPHS) was originally discovered in prokaryotes as an enzyme that catalyzes selenophosphate synthesis using inorganic selenium and ATP as substrates. However, in contrast to prokaryotes, two paralogs, SEPHS1 and SEPHS2, occur in many eukaryotes. Prokaryotic SEPHS, also known as SelD, contains either cysteine (Cys) or selenocysteine (Sec) in the catalytic domain. In eukaryotes, only SEPHS2 carries out selenophosphate synthesis and contains Sec at the active site. However, SEPHS1 contains amino acids other than Sec or Cys at the catalytic position. Phylogenetic analysis of SEPHSs reveals that the ancestral SEPHS contains both selenophosphate synthesis and another unknown activity, and that SEPHS1 lost the selenophosphate synthesis activity. The three-dimensional structure of SEPHS1 suggests that its homodimer is unable to form selenophosphate, but retains ATPase activity to produce ADP and inorganic phosphate. The most prominent function of SEPHS1 is that it is implicated in the regulation of cellular redox homeostasis. Deficiency of SEPHS1 leads to the disturbance in the expression of genes involved in redox homeostasis. Different types of reactive oxygen species (ROS) are accumulated in response to SEPHS deficiency depending on cell or tissue types. The accumulation of ROS causes pleiotropic effects such as growth retardation, apoptosis, DNA damage, and embryonic lethality. SEPHS1 deficiency in mouse embryos affects retinoic signaling and other related signaling pathways depending on the embryonal stage until the embryo dies at E11.5. Dysregulated SEPHS1 is associated with the pathogenesis of various diseases including cancer, Crohn’s disease, and osteoarthritis. More Information

Bohan Zhang, Andrei E. Tarkhov, Wil Ratzan, Kejun Ying, Mahdi Moqri, Jesse R. Poganik, Benjamin Barre, Alexandre Trapp, Joseph A. Zoller, Amin Haghani, Steve Horvath, Leonid Peshkin, Vadim N. Gladyshev(2022) Epigenetic profiling and incidence of disrupted development point to gastrulation as aging ground zero in Xenopus laevis. bioRxive doi: https://doi.org/10.1101/2022.08.02.502559

AbstractRecent studies suggest the existence of a natural rejuvenation event during early embryonic development of mice, followed by epigenetic aging. Here, by profiling embryonic DNA methylation in the African clawed frog, Xenopus laevis, we found that the epigenetic entropy basepoint maps to the gastrulation stage of embryogenesis and corresponds to a rapid increase in embryo transcript abundance. We further developed a frog aging clock, revealing that this species epigenetically ages. Application of this clock to developmental stages identified a decrease in epigenetic age during early embryogenesis, with the minimal age reached around gastrulation. By examining individual developmental trajectories of 6,457 embryos, we found that this stage is also accompanied by a higher incidence of disrupted development. Taken together, our data point to gastrulation as a critical stage for aging and natural rejuvenation, characterized by the lowest epigenetic age, increased mortality, nadir of DNA methylation entropy and rapid increase in embryo transcript abundance, defining aging ground zero as the basepoint where rejuvenation ends and the aging process begins. More Information

Jesse R. Poganik, Bohan Zhang, Gurpreet S. Baht, Csaba Kerepesi, Sun Hee Yim, Ake T. Lu, Amin Haghani, Tong Gong, Anna M. Hedman, Ellika Andolf, Göran Pershagen, Catarina Almqvist, James P. White, Steve Horvath, Vadim N. Gladyshev (2022) Biological age is increased by stress and restored upon recovery. bioRxive doi:https://doi.org/10.1101/2022.05.04.490686

AbstractAging is classically conceptualized as an ever-increasing trajectory of damage accumulation and loss of function, leading to increases in morbidity and mortality. However, recent in vitro studies have raised the possibility of age reversal. Here, we report that biological age is fluid and exhibits rapid changes in both directions. By applying advanced epigenetic aging clocks, we find that the biological age of young mice is increased by heterochronic parabiosis and restored following surgical detachment of animals. We also identify transient changes in biological age during major surgery, pregnancy, and severe COVID-19 in humans and/or mice. Together, these data show that biological age undergoes a rapid increase in response to diverse forms of stress, which is reversed following recovery from stress. Our study uncovers a new layer of aging dynamics that should be considered in future studies. Elevation of biological age by stress may be a quantifiable and actionable target for future interventions. More Information

Shindyapina AV, Cho Y, Kaya A, Tyshkovskiy A, Castro JP, Deik A, Gordevicius J, Poganik JR, Clish CB, Horvath S, Peshkin L, Gladyshev VN (2022) Rapamycin treatment during development extends life span and health span of male mice and Daphnia magna. Science Advances 8, 37.

AbstractDevelopment is tightly connected to aging, but whether pharmacologically targeting development can extend life remains unknown. Here, we subjected genetically diverse UMHET3 mice to rapamycin for the first 45 days of life. The mice grew slower and remained smaller than controls for their entire lives. Their reproductive age was delayed without affecting offspring numbers. The treatment was sufficient to extend the median life span by 10%, with the strongest effect in males, and helped to preserve health as measured by frailty index scores, gait speed, and glucose and insulin tolerance tests. Mechanistically, the liver transcriptome and epigenome of treated mice were younger at the completion of treatment. Analogous to mice, rapamycin exposure during development robustly extended the life span of Daphnia magna and reduced its body size. Overall, the results demonstrate that short-term rapamycin treatment during development is a novel longevity intervention that acts by slowing down development and aging, suggesting that aging may be targeted already early in life. More Information

Kerepesi C, Meer MV, Ablaeva J, Amoroso VG, Lee SG, Zhang B, Gerashchenko MV, Trapp A, Yim SH, Lu AT, Levine ME, Seluanov A, Horvath S, Park TJ, Gorbunova V, Gladyshev VN (2022) Epigenetic aging of the demographically non-aging naked mole-rat. Nature Communications 13, 355.

AbstractThe naked mole-rat (NMR) is an exceptionally long-lived rodent that shows no increase of mortality with age, defining it as a demographically non-aging mammal. Here, we perform bisulfite sequencing of the blood of > 100 NMRs, assessing > 3 million common CpG sites. Unsupervised clustering based on sites whose methylation correlates with age reveals an age-related methylome remodeling, and we also observe a methylome information loss, suggesting that NMRs age. We develop an epigenetic aging clock that accurately predicts the NMR age. We show that these animals age much slower than mice and much faster than humans, consistent with their known maximum lifespans. Interestingly, patterns of age-related changes of clock sites in Tert and Prpf19 differ between NMRs and mice, but there are also sites conserved between the two species. Together, the data indicate that NMRs, like other mammals, epigenetically age even in the absence of demographic aging of this species. More Information

Zhang B, Trapp A, Kerepesi C, Gladyshev VN (2022) Emerging rejuvenation strategies-Reducing the biological age. Aging Cell 21, e13538.

Abstract Several interventions have recently emerged that were proposed to reverse rather than just attenuate aging, but the criteria for what it takes to achieve rejuvenation remain controversial. Distinguishing potential rejuvenation therapies from other longevity interventions, such as those that slow down aging, is challenging, and these anti-aging strategies are often referred to interchangeably. We suggest that the prerequisite for a rejuvenation intervention is a robust, sustained, and systemic reduction in biological age, which can be assessed by biomarkers of aging, such as epigenetic clocks. We discuss known and putative rejuvenation interventions and comparatively analyze them to explore underlying mechanisms. More Information

Mariotti M, Kerepesi C, Oliveros W, Mele M, Gladyshev VN (2022) Deterioration of the human transcriptome with age due to increasing intron retention and spurious splicing. bioRxiv, 10.1101/2022.03.14.484341

Abstract Adult aging is characterized by a progressive deterioration of biological functions at physiological, cellular and molecular levels, but its damaging effects on the transcriptome are not well characterized. Here, by analyzing splicing patterns in ∼1,000 human subjects sampled across multiple tissues, we found that splicing fidelity declines with age. Most prominently, genuine introns fail to be spliced out, manifesting as a broad surge in intron retention, and this is exacerbated by the increase in diverse spurious exon-exon junctions with age. Both of these effects are prominently detected in the majority of human tissues. Collectively, they result in the progressive deterioration of the active transcriptome, wherein functional mRNAs are increasingly diluted with non-functional splicing isoforms. We discuss the concept of “splicing damage” and formulate methods to quantify it. Using these tools, we show that splicing damage increases both with age and with the incidence of diseases. Altogether, this work uncovers transcriptome damage as a critical molecular indicator of human aging and healthspan. More Information

Meron E, Thaysen M, Angeli S, Antebi A, Barzilai N, Baur JA, Bekker-Jensen S, Birkisdottir M, Bischof E, Bruening J, Brunet A, Buchwalter A, Cabreiro F, Fortney K, Freund A, Georgievskaya A, Gladyshev VN, Glass D, Golato T, Gorbunova V, Hoejimakers J, Houtkooper RH, Jager S, Jaksch F, Janssens G, Jensen MB, Kaeberlein M, Karsenty G, de Keizer P, Kennedy B, Kirkland JL, Kjaer M, Kroemer G, Lee KF, Lemaitre JM, Liaskos D, Longo VD, Lu YX, MacArthur MR, Maier AB, Manakanatas C, Mitchell SJ, Moskalev A, Niedernhofer L, Ozerov I, Partridge L, Passegué E, Petr MA, Peyer J, Radenkovic D, Rando TA, Rattan S, Riedel CG, Rudolph L, Ai R, Serrano M, Schumacher B, Sinclair DA, Smith R, Suh Y, Taub P, Trapp A, Trendelenburg AU, Valenzano DR, Verburgh K, Verdin E, Vijg J, Westendorp RGJ, Zonari A, Bakula D, Zhavoronkov A, Scheibye-Knudsen M (2022) Meeting Report: Aging Research and Drug Discovery. Aging 14, 530-543.

Abstract Aging is the single largest risk factor for most chronic diseases, and thus possesses large socioeconomic interest to continuously aging societies. Consequently, the field of aging research is expanding alongside a growing focus from the industry and investors in aging research. This year’s 8th Annual Aging Research and Drug Discovery (ARDD) meeting was organized as a hybrid meeting from August 30th to September 3rd 2021 with more than 130 attendees participating on-site at the Ceremonial Hall at University of Copenhagen, Denmark, and 1800 engaging online. The conference comprised of presentations from 75 speakers focusing on new research in topics including mechanisms of aging and how these can be modulated as well as the use of AI and new standards of practices within aging research. This year, a longevity workshop was included to build stronger connections with the clinical community. More Information

Kang D, Lee J, Jung J, Carlson BA, Chang MJ, Chang CB, Kang SB, Lee BC, Gladyshev VN, Hatfield DL, Lee BJ, Kim JH (2022) Selenophosphate synthetase 1 deficiency exacerbates osteoarthritis by dysregulating redox homeostasis. Nature Communications 13, 779.

AbstractAging and mechanical overload are prominent risk factors for osteoarthritis (OA), which lead to an imbalance in redox homeostasis. The resulting state of oxidative stress drives the pathological transition of chondrocytes during OA development. However, the specific molecular pathways involved in disrupting chondrocyte redox homeostasis remain unclear. Here, we show that selenophosphate synthetase 1 (SEPHS1) expression is downregulated in human and mouse OA cartilage. SEPHS1 downregulation impairs the cellular capacity to synthesize a class of selenoproteins with oxidoreductase functions in chondrocytes, thereby elevating the level of reactive oxygen species (ROS) and facilitating chondrocyte senescence. Cartilage-specific Sephs1 knockout in adult mice causes aging-associated OA, and augments post-traumatic OA, which is rescued by supplementation of N-acetylcysteine (NAC). Selenium-deficient feeding and Sephs1 knockout have synergistic effects in exacerbating OA pathogenesis in mice. Therefore, we propose that SEPHS1 is an essential regulator of selenium metabolism and redox homeostasis, and its dysregulation governs the progression of OA. More Information

Lee HM, Choi DW, Kim S, Lee A, Kim M, Roh YJ, Jo YH, Cho HY, Lee HJ, Lee SR, Tarrago L, Gladyshev VN, Kim JH, Lee BC (2022) Biosensor-Linked Immunosorbent Assay for the Quantification of Methionine Oxidation in Target Proteins. ACS Sensors 7, 131-141.

Abstract Methionine oxidation is involved in regulating the protein activity and often leads to protein malfunction. However, tools for quantitative analyses of protein-specific methionine oxidation are currently unavailable. In this work, we developed a biological sensor that quantifies oxidized methionine in the form of methionine-R-sulfoxide in target proteins. The biosensor “tpMetROG” consists of methionine sulfoxide reductase B (MsrB), circularly permuted yellow fluorescent protein (cpYFP), thioredoxin, and protein G. Protein G binds to the constant region of antibodies against target proteins, specifically capturing them. Then, MsrB reduces the oxidized methionine in these proteins, leading to cpYFP fluorescence changes. We assessed this biosensor for quantitative analysis of methionine-R-sulfoxide in various proteins, such as calmodulin, IDLO, LegP, Sacde, and actin. We further developed an immunosorbent assay using the biosensor to quantify methionine oxidation in specific proteins such as calmodulin in animal tissues. The biosensor-linked immunosorbent assay proves to be an indispensable tool for detecting methionine oxidation in a protein-specific manner. This is a versatile tool for studying the redox biology of methionine oxidation in proteins. More Information

Tian R, Han K, Geng Y, Yang C, Shi C, Thomas PB, Pearce C, Moffatt K, Ma S, Xu S, Yang G, Zhou X, Gladyshev VN, Liu X, Fisher DO, Chopin LK, Leiner NO, Baker AM, Fan G, Seim I (2022) A chromosome-level genome of Antechinus flavipes provides a reference for an Australian marsupial genus with male death after mating. Molecular Ecology Resources 22, 740-754.

Abstract The 15 species of small carnivorous marsupials that comprise the genus Antechinus exhibit semelparity, a rare life-history strategy in mammals where synchronized death occurs after one breeding season. Antechinus males, but not females, age rapidly (demonstrate organismal senescence) during the breeding season and show promise as new animal models of ageing. Some antechinus species are also threatened or endangered. Here, we report a chromosome-level genome of a male yellow-footed antechinus Antechinus flavipes. The genome assembly has a total length of 3.2 Gb with a contig N50 of 51.8 Mb and a scaffold N50 of 636.7 Mb. We anchored and oriented 99.7% of the assembly on seven pseudochromosomes and found that repetitive DNA sequences occupy 51.8% of the genome. Draft genome assemblies of three related species in the subfamily Phascogalinae, two additional antechinus species (Antechinus argentus and A. arktos) and the iteroparous sister species Murexia melanurus, were also generated. Preliminary demographic analysis supports the hypothesis that climate change during the Pleistocene isolated species in Phascogalinae and shaped their population size. A transcriptomic profile across the A. flavipes breeding season allowed us to identify genes associated with aspects of the male die-off. The chromosome-level A. flavipes genome provides a steppingstone to understanding an enigmatic life-history strategy and a resource to assist the conservation of antechinuses. More Information

2021 Articles

Gladyshev VN, Kritchevsky SB, Clarke SG, Cuervo AM, Fiehn O, de Magalhães JP, Mau T, Maes M, Moritz RL, Niedernhofer LJ, Schaftingen EV, Tranah GJ, Walsh K, Yura Y, Zhang B, Cummings SR (2021) Molecular damage in aging. Nature Aging, 1, 1096–1106

Abstract Cellular metabolism and environmental interactions generate molecular damage affecting all levels of biological organization. Accumulation of this damage over time is thought to have a central role in the aging process. Insufficient attention has been paid to the role of molecular damage in aging-related phenotypes, particularly in humans, in part because of the difficulty in measuring its various forms. Recently, omics approaches have been developed that begin to address this challenge, because they can assess a sizable proportion of age-related damage at the level of small molecules, proteins, RNA, DNA, organelles and cells. This Review describes the concept of molecular damage in aging and discusses its diverse aspects from theoretical models to experimental approaches. Measurement of multiple types of damage enables studies of the role of damage in aging and lays a foundation for testing interventions that reduce the burden of molecular damage, thereby targeting aging. More Information

Trapp A, Kerepesi C & Gladyshev VN (2021) Profiling epigenetic age in single cells. Nature Aging, 1, 1189–1201

Abstract DNA methylation of a defined set of CpG dinucleotides emerged as a critical and precise biomarker of the aging process. Multi-variate machine learning models, known as epigenetic clocks, can exploit quantitative changes in the methylome to predict the age of bulk tissue with remarkable accuracy. However, intrinsic sparsity and digitized methylation in individual cells have so far precluded the assessment of aging in single cell data. Here, we present scAge, a probabilistic approach to determine the epigenetic age of single cells, and validate our results in mice. scAge tissue-specific and multi-cell type single cell clocks correctly recapitulate chronological age of the original tissue, while uncovering the inherent heterogeneity that exists at the single-cell level. The data suggest that while tissues age in a coordinated fashion, some cells age more or less rapidly than others. We show that individual embryonic stem cells exhibit an age close to zero, that certain stem cells in a tissue show a reduced age compared to their chronological age, and that early embryogenesis is associated with the reduction of epigenetic age of individual cells, the latter supporting a natural rejuvenation event during gastrulation. scAge is both robust against the low coverage that is characteristic of single cell sequencing techniques and is flexible for studying any cell type and vertebrate organism of interest. This study demonstrates for the first time the potential for accurate epigenetic age profiling at single-cell resolution. More Information

Zhang B, Lee DE, Trapp A, Tyshkovskiy A, Lu AT, Bareja A, Kerepesi C, Katz LH, Shindyapina AV, Dmitriev SE, Baht GS, Horvath S, Gladyshev VN (2021) Multi-omic rejuvenation and lifespan extension upon exposure to youthful circulation. bioRxiv, 10.1101/2021.11.11.468258

Abstract Heterochronic parabiosis (HPB) is known for its functional rejuvenation effects across several mouse tissues. However, its impact on the biological age of organisms and their long-term health remains unknown. Here, we performed extended (3-month) HPB, followed by a 2-month detachment period of anastomosed pairs. Old detached mice exhibited improved physiological parameters and lived longer than control isochronic mice. HPB drastically reduced the biological age of blood and liver based on epigenetic analyses across several clock models on two independent platforms; remarkably, this rejuvenation effect persisted even after 2 months of detachment. Transcriptomic and epigenomic profiles of anastomosed mice showed an intermediate phenotype between old and young, suggesting a comprehensive multi-omic rejuvenation effect. In addition, old HPB mice showed transcriptome changes opposite to aging, but akin to several lifespan-extending interventions. Altogether, we reveal that long-term HPB can decrease the biological age of mice, in part through long-lasting epigenetic and transcriptome remodeling, culminating in the extension of lifespan and healthspan. More Information

Trapp A & Gladyshev VN (2021) Cost-effective epigenetic age profiling in shallow methylation sequencing data. bioRxiv, 10.1101/2021.10.25.465778

Abstract There is a critical need for robust, high-throughput assays of biological aging trajectories. Among various approaches, epigenetic aging clocks emerged as reliable molecular trackers of the aging process. However, current methods for epigenetic age profiling are inherently costly and lack throughput. Here, we leverage the scAge framework for accurate prediction of biological age from very few bisulfite sequencing reads in bulk samples, thereby enabling drastic (100-1,000-fold) reduction in sequencing costs per sample. Our approach permits age assessment based on distinct assortments of CpG sites in different samples, without the need for targeted site enrichment or specialized reagents. We demonstrate the efficacy of this method to quantify the age of mouse blood samples across independent cohorts, identify the effect of calorie restriction as an attenuator of the aging process, and discern rejuvenation upon cellular reprogramming. We propose that this framework may be used for epigenetic age prediction in extremely high-throughput applications, enabling robust, large-scale and inexpensive interventions testing and age profiling. More Information

Griffin PT, Kane AE, Trapp A, Li J, McNamara MS, Meer MV, MacArthur MR, Mitchell SJ, Mueller AL, Colleen C, Vera DL, Kerepesi C, Hooten NN, Mitchell JR, Evans MK, Gladyshev VN, Sinclair DA (2021) Ultra-cheap and scalable epigenetic age predictions with TIME-Seq. bioRxiv, 10.1101/2021.10.25.465725

Abstract Epigenetic “clocks” based on DNA methylation (DNAme) are the most robust and widely employed aging biomarker. They have been built for numerous species and reflect gold-standard interventions that extend lifespan. However, conventional methods for measuring epigenetic clocks are expensive and low-throughput. Here, we describe Tagmentation-based Indexing for Methylation Sequencing (TIME-Seq) for ultra-cheap and scalable targeted methylation sequencing of epigenetic clocks and other DNAme biomarkers. Using TIME-Seq, we built and validated inexpensive epigenetic clocks based on genomic and ribosomal DNAme in hundreds of mice and human samples. We also discover it is possible to accurately predict age from extremely low-cost shallow sequencing (e.g., 10,000 reads) of TIME-Seq libraries using scAge, a probabilistic age-prediction algorithm originally applied to single cells. Together, these methods reduce the cost of DNAme biomarker analysis by more than two orders of magnitude, thereby expanding and democratizing their use in aging research, clinical trials, and disease diagnosis. More Information

Kerepesi C, Zhang B, Lee SL, Trapp A & Gladyshev VN (2021) Epigenetic clocks reveal a rejuvenation event during embryogenesis followed by aging. Science Advances 7, eabg6082.

Abstract The notion that the germ line does not age goes back to the 19th-century ideas of August Weismann. However, being metabolically active, the germ line accumulates damage and other changes over time, i.e., it ages. For new life to begin in the same young state, the germ line must be rejuvenated in the offspring. Here, we developed a multi-tissue epigenetic clock and applied it, together with other aging clocks, to track changes in biological age during mouse and human prenatal development. This analysis revealed a significant decrease in biological age, i.e., rejuvenation, during early stages of embryogenesis, followed by an increase in later stages. We further found that pluripotent stem cells do not age even after extensive passaging and that the examined epigenetic age dynamics is conserved across species. Overall, this study uncovers a natural rejuvenation event during embryogenesis and suggests that the minimal biological age (ground zero) marks the beginning of organismal aging. More Information

Shindyapina AV, Castro JP, Barbieri A, Strelkova OS, Paulo JA, Kerepesi C, Petrashen AP, Mariotti M, Meer M, Hu Y, Losyev G, Indzhykulian AA, Gygi SP, Sedivy JM, Manis JP & Gladyshev VN (2021) Aging predisposes B cells to malignancy by activating c-Myc and perturbing the genome and epigenome. bioRxiv, 10.1101/2021.02.23.432500.

Abstract Age is the single major risk factor for human cancer; however, naturally occurring cancers are rarely studied in aged animal models. Laboratory mouse strains spontaneously develop cancer with age and some predominantly die from B-cell lymphoma. Here, we uncover how B-cell lymphoma develops as a consequence of the aging immune system. We found that aged B cells undergo clonal expansions driven by genetic and epigenetic changes and established cell and spleen size as early markers of malignant transformation. High-throughput and omics assays of aged B cells and the use of mouse models revealed that c-Myc is a master regulator of B cell size and clonal expansion. A single-cell RNA-seq analysis suggested that clonal B cells originate from age-associated B cells, memory B cells that accumulate during aging. Further studies showed that c-Myc becomes activated in B cells in response to the aging microenvironment. Thus, c-Myc, aging environment, somatic mutations and the epigenome cooperate to give rise to clonal age-accelerated B cells, which we named Myc+ cells. We further show the relevance of this model to aged human B cells in blood and spleen. This study characterized a first mouse model that captures a natural transition of B cells to a prevalent type of cancer during aging. More Information

Ying K, Zhai R, Pyrkov TV, Shindyapina AV, Mariotti M, Fedichev PO, Shen X &; Gladyshev VN (2021) Genetic and phenotypic analysis of the causal relationship between aging and COVID-19. Communications Medicine 5, e2144.

Abstract Epidemiological studies revealed that the elderly and those with comorbidities are most affected by COVID-19, but it is important to investigate shared genetic mechanisms between COVID-19 risk and aging. We conducted a multi-instrument Mendelian Randomization analysis of multiple lifespan-related traits and COVID-19. Aging clock models were applied to the subjects with different COVID-19 conditions in the UK-Biobank cohort. We performed a bivariate genomic scan for age-related COVID-19 and Mendelian Randomization analysis of 389 immune cell traits to investigate their effect on lifespan and COVID-19 risk. We show that the genetic variation that supports longer life is significantly associated with the lower risk of COVID-19 infection and hospitalization. The odds ratio is 0.31 (P = 9.7 × 10−6) and 0.46 (P = 3.3 × 10−4), respectively, per additional 10 years of life. We detect an association between biological age acceleration and future incidence and severity of COVID-19 infection. Genetic profiling of age-related COVID-19 infection indicates key contributions of Notch signaling and immune system development. We reveal a negative correlation between the effects of immune cell traits on lifespan and COVID-19 risk. We find that lower B-cell CD19 levels are indicative of an increased risk of COVID-19 and decreased life expectancy, which is further validated by COVID-19 clinical data. Our analysis suggests that the factors that accelerate aging lead to an increased COVID-19 risk and point to the importance of Notch signaling and B cells in both. Interventions that target these factors to reduce biological age may reduce the risk of COVID-19. More Information

Jin L, Tang Q, Hu S, Chen Z, Zhou X, Zeng B, Wang Y, He M, Li Y, Gui L, Shen L, Long K, Ma J, Wang X, Chen Z, Jiang Y, Tang G, Zhu L, Liu F, Zhang B, Huang Z, Li G, Li D, Gladyshev VN, Yin J, Gu Y, Li X & Li M (2021) A pig BodyMap transcriptome reveals diverse tissue physiologies and evolutionary dynamics of transcription. Nature Communications 12, 3715.

Abstract A comprehensive transcriptomic survey of pigs can provide a mechanistic understanding of tissue specialization processes underlying economically valuable traits and accelerate their use as a biomedical model. Here we characterize four transcript types (lncRNAs, TUCPs, miRNAs, and circRNAs) and protein-coding genes in 31 adult pig tissues and two cell lines. We uncover the transcriptomic variability among 47 skeletal muscles, and six adipose depots linked to their different origins, metabolism, cell composition, physical activity, and mitochondrial pathways. We perform comparative analysis of the transcriptomes of seven tissues from pigs and nine other vertebrates to reveal that evolutionary divergence in transcription potentially contributes to lineage-specific biology. Long-range promoter-enhancer interaction analysis in subcutaneous adipose tissues across species suggests evolutionarily stable transcription patterns likely attributable to redundant enhancers buffering gene expression patterns against perturbations, thereby conferring robustness during speciation. This study can facilitate adoption of the pig as a biomedical model for human biology and disease and uncovers the molecular bases of valuable traits. More Information

Zhang L, Dong X, Tian X, Lee M, Ablaeva J, Firsanov D, Lee SG, Maslov AY, Gladyshev VN, Seluanov A, Gorbunova V & Vijg J (2021) Maintenance of genome sequence integrity in long- and short-lived rodent species. Science Advances 7, eabj3284.

Abstract DNA mutations in somatic cells have been implicated in the causation of aging, with longer-lived species having a higher capacity to maintain genome sequence integrity than shorter-lived species. In an attempt to directly test this hypothesis, we used single-cell whole-genome sequencing to analyze spontaneous and bleomycin-induced somatic mutations in lung fibroblasts of four rodent species with distinct maximum life spans, including mouse, guinea pig, blind mole-rat, and naked mole-rat, as well as humans. As predicted, the mutagen-induced mutation frequencies inversely correlated with species-specific maximum life span, with the greatest difference observed between the mouse and all other species. These results suggest that long-lived species are capable of processing DNA damage in a more accurate way than short-lived species. More Information

Emmrich S, Tolibzoda Zakusilo F, Trapp A, Zhou X, Zhang Q, Irving EM, Drage MG, Zhang Z, Gladyshev VN, Seluanov A & Gorbunova V (2021) Ectopic cervical thymi and no thymic involution until midlife in naked mole rats. Aging Cell 20, e13477.

Abstract Immunosenescence is a hallmark of aging and manifests as increased susceptibility to infection, autoimmunity, and cancer in the elderly. One component of immunosenescence is thymic involution, age-associated shrinkage of the thymus, observed in all vertebrates studied to date. The naked mole rat (Heterocephalus glaber) has become an attractive animal model in aging research due to its extreme longevity and resistance to disease. Here, we show that naked mole rats display no thymic involution up to 11 years of age. Furthermore, we found large ectopic cervical thymi in addition to the canonical thoracic thymus, both being identical in their cell composition. The developmental landscape in naked mole rat thymi revealed overt differences from the murine T-cell compartment, most notably a decrease of CD4+ /CD8+ double-positive cells and lower abundance of cytotoxic effector T cells. Our observations suggest that naked mole rats display a delayed immunosenescence. Therapeutic interventions aimed at reversing thymic aging remain limited, underscoring the importance of understanding the cellular and molecular mechanisms behind a sustained immune function in the naked mole rat. More Information

Gladyshev VN (2021) The Ground Zero of Organismal Life and Aging. Trends in Molecular Medicine, 27, 11-19

Abstract Cells may naturally proceed or be forced to transition to a state with a radically lower biological age, that is, be rejuvenated. Examples are the conversion of somatic cells to induced pluripotent stem cells and rejuvenation of the germline with each generation. We posit that these processes converge to the same ‘ground zero’, the mid-embryonic state characterized by the lowest biological age where both organismal life and aging begin. It may also be related to the phylotypic state. The ground zero model clarifies the relationship between aging, development, rejuvenation, and de-differentiation, which are distinct throughout life. By extending the rejuvenation phase during early embryogenesis and editing the genome, it may be possible to achieve the biological age at the ground zero lower than that achieved naturally. More Information

Ristow M, Lee CH, De Bock K, Gladyshev VN, Hotamisligil GS & Manning BD (2021) James R. Mitchell (1971-2020). Cell Metabolism 33, 458-461

Abstract We are deeply saddened by the task of writing this memoriam for our beloved friend and colleague James (Jay) Mitchell, who passed away after a cycling accident on November 17 at the age of 49. Jay was absolutely unmatched in his creativity and ability to cross fields from molecular biology to physiology, from nutrition to genetics, and from epidemiology to surgery. He was among the first to crack the molecular code of the benefits of dietary restriction and defined unique pathways that opened many translational opportunities. He was a “science whisperer” in all stages of his career; this was his magical trademark. He was soft spoken but fiercely determined, incredibly resilient yet very kind and thoughtful, expressed strength with humility and content, and could relate to anyone in any walk of life and in any intellectual or artistic domain. He leaves behind his wife Elisabeth; his three sons, Lucas, Darius, and Ian; and an irreplaceable legacy as a husband, father, brother, son, friend, teacher, colleague, and most importantly, an invaluable human treasure. More Information

Augereau A, Mariotti M, Pousse M, Filipponi D, Libert F, Beck B, Gorbunova V, Gilson E & Gladyshev VN (2021) Naked mole rat TRF1 safeguards glycolytic capacity and telomere replication under low oxygen. Science Advances , 7, eabe0174

Abstract The naked mole rat (NMR), a long-lived and cancer-resistant rodent, is highly resistant to hypoxia. Here, using robust cellular models wherein the mouse telomeric protein TRF1 is substituted by NMR TRF1 or its mutant forms, we show that TRF1 supports maximal glycolytic capacity under low oxygen, shows increased nuclear localization and association with telomeres, and protects telomeres from replicative stress. We pinpoint this evolutionary gain of metabolic function to specific amino acid changes in the homodimerization domain of this protein. We further find that NMR TRF1 accelerates telomere shortening. These findings reveal an evolutionary strategy to adapt telomere biology for metabolic control under an extreme environment. More Information

Kaya A, Phua CZJ, Lee M, Wang L, Tyshkovskiy A, Ma S, Barre B, Liu W, Harrison BR, Zhao X, Zhou X, Wasko BM, Bammler TK, Promislow DE, Kaeberlein M & Gladyshev VN (2021) Evolution of natural lifespan variation and molecular strategies of extended lifespan. eLife, 10:e64860.

Abstract To understand the genetic basis and selective forces acting on longevity, it is useful to examine lifespan variation among closely related species, or ecologically diverse isolates of the same species, within a controlled environment. In particular, this approach may lead to understanding mechanisms underlying natural variation in lifespan. Here, we analyzed 76 ecologically diverse wild yeast isolates and discovered a wide diversity of replicative lifespan. Phylogenetic analyses pointed to genes and environmental factors that strongly interact to modulate the observed aging patterns. We then identified genetic networks causally associated with natural variation in replicative lifespan across wild yeast isolates, as well as genes, metabolites and pathways, many of which have never been associated with yeast lifespan in laboratory settings. In addition, a combined analysis of lifespan-associated metabolic and transcriptomic changes revealed unique adaptations to interconnected amino acid biosynthesis, glutamate metabolism and mitochondrial function in long-lived strains. Overall, our multi-omic and lifespan analyses across diverse isolates of the same species shows how gene-environment interactions shape cellular processes involved in phenotypic variation such as lifespan. More Information

Tian R, Han K, Geng Y, Yang C, Guo H, Shi C, Xu S, Yang G, Zhou X, Gladyshev VN, Liu X, Chopin LK, Fisher DO, Baker AM, Leiner NO, Fan G & Seim I (2021) A Chromosome-Level Genome of the Agile Gracile Mouse Opossum (Gracilinanus agilis). Molecular Biology and Evolution 13, evab162.

Abstract There are more than 100 species of American didelphid marsupials (opossums and mouse opossums). Limited genomic resources for didelphids exists, with only two publicly available genome assemblies compared with dozens in the case of their Australasian counterparts. This discrepancy impedes evolutionary and ecological research. To address this gap, we assembled a high-quality chromosome-level genome of the agile gracile mouse opossum (Gracilinanus agilis) using a combination of stLFR sequencing, polishing with mate-pair data, and anchoring onto pseudochromosomes using Hi-C. This species employs a rare life-history strategy, semelparity, and all G. agilis males and most females die at the end of their first breeding season after succumbing to stress and exhaustion. The 3.7-Gb chromosome-level assembly, with 92.6% anchored onto pseudochromosomes, has a scaffold N50 of 683.5 Mb and a contig N50 of 56.9 kb. The genome assembly shows high completeness, with a mammalian BUSCO score of 88.1%. Around 49.7% of the genome contains repetitive elements. Gene annotation yielded 24,425 genes, of which 83.9% were functionally annotated. The G. agilis genome is an important resource for future studies of marsupial biology, evolution, and conservation. More Information

Khera N, Santesmasses D, Kerepesi C & Gladyshev VN (2021) COVID-19 mortality rate in children is U-shaped. Aging 13, 19954-19962.

Abstract Children are known to be better protected from COVID-19 than adults, but their susceptibility patterns and the risk relative to other diseases are insufficiently defined. Here, we found that the COVID-19 mortality rate is U-shaped in childhood: it initially decreases, reaching the minimum at the ages 3-10 years, and then increases throughout life. All-cause mortality and mortality from other diseases, such as pneumonia and influenza, show a similar pattern; however, childhood mortality rates from COVID-19 are considerably lower than from other diseases, with the best relative protection achieved at the youngest ages. Consistent with this, the fraction of COVID-19 deaths among all deaths increases as a function of age throughout childhood and the entire life. We discuss implications of the elevated postnatal COVID-19 risk and lower childhood COVID-19 mortality compared to other diseases. More Information

Tian R, Han K, Geng Y, Yang C, Shi C, Thomas PB, Pearce C, Moffatt K, Ma S, Xu S, Yang G, Zhou X, Gladyshev VN, Liu X, Fisher DO, Chopin LK, Leiner NO, Baker AM, Fan G & Seim I (2021) A chromosome-level genome of Antechinus flavipes provides a reference for an Australian marsupial genus with male death after mating. Molecular Ecology Resources, 22 740-754.

Moldakozhayev A, Tskhay A & Gladyshev VN (2021) Applying deductive reasoning and the principles of particle physics to aging research. Aging, 13 22611-22622.

Abstract Aging is debatably one of the biggest mysteries for humanity, a process consisting of myriads of genetic, molecular, environmental, and stochastic deleterious events, leading to a progressive loss of organism functionality. Aging research currently lacks a common conceptual framework, and one challenge in establishing it is the fact that aging is a highly complex process. To help develop a framework of standard aging rules, we suggest the use of deductive reasoning based on particle physics’ principles. Specifically, the principles that we suggest applying to study aging are discreteness of processes, transformation as a result of interaction, and understanding of threshold. Using this framework, biological aging may be described as a sequence of highly discrete molecular transformations caused by a combination of various specific internal and external factors. Internal organismal function and interaction of an organism with the environment result in chronic accumulation of molecular damage and other deleterious consequences of metabolism and the consequent loss of system’s functionality. The loss of functionality occurs as a series of thresholds the organism reaches before it turns into an utterly non-functional state. We discuss how having a common ground may benefit aging research, introduce the logic of new principles and analyze specific examples of how this framework could be used to study aging and design longevity interventions. More Information

Egorov AA, Alexandrov AI, Urakov VN, Makeeva DS, Edakin RO, Kushchenko AS, Gladyshev VN, Kulakovskiy IV & Dmitriev SE. (2021) A standard knockout procedure alters expression of adjacent loci at the translational level. International Journal of Molecular Sciences49 11134.

Abstract The Saccharomyces cerevisiae gene deletion collection is widely used for functional gene annotation and genetic interaction analyses. However, the standard G418-resistance cassette used to produce knockout mutants delivers strong regulatory elements into the target genetic loci. To date, its side effects on the expression of neighboring genes have never been systematically assessed. Here, using ribosome profiling data, RT-qPCR, and reporter expression, we investigated perturbations induced by the KanMX module. Our analysis revealed significant alterations in the transcription efficiency of neighboring genes and, more importantly, severe impairment of their mRNA translation, leading to changes in protein abundance. In the ‘head-to-head’ orientation of the deleted and neighboring genes, knockout often led to a shift of the transcription start site of the latter, introducing new uAUG codon(s) into the expanded 5′ untranslated region (5′ UTR). In the ‘tail-to-tail’ arrangement, knockout led to activation of alternative polyadenylation signals in the neighboring gene, thus altering its 3′ UTR. These events may explain the so-called neighboring gene effect (NGE), i.e. false genetic interactions of the deleted genes. We estimate that in as much as ∼1/5 of knockout strains the expression of neighboring genes may be substantially (>2-fold) deregulated at the level of translation. More Information

Canter JA, Ernst SE, Peters KM, Carlson BA, Thielman NRJ, Grysczyk L, Udofe P, Yu Y, Cao L, Davis CD, Gladyshev VN, Hatfield DL & Tsuji PA (2021) Selenium and the 15kDa Selenoprotein Impact Colorectal Tumorigenesis by Modulating Intestinal Barrier Integrity. International Journal of Molecular Sciences, 22 10651.

Abstract Selenoproteins play important roles in many cellular functions and biochemical pathways in mammals. Our previous study showed that the deficiency of the 15 kDa selenoprotein (Selenof) significantly reduced the formation of aberrant crypt foci (ACF) in a mouse model of azoxymethane (AOM)-induced colon carcinogenesis. The objective of this study was to examine the effects of Selenof on inflammatory tumorigenesis, and whether dietary selenium modified these effects. For 20 weeks post-weaning, Selenof-knockout (KO) mice and littermate controls were fed diets that were either deficient, adequate or high in sodium selenite. Colon tumors were induced with AOM and dextran sulfate sodium. Surprisingly, KO mice had drastically fewer ACF but developed a similar number of tumors as their littermate controls. Expression of genes important in inflammatory colorectal cancer and those relevant to epithelial barrier function was assessed, in addition to structural differences via tissue histology. Our findings point to Selenof’s potential role in intestinal barrier integrity and structural changes in glandular and mucin-producing goblet cells in the mucosa and submucosa, which may determine the type of tumor developing. More Information

Omotoso O, Gladyshev VN & Zhou X (2021) Lifespan Extension in Long-Lived Vertebrates Rooted in Ecological Adaptation. Frontiers in Cell and Developmental Biology, 9 704966.

Abstract Contemporary studies on aging and longevity have largely overlooked the role that adaptation plays in lifespan variation across species. Emerging evidence indicates that the genetic signals of extended lifespan may be maintained by natural selection, suggesting that longevity could be a product of organismal adaptation. The mechanisms of adaptation in long-lived animals are believed to account for the modification of physiological function. Here, we first review recent progress in comparative biology of long-lived animals, together with the emergence of adaptive genetic factors that control longevity and disease resistance. We then propose that hitchhiking of adaptive genetic changes is the basis for lifespan changes and suggest ways to test this evolutionary model. As individual adaptive or adaptation-linked mutations/substitutions generate specific forms of longevity effects, the cumulative beneficial effect is largely nonrandom and is indirectly favored by natural selection. We consider this concept in light of other proposed theories of aging and integrate these disparate ideas into an adaptive evolutionary model, highlighting strategies in decoding genetic factors of lifespan control. More Information

Zhang Q, Tombline G, Ablaeva J, Zhang L, Zhou X, Smith Z, Zhao Y, Xiaoli AM, Wang Z, Lin JR, Jabalameli MR, Mitra J, Nguyen N, Vijg J, Seluanov A, Gladyshev VN, Gorbunova V & Zhang ZD (2021) Genomic expansion of Aldh1a1 protects beavers against high metabolic aldehydes from lipid oxidation. Cell Reports, 37 109965.

Abstract The North American beaver is an exceptionally long-lived and cancer-resistant rodent species. Here, we report the evolutionary changes in its gene coding sequences, copy numbers, and expression. We identify changes that likely increase its ability to detoxify aldehydes, enhance tumor suppression and DNA repair, and alter lipid metabolism, potentially contributing to its longevity and cancer resistance. Hpgd, a tumor suppressor gene, is uniquely duplicated in beavers among rodents, and several genes associated with tumor suppression and longevity are under positive selection in beavers. Lipid metabolism genes show positive selection signals, changes in copy numbers, or altered gene expression in beavers. Aldh1a1, encoding an enzyme for aldehydes detoxification, is particularly notable due to its massive expansion in beavers, which enhances their cellular resistance to ethanol and capacity to metabolize diverse aldehyde substrates from lipid oxidation and their woody diet. We hypothesize that the amplification of Aldh1a1 may contribute to the longevity of beavers. More Information

Santesmasses D & Gladyshev VN (2021) Pathogenic Variants in Selenoproteins and Selenocysteine Biosynthesis Machinery. International Journal of Molecular Sciences, 22 11593.

Abstract Selenium is incorporated into selenoproteins as the 21st amino acid selenocysteine (Sec). There are 25 selenoproteins encoded in the human genome, and their synthesis requires a dedicated machinery. Most selenoproteins are oxidoreductases with important functions in human health. A number of disorders have been associated with deficiency of selenoproteins, caused by mutations in selenoprotein genes or Sec machinery genes. We discuss mutations that are known to cause disease in humans and report their allele frequencies in the general population. The occurrence of protein-truncating variants in the same genes is also presented. We provide an overview of pathogenic variants in selenoproteins genes from a population genomics perspective. More Information

Bang J, Han M, Yoo TJ, Qiao L, Jung J, Na J, Carlson BA, Gladyshev VN, Hatfield DL, Kim JH, Kim LK & Lee BJ. (2021) Identification of Signaling Pathways for Early Embryonic Lethality and Developmental Retardation in Sephs1^-/- Mice. International Journal of Molecular Sciences, 22 11647.

Abstract Selenophosphate synthetase 1 (SEPHS1) plays an essential role in cell growth and survival. However, the underlying molecular mechanisms remain unclear. In the present study, the pathways regulated by SEPHS1 during gastrulation were determined by bioinformatical analyses and experimental verification using systemic knockout mice targeting Sephs1. We found that the coagulation system and retinoic acid signaling were most highly affected by SEPHS1 deficiency throughout gastrulation. Gene expression patterns of altered embryo morphogenesis and inhibition of Wnt signaling were predicted with high probability at E6.5. These predictions were verified by structural abnormalities in the dermal layer of Sephs1^-/- embryos. At E7.5, organogenesis and activation of prolactin signaling were predicted to be affected by Sephs1 knockout. Delay of head fold formation was observed in the Sephs1^-/- embryos. At E8.5, gene expression associated with organ development and insulin-like growth hormone signaling that regulates organ growth during development was altered. Consistent with these observations, various morphological abnormalities of organs and axial rotation failure were observed. We also found that the gene sets related to redox homeostasis and apoptosis were gradually enriched in a time-dependent manner until E8.5. However, DNA damage and apoptosis markers were detected only when the Sephs1^-/- embryos aged to E9.5. Our results suggest that SEPHS1 deficiency causes a gradual increase of oxidative stress which changes signaling pathways during gastrulation, and afterwards leads to apoptosis. More Information

Gerashchenko MV, Peterfi Z, Yim SH & Gladyshev VN (2021) Translation elongation rate varies among organs and decreases with age. Nucleic Acids Research, 49 e9

Abstract There has been a surge of interest towards targeting protein synthesis to treat diseases and extend lifespan. Despite the progress, few options are available to assess translation in live animals, as their complexity limits the repertoire of experimental tools to monitor and manipulate processes within organs and individual cells. It this study, we developed a labeling-free method for measuring organ- and cell-type-specific translation elongation rates in vivo. It is based on time-resolved delivery of translation initiation and elongation inhibitors in live animals followed by ribosome profiling. It also reports translation initiation sites in an organ-specific manner. Using this method, we found that the elongation rates differ more than 50% among mouse organs and determined them to be 6.8, 5.0 and 4.3 amino acids per second for liver, kidney, and skeletal muscle, respectively. We further found that the elongation rate is reduced by 20% between young adulthood and mid-life. Thus, translation, a major metabolic process in cells, is tightly regulated at the level of elongation of nascent polypeptide chains. More Information

Vorontsov IE, Egorov AA, Anisimova AS, Eliseeva IA, Makeev VJ, Gladyshev VN, Dmitriev SE & Kulakovskiy IV (2021) Assessing Ribosome Distribution Along Transcripts with Polarity Scores and Regression Slope Estimates. Methods in Molecular Medicine, 2252:269-294. doi: 10.1007/978-1-0716-1150-0_13

Abstract During translation, the rate of ribosome movement along mRNA varies. This leads to a non-uniform ribosome distribution along the transcript, depending on local mRNA sequence, structure, tRNA availability, and translation factor abundance, as well as the relationship between the overall rates of initiation, elongation, and termination. Stress, antibiotics, and genetic perturbations affecting composition and properties of translation machinery can alter the ribosome positional distribution dramatically. Here, we offer a computational protocol for analyzing positional distribution profiles using ribosome profiling (Ribo-Seq) data. The protocol uses papolarity, a new Python toolkit for the analysis of transcript-level short read coverage profiles. For a single sample, for each transcript papolarity allows for computing the classic polarity metric which, in the case of Ribo-Seq, reflects ribosome positional preferences. For comparison versus a control sample, papolarity estimates an improved metric, the relative linear regression slope of coverage along transcript length. This involves de-noising by profile segmentation with a Poisson model and aggregation of Ribo-Seq coverage within segments, thus achieving reliable estimates of the regression slope. The papolarity software and the associated protocol can be conveniently used for Ribo-Seq data analysis in the command-line Linux environment. Papolarity package is available through Python pip package manager. The source code is available at https://github.com/autosome-ru/papolarity. More Information

Mammalian Methylation Consortium (2021) Universal DNA methylation age across mammalian tissues. bioRxiv, 10.1101/2021.01.18.426733

Abstract Aging is often perceived as a degenerative process caused by random accrual of cellular damage over time. In spite of this, age can be accurately estimated by epigenetic clocks based on DNA methylation profiles from almost any tissue of the body. Since such pan-tissue epigenetic clocks have been successfully developed for several different species, it is difficult to ignore the likelihood that a defined and shared mechanism instead, underlies the aging process. To address this, we generated 10,000 methylation arrays, each profiling up to 37,000 cytosines in highly-conserved stretches of DNA, from over 59 tissue-types derived from 128 mammalian species. From these, we identified and characterized specific cytosines, whose methylation levels change with age across mammalian species. Genes associated with these cytosines are greatly enriched in mammalian developmental processes and implicated in age-associated diseases. From the methylation profiles of these age-related cytosines, we successfully constructed three highly accurate universal mammalian clocks for eutherians, and one universal clock for marsupials. The universal clocks for eutherians are similarly accurate for estimating ages (r>0.96) of any mammalian species and tissue with a single mathematical formula. Collectively, these new observations support the notion that aging is indeed evolutionarily conserved and coupled to developmental processes across all mammalian species – a notion that was long-debated without the benefit of this new and compelling evidence. More Information

Haghani A, Lu AT, Li CZ, Robeck TR, Belov K, Breeze CE, Brooke RT, Clarke S, Faulkes CG, Fei Z, Ferguson SH, Finno CJ, Gladyshev VN, Gorbunova V, Goya RG, Hogan AN, Hogg CJ, Hore TA, Kiaris H, Kordowitzki P, Banks G, Koski WR, Mozhui K, Naderi A, Ostrander EA, Parsons KM, Plassais J, Robbins J, Sears KE, Seluanov A, Steinman KJ, Szladovits B, Thompson MJ, Villar D, Wang N, Wilkinson GS, Young BG, Zhang J, Zoller JA, Ernst J, Yang XW, Raj K & S. Horvath. (2021) DNA Methylation Networks Underlying Mammalian Traits. bioRxiv, 10.1101/2021.03.16.435708.

Abstract Epigenetics has hitherto been studied and understood largely at the level of individual organisms. Here, we report a multi-faceted investigation of DNA methylation across 11,117 samples from 176 different species. We performed an unbiased clustering of individual cytosines into 55 modules and identified 31 modules related to primary traits including age, species lifespan, sex, adult species weight, tissue type and phylogenetic order. Analysis of the correlation between DNA methylation and species allowed us to construct phyloepigenetic trees for different tissues that parallel the phylogenetic tree. In addition, while some stable cytosines reflect phylogenetic signatures, others relate to age and lifespan, and in many cases responding to anti-aging interventions in mice such as caloric restriction and ablation of growth hormone receptors. Insights uncovered by this investigation have important implications for our understanding of the role of epigenetics in mammalian evolution, aging and lifespan. More Information

Gerashchenko MV & Gladyshev VN (2021) Measuring Organ-Specific Translation Elongation Rate in Mice. Methods in Molecular Biology 2252, 189-200.

Abstract Modern methods of genome editing enable the rapid generation of mouse models to study the regulation of protein synthesis. At the same time, few options are available to study translation in rodents as the animal’s complexity severely limits the repertoire of experimental tools. Here we describe a method to monitor translation in mice and other small animals. The technique is based on a ribosome profiling and specifically tailored toward measuring translation elongation. However, it can be easily applied for short upstream reading frames discovery. The advantage of this method is the ability to study translation in fully developed animals without extracting and subculturing cells, therefore, maintaining unperturbed physiological conditions. More Information

Choi DW, Roh YJ, Kim S, Lee HM, Kim M, Shin D, Park JH, Cho Y, Park HH, Ok YS, Kang D, Kim JH, Tarrago L, Danial NN, Gladyshev VN, Min PK & Lee BC (2021) Development of a novel fluorescent biosensor for dynamic monitoring of metabolic methionine redox status in cells and tissues. Biosensors and Bioelectronics 178, 113031.

Abstract Aberrant production of reactive oxygen species (ROS) leads to tissue damage accumulation, which is associated with a myriad of human pathologies. Although several sensors have been developed for ROS quantification, their applications for ROS-related human physiologies and pathologies still remain problematic due to the unstable nature of ROS. Herein, we developed Trx1-cpYFP-fRMsr (TYfR), a genetically-encoded fluorescent biosensor with the remarkable specificity and sensitivity toward fMetRO (free Methionine-R-sulfoxide), allowing for dynamic quantification of physiological levels of fMetRO, a novel indicator of ROS and methionine redox status in vitro and in vivo. Moreover, using the sensor, we observed a significant fMetRO enrichment in serum from patients with acute coronary syndrome, one of the most severe cardiovascular diseases, which becomes more evident following percutaneous coronary intervention. Collectively, this study proposes that fMetRO is a novel biomarker of tissue damage accumulation in ROS-associated human pathologies, and that TYfR is a promising tool for quantifying fMetRO with potentials in versatile applications. More Information

2020 Articles

Lu Y, Brommer B, Tian X, Krishnan A, Meer M, Wang C, Vera DL, Zeng Q, Yu D, Bonkowski MS, Yang JH, Zhou S, Hoffmann EM, Karg MM, Schultz MB, Kane AE, Davidsohn N, Korobkina E, Chwalek K, Rajman LA, Church GM, Hochedlinger K, Gladyshev VN, Horvath S, Levine ME, Gregory-Ksander MS, Ksander BR, He Z, Sinclair DA. (2020) Reprogramming to recover youthful epigenetic information and restore vision. Nature. 588(7836)

AbstractAgeing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1-3. Changes to DNA methylation patterns over time form the basis of ageing clocks4, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity5-7. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo. More Information

Zoonomia Consortium. (2020) A comparative genomics multitool for scientific discovery and conservation. Nature. 587(7833)

AbstractThe Zoonomia Project is investigating the genomics of shared and specialized traits in eutherian mammals. Here we provide genome assemblies for 131 species, of which all but 9 are previously uncharacterized, and describe a whole-genome alignment of 240 species of considerable phylogenetic diversity, comprising representatives from more than 80% of mammalian families. We find that regions of reduced genetic diversity are more abundant in species at a high risk of extinction, discern signals of evolutionary selection at high resolution and provide insights from individual reference genomes. By prioritizing phylogenetic diversity and making data available quickly and without restriction, the Zoonomia Project aims to support biological discovery, medical research and the conservation of biodiversity. More Information

Mkrtchyan GV, Abdelmohsen K, Andreux P, Bagdonaite I, Barzilai N, Brunak S, Cabreiro F, de Cabo R, Campisi J, Cuervo AM, Demaria M, Ewald CY, Fang EF, Faragher R, Ferrucci L, Freund A, Silva-García CG, Georgievskaya A, Gladyshev VN, Glass DJ, Gorbunova V, de Grey A, He WW, Hoeijmakers J, Hoffmann E, Horvath S, Houtkooper RH, Jensen MK, Jensen MB, Kane A, Kassem M, de Keizer P, Kennedy B, Karsenty G, Lamming DW, Lee KF, MacAulay N, Mamoshina P, Mellon J, Molenaars M, Moskalev A, Mund A, Niedernhofer L, Osborne B, Pak HH, Parkhitko A, Raimundo N, Rando TA, Rasmussen LJ, Reis C, Riedel CG, Franco-Romero A, Schumacher B, Sinclair DA, Suh Y, Taub PR, Toiber D, Treebak JT, Valenzano DR, Verdin E, Vijg J, Young S, Zhang L, Bakula D, Zhavoronkov A, Scheibye-Knudsen M. (2020) ARDD 2020: from aging mechanisms to interventions. Aging. 12(24)

AbstractAging is emerging as a druggable target with growing interest from academia, industry and investors. New technologies such as artificial intelligence and advanced screening techniques, as well as a strong influence from the industry sector may lead to novel discoveries to treat age-related diseases. The present review summarizes presentations from the 7th Annual Aging Research and Drug Discovery (ARDD) meeting, held online on the 1st to 4th of September 2020. The meeting covered topics related to new methodologies to study aging, knowledge about basic mechanisms of longevity, latest interventional strategies to target the aging process as well as discussions about the impact of aging research on society and economy. More than 2000 participants and 65 speakers joined the meeting and we already look forward to an even larger meeting next year. Please mark your calendars for the 8th ARDD meeting that is scheduled for the 31st of August to 3rd of September, 2021, at Columbia University, USA. More Information

Gerashchenko MV, Peterfi Z, Yim SH, Gladyshev VN. (2020) Translation elongation rate varies among organs and decreases with age. Nucleic Acids Res.

AbstractThere has been a surge of interest towards targeting protein synthesis to treat diseases and extend lifespan. Despite the progress, few options are available to assess translation in live animals, as their complexity limits the repertoire of experimental tools to monitor and manipulate processes within organs and individual cells. It this study, we developed a labeling-free method for measuring organ- and cell-type-specific translation elongation rates in vivo. It is based on time-resolved delivery of translation initiation and elongation inhibitors in live animals followed by ribosome profiling. It also reports translation initiation sites in an organ-specific manner. Using this method, we found that the elongation rates differ more than 50% among mouse organs and determined them to be 6.8, 5.0 and 4.3 amino acids per second for liver, kidney, and skeletal muscle, respectively. We further found that the elongation rate is reduced by 20% between young adulthood and mid-life. Thus, translation, a major metabolic process in cells, is tightly regulated at the level of elongation of nascent polypeptide chains. More Information

Santesmasses D, Castro JP, Zenin AA, Shindyapina AV, Gerashchenko MV, Zhang B, Kerepesi C, Yim SH, Fedichev PO, Gladyshev VN. (2020) COVID-19 is an emergent disease of aging. Aging Cell. 19(10)

AbstractCOVID-19 is an ongoing pandemic caused by the SARS-CoV-2 coronavirus that poses one of the greatest challenges to public health in recent years. SARS-CoV-2 is known to preferentially target older subjects and those with pre-existing conditions, but the reason for this age dependence is unclear. Here, we found that the case fatality rate for COVID-19 grows exponentially with age in all countries tested, with the doubling time approaching that of all-cause human mortality. In addition, men and those with multiple age-related diseases are characterized by increased mortality. Moreover, similar mortality patterns were found for all-cause pneumonia. We further report that the gene expression of ACE2, the SARS-CoV-2 receptor, grows in the lung with age, except for subjects on a ventilator. Together, our findings establish COVID-19 as an emergent disease of aging, and age and age-related diseases as its major risk factors. In turn, this suggests that COVID-19, and deadly respiratory diseases in general, may be targeted, in addition to antiviral approaches, by approaches that target the aging process. More Information

Gerashchenko MV, Nesterchuk MV, Smekalova EM, Paulo JA, Kowalski PS, Akulich KA, Bogorad R, Dmitriev SE, Gygi S, Zatsepin T, Anderson DG, Gladyshev VN, Koteliansky VE. (2020) Translation elongation factor 2 depletion by siRNA in mouse liver leads to mTOR-independent translational upregulation of ribosomal protein genes. Scientific Reports. 10(1), 15473.

AbstractDue to breakthroughs in RNAi and genome editing methods in the past decade, it is now easier than ever to study fine details of protein synthesis in animal models. However, most of our understanding of translation comes from unicellular organisms and cultured mammalian cells. In this study, we demonstrate the feasibility of perturbing protein synthesis in a mouse liver by targeting translation elongation factor 2 (eEF2) with RNAi. We were able to achieve over 90% knockdown efficacy and maintain it for 2 weeks effectively slowing down the rate of translation elongation. As the total protein yield declined, both proteomics and ribosome profiling assays showed robust translational upregulation of ribosomal proteins relative to other proteins. Although all these genes bear the TOP regulatory motif, the branch of the mTOR pathway responsible for translation regulation was not activated. Paradoxically, coordinated translational upregulation of ribosomal proteins only occurred in the liver but not in murine cell culture. Thus, the upregulation of ribosomal transcripts likely occurred via passive mTOR-independent mechanisms. Impaired elongation sequesters ribosomes on mRNA and creates a shortage of free ribosomes. This leads to preferential translation of transcripts with high initiation rates such as ribosomal proteins. Furthermore, severe eEF2 shortage reduces the negative impact of positively charged amino acids frequent in ribosomal proteins on ribosome progression. More Information

Pawelec G, Bronikowski A, Cunnane SC, Ferrucci L, Franceschi C, Fülöp T, Gaudreau P, Gladyshev VN, Gonos ES, Gorbunova V, Kennedy BK, Larbi A, Lemaître JF, Liu GH, Maier AB, Morais JA, Nóbrega OT, Moskalev A, Rikkert MO, Seluanov A, Senior AM, Ukraintseva S, Van Haelen Q, Witkowski J, Cohen AA. (2020) The conundrum of human immune system “senescence”. Mechanisms of ageing and development. 192, 10.1016/j.mad.2020.111357.

AbstractThere is a great deal of debate on the question of whether or not we know what ageing is (Ref. Cohen et al). Here, we consider what we believe to be the especially confused and confusing case of the ageing of the human immune system, commonly referred to as “immunosenescence”. But what exactly is meant by this term? It has been used loosely in the literature, resulting in a certain degree of confusion as to its definition and implications. Here, we argue that only those differences in immune parameters between younger and older adults that are associated in some definitive manner with detrimental health outcomes and/or impaired survival prospects should be classed as indicators of immunosenescence in the strictest sense of the word, and that in humans we know remarkably little about their identity. Such biomarkers of immunosenescence may nonetheless indicate beneficial effects in other contexts, consistent with the notion of antagonistic pleiotropy. Identifying what could be true immunosenescence in this respect requires examining: (1) what appears to correlate with age, though generality across human populations is not yet confirmed; (2) what clearly is part of a suite of canonical changes in the immune system that happen with age; (3) which subset of those changes accelerates rather than slows aging; and (4) all changes, potentially population-specific, that accelerate agig. This remains an immense challenge. These questions acquire an added urgency in the current SARS-CoV-2 pandemic, given the clearly greater susceptibility of older adults to COVID-19. More Information

Konstantinidis K, Bezzerides VJ, Lai L, Isbell HM, Wei AC, Wu Y, Viswanathan MC, Blum ID, Granger JM, Heims-Waldron D, Zhang D, Luczak ED, Murphy KR, Lu F, Gratz DH, Manta B, Wang Q, Wang Q, Kolodkin AL, Gladyshev VN, Hund TJ, Pu WT, Wu MN, Cammarato A, Bianchet MA, Shea MA, Levine RL, Anderson ME. (2020) MICAL1 constrains cardiac stress responses and protects against disease by oxidizing CaMKII. J Clin Invest. 130(9), 4663-4687.

AbstractOxidant stress can contribute to health and disease. Here we show that invertebrates and vertebrates share a common stereospecific redox pathway that protects against pathological responses to stress, at the cost of reduced physiological performance, by constraining Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. MICAL1, a methionine monooxygenase thought to exclusively target actin, and MSRB, a methionine reductase, control the stereospecific redox status of M308, a highly conserved residue in the calmodulin-binding (CaM-binding) domain of CaMKII. Oxidized or mutant M308 (M308V) decreased CaM binding and CaMKII activity, while absence of MICAL1 in mice caused cardiac arrhythmias and premature death due to CaMKII hyperactivation. Mimicking the effects of M308 oxidation decreased fight-or-flight responses in mice, strikingly impaired heart function in Drosophila melanogaster, and caused disease protection in human induced pluripotent stem cell-derived cardiomyocytes with catecholaminergic polymorphic ventricular tachycardia, a CaMKII-sensitive genetic arrhythmia syndrome. Our studies identify a stereospecific redox pathway that regulates cardiac physiological and pathological responses to stress across species. More Information

Zhou X, Dou Q, Fan G, Zhang Q, Sanderford M, Kaya A, Johnson J, Karlsson EK, Tian X, Mikhalchenko A, Kumar S, Seluanov A, Zhang ZD, Gorbunova V, Liu X, Gladyshev VN. (2020) Beaver and Naked Mole Rat Genomes Reveal Common Paths to Longevity. Cell Rep. 32(4), 107949.

AbstractLong-lived rodents have become an attractive model for the studies on aging. To understand evolutionary paths to long life, we prepare chromosome-level genome assemblies of the two longest-lived rodents, Canadian beaver (Castor canadensis) and naked mole rat (NMR, Heterocephalus glaber), which were scaffolded with in vitro proximity ligation and chromosome conformation capture data and complemented with long-read sequencing. Our comparative genomic analyses reveal that amino acid substitutions at “disease-causing” sites are widespread in the rodent genomes and that identical substitutions in long-lived rodents are associated with common adaptive phenotypes, e.g., enhanced resistance to DNA damage and cellular stress. By employing a newly developed substitution model and likelihood ratio test, we find that energy and fatty acid metabolism pathways are enriched for signals of positive selection in both long-lived rodents. Thus, the high-quality genome resource of long-lived rodents can assist in the discovery of genetic factors that control longevity and adaptive evolution. More Information

Cohen AA, Kennedy BK, Anglas U, Bronikowski AM, Deelen J, Dufour F, Ferbeyre G, Ferrucci L, Franceschi C, Frasca D, Friguet B, Gaudreau P, Gladyshev VN, Gonos ES, Gorbunova V, Gut P, Ivanchenko M, Legault V, Lemaître JF, Liontis T, Liu GH, Liu M, Maier AB, Nóbrega OT, Olde Rikkert MGM, Pawelec G, Rheault S, Senior AM, Simm A, Soo S, Traa A, Ukraintseva S, Vanhaelen Q, Van Raamsdonk JM, Witkowski JM, Yashin AI, Ziman R, Fülöp T. (2020) Lack of consensus on an aging biology paradigm? A global survey reveals an agreement to disagree, and the need for an interdisciplinary framework. Mech Ageing Dev. 191, 111316.

AbstractAt a recent symposium on aging biology, a debate was held as to whether or not we know what biological aging is. Most of the participants were struck not only by the lack of consensus on this core question, but also on many basic tenets of the field. Accordingly, we undertook a systematic survey of our 71 participants on key questions that were raised during the debate and symposium, eliciting 37 responses. The results confirmed the impression from the symposium: there is marked disagreement on the most fundamental questions in the field, and little consensus on anything other than the heterogeneous nature of aging processes. Areas of major disagreement included what participants viewed as the essence of aging, when it begins, whether aging is programmed or not, whether we currently have a good understanding of aging mechanisms, whether aging is or will be quantifiable, whether aging will be treatable, and whether many non-aging species exist. These disagreements lay bare the urgent need for a more unified and cross-disciplinary paradigm in the biology of aging that will clarify both areas of agreement and disagreement, allowing research to proceed more efficiently. We suggest directions to encourage the emergence of such a paradigm. More Information

Anisimova AS, Meerson MB, Gerashchenko MV, Kulakovskiy IV, Dmitriev SE, Gladyshev VN. (2020) Multifaceted deregulation of gene expression and protein synthesis with age. Proc Natl Acad Sci U S A. 117(27), 15581-15590.

AbstractProtein synthesis represents a major metabolic activity of the cell. However, how it is affected by aging and how this in turn impacts cell function remains largely unexplored. To address this question, herein we characterized age-related changes in both the transcriptome and translatome of mouse tissues over the entire life span. We showed that the transcriptome changes govern those in the translatome and are associated with altered expression of genes involved in inflammation, extracellular matrix, and lipid metabolism. We also identified genes that may serve as candidate biomarkers of aging. At the translational level, we uncovered sustained down-regulation of a set of 5′-terminal oligopyrimidine (5′-TOP) transcripts encoding protein synthesis and ribosome biogenesis machinery and regulated by the mTOR pathway. For many of them, ribosome occupancy dropped twofold or even more. Moreover, with age, ribosome coverage gradually decreased in the vicinity of start codons and increased near stop codons, revealing complex age-related changes in the translation process. Taken together, our results reveal systematic and multidimensional deregulation of protein synthesis, showing how this major cellular process declines with age. More Information

Lashkevich KA, Shlyk VI, Kushchenko AS, Gladyshev VN, Alkalaeva EZ, Dmitriev SE. (2020) CTELS: A Cell-Free System for the Analysis of Translation Termination Rate. Biomolecules. 10(6), 911.

AbstractTranslation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3′ UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a “leaky” stop codon context, which likely defines the basis of nonsense suppression. More Information

Sanchez-Lopez C, Labadie N, Lombardo V, Biglione F, Manta B, Jacob R, Gladyshev VN, Abdelilah-Seyfried S, Selenko P, Binolfi A. (2020) An NMR-based biosensor to measure stereo-specific methionine sulfoxide reductase (MSR) activities in vitro and in vivo. Chemistry.

AbstractOxidation of protein methionines to methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to methionine by two families of conserved methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the S – or R -diastereoisomers of methionine-sulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups. We demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments, including cell lysates and live zebrafish embryos. Thereby, we establish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOx as a highly sensitive tool for studying therapeutic targets of oxidative stress-related human diseases and redox regulated signaling pathways. More Information

Galkin F, Mamoshina P, Aliper A, Putin E, Moskalev V, Gladyshev VN, Zhavoronkov A. (2020) Human Gut Microbiome Aging Clock Based on Taxonomic Profiling and Deep Learning. iScience. 23(6), 101199.

AbstractThe human gut microbiome is a complex ecosystem that both affects and is affected by its host status. Previous metagenomic analyses of gut microflora revealed associations between specific microbes and host age. Nonetheless there was no reliable way to tell a host’s age based on the gut community composition. Here we developed a method of predicting hosts’ age based on microflora taxonomic profiles using a cross-study dataset and deep learning. Our best model has an architecture of a deep neural network that achieves the mean absolute error of 5.91 years when tested on external data. We further advance a procedure for inferring the role of particular microbes during human aging and defining them as potential aging biomarkers. The described intestinal clock represents a unique quantitative model of gut microflora aging and provides a starting point for building host aging and gut community succession into a single narrative. More Information

Liberman N, O’Brown ZK, Earl AS, Boulias K, Gerashchenko MV, Wang SY, Fritsche C, Fady PE, Dong A, Gladyshev VN, Greer EL. (2020) N6-adenosine methylation of ribosomal RNA affects lipid oxidation and stress resistance. Sci Adv. 6(17), eaaz4370.

AbstractDuring stress, global translation is reduced, but specific transcripts are actively translated. How stress-responsive mRNAs are selectively translated is unknown. We show that METL-5 methylates adenosine 1717 on 18S ribosomal RNA in C. elegans, enhancing selective ribosomal binding and translation of specific mRNAs. One of these mRNAs, CYP-29A3, oxidizes the omega-3 polyunsaturated fatty acid eicosapentaenoic acid to eicosanoids, key stress signaling molecules. While metl-5-deficient animals grow normally under homeostatic conditions, they are resistant to a variety of stresses. metl-5 mutant worms also show reduced bioactive lipid eicosanoids and dietary supplementation of eicosanoid products of CYP-29A3 restores stress sensitivity of metl-5 mutant worms. Thus, methylation of a specific residue of 18S rRNA by METL-5 selectively enhances translation of cyp-29A3 to increase production of eicosanoids, and blocking this pathway increases stress resistance. This study suggests that ribosome methylation can facilitate selective translation, providing another layer of regulation of the stress response. More Information

Bozaykut P, Ekren R, Sezerman OU, Gladyshev VN, Ozer NK. (2020) High-throughput profiling reveals perturbation of endoplasmic reticulum stress-related genes in atherosclerosis induced by high-cholesterol diet and the protective role of vitamin E. Biofactors.

AbstractFormation of atherosclerotic plaques, called atherogenesis, is a complex process affected by genetic and environmental factors. It was proposed that endoplasmic reticulum (ER) stress is an important factor in the pathogenesis of atherosclerosis and that vitamin E affects atherosclerotic plaque formation via its antioxidant properties. Here, we investigated ER stress-related molecular mechanisms in high-cholesterol diet (HCD, 2%)-induced atherosclerosis model and the role of vitamin E supplementation in it, beyond its antioxidant properties. The consequences of HCD and vitamin E supplementation were examined by determining protein levels of ER stress markers in aortic tissues. As vitamin E supplementation acts on several unfolded protein response (UPR) factors, it decreased ER stress induced by HCD. To elucidate the associated pathways, gene expression profiling was performed, revealing differentially expressed genes enriched in ER stress-related pathways such as the proteasome and the apoptosis pathways. We further assessed the proteasomal activity impaired by HCD in the aorta and showed that vitamin E reversed it to that of control animals. Overall, the study characterized the effects of HCD and vitamin E on ER stress-related gene expression, revealing the role of proteolytic systems during atherogenesis. More Information

Jedrychowski MP, Lu GZ, Szpyt J, Mariotti M, Garrity R, Paulo JA, Schweppe DK, Laznik-Bogoslavski D, Kazak L, Murphy MP, Gladyshev VN, Gygi SP, Chouchani ET, Spiegelman BM. (2020) Facultative protein selenation regulates redox sensitivity, adipose tissue thermogenesis, and obesity. Proc Natl Acad Sci U S A. 117(20), 10789-10796.

AbstractOxidation of cysteine thiols by physiological reactive oxygen species (ROS) initiates thermogenesis in brown and beige adipose tissues. Cellular selenocysteines, where sulfur is replaced with selenium, exhibit enhanced reactivity with ROS. Despite their critical roles in physiology, methods for broad and direct detection of proteogenic selenocysteines are limited. Here we developed a mass spectrometric method to interrogate incorporation of selenium into proteins. Unexpectedly, this approach revealed facultative incorporation of selenium as selenocysteine or selenomethionine into proteins that lack canonical encoding for selenocysteine. Selenium was selectively incorporated into regulatory sites on key metabolic proteins, including as selenocysteine-replacing cysteine at position 253 in uncoupling protein 1 (UCP1). This facultative utilization of selenium was initiated by increasing cellular levels of organic, but not inorganic, forms of selenium. Remarkably, dietary selenium supplementation elevated facultative incorporation into UCP1, elevated energy expenditure through thermogenic adipose tissue, and protected against obesity. Together, these findings reveal the existence of facultative protein selenation, which correlates with impacts on thermogenic adipocyte function and presumably other biological processes as well. More Information

Zhang B, Podolskiy DI, Mariotti M, Seravalli J, Gladyshev VN. (2020) Systematic age-, organ-, and diet-associated ionome remodeling and the development of ionomic aging clocks. Aging Cell. 19(5), e13119.

AbstractAging involves coordinated yet distinct changes in organs and systems throughout life, including changes in essential trace elements. However, how aging affects tissue element composition (ionome) and how these changes lead to dysfunction and disease remain unclear. Here, we quantified changes in the ionome across eight organs and 16 age groups of mice. This global profiling revealed novel interactions between elements at the level of tissue, age, and diet, and allowed us to achieve a broader, organismal view of the aging process. We found that while the entire ionome steadily transitions along the young-to-old trajectory, individual organs are characterized by distinct element changes. The ionome of mice on calorie restriction (CR) moved along a similar but shifted trajectory, pointing that at the organismal level this dietary regimen changes metabolism in order to slow down aging. However, in some tissues CR mimicked a younger state of control mice. Even though some elements changed with age differently in different tissues, in general aging was characterized by the reduced levels of elements as well as their increased variance. The dataset we prepared also allowed to develop organ-specific, ionome-based markers of aging that could help monitor the rate of aging. In some tissues, these markers reported the lifespan-extending effect of CR. These aging biomarkers have the potential to become an accessible tool to test the age-modulating effects of interventions. More Information

Zhang B, Gladyshev VN. (2020) How can aging be reversed? Exploring rejuvenation from a damage‐based perspective. Advanced Genetics. 1(1), e10025.

AbstractAdvanced age is associated with accumulation of damage and other deleterious changes and a consequential systemic decline of function. This decline affects all organs and systems in an organism, leading to their inadaptability to the environment, and therefore is thought to be inevitable for humans and most animal species. However, in vitro and in vivo application of reprogramming strategies, which convert somatic cells to induced pluripotent stem cells, has demonstrated that the aged cells can be rejuvenated. Moreover, the data and theoretical considerations suggest that reversing the biological age of somatic cells (from old to young) and de‐differentiating somatic cells into stem cells represent two distinct processes that take place during rejuvenation, and thus they may be differently targeted. We advance a stemness‐function model to explain these data and discuss a possibility of rejuvenation from the perspective of damage accumulation. In turn, this suggests approaches to achieve rejuvenation of cells in vitro and in vivo. More Information

Galkin F, Mamoshina P, Aliper A, de Magalhães JP, Gladyshev VN, Zhavoronkov A. (2020) Biohorology and biomarkers of aging: Current state-of-the-art, challenges and opportunities. Ageing Res Rev. 60, 101050.

AbstractThe aging process results in multiple traceable footprints, which can be quantified and used to estimate an organism’s age. Examples of such aging biomarkers include epigenetic changes, telomere attrition, and alterations in gene expression and metabolite concentrations. More than a dozen aging clocks use molecular features to predict an organism’s age, each of them utilizing different data types and training procedures. Here, we offer a detailed comparison of existing mouse and human aging clocks, discuss their technological limitations and the underlying machine learning algorithms. We also discuss promising future directions of research in biohorology – the science of measuring the passage of time in living systems. Overall, we expect deep learning, deep neural networks and generative approaches to be the next power tools in this timely and actively developing field. More Information

Shindyapina AV, Zenin AA, Tarkhov AE, Santesmasses D, Fedichev PO, Gladyshev VN. (2020) Germline burden of rare damaging variants negatively affects human healthspan and lifespan. eLife 9, 53449.

AbstractHeritability of human lifespan is 23-33% as evident from twin studies. Genome-wide association studies explored this question by linking particular alleles to lifespan traits. However, genetic variants identified so far can explain only a small fraction of lifespan heritability in humans. Here, we report that the burden of rarest protein-truncating variants (PTVs) in two large cohorts is negatively associated with human healthspan and lifespan, accounting for 0.4 and 1.3 years of their variability, respectively. In addition, longer-living individuals possess both fewer rarest PTVs and less damaging PTVs. We further estimated that somatic accumulation of PTVs accounts for only a small fraction of mortality and morbidity acceleration and hence is unlikely to be causal in aging. We conclude that rare damaging mutations, both inherited and accumulated throughout life, contribute to the aging process, and that burden of ultra-rare variants in combination with common alleles better explain apparent heritability of human lifespan. More Information

Fernando R, Wardelmann K, Deubel S, Kehm R, Jung T, Mariotti M, Vasilaki A, Gladyshev VN, Kleinridders A, Grune T, Castro JP. (2020) Low steady-state oxidative stress inhibits adipogenesis by altering mitochondrial dynamics and decreasing cellular respiration. Redox Biol. 32, 101507.

AbstractAdipogenesis is a fundamental process of white adipose tissue function, supporting lipid storage and release, while avoiding its spillover and ectopic accumulation in tissues and organs. During aging adipogenesis is impaired and among other factors, oxidative stress contributes to this process. Adipogenesis requires functional and dynamic mitochondria; however, this organelle itself becomes dysfunctional during aging and accounts for most of reactive oxygen species (ROS) production. Here, we evaluated whether oxidative stress impairs adipogenesis through functional impairment of mitodynamics by utilizing hyperoxia as a continuous source of oxidative stress while maintaining cellular viability. This negatively impacted mitochondrial function, including respiration and dynamics and ultimately blocked adipogenesis. Interestingly, this state was reversible by using the antidiabetic drug, Rosiglitazone, which reduced oxidative stress, restored mitochondrial dynamics and respiration and augmented adipogenesis. Moreover, in vitro results were in agreement with in vivo models of oxidative stress and aging, in which mice depleted of the superoxide dismutase enzyme 1 (SOD1) and old wild-type C57BL/6JRj mice demonstrated the same trend of adipogenic potential. Importantly, in humans the results follow the same pattern, showing a downregulation of adipogenic markers during aging. Since the levels of oxidative stress and peripheral insulin resistance increase with age, while adipogenesis decreases during aging, our model helps to understand a possible way to overcome physiologically low, steady stress conditions and restore adipogenesis, avoiding accumulation of deleterious hypertrophic adipocytes in favor of beneficial hyperplasia. More Information

Santesmasses D, Mariotti M, Gladyshev VN. (2020) Bioinformatics of Selenoproteins. Antioxid Redox Signal. 33(7), 525-536.

AbstractSignificance: Bioinformatics has brought important insights into the field of selenium research. The progress made in the development of computational tools in the last two decades, coordinated with growing genome resources, provided new opportunities to study selenoproteins. The present review discusses existing tools for selenoprotein gene finding and other bioinformatic approaches to study the biology of selenium. Recent Advances: The availability of complete selenoproteomes allowed assessing a global distribution of the use of selenocysteine (Sec) across the tree of life, as well as studying the evolution of selenoproteins and their biosynthetic pathway. Beyond gene identification and characterization, human genetic variants in selenoprotein genes were used to examine adaptations to selenium levels in diverse human populations and to estimate selective constraints against gene loss. Critical Issues: The synthesis of selenoproteins is essential for development in mice. In humans, several mutations in selenoprotein genes have been linked to rare congenital disorders. And yet, the mechanism of Sec insertion and the regulation of selenoprotein synthesis in mammalian cells are not completely understood. Future Directions: Omics technologies offer new possibilities to study selenoproteins and mechanisms of Sec incorporation in cells, tissues, and organisms. More Information

Kaya A, Mariotti M, Tyshkovskiy A, Zhou X, Hulke ML, Ma S, Gerashchenko MV, Koren A, Gladyshev VN. (2020) Molecular signatures of aneuploidy-driven adaptive evolution. Nat Commun. 11(1), 588.

AbstractAlteration of normal ploidy (aneuploidy) can have a number of opposing effects, such as unbalancing protein abundances and inhibiting cell growth but also accelerating genetic diversification and rapid adaptation. The interplay of these detrimental and beneficial effects remains puzzling. Here, to understand how cells develop tolerance to aneuploidy, we subject disomic (i.e. with an extra chromosome copy) strains of yeast to long-term experimental evolution under strong selection, by forcing disomy maintenance and daily population dilution. We characterize mutations, karyotype alterations and gene expression changes, and dissect the associated molecular strategies. Cells with different extra chromosomes accumulated mutations at distinct rates and displayed diverse adaptive events. They tended to evolve towards normal ploidy through chromosomal DNA loss and gene expression changes. We identify genes with recurrent mutations and altered expression in multiple lines, revealing a variant that improves growth under genotoxic stresses. These findings support rapid evolvability of disomic strains that can be used to characterize fitness effects of mutations under different stress conditions. More Information

Smekalova EM, Gerashchenko MV, O’Connor PBF, Whittaker CA, Kauffman KJ, Fefilova AS, Zatsepin TS, Bogorad RL, Baranov PV, Langer R, Gladyshev VN, Anderson DG, Koteliansky V. (2020) In Vivo RNAi-Mediated eIF3m Knockdown Affects Ribosome Biogenesis and Transcription but Has Limited Impact on mRNA-Specific Translation. Mol Ther Nucleic Acids. 19, 252-266.

AbstractTranslation is an essential biological process, and dysregulation is associated with a range of diseases including ribosomopathies, diabetes, and cancer. Here, we examine translation dysregulation in vivo using RNAi to knock down the m-subunit of the translation initiation factor eIF3 in the mouse liver. Transcriptome sequencing, ribosome profiling, whole proteome, and phosphoproteome analyses show that eIF3m deficiency leads to the transcriptional response and changes in cellular translation that yield few detectable differences in the translation of particular mRNAs. The transcriptional response fell into two main categories: ribosome biogenesis (increased transcription of ribosomal proteins) and cell metabolism (alterations in lipid, amino acid, nucleic acid, and drug metabolism). Analysis of ribosome biogenesis reveals inhibition of rRNA processing, highlighting decoupling of rRNA synthesis and ribosomal protein gene transcription in response to eIF3m knockdown. Interestingly, a similar reduction in eIF3m protein levels is associated with induction of the mTOR pathway in vitro but not in vivo. Overall, this work highlights the utility of a RNAi-based in vivo approach for studying the regulation of mammalian translation in vivo. More Information

Santesmasses D, Mariotti M, Gladyshev VN. (2020) Tolerance to Selenoprotein Loss Differs between Human and Mouse. Mol Biol Evol. 37(2), 341-354.

AbstractMouse has emerged as the most common model organism in biomedicine. Here, we analyzed the tolerance to the loss-of-function (LoF) of selenoprotein genes, estimated from mouse knockouts and the frequency of LoF variants in humans. We found not only a general correspondence in tolerance (e.g., GPX1, GPX2) and intolerance (TXNRD1, SELENOT) to gene LoF between humans and mice but also important differences. Notably, humans are intolerant to the loss of iodothyronine deiodinases, whereas their deletion in mice leads to mild phenotypes, and this is consistent with phenotype differences in selenocysteine machinery loss between these species. In contrast, loss of TXNRD2 and GPX4 is lethal in mice but may be tolerated in humans. We further identified the first human SELENOP variants coding for proteins varying in selenocysteine content. Finally, our analyses suggested that premature termination codons in selenoprotein genes trigger nonsense-mediated decay, but do this inefficiently when UGA codon is gained. Overall, our study highlights differences in the physiological importance of selenoproteins between human and mouse. More Information

2019 Articles

Kinzina ED, Podolskiy DI, Dmitriev SE, Gladyshev VN. (2019) Patterns of Aging Biomarkers, Mortality, and Damaging Mutations Illuminate the Beginning of Aging and Causes of Early-Life Mortality. Cell Rep. 29(13), 4276-4284.

AbstractAn increase in the probability of death has been a defining feature of aging, yet human perinatal mortality starts high and decreases with age. Previous evolutionary models suggested that organismal aging begins after the onset of reproduction. However, we find that mortality and incidence of diseases associated with aging follow a U-shaped curve with the minimum before puberty, whereas quantitative biomarkers of aging, including somatic mutations and DNA methylation, do not, revealing that aging starts early but is masked by early-life mortality. Moreover, our genetic analyses point to the contribution of damaging mutations to early mortality. We propose that mortality patterns are governed, in part, by negative selection against damaging mutations in early life, manifesting after the corresponding genes are first expressed. Deconvolution of mortality patterns suggests that deleterious changes rather than mortality are the defining characteristic of aging and that aging begins in very early life. More Information

Bakula D, Ablasser A, Aguzzi A, Antebi A, Barzilai N, Bittner MI, Jensen MB, Calkhoven CF, Chen D, de Grey ADNJ, Feige JN, Georgievskaya A, Gladyshev VN, Golato T, Gudkov AV, Hoppe T, Kaeberlein M, Katajisto P, Kennedy BK, Lal U, Martin-Villalba A, Moskalev AA, Ozerov I, Petr MA, Reason, Rubinsztein DC, Tyshkovskiy A, Vanhaelen Q 31, Zhavoronkov A, Scheibye-Knudsen M. (2019) Latest advances in aging research and drug discovery. Aging (Albany NY). 11(22), 9971-9981.

AbstractAn increasing aging population poses a significant challenge to societies worldwide. A better understanding of the molecular, cellular, organ, tissue, physiological, psychological, and even sociological changes that occur with aging is needed in order to treat age-associated diseases. The field of aging research is rapidly expanding with multiple advances transpiring in many previously disconnected areas. Several major pharmaceutical, biotechnology, and consumer companies made aging research a priority and are building internal expertise, integrating aging research into traditional business models and exploring new go-to-market strategies. Many of these efforts are spearheaded by the latest advances in artificial intelligence, namely deep learning, including generative and reinforcement learning. To facilitate these trends, the Center for Healthy Aging at the University of Copenhagen and Insilico Medicine are building a community of Key Opinion Leaders (KOLs) in these areas and launched the annual conference series titled “Aging Research and Drug Discovery (ARDD)” held in the capital of the pharmaceutical industry, Basel, Switzerland (www.agingpharma.org). This ARDD collection contains summaries from the 6th annual meeting that explored aging mechanisms and new interventions in age-associated diseases. The 7th annual ARDD exhibition will transpire 2nd-4th of September, 2020, in Basel. More Information

Bell CG, Lowe R, Adams PD, Baccarelli AA, Beck S, Bell JT, Christensen BC, Gladyshev VN, Heijmans BT, Horvath S, Ideker T, Issa JPJ, Kelsey KT, Marioni RE, Reik W, Relton CL, Schalkwyk LC, Teschendorff AE, Wagner W, Zhang K, Rakyan VK. (2019) DNA methylation aging clocks: challenges and recommendations. Genome Biol. 20(1), 249.

AbstractEpigenetic clocks comprise a set of CpG sites whose DNA methylation levels measure subject age. These clocks are acknowledged as a highly accurate molecular correlate of chronological age in humans and other vertebrates. Also, extensive research is aimed at their potential to quantify biological aging rates and test longevity or rejuvenating interventions. Here, we discuss key challenges to understand clock mechanisms and biomarker utility. This requires dissecting the drivers and regulators of age-related changes in single-cell, tissue- and disease-specific models, as well as exploring other epigenomic marks, longitudinal and diverse population studies, and non-human models. We also highlight important ethical issues in forensic age determination and predicting the trajectory of biological aging in an individual. More Information

Baclaocos J, Santesmasses D, Mariotti M, Bierła K, Vetick MB, Lynch S, McAllen R, Mackrill JJ, Loughran G, Guigó R, Szpunar J, Copeland PR, Gladyshev VN, Atkins JF. (2019) Processive Recoding and Metazoan Evolution of Selenoprotein P: Up to 132 UGAs in Molluscs. J Mol Biol. 431(22), 4381-4407.

AbstractSelenoproteins typically contain a single selenocysteine, the 21st amino acid, encoded by a context-redefined UGA. However, human selenoprotein P (SelenoP) has a redox-functioning selenocysteine in its N-terminal domain and nine selenium transporter-functioning selenocysteines in its C-terminal domain. Here we show that diverse SelenoP genes are present across metazoa with highly variable numbers of Sec-UGAs, ranging from a single UGA in certain insects, to 9 in common spider, and up to 132 in bivalve molluscs. SelenoP genes were shaped by a dynamic evolutionary process linked to selenium usage. Gene evolution featured modular expansions of an ancestral multi-Sec domain, which led to particularly Sec-rich SelenoP proteins in many aquatic organisms. We focused on molluscs, and chose Pacific oyster Magallana gigas as experimental model. We show that oyster SelenoP mRNA with 46 UGAs is translated full-length in vivo. Ribosome profiling indicates that selenocysteine specification occurs with ∼5% efficiency at UGA1 and approaches 100% efficiency at distal 3′ UGAs. We report genetic elements relevant to its expression, including a leader open reading frame and an RNA structure overlapping the initiation codon that modulates ribosome progression in a selenium-dependent manner. Unlike their mammalian counterparts, the two SECIS elements in oyster SelenoP (3’UTR recoding elements) do not show functional differentiation in vitro. Oysters can increase their tissue selenium level up to 50-fold upon supplementation, which also results in extensive changes in selenoprotein expression. More Information

Tyshkovskiy A, Bozaykut P, Borodinova AA, Gerashchenko MV, Ables GP, Garratt M, Khaitovich P, Clish CB, Miller RA, Gladyshev VN. (2019) Identification and Application of Gene Expression Signatures Associated with Lifespan Extension. Cell Metab. 30(3), 573-593.

AbstractSeveral pharmacological, dietary, and genetic interventions that increase mammalian lifespan are known, but general principles of lifespan extension remain unclear. Here, we performed RNA sequencing (RNA-seq) analyses of mice subjected to 8 longevity interventions. We discovered a feminizing effect associated with growth hormone regulation and diminution of sex-related differences. Expanding this analysis to 17 interventions with public data, we observed that many interventions induced similar gene expression changes. We identified hepatic gene signatures associated with lifespan extension across interventions, including upregulation of oxidative phosphorylation and drug metabolism, and showed that perturbed pathways may be shared across tissues. We further applied the discovered longevity signatures to identify new lifespan-extending candidates, such as chronic hypoxia, KU-0063794, and ascorbyl-palmitate. Finally, we developed GENtervention, an app that visualizes associations between gene expression changes and longevity. Overall, this study describes general and specific transcriptomic programs of lifespan extension in mice and provides tools to discover new interventions. More Information

Yim SH, Clish CB, Gladyshev VN. (2019) Selenium Deficiency Is Associated with Pro-longevity Mechanisms. Cell Rep. 27(9), 2785-2797.

AbstractSelenium (Se) is an essential trace element because of its presence in selenoproteins in the form of selenocysteine residue. Both Se deficiency, which compromises selenoprotein functions, and excess Se, which is toxic, have been associated with altered redox homeostasis and adverse health conditions. Surprisingly, we found that, although Se deficiency led to a drastic decline in selenoprotein expression, mice subjected to this dietary regimen for their entire life had normal lifespans. To understand the molecular mechanisms involved, we performed systemic analyses at the level of metabolome, transcriptome, and microRNA profiling. These analyses revealed that Se deficiency reduced amino acid levels, elevated mononucleotides, altered metabolism, and activated signaling pathways linked to longevity-related nutrient sensing. The data show that the metabolic control associated with nutrient sensing coordinately responds to suppressed selenoprotein functions, resulting in normal lifespan under Se deficiency. More Information

Mariotti M, Salinas G, Gabaldón T, Gladyshev VN. (2019) Utilization of selenocysteine in early-branching fungal phyla. Nat Microbiol. 4(5), 759-765.

AbstractSelenoproteins are a diverse group of proteins containing selenocysteine (Sec)-the twenty-first amino acid-incorporated during translation via a unique recoding mechanism1,2. Selenoproteins fulfil essential roles in many organisms1, yet are not ubiquitous across the tree of life3-7. In particular, fungi were deemed devoid of selenoproteins4,5,8. However, we show here that Sec is utilized by nine species belonging to diverse early-branching fungal phyla, as evidenced by the genomic presence of both Sec machinery and selenoproteins. Most fungal selenoproteins lack consensus Sec recoding signals (SECIS elements9) but exhibit other RNA structures, suggesting altered mechanisms of Sec insertion in fungi. Phylogenetic analyses support a scenario of vertical inheritance of the Sec trait within eukaryotes and fungi. Sec was then lost in numerous independent events in various fungal lineages. Notably, Sec was lost at the base of Dikarya, resulting in the absence of selenoproteins in Saccharomyces cerevisiae and other well-studied fungi. Our results indicate that, despite scattered occurrence, selenoproteins are found in all kingdoms of life. More Information

Simon M, Van Meter M, Ablaeva J, Ke Z, Gonzalez RS, Taguchi T, De Cecco M, Leonova KI, Kogan V, Helfand SL, Neretti N, Roichman A, Cohen HY, Meer MV, Gladyshev VN, Antoch MP, Gudkov AV, Sedivy JM, Seluanov A, Gorbunova V. (2019) LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation. Cell Metab. 29, e5.

AbstractMice deficient for SIRT6 exhibit a severely shortened lifespan, growth retardation, and highly elevated LINE1 (L1) activity. Here we report that SIRT6-deficient cells and tissues accumulate abundant cytoplasmic L1 cDNA, which triggers strong type I interferon response via activation of cGAS. Remarkably, nucleoside reverse-transcriptase inhibitors (NRTIs), which inhibit L1 retrotransposition, significantly improved health and lifespan of SIRT6 knockout mice and completely rescued type I interferon response. In tissue culture, inhibition of L1 with siRNA or NRTIs abrogated type I interferon response, in addition to a significant reduction of DNA damage markers. These results indicate that L1 activation contributes to the pathologies of SIRT6 knockout mice. Similarly, L1 transcription, cytoplasmic cDNA copy number, and type I interferons were elevated in the wild-type aged mice. As sterile inflammation is a hallmark of aging, we propose that modulating L1 activity may be an important strategy for attenuating age-related pathologies. More Information

Egorov AA, Sakharova EA, Anisimova AS, Dmitriev SE, Gladyshev VN, Kulakovskiy IV. (2019) svis4get: a simple visualization tool for genomic tracks from sequencing experiments. BMC Bioinformatics. 20, 113.

AbstractHigh-throughput sequencing often provides a foundation for experimental analyses in the life sciences. For many such methods, an intermediate layer of bioinformatics data analysis is the genomic signal track constructed by short read mapping to a particular genome assembly. There are many software tools to visualize genomic tracks in a web browser or with a stand-alone graphical user interface. However, there are only few command-line applications suitable for automated usage or production of publication-ready visualizations. More Information

O’Connell AE, Gerashchenko MV, O’Donohue MF, Rosen SM, Huntzinger E, Gleeson D, Galli A, Ryder E, Cao S, Murphy Q, Kazerounian S, Morton SU, Schmitz-Abe K, Gladyshev VN, Gleizes PE, Séraphin B, Agrawal PB. (2019) Mammalian Hbs1L deficiency causes congenital anomalies and developmental delay associated with Pelota depletion and 80S monosome accumulation. PLoS Genet. 15, e1007917.

AbstractHbs1 has been established as a central component of the cell’s translational quality control pathways in both yeast and prokaryotic models; however, the functional characteristics of its human ortholog (Hbs1L) have not been well-defined. We recently reported a novel human phenotype resulting from a mutation in the critical coding region of the HBS1L gene characterized by facial dysmorphism, severe growth restriction, axial hypotonia, global developmental delay and retinal pigmentary deposits. Here we further characterize downstream effects of the human HBS1L mutation. HBS1L has three transcripts in humans, and RT-PCR demonstrated reduced mRNA levels corresponding with transcripts V1 and V2 whereas V3 expression was unchanged. Western blot analyses revealed Hbs1L protein was absent in the patient cells. Additionally, polysome profiling revealed an abnormal aggregation of 80S monosomes in patient cells under baseline conditions. RNA and ribosomal sequencing demonstrated an increased translation efficiency of ribosomal RNA in Hbs1L-deficient fibroblasts, suggesting that there may be a compensatory increase in ribosome translation to accommodate the increased 80S monosome levels. This enhanced translation was accompanied by upregulation of mTOR and 4-EBP protein expression, suggesting an mTOR-dependent phenomenon. Furthermore, lack of Hbs1L caused depletion of Pelota protein in both patient cells and mouse tissues, while PELO mRNA levels were unaffected. Inhibition of proteasomal function partially restored Pelota expression in human Hbs1L-deficient cells. We also describe a mouse model harboring a knockdown mutation in the murine Hbs1l gene that shared several of the phenotypic elements observed in the Hbs1L-deficient human including facial dysmorphism, growth restriction and retinal deposits. The Hbs1lKO mice similarly demonstrate diminished Pelota levels that were rescued by proteasome inhibition. More Information

Tian X, Firsanov D, Zhang Z, Cheng Y, Luo L, Tombline G, Tan R, Simon M, Henderson S, Steffan J, Goldfarb A, Tam J, Zheng K, Cornwell A, Johnson A, Yang JN, Mao Z, Manta B, Dang W, Zhang Z, Vijg J, Wolfe A, Moody K, Kennedy BK, Bohmann D, Gladyshev VN, Seluanov, Gorbunova V. (2019) SIRT6 Is Responsible for More Efficient DNA Double-Strand Break Repair in Long-Lived Species. Cell. 177, 622-638.e22.

AbstractDNA repair has been hypothesized to be a longevity determinant, but the evidence for it is based largely on accelerated aging phenotypes of DNA repair mutants. Here, using a panel of 18 rodent species with diverse lifespans, we show that more robust DNA double-strand break (DSB) repair, but not nucleotide excision repair (NER), coevolves with longevity. Evolution of NER, unlike DSB, is shaped primarily by sunlight exposure. We further show that the capacity of the SIRT6 protein to promote DSB repair accounts for a major part of the variation in DSB repair efficacy between short- and long-lived species. We dissected the molecular differences between a weak (mouse) and a strong (beaver) SIRT6 protein and identified five amino acid residues that are fully responsible for their differential activities. Our findings demonstrate that DSB repair and SIRT6 have been optimized during the evolution of longevity, which provides new targets for anti-aging interventions. More Information

Ogrodnik M, Salmonowicz H, Gladyshev VN. (2019) Integrating cellular senescence with the concept of damage accumulation in aging: Relevance for clearance of senescent cells. Agin Cell. 18, e12841.

AbstractUnderstanding the aging process and ways to manipulate it is of major importance for biology and medicine. Among the many aging theories advanced over the years, the concept most consistent with experimental evidence posits the buildup of numerous forms of molecular damage as a foundation of the aging process. Here, we discuss that this concept integrates well with recent findings on cellular senescence, offering a novel view on the role of senescence in aging and age-related disease. Cellular senescence has a well-established role in cellular aging, but its impact on the rate of organismal aging is less defined. One of the most prominent features of cellular senescence is its association with macromolecular damage. The relationship between cell senescence and damage concerns both damage as a molecular signal of senescence induction and accelerated accumulation of damage in senescent cells. We describe the origin, regulatory mechanisms, and relevance of various damage forms in senescent cells. This view on senescent cells as carriers and inducers of damage puts new light on senescence, considering it as a significant contributor to the rise in organismal damage. Applying these ideas, we critically examine current evidence for a role of cellular senescence in aging and age-related diseases. We also discuss the differential impact of longevity interventions on senescence burden and other types of age-related damage. Finally, we propose a model on the role of aging-related damage accumulation and the rate of aging observed upon senescent cell clearance. More Information

Galkin F, Zhang B, Dmitriev SE, Gladyshev VN. (2019) Reversibility of irreversible aging. Ageing Res Rev. 49, 104-114.

AbstractMost multicellular organisms are known to age, due to accumulation of damage and other deleterious changes over time. These changes are often irreversible, as organisms, humans included, evolved fully differentiated, irreplaceable cells (e.g. neurons) and structures (e.g. skeleton). Hence, deterioration or loss of at least some cells and structures should lead to inevitable aging of these organisms. Yet, some cells may escape this fate: adult somatic cells may be converted to partially reprogrammed cells or induced pluripotent stem cells (iPSCs). By their nature, iPSCs are the cells representing the early stages of life, indicating a possibility of reversing the age of cells within the organism. Reprogramming strategies may be accomplished both in vitro and in vivo, offering opportunities for rejuvenation in the context of whole organisms. Similarly, older organs may be replaced with the younger ones prepared ex vivo, or grown within other organisms or even other species. How could the irreversibility of aging of some parts of the organism be reconciled with the putative reversal of aging of the other parts of the same organism? Resolution of this question holds promise for dramatically extending lifespan, which is currently not possible with traditional genetic, dietary and pharmacological approaches. Critical issues in this challenge are the nature of aging, relationship between aging of an organism and aging of its parts, relationship between cell dedifferentiation and rejuvenation, and increased risk of cancer that goes hand in hand with rejuvenation approaches. More Information

2018 Articles

Meer MV, Podolskiy DI, Tyshkovskiy A, Gladyshev VN. (2018) A whole lifespan mouse multi-tissue DNA methylation clock. eLife.7, e40675.

AbstractAge predictors based on DNA methylation levels at a small set of CpG sites, DNAm clocks, have been developed for humans and extended to several other species. Three currently available versions of mouse DNAm clocks were either created for individual tissues or tuned towards young ages. Here, we constructed a robust multi-tissue age predictor based on 435 CpG sites, which covers the entire mouse lifespan and remains unbiased with respect to any particular age group. It can successfully detect the effects of certain lifespan-modulating interventions on DNAm age as well as the rejuvenation effect related to the transition from fibroblasts to iPSCs. We have carried out comparative analyses of available mouse DNAm clocks, which revealed their broad applicability, but also certain limitations to the use of tissue-specific and multi-tissue age predictors. Together, these tools should help address diverse questions in aging research. More Information

Bakula D, Aliper AM, Mamoshina P, Petr MA, Teklu A, Baur JA, Campisi J, Ewald CY, Georgievskaya A, Gladyshev VN, Kovalchuk O, Lamming DW, Luijsterburg MS, Martín-Montalvo A, Maudsley S, Mkrtchyan GV, Moskalev A, Olshansky SJ, Ozerov IV, Pickett A, Ristow M, Zhavoronkov A, Scheibye-Knudsen M. (2018) Aging and drug discovery. Aging (Albany NY). 11, 3079-3088.

AbstractMultiple interventions in the aging process have been discovered to extend the healthspan of model organisms. Both industry and academia are therefore exploring possible transformative molecules that target aging and age-associated diseases. In this overview, we summarize the presented talks and discussion points of the 5th Annual Aging and Drug Discovery Forum 2018 in Basel, Switzerland. Here academia and industry came together, to discuss the latest progress and issues in aging research. The meeting covered talks about the mechanistic cause of aging, how longevity signatures may be highly conserved, emerging biomarkers of aging, possible interventions in the aging process and the use of artificial intelligence for aging research and drug discovery. Importantly, a consensus is emerging both in industry and academia, that molecules able to intervene in the aging process may contain the potential to transform both societies and healthcare. More Information

Peters KM, Carlson BA, Gladyshev VN, Tsuji PA. (2018) Selenoproteins in colon cancer. Free Radic Biol Med.127, 14-25.

AbstractSelenocysteine-containing proteins (selenoproteins) have been implicated in the regulation of various cell signaling pathways, many of which are linked to colorectal malignancies. In this in-depth excurse into the selenoprotein literature, we review possible roles for human selenoproteins in colorectal cancer, focusing on the typical hallmarks of cancer cells and their tumor-enabling characteristics. Human genome studies of single nucleotide polymorphisms in various genes coding for selenoproteins have revealed potential involvement of glutathione peroxidases, thioredoxin reductases, and other proteins. Cell culture studies with targeted down-regulation of selenoproteins and studies utilizing knockout/transgenic animal models have helped elucidate the potential roles of individual selenoproteins in this malignancy. Those selenoproteins, for which strong links to development or progression of colorectal cancer have been described, may be potential future targets for clinical interventions. More Information

Lee SG, Mikhalchenko AE, Yim SH, Gladyshev VN. (2018) A naked mole rat iPSC line expressing drug-inducible mouse pluripotency factors developed from embryonic fibroblasts. Stem Cell Res. 10, 197-200.

AbstractNaked mole rats (NMRs, Heterocephalus glaber) are long-lived, cancer-resistant rodents. Here, we report the development of an induced pluripotent stem cell (iPSC) line generated from immortalized NMR embryonic fibroblasts transduced with a doxycycline-inducible mouse OSKM polycistronic vector. This iPSC line was shown to express pluripotency-associated markers, form embryoid bodies, differentiate in vitro to the derivatives of three germ layers, and exhibit normal karyotype. The ability of iPSCs to differentiate in vivo was supported by the contribution to interspecific chimera upon injection into mouse blastocysts. This NMR iPSC line may be a useful tool in cancer and aging research. More Information

Zhang Y, Lee JH, Paull TT, Gehrke S, D’Alessandro A, Dou Q, Gladyshev VN, Schroeder EA, Steyl SK, Christian BE, Shadel GS. (2018) Mitochondrial redox sensing by the kinase ATM maintains cellular antioxidant capacity. Sci Signal. 11, 538.

AbstractMitochondria are integral to cellular energy metabolism and ATP production and are involved in regulating many cellular processes. Mitochondria produce reactive oxygen species (ROS), which not only can damage cellular components but also participate in signal transduction. The kinase ATM, which is mutated in the neurodegenerative, autosomal recessive disease ataxia-telangiectasia (A-T), is a key player in the nuclear DNA damage response. However, ATM also performs a redox-sensing function mediated through formation of ROS-dependent disulfide-linked dimers. We found that mitochondria-derived hydrogen peroxide promoted ATM dimerization. In HeLa cells, ATM dimers were localized to the nucleus and inhibited by the redox regulatory protein thioredoxin 1 (TRX1), suggesting the existence of a ROS-mediated, stress-signaling relay from mitochondria to the nucleus. ATM dimer formation did not affect its association with chromatin in the absence or presence of nuclear DNA damage, consistent with the separation of its redox and DNA damage signaling functions. Comparative analysis of U2OS cells expressing either wild-type ATM or the redox sensing-deficient C2991L mutant revealed that one function of ATM redox sensing is to promote glucose flux through the pentose phosphate pathway (PPP) by increasing the abundance and activity of glucose-6-phosphate dehydrogenase (G6PD), thereby increasing cellular antioxidant capacity. The PPP produces the coenzyme NADPH needed for a robust antioxidant response, including the regeneration of TRX1, indicating the existence of a regulatory feedback loop involving ATM and TRX1. We propose that loss of the mitochondrial ROS-sensing function of ATM may cause cellular ROS accumulation and oxidative stress in A-T. More Information

Yim SH, Everley RA, Schildberg FA, Lee SG, Orsi A, Barbati ZR, Karatepe K, Fomenko DE, Tsuji PA, Luo HR, Gygi SP, Sitia R, Sharpe AH, Hatfield DL, Gladyshev VN. (2018) Role of Selenof as a Gatekeeper of Secreted Disulfide-Rich Glycoproteins. Cell Rep. 23, 1387-1398.

AbstractSelenof (15-kDa selenoprotein; Sep15) is an endoplasmic reticulum (ER)-resident thioredoxin-like oxidoreductase that occurs in a complex with UDP-glucose:glycoprotein glucosyltransferase. We found that Selenof deficiency in mice leads to elevated levels of non-functional circulating plasma immunoglobulins and increased secretion of IgM during in vitro splenic B cell differentiation. However, Selenof knockout animals show neither enhanced bacterial killing capacity nor antigen-induced systemic IgM activity, suggesting that excess immunoglobulins are not functional. In addition, ER-to-Golgi transport of a target glycoprotein was delayed in Selenof knockout embryonic fibroblasts, and proteomic analyses revealed that Selenof deficiency is primarily associated with antigen presentation and ER-to-Golgi transport. Together, the data suggest that Selenof functions as a gatekeeper of immunoglobulins and, likely, other client proteins that exit the ER, thereby supporting redox quality control of these proteins. More Information

Samokhin AO, Stephens T, Wertheim BM, Wang RS, Vargas SO, Yung LM, Cao M, Brown M, Arons E, Dieffenbach PB, Fewell JG, Matar M, Bowman FP, Haley KJ, Alba GA, Marino SM, Kumar R, Rosas IO, Waxman AB, Oldham WM, Khanna D, Graham BB, Seo S, Gladyshev VN, Yu PB, Fredenburgh LE, Loscalzo J, Leopold JA, Maron BA. (2018) NEDD9 targets COL3A1 to promote endothelial fibrosis and pulmonary arterial hypertension. Sci Transl Med. 10, 445.

AbstractGermline mutations involving small mothers against decapentaplegic-transforming growth factor-β (SMAD-TGF-β) signaling are an important but rare cause of pulmonary arterial hypertension (PAH), which is a disease characterized, in part, by vascular fibrosis and hyperaldosteronism (ALDO). We developed and analyzed a fibrosis protein-protein network (fibrosome) in silico, which predicted that the SMAD3 target neural precursor cell expressed developmentally down-regulated 9 (NEDD9) is a critical ALDO-regulated node underpinning pathogenic vascular fibrosis. Bioinformatics and microscale thermophoresis demonstrated that oxidation of Cys18 in the SMAD3 docking region of NEDD9 impairs SMAD3-NEDD9 protein-protein interactions in vitro. This effect was reproduced by ALDO-induced oxidant stress in cultured human pulmonary artery endothelial cells (HPAECs), resulting in impaired NEDD9 proteolytic degradation, increased NEDD9 complex formation with Nk2 homeobox 5 (NKX2-5), and increased NKX2-5 binding to COL3A1 Up-regulation of NEDD9-dependent collagen III expression corresponded to changes in cell stiffness measured by atomic force microscopy. HPAEC-derived exosomal signaling targeted NEDD9 to increase collagen I/III expression in human pulmonary artery smooth muscle cells, identifying a second endothelial mechanism regulating vascular fibrosis. ALDO-NEDD9 signaling was not affected by treatment with a TGF-β ligand trap and, thus, was not contingent on TGF-β signaling. Colocalization of NEDD9 with collagen III in HPAECs was observed in fibrotic pulmonary arterioles from PAH patients. Furthermore, NEDD9 ablation or inhibition prevented fibrotic vascular remodeling and pulmonary hypertension in animal models of PAH in vivo. These data identify a critical TGF-β-independent posttranslational modification that impairs SMAD3-NEDD9 binding in HPAECs to modulate vascular fibrosis and promote PAH. More Information

Na J, Jung J, Bang J, Lu Q, Carlson BA, Guo X, Gladyshev VN, Kim J, Hatfield DL, Lee BJ. (2018) Selenophosphate synthetase 1 and its role in redox homeostasis, defense and proliferation. Free Radic Biol Med. 127,190-197.

AbstractSelenophosphate synthetase (SEPHS) synthesizes selenophosphate, the active selenium donor, using ATP and selenide as substrates. SEPHS was initially identified and isolated from bacteria and has been characterized in many eukaryotes and archaea. Two SEPHS paralogues, SEPHS1 and SEPHS2, occur in various eukaryotes, while prokaryotes and archaea have only one form of SEPHS. Between the two isoforms in eukaryotes, only SEPHS2 shows catalytic activity during selenophosphate synthesis. Although SEPHS1 does not contain any significant selenophosphate synthesis activity, it has been reported to play an essential role in regulating cellular physiology. Prokaryotic SEPHS contains a cysteine or selenocysteine (Sec) at the catalytic domain. However, in eukaryotes, SEPHS1 contains other amino acids such as Thr, Arg, Gly, or Leu at the catalytic domain, and SEPHS2 contains only a Sec. Sequence comparisons, crystal structure analyses, and ATP hydrolysis assays suggest that selenophosphate synthesis occurs in two steps. In the first step, ATP is hydrolyzed to produce ADP and gamma-phosphate. In the second step, ADP is further hydrolyzed and selenophosphate is produced using gamma-phosphate and selenide. Both SEPHS1 and SEPHS2 have ATP hydrolyzing activities, but Cys or Sec is required in the catalytic domain for the second step of reaction. The gene encoding SEPHS1 is divided by introns, and five different splice variants are produced by alternative splicing in humans. SEPHS1 mRNA is abundant in rapidly proliferating cells such as embryonic and cancer cells and its expression is induced by various stresses including oxidative stress and salinity stress. The disruption of the SEPHS1 gene in mice or Drosophila leads to the inhibition of cell proliferation, embryonic lethality, and morphological changes in the embryos. Targeted removal of SEPHS1 mRNA in insect, mouse, and human cells also leads to common phenotypic changes similar to those observed by in vivo gene knockout: the inhibition of cell growth/proliferation, the accumulation of hydrogen peroxide in mammals and an unidentified reactive oxygen species (ROS) in Drosophila, and the activation of a defense system. Hydrogen peroxide accumulation in SEPHS1-deficient cells is mainly caused by the down-regulation of genes involved in ROS scavenging, and leads to the inhibition of cell proliferation and survival. However, the mechanisms underlying SEPHS1 regulation of redox homeostasis are still not understood. More Information

Zhou X, Guang X, Sun D, Xu S, Li M, Seim I, Jie W, Yang L, Zhu Q, Xu J, Gao Q, Kaya A, Dou Q, Chen B, Ren W, Li S, Zhou K, Gladyshev VN, Nielsen R, Fang X, Yang G. (2018) Population genomics of finless porpoises reveal an incipient cetacean species adapted to freshwater. Nat Commun. 9, 1276.

AbstractCetaceans (whales, dolphins, and porpoises) are a group of mammals adapted to various aquatic habitats, from oceans to freshwater rivers. We report the sequencing, de novo assembly and analysis of a finless porpoise genome, and the re-sequencing of an additional 48 finless porpoise individuals. We use these data to reconstruct the demographic history of finless porpoises from their origin to the occupation into the Yangtze River. Analyses of selection between marine and freshwater porpoises identify genes associated with renal water homeostasis and urea cycle, such as urea transporter 2 and angiotensin I-converting enzyme 2, which are likely adaptations associated with the difference in osmotic stress between ocean and rivers. Our results strongly suggest that the critically endangered Yangtze finless porpoises are reproductively isolated from other porpoise populations and harbor unique genetic adaptations, supporting that they should be considered a unique incipient species. More Information

Tarrago L, Oheix E, Péterfi Z, Gladyshev VN. (2018) Monitoring of Methionine Sulfoxide Content and Methionine Sulfoxide Reductase Activity. Methods Mol Biol. 1661, 285-299.

AbstractThe sulfur-containing amino acid methionine (Met) plays critical roles in protein synthesis, methylation, and sulfur metabolism. Both in its free form and in the form of an amino acid residue, it can be oxidized to the R and S diastereomers of methionine sulfoxide (MetO). Organisms evolved methionine sulfoxide reductases (MSRs) to reduce MetO to Met, with the MSRs type A (MSRA) and type B (MSRB) being specific for the S and R forms of MetO, respectively. In mammals, the selenoprotein MSRB1 plays an important protein repair function, and its expression is tightly regulated by dietary selenium. In this chapter, we describe a protocol for determining the concentration of protein-based Met-R-O and its analysis in HEK293 cells using a genetically encoded ratiometric fluorescent biosensor MetROx. We also describe the procedure for quantifying MSR activities in cell extracts using specific substrates and a reverse phase HPLC-based method. More Information

Carlson BA, Lee BJ, Tsuji PA, Copeland PR, Schweizer U, Gladyshev VN, Hatfield DL. (2018) Selenocysteine tRNA[Ser]Sec, the Central Component of Selenoprotein Biosynthesis: Isolation, Identification, Modification, and Sequencing. Methods Mol Bio. 1661, 43-60.

AbstractThe selenocysteine (Sec) tRNA[Ser]Sec population consists of two isoforms that differ from each other by a single 2′-O-methylribosyl moiety at position 34 (Um34). These two isoforms, which are encoded in a single gene, Trsp, and modified posttranscriptionally, are involved individually in the synthesis of two subclasses of selenoproteins, designated housekeeping and stress-related selenoproteins. Techniques used in obtaining these isoforms for their characterization include extraction of RNA from mammalian cells and tissues, purifying the tRNA[Ser]Sec population by one or more procedures, and finally resolving the two isoforms from each other. Since some of the older techniques for isolating tRNA[Ser]Sec and resolving the isoforms are used in only a few laboratories, these procedures will be discussed briefly and references provided for more detailed information, while the more recently developed procedures are discussed in detail. In addition, a novel technique that was developed in sequencing tRNA[Ser]Sec for identifying their occurrence in other organisms is also presented. More Information

Golubev A, Hanson AD, Gladyshev VN. (2018) A tale of two concepts: Harmonizing the free radical and antagonistic pleiotropy theories of aging. Antioxid Redox Signal. 29,1003-1017.

AbstractThe two foremost concepts of aging are the mechanistic free radical theory (FRT) of how we age, and the evolutionary antagonistic pleiotropy theory (APT) of why we age. Both date from the late 1950s. The FRT holds that reactive oxygen species (ROS) are the principal contributors to the lifelong cumulative damage suffered by cells, whereas the APT is generally understood as positing that genes that are good for young organisms can take over a population even if they are bad for the old ones. Being related to two sides of the same phenomenon, these theories should be compatible. However, the interface between them is obscured by the FRT mistaking a subset of damaging processes for the whole, and the APT mistaking a cumulative quantitative process for a qualitative switch. Here, we provide a common ground for the two theories by showing how aging can result from the inherent chemical reactivity of many biomolecules, not just ROS, which imposes a fundamental constraint on biological evolution. Chemically reactive metabolites spontaneously modify slowly renewable macromolecules in a continuous way over time; the resulting buildup of damage wrought by the genes coding for enzymes that generate such small molecules eventually masquerades as late-acting pleiotropic effects. In aerobic organisms, ROS are major agents of this damage but they are far from alone. The manifestations of ROS-mediated cumulative chemical damage at the population level may include the often-observed negative correlation between fitness and the rate of its decline with increasing age, further linking FRT and APT. More Information

Seluanov A, Gladyshev VN, Vijg J, Gorbunova V. (2018) Mechanisms of cancer resistance in long-lived mammals. Nat Rev Cancer. 18, 433-441.

AbstractCancer researchers have traditionally used the mouse and the rat as staple model organisms. These animals are very short-lived, reproduce rapidly and are highly prone to cancer. They have been very useful for modelling some human cancer types and testing experimental treatments; however, these cancer-prone species offer little for understanding the mechanisms of cancer resistance. Recent technological advances have expanded bestiary research to non-standard model organisms that possess unique traits of very high value to humans, such as cancer resistance and longevity. In recent years, several discoveries have been made in non-standard mammalian species, providing new insights on the natural mechanisms of cancer resistance. These include mechanisms of cancer resistance in the naked mole rat, blind mole rat and elephant. In each of these species, evolution took a different path, leading to novel mechanisms. Many other long-lived mammalian species display cancer resistance, including whales, grey squirrels, microbats, cows and horses. Understanding the molecular mechanisms of cancer resistance in all these species is important and timely, as, ultimately, these mechanisms could be harnessed for the development of human cancer therapies. More Information

Zhou X, Sun D, Guang X, Ma S, Fang X, Mariotti M, Nielsen R, Gladyshev VN, Yang G. (2018) Molecular Footprints of Aquatic Adaptation Including Bone Mass Changes in Cetaceans. Genome Biol Evol. 10, 967-975.

AbstractCetaceans (whales, dolphins, and porpoises) are a group of specialized mammals that evolved from terrestrial ancestors and are fully adapted to aquatic habitats. Taking advantage of the recently sequenced finless porpoise genome, we conducted comparative analyses of the genomes of seven cetaceans and related terrestrial species to provide insight into the molecular bases of adaptation of these aquatic mammals. Changes in gene sequences were identified in main lineages of cetaceans, offering an evolutionary picture of cetacean genomes that reveal new pathways that could be associated with adaptation to aquatic lifestyle. We profiled bone microanatomical structures across 28 mammals, including representatives of cetaceans, pinnipeds, and sirenians. Subsequent phylogenetic comparative analyses revealed genes (including leptin, insulin-like growth factor 1, and collagen type I alpha 2 chain) with the root-to-tip substitution rate significantly correlated with bone compactness, implicating these genes could be involved in bone mass control. Overall, this study described adjustments of the genomes of cetaceans according to lifestyle, phylogeny, and bone mass. More Information

Sziráki A, Tyshkovskiy A, Gladyshev VN. (2018) Global remodeling of the mouse DNA methylome during aging and in response to calorie restriction. Aging Cell. 17, e12738.

AbstractAging is characterized by numerous molecular changes, such as accumulation of molecular damage and altered gene expression, many of which are linked to DNA methylation. Here, we characterize the blood DNA methylome across 16 age groups of mice and report numerous global, region- and site-specific features, as well as the associated dynamics of methylation changes. Transition of the methylome throughout lifespan was not uniform, with many sites showing accelerated changes in late life. The associated genes and promoters were enriched for aging-related pathways, pointing to a fundamental link between DNA methylation and control of the aging process. Calorie restriction both shifted the overall methylation pattern and was accompanied by its gradual age-related remodeling, the latter contributing to the lifespan-extending effect. With age, both highly and poorly methylated sites trended toward intermediate levels, and aging was accompanied by an accelerated increase in entropy, consistent with damage accumulation. However, the entropy effects differed for the sites that increased, decreased and did not change methylation with age. Many sites trailed behind, whereas some followed or even exceeded the entropy trajectory and altered the developmental DNA methylation pattern. The patterns we observed in certain genomic regions were conserved between humans and mice, suggesting common principles of functional DNA methylome remodeling and its critical role in aging. The highly resolved DNA methylome remodeling provides an excellent model for understanding systemic changes that characterize the aging process. More Information

Zhao Y, Tyshkovskiy A, Muñoz-Espín D, Tian X, Serrano M, de Magalhaes JP, Nevo E, Gladyshev VN, Seluanov A, Gorbunova V. (2018) Naked mole rats can undergo developmental, oncogene-induced and DNA damage-induced cellular senescence. Proc Natl Acad Sci U S A. 115, 1801-1806.

AbstractCellular senescence is an important anticancer mechanism that restricts proliferation of damaged or premalignant cells. Cellular senescence also plays an important role in tissue remodeling during development. However, there is a trade-off associated with cellular senescence as senescent cells contribute to aging pathologies. The naked mole rat (NMR) (Heterocephalus glaber) is the longest-lived rodent that is resistant to a variety of age-related diseases. Remarkably, NMRs do not show aging phenotypes until very late stages of their lives. Here, we tested whether NMR cells undergo cellular senescence. We report that the NMR displays developmentally programmed cellular senescence in multiple tissues, including nail bed, skin dermis, hair follicle, and nasopharyngeal cavity. NMR cells also underwent cellular senescence when transfected with oncogenic Ras. In addition, cellular senescence was detected in NMR embryonic and skin fibroblasts subjected to γ-irradiation (IR). However, NMR cells required a higher dose of IR for induction of cellular senescence, and NMR fibroblasts were resistant to IR-induced apoptosis. Gene expression analyses of senescence-related changes demonstrated that, similar to mice, NMR cells up-regulated senescence-associated secretory phenotype genes but displayed more profound down-regulation of DNA metabolism, transcription, and translation than mouse cells. We conclude that the NMR displays the same types of cellular senescence found in a short-lived rodent. More Information

Lee BC, Lee HM, Kim S, Avanesov AS, Lee A, Chun BH, Vorbruggen G, Gladyshev VN. (2018) Expression of the methionine sulfoxide reductase lost during evolution extends Drosophila lifespan in a methionine-dependent manner. Sci Rep. 8, 1010.

AbstractAccumulation of oxidized amino acids, including methionine, has been implicated in aging. The ability to reduce one of the products of methionine oxidation, free methionine-R-sulfoxide (Met-R-SO), is widespread in microorganisms, but during evolution this function, conferred by the enzyme fRMsr, was lost in metazoa. We examined whether restoration of the fRMsr function in an animal can alleviate the consequences of methionine oxidation. Ectopic expression of yeast fRMsr supported the ability of Drosophila to catalyze free Met-R-SO reduction without affecting fecundity, food consumption, and response to starvation. fRMsr expression also increased resistance to oxidative stress. Moreover, it extended lifespan of flies in a methionine-dependent manner. Thus, expression of an oxidoreductase lost during evolution can enhance metabolic and redox functions and lead to an increase in lifespan in an animal model. More broadly, our study exposes the potential of a combination of genetic and nutritional strategies in lifespan control. More Information

Yordanova MM, Loughran G, Zhdanov AV, Mariotti M, Kiniry SJ, O’Connor PBF, Andreev DE, Tzani I, Saffert P, Michel AM, Gladyshev VN, Papkovsky DB, Atkins JF, Baranov PV. (2018) AMD1 mRNA employs ribosome stalling as a mechanism for molecular memory formation. Nature. 553, 356-360.

AbstractIn addition to acting as template for protein synthesis, messenger RNA (mRNA) often contains sensory sequence elements that regulate this process. Here we report a new mechanism that limits the number of complete protein molecules that can be synthesized from a single mRNA molecule of the human AMD1 gene encoding adenosylmethionine decarboxylase 1 (AdoMetDC). A small proportion of ribosomes translating AMD1 mRNA stochastically read through the stop codon of the main coding region. These readthrough ribosomes then stall close to the next in-frame stop codon, eventually forming a ribosome queue, the length of which is proportional to the number of AdoMetDC molecules that were synthesized from the same AMD1 mRNA. Once the entire spacer region between the two stop codons is filled with queueing ribosomes, the queue impinges upon the main AMD1 coding region halting its translation. Phylogenetic analysis suggests that this mechanism is highly conserved in vertebrates and existed in their common ancestor. We propose that this mechanism is used to count and limit the number of protein molecules that can be synthesized from a single mRNA template. It could serve to safeguard from dysregulated translation that may occur owing to errors in transcription or mRNA damage. More Information

Ma S, Avanesov AS, Porter E, Lee BC, Mariotti M, Zemskaya N, Guigo R, Moskalev AA, Gladyshev VN. (2018) Comparative transcriptomics across 14 Drosophila species reveals signatures of longevity. Aging Cell. e12740.

AbstractLifespan varies dramatically among species, but the biological basis is not well understood. Previous studies in model organisms revealed the importance of nutrient sensing, mTOR, NAD/sirtuins, and insulin/IGF1 signaling in lifespan control. By studying life-history traits and transcriptomes of 14 Drosophila species differing more than sixfold in lifespan, we explored expression divergence and identified genes and processes that correlate with longevity. These longevity signatures suggested that longer-lived flies upregulate fatty acid metabolism, downregulate neuronal system development and activin signaling, and alter dynamics of RNA splicing. Interestingly, these gene expression patterns resembled those of flies under dietary restriction and several other lifespan-extending interventions, although on the individual gene level, there was no significant overlap with genes previously reported to have lifespan-extension effects. We experimentally tested the lifespan regulation potential of several candidate genes and found no consistent effects, suggesting that individual genes generally do not explain the observed longevity patterns. Instead, it appears that lifespan regulation across species is modulated by complex relationships at the system level represented by global gene expression. More Information

2017 Articles

Tang Q, Gu Y, Zhou X, Jin L, Guan J, Liu R, Li J, Long K, Tian S, Che T, Hu S, Liang Y, Yang X, Tao X, Zhong Z, Wang G, Chen X, Li D, Ma J, Wang X, Mai M, Jiang A, Luo X, Lv X, Gladyshev VN, Li X, Li M. (2017) Comparative transcriptomics of 5 high-altitude vertebrates and their low-altitude relatives. Gigascience. 6, 1–9.

AbstractBackground – Species living at high altitude are subject to strong selective pressures due to inhospitable environments (e.g., hypoxia, low temperature, high solar radiation, and lack of biological production), making these species valuable models for comparative analyses of local adaptation. Studies that have examined high-altitude adaptation have identified a vast array of rapidly evolving genes that characterize the dramatic phenotypic changes in high-altitude animals. However, how high-altitude environment shapes gene expression programs remains largely unknown.
Findings – We generated a total of 910 Gb of high-quality RNA-seq data for 180 samples derived from 6 tissues of 5 agriculturally important high-altitude vertebrates (Tibetan chicken, Tibetan pig, Tibetan sheep, Tibetan goat, and yak) and their cross-fertile relatives living in geographically neighboring low-altitude regions. Of these, ∼75% reads could be aligned to their respective reference genomes, and on average ∼60% of annotated protein coding genes in each organism showed FPKM expression values greater than 0.5. We observed a general concordance in topological relationships between the nucleotide alignments and gene expression–based trees. Tissue and species accounted for markedly more variance than altitude based on either the expression or the alternative splicing patterns. Cross-species clustering analyses showed a tissue-dominated pattern of gene expression and a species-dominated pattern for alternative splicing. We also identified numerous differentially expressed genes that could potentially be involved in phenotypic divergence shaped by high-altitude adaptation.
Conclusions – These data serve as a valuable resource for examining the convergence and divergence of gene expression changes between species as they adapt or acclimatize to high-altitude environments. More Information

Kim KY, Kwak GH, Singh MP, Gladyshev VN, Kim HY. (2017) Monitoring of Methionine Sulfoxide Content and Methionine Sulfoxide Reductase Activity. Arch Biochem Biophys. 634, 69-75.

AbstractAcetaminophen (APAP) overdose induces acute liver damage and failure via reactive oxygen species production and glutathione (GSH) depletion. Methionine sulfoxide reductase B1 (MsrB1) is an antioxidant selenoenzyme that specifically catalyzes the reduction of methionine R-sulfoxide residues. In this study, we used MsrB1 gene-knockout mice and primary hepatocytes to investigate the effect of MsrB1 on APAP-induced hepatotoxicity. Analyses of histological alterations and serum indicators of liver damage showed that MsrB1-/- mice were more susceptible to APAP-induced acute liver injury than wild-type (MsrB1+/+) mice. Consistent with the in vivo results, primary MsrB1-/- hepatocytes displayed higher susceptibility to APAP-induced cytotoxicity than MsrB1+/+ cells. MsrB1 deficiency increased hepatic oxidative stress after APAP challenge such as hydrogen peroxide production, lipid peroxidation, and protein oxidation levels. Additionally, basal and APAP-induced ratios of reduced-to-oxidized GSH (GSH/GSSG) were significantly lower in MsrB1-/- than in MsrB1+/+ livers. Nrf2 nuclear accumulation and heme oxygenase-1 expression levels after APAP challenge were lower in MsrB1-/- than in MsrB1+/+ livers, suggesting that MsrB1 deficiency attenuates the APAP-induced activation of Nrf2. Collectively, the results of this study suggest that selenoprotein MsrB1 plays a protective role against APAP-induced hepatotoxicity via its antioxidative function. More Information

Ma S, Gladyshev VN. (2017) Molecular signatures of longevity: Insights from cross-species comparative studies. Semin Cell Dev Biol. 70, 190-203.

AbstractMuch of the current research on longevity focuses on the aging process within a single species. Several molecular players (e.g. IGF1 and MTOR), pharmacological compounds (e.g. rapamycin and metformin), and dietary approaches (e.g. calorie restriction and methionine restriction) have been shown to be important in regulating and modestly extending lifespan in model organisms. On the other hand, natural lifespan varies much more significantly across species. Within mammals alone, maximum lifespan differs more than 100 fold, but the underlying regulatory mechanisms remain poorly understood. Recent comparative studies are beginning to shed light on the molecular signatures associated with exceptional longevity. These include genome sequencing of microbats, naked mole rat, blind mole rat, bowhead whale and African turquoise killifish, and comparative analyses of gene expression, metabolites, lipids and ions across multiple mammalian species. Together, they point towards several putative strategies for lifespan regulation and cancer resistance, as well as the pathways and metabolites associated with longevity variation. In particular, longevity may be achieved by both lineage-specific adaptations and common mechanisms that apply across the species. Comparing the resulting cross-species molecular signatures with the within-species lifespan extension strategies will improve our understanding of mechanisms of longevity control and provide a starting point for novel and effective interventions. More Information

Kaya A, Mariotti M, Gladyshev VN. (2017) Cytochrome c peroxidase facilitates the beneficial use of H2O2 in prokaryotes. Proc Natl Acad Sci U S A. 144, 8678-8680.

AbstractA new exciting study reports the discovery of a beneficial function of H2O2 in bacteria (1). Khademian and Imlay show that Escherichia coli can use cytochrome c peroxidase (Ccp) as a respiratory enzyme, wherein H2O2 acts as an electron acceptor under anoxic conditions (Fig. 1A). This finding may impact our understanding of microbial metabolism and the role of H2O2 in prokaryotes. The use of molecular oxygen by organisms is often associated with the phenomenon known as oxidative stress, a deleterious state counteracted by an arsenal of specialized enzymes that ensure that this threat is contained and the intracellular milieu is protected (2). This function is supported by oxidation and reduction reactions that alleviate the deleterious effects of partially reduced species of molecular oxygen, also known as reactive oxygen species (ROS). We also know that, in eukaryotes, ROS may not only induce “collateral damage,” but also have fundamental roles in cellular physiology, supporting such processes as the immune response, signal transduction, and cell proliferation (3). Organisms tightly control the levels of ROS and their spatiotemporal characteristics, so that they could be used as second messengers or agents for bacterial killing, yet would not significantly damage cellular components. Mitochondria are considered as a major intracellular source of H2O2, an abundant and physiologically relevant form of ROS in cells that originates from the superoxide anion (O2.−) during aerobic respiration (4). Cells use several types of enzymes to fine-tune the rate of H2O2 release from this compartment to cytoplasm. H2O2 is also a product of various enzymes that use molecular oxygen for two-electron oxidation reactions (5), further supporting the physiological relevance of H2O2 for cellular life. However, because the purposeful use of H2O2 has only been described for eukaryotes, a question arises as to when, during the evolution of life, the cells adopted the toxic H2O2 for a beneficial use. More Information

Petkovich DA, Podolskiy DI, Lobanov AV, Lee SG, Miller RA, Gladyshev VN. (2017) Using DNA Methylation Profiling to Evaluate Biological Age and Longevity Interventions. Cell Metabolism 25, 954-960.

AbstractThe DNA methylation levels of certain CpG sites are thought to reflect the pace of human aging. Here, we developed a robust predictor of mouse biological age based on 90 CpG sites derived from partial blood DNA methylation profiles. The resulting clock correctly determines the age of mouse cohorts, detects the longevity effects of calorie restriction and gene knockouts, and reports rejuvenation of fibroblast-derived iPSCs. The data show that mammalian DNA methylomes are characterized by CpG sites that may represent the organism’s biological age. They are scattered across the genome, they are distinct in human and mouse, and their methylation gradually changes with age. The clock derived from these sites represents a biomarker of aging and can be used to determine the biological age of organisms and evaluate interventions that alter the rate of aging. More Information

Ke Z, Mallik P, Johnson AB, Luna F, Nevo E, Zhang ZD, Gladyshev VN, Seluanov A, Gorbunova V. (2017) Translation fidelity coevolves with longevity. Aging Cell. 5, 988-993.

AbstractWhether errors in protein synthesis play a role in aging has been a subject of intense debate. It has been suggested that rare mistakes in protein synthesis in young organisms may result in errors in the protein synthesis machinery, eventually leading to an increasing cascade of errors as organisms age. Studies that followed generally failed to identify a dramatic increase in translation errors with aging. However, whether translation fidelity plays a role in aging remained an open question. To address this issue, we examined the relationship between translation fidelity and maximum lifespan across 17 rodent species with diverse lifespans. To measure translation fidelity, we utilized sensitive luciferase-based reporter constructs with mutations in an amino acid residue critical to luciferase activity, wherein misincorporation of amino acids at this mutated codon re-activated the luciferase. The frequency of amino acid misincorporation at the first and second codon positions showed strong negative correlation with maximum lifespan. This correlation remained significant after phylogenetic correction, indicating that translation fidelity coevolves with longevity. These results give new life to the role of protein synthesis errors in aging: Although the error rate may not significantly change with age, the basal rate of translation errors is important in defining lifespan across mammals. More Information

Lee BC, Lee SG, Choo MK, Kim JH, Lee HM, Kim S, Fomenko DE, Kim HY, Park JM, Gladyshev VN. (2017) Selenoprotein MsrB1 promotes anti-inflammatory cytokine gene expression in macrophages and controls immune response in vivo. Sci Rep. 7, 5119.

AbstractPost-translational redox modification of methionine residues often triggers a change in protein function. Emerging evidence points to this reversible protein modification being an important regulatory mechanism under various physiological conditions. Reduction of oxidized methionine residues is catalyzed by methionine sulfoxide reductases (Msrs). Here, we show that one of these enzymes, a selenium-containing MsrB1, is highly expressed in immune-activated macrophages and contributes to shaping cellular and organismal immune responses. In particular, lipopolysaccharide (LPS) induces expression of MsrB1, but not other Msrs. Genetic ablation of MsrB1 did not preclude LPS-induced intracellular signaling in macrophages, but resulted in attenuated induction of anti-inflammatory cytokines, such as interleukin (IL)-10 and the IL-1 receptor antagonist. This anomaly was associated with excessive pro-inflammatory cytokine production as well as an increase in acute tissue inflammation in mice. Together, our findings suggest that MsrB1 controls immune responses by promoting anti-inflammatory cytokine expression in macrophages. MsrB1-dependent reduction of oxidized methionine in proteins may be a heretofore unrecognized regulatory event underlying immunity and inflammatory disease, and a novel target for clinical applications. More Information

Renko K, Martitz J, Hybsier S, Heynisch B, Voss L, Everley RA, Gygi SP, Stoedter M, Wisniewska M, Köhrle J, Gladyshev VN, Schomburg L. (2017) Aminoglycoside-driven biosynthesis of selenium-deficient Selenoprotein P. Sci Rep. 7, 4391.

AbstractSelenoprotein biosynthesis relies on the co-translational insertion of selenocysteine in response to UGA codons. Aminoglycoside antibiotics interfere with ribosomal function and may cause codon misreading. We hypothesized that biosynthesis of the selenium (Se) transporter selenoprotein P (SELENOP) is particularly sensitive to antibiotics due to its ten in frame UGA codons. As liver regulates Se metabolism, we tested the aminoglycosides G418 and gentamicin in hepatoma cell lines (HepG2, Hep3B and Hepa1-6) and in experimental mice. In vitro, SELENOP levels increased strongly in response to G418, whereas expression of the glutathione peroxidases GPX1 and GPX2 was marginally affected. Se content of G418-induced SELENOP was dependent on Se availability, and was completely suppressed by G418 under Se-poor conditions. Selenocysteine residues were replaced mainly by cysteine, tryptophan and arginine in a codon-specific manner. Interestingly, in young healthy mice, antibiotic treatment failed to affect Selenop biosynthesis to a detectable degree. These findings suggest that the interfering activity of aminoglycosides on selenoprotein biosynthesis can be severe, but depend on the Se status, and other parameters likely including age and general health. Focused analyses with aminoglycoside-treated patients are needed next to evaluate a possible interference of selenoprotein biosynthesis by the antibiotics and elucidate potential side effects. More Information

Salinas G, Gao W, Wang Y, Bonilla M, Yu L, Novikov A, Virginio VG, Ferreira H, Vieites M, Gladyshev VN, Gambino D, Dai S. (2017) The enzymatic and structural basis for inhibition of Echinococcus granulosus thioredoxin glutathione reductase by gold(I). Antioxid Redox Signal. 27,1491-1504

AbstractAIMS: New drugs are needed to treat flatworm infections that cause severe human diseases such as schistosomiasis. The unique flatworm enzyme thioredoxin glutathione reductase (TGR), structurally different from the human enzyme, is a key drug target. Structural studies of the flatworm Echinococcus granulosus TGR, free and complexed with AuI-MPO, a novel gold inhibitor, together with inhibition assays were performed.
RESULTS: AuI-MPO is a potent TGR inhibitor that achieves 75% inhibition at a 1:1 TGR:Au ratio and efficiently kills E. granulosus in vitro. The structures revealed salient insights: i) unique monomer-monomer interactions, ii) distinct binding sites for thioredoxin and the glutaredoxin domain, iii) a single glutathione disulfide reduction site in the glutaredoxin domain, iv) rotation of the glutaredoxin domain towards the Sec-containing redox active site, v) a single gold atom bound to Cys519 and Cys573 in the AuI-TGR complex. Structural modeling suggests that these residues are involved in the stabilization of the Sec-containing C-terminus. Consistently, Cys→Ser mutations in these residues decreased TGR activities. Mass spectroscopy confirmed these cysteines are the primary binding site.
INNOVATION: The identification of a primary site for gold binding and the structural model provide a basis for gold compound optimization through scaffold adjustments.
CONCLUSIONS: The structural study revealed that TGR functions are achieved not only through a mobile Sec-containing redox center, but also by rotation of the glutaredoxin domain and distinct binding sites for glutaredoxin domain and thioredoxin. The conserved Cys519 and Cys573 residues targeted by gold assist catalysis through stabilization of the Sec-containing redox center. More Information

Lee SG, Kaya A, Avanesov AS, Podolskiy DI, Song EJ, Go DM, Jin GD, Hwang JY, Kim EB, Kim DY, Gladyshev VN. (2017) Age-associated molecular changes are deleterious and may modulate life span through diet. Sci Adv. 3, e1601833.

AbstractTransition through life span is accompanied by numerous molecular changes, such as dysregulated gene expression, altered metabolite levels, and accumulated molecular damage. These changes are thought to be causal factors in aging; however, because they are numerous and are also influenced by genotype, environment, and other factors in addition to age, it is difficult to characterize the cumulative effect of these molecular changes on longevity. We reasoned that age-associated changes, such as molecular damage and tissue composition, may influence life span when used in the diet of organisms that are closely related to those that serve as a dietary source. To test this possibility, we used species-specific culture media and diets that incorporated molecular extracts of young and old organisms and compared the influence of these diets on the life span of yeast, fruitflies, and mice. In each case, the “old” diet or medium shortened the life span for one or both sexes. These findings suggest that age-associated molecular changes, such as cumulative damage and altered dietary composition, are deleterious and causally linked with aging and may affect life span through diet. More Information

Golubev A, Hanson AD, Gladyshev VN. (2017) Non-Enzymatic Molecular Damage as a Prototypic Driver of Aging. J Biol Chem. 292, 6029-6038.

AbstractThe chemical potentialities of metabolites far exceed metabolic requirements. The required potentialities are realized mostly through enzymatic catalysis. The rest are realized spontaneously through organic reactions that (i) occur wherever appropriate reactants come together, (ii) are so typical that many have proper names (e.g. Michael addition, Amadori rearrangement, Pictet-Spengler reaction), and (iii) often have damaging consequences. There are many more causes of non-enzymatic damage to metabolites than reactive oxygen species and free radical processes (the “usual suspects”). Endogenous damae accumulation in non-renewable macromolecules and spontaneously polymerized material is sufficient to account for aging and differentiates aging from wear-and-tear of inanimate objects by deriving it from metabolism, the essential attribute of life. More Information

Manta B, Gladyshev VN. (2017) Regulated methionine oxidation by monooxygenases. Free Radic Biol Med. 109, 141-155.

AbstractProtein function can be regulated via post-translational modifications by numerous enzymatic and non-enzymatic mechanisms, including oxidation of cysteine and methionine residues. Redox-dependent regulatory mechanisms have been identified for nearly every cellular process, but the major paradigm has been that cellular components are oxidized (damaged) by reactive oxygen species (ROS) in a relatively unspecific way, and then reduced (repaired) by designated reductases. While this scheme may work with cysteine, it cannot be ascribed to other residues, such as methionine, whose reaction with ROS is too slow to be biologically relevant. However, methionine is clearly oxidized in vivo and enzymes for its stereoselective reduction are present in all three domains of life. Here, we revisit the chemistry and biology of methionine oxidation, with emphasis on its generation by enzymes from the monooxygenase family. Particular attention is placed on MICALs, a recently discovered family of proteins that harbor an unusual flavin-monooxygenase domain with an NADPH-dependent methionine sulfoxidase activity. Based on the structural and kinetic information we provide a rational framework to explain MICAL mechanism, inhibition, and regulation. Methionine residues that are targeted by MICALs are reduced back by methionine sulfoxide reductases, suggesting that reversible methionine oxidation may be a general mechanism analogous to the regulation by phosphorylation by kinases/phosphatases. The identification of new enzymes that catalyze the oxidation of methionine will open a new area of research at the forefront of redox signaling. More Information

Payne NC, Geissler A, Button A, Sasuclark AR, Schroll AL, Ruggles EL, Gladyshev VN, Hondal RJ. (2017) Comparison of the redox chemistry of sulfur- and selenium-containing analogs of uracil. Free Radic Biol Med. 104, 249-261

AbstractSelenium is present in proteins in the form of selenocysteine, where this amino acid serves catalytic oxidoreductase functions. The use of selenocysteine in nature is strongly associated with redox catalysis. However, selenium is also found in a 2-selenouridine moiety at the wobble position of tRNAGlu, tRNAGln and tRNALys. It is thought that the modifications of the wobble position of the tRNA improves the selectivity of the codon-anticodon pair as a result of the physico-chemical changes that result from substitution of sulfur and selenium for oxygen. Both selenocysteine and 2-selenouridine have widespread analogs, cysteine and thiouridine, where sulfur is used instead. To examine the role of selenium in 2-selenouridine, we comparatively analyzed the oxidation reactions of sulfur-containing 2-thiouracil-5-carboxylic acid (s2c5Ura) and its selenium analog 2-selenouracil-5-carboxylic acid (se2c5Ura) using 1H-NMR spectroscopy, 77Se-NMR spectroscopy, and liquid chromatography-mass spectrometry. Treatment of s2c5Ura with hydrogen peroxide led to oxidized intermediates, followed by irreversible desulfurization to form uracil-5-carboxylic acid (c5Ura). In contrast, se2c5Ura oxidation resulted in a diselenide intermediate, followed by conversion to the seleninic acid, both of which could be readily reduced by ascorbate and glutathione. Glutathione and ascorbate only minimally prevented desulfurization of s2c5Ura, whereas very little deselenization of se2c5Ura occurred in the presence of the same antioxidants. In addition, se2c5Ura but not s2c5Ura showed glutathione peroxidase activity, further suggesting that oxidation of se2c5Ura is readily reversible, while oxidation of s2c5Ura is not. The results of the study of these model nucleobases suggest that the use of 2-selenouridine is related to resistance to oxidative inactivation that otherwise characterizes 2-thiouridine. As the use of selenocysteine in proteins also confers resistance to oxidation, our findings suggest a common mechanism for the use of selenium in biology. More Information

Moskalev A, Anisimov V, Aliper A, Artemov A, Asadullah K, Belsky D, Baranova A, de Grey A, Dixit VD, Debonneuil E, Dobrovolskaya E, Fedichev P, Fedintsev A, Fraifeld V, Franceschi C, Freer R, Fülöp T, Feige J, Gems D, Gladyshev V, Gorbunova V, Irincheeva I, Jager S, Jazwinski SM, Kaeberlein M, Kennedy B, Khaltourina D, Kovalchuk I, Kovalchuk O, Kozin S, Kulminski A, Lashmanova E, Lezhnina K, Liu GH, Longo V, Mamoshina P, Maslov A, Pedro de Magalhaes J, Mitchell J, Mitnitski A, Nikolsky Y, Ozerov I, Pasyukova E, Peregudova D, Popov V, Proshkina E, Putin E, Rogaev E, Rogina B, Schastnaya J, Seluanov A, Shaposhnikov M, Simm A, Skulachev V, Skulachev M, Solovev I, Spindler S, Stefanova N, Suh Y, Swick A, Tower J, Gudkov AV, Vijg J, Voronkov A, West M, Wagner W, Yashin A, Zemskaya N, Zhumadilov Z, Zhavoronkov A. (2017) A review of the biomedical innovations for healthy longevity. Aging (Albany NY) 9, 7-25.

AbstractKEYWORDS: aging; biomarkers; epigenetics; geroprotectors; longevity; transcriptomics More Information

Lobanov AV, Heaphy SM, Turanov AA, Gerashchenko MV, Pucciarelli S, Devaraj RR, Xie F, Petyuk VA, Smith RD, Klobutcher LA, Atkins JF, Miceli C, Hatfield DL, Baranov PV, Gladyshev VN. (2017) Position-dependent termination and widespread obligatory frameshifting in Euplotes translation. Nat Struct Mol Biol. 24, 61-68.

AbstractThe ribosome can change its reading frame during translation in a process known as programmed ribosomal frameshifting. These rare events are supported by complex mRNA signals. However, we found that the ciliates Euplotes crassus and Euplotes focardii exhibit widespread frameshifting at stop codons. 47 different codons preceding stop signals resulted in either +1 or +2 frameshifts, and +1 frameshifting at AAA was the most frequent. The frameshifts showed unusual plasticity and rapid evolution, and had little influence on translation rates. The proximity of a stop codon to the 3′ mRNA end, rather than its occurrence or sequence context, appeared to designate termination. Thus, a ‘stop codon’ is not a sufficient signal for translation termination, and the default function of stop codons in Euplotes is frameshifting, whereas termination is specific to certain mRNA positions and probably requires additional factors. More Information

Heo JY, Cha HN, Kim KY, Lee E, Kim SJ, Kim YW, Kim JY, Lee IK, Gladyshev VN, Kim HY, Park SY. (2017) Methionine sulfoxide reductase B1 deficiency does not increase high-fat diet-induced insulin resistance in mice. Free Radic Res. 51, 24-37.

AbstractMethionine-S-sulfoxide reductase (MsrA) protects against high-fat diet-induced insulin resistance due to its antioxidant effects. To determine whether its counterpart, methionine-R-sulfoxide reductase (MsrB) has similar effects, we compared MsrB1 knockout and wild-type mice using a hyperinsulinemic-euglycemic clamp technique. High-fat feeding for 8 weeks increased body weights, fat masses, and plasma levels of glucose, insulin, and triglycerides to similar extents in wild-type and MsrB1 knockout mice. Intraperitoneal glucose tolerance test showed no difference in blood glucose levels between the two genotypes after 8 weeks on the high-fat diet. The hyperglycemic-euglycemic clamp study showed that glucose infusion rates and whole body glucose uptakes were decreased to similar extents by the high-fat diet in both wild-type and MsrB1 knockout mice. Hepatic glucose production and glucose uptake of skeletal muscle were unaffected by MsrB1 deficiency. The high-fat diet-induced oxidative stress in skeletal muscle and liver was not aggravated in MsrB1-deficient mice. Interestingly, whereas MsrB1 deficiency reduced JNK protein levels to a great extent in skeletal muscle and liver, it markedly elevated phosphorylation of JNK, suggesting the involvement of MsrB1 in JNK protein activation. However, this JNK phosphorylation based on a p-JNK/JNK level did not positively correlate with insulin resistance in MsrB1-deficient mice. Taken together, our results show that, in contrast to MsrA deficiency, MsrB1 deficiency does not increase high-fat diet-induced insulin resistance in mice. More Information

Gerashchenko MV, Gladyshev VN. (2017) Ribonuclease selection for ribosome profiling. Nucleic Acids Res. 45, e6.

AbstractRibosome profiling has emerged as a powerful method to assess global gene translation, but methodological and analytical challenges often lead to inconsistencies across labs and model organisms. A critical issue in ribosome profiling is nuclease treatment of ribosome-mRNA complexes, as it is important to ensure both stability of ribosomal particles and complete conversion of polysomes to monosomes. We performed comparative ribosome profiling in yeast and mice with various ribonucleases including I, A, S7 and T1, characterized their cutting preferences, trinucleotide periodicity patterns and coverage similarities across coding sequences, and showed that they yield comparable estimations of gene expression when ribosome integrity is not compromised. However, ribosome coverage patterns of individual transcripts had little in common between the ribonucleases. We further examined their potency at converting polysomes to monosomes across other commonly used model organisms, including bacteria, nematodes and fruit flies. In some cases, ribonuclease treatment completely degraded ribosome populations. Ribonuclease T1 was the only enzyme that preserved ribosomal integrity while thoroughly converting polysomes to monosomes in all examined species. This study provides a guide for ribonuclease selection in ribosome profiling experiments across most common model systems. More Information

Li M, Chen L, Tian S, Lin Y, Tang Q, Zhou X, Li D, Yeung CK, Che T, Jin L, Fu Y, Ma J, Wang X, Jiang A, Lan J, Pan Q, Liu Y, Luo Z, Guo Z, Liu H, Zhu L, Shuai S, Tang G, Zhao J, Jiang Y, Bai L, Zhang S, Mai M, Li C, Wang D, Gu Y, Wang G, Lu H, Li Y, Zhu H, Li Z, Li M, Gladyshev VN, Jiang Z, Zhao S, Wang J, Li R, Li X. (2017) Comprehensive variation discovery and recovery of missing sequence in the pig genome using multiple de novo assemblies. Genome Res. 27, 865-874.

AbstractUncovering genetic variation through resequencing is limited by the fact that only sequences with similarity to the reference genome are examined. Reference genomes are often incomplete and cannot represent the full range of genetic diversity as a result of geographical divergence and independent demographic events. To more comprehensively characterize genetic variation of pigs (Sus scrofa), we generated de novo assemblies of nine geographically and phenotypically representative pigs from Eurasia. By comparing them to the reference pig assembly, we uncovered a substantial number of novel SNPs, structural variations, as well as 137.02 Mb sequences harboring 1,737 protein coding genes that were absent in the reference assembly, revealing variants left by selection. Our results illustrate the power of whole-genome de novo sequencing relative to resequencing, and provide valuable genetic resources that enable effective use of pigs in both agricultural production and biomedical research. More Information

2016 Articles

Seim I, Ma S, Gladyshev VN. (2016) Gene expression signatures of human cell and tissue longevity. NPJ Aging Mech Dis. 7, 16014.

AbstractDifferent cell types within the body exhibit substantial variation in the average time they live, ranging from days to the lifetime of the organism. The underlying mechanisms governing the diverse lifespan of different cell types are not well understood. To examine gene expression strategies that support the lifespan of different cell types within the human body, we obtained publicly available RNA-seq data sets and interrogated transcriptomes of 21 somatic cell types and tissues with reported cellular turnover, a bona fide estimate of lifespan, ranging from 2 days (monocytes) to a lifetime (neurons). Exceptionally long-lived neurons presented a gene expression profile of reduced protein metabolism, consistent with neuronal survival and similar to expression patterns induced by longevity interventions such as dietary restriction. Across different cell lineages, we identified a gene expression signature of human cell and tissue turnover. In particular, turnover showed a negative correlation with the energetically costly cell cycle and factors supporting genome stability, concomitant risk factors for aging-associated pathologies. In addition, the expression of p53 was negatively correlated with cellular turnover, suggesting that low p53 activity supports the longevity of post-mitotic cells with inherently low risk of developing cancer. Our results demonstrate the utility of comparative approaches in unveiling gene expression differences among cell lineages with diverse cell turnover within the same organism, providing insights into mechanisms that could regulate cell longevity. More Information

Podolskiy DI, Lobanov AV, Kryukov GV, Gladyshev VN. (2016) Analysis of cancer genomes reveals basic features of human aging and its role in cancer development. Nature Commun. 7, 12157.

AbstractSomatic mutations have long been implicated in aging and disease, but their impact on fitness and function is difficult to assess. Here by analysing human cancer genomes we identify mutational patterns associated with aging. Our analyses suggest that age-associated mutation load and burden double approximately every 8 years, similar to the all-cause mortality doubling time. This analysis further reveals variance in the rate of aging among different human tissues, for example, slightly accelerated aging of the reproductive system. Age-adjusted mutation load and burden correlate with the corresponding cancer incidence and precede it on average by 15 years, pointing to pre-clinical cancer development times. Behaviour of mutation load also exhibits gender differences and late-life reversals, explaining some gender-specific and late-life patterns in cancer incidence rates. Overall, this study characterizes some features of human aging and offers a mechanism for age being a risk factor for the onset of cancer. More Information

Ma S, Upneja A, Galecki A, Tsai YM, Burant CF, Raskind S, Zhang Q, Zhang ZD, Seluanov A, Gorbunova V, Clish CB, Miller RA, Gladyshev VN. (2016) Cell culture-based profiling across mammals reveals DNA repair and metabolism as determinants of species longevity. Elife 5, e19130.

AbstractMammalian lifespan differs by >100-fold, but the mechanisms associated with such longevity differences are not understood. Here, we conducted a study on primary skin fibroblasts isolated from 16 species of mammals and maintained under identical cell culture conditions. We developed a pipeline for obtaining species-specific ortholog sequences, profiled gene expression by RNA-seq and small molecules by metabolite profiling, and identified genes and metabolites correlating with species longevity. Cells from longer-lived species up-regulated genes involved in DNA repair and glucose metabolism, down-regulated proteolysis and protein transport, and showed high levels of amino acids but low levels of lysophosphatidylcholine and lysophosphatidylethanolamine. The amino acid patterns were recapitulated by further analyses of primate and bird fibroblasts. The study suggests that fibroblast profiling captures differences in longevity across mammals at the level of global gene expression and metabolite levels and reveals pathways that define these differences. More Information

Gladyshev TV, Gladyshev VN. (2016) A Disease or Not a Disease? Aging As a Pathology. Trends Mol Med. S1471-4914, 30142-30143.

AbstractThe debate on the relationship between aging and disease is centered on whether aging is a normal/natural/physiological process or it represents a pathology. Considering this relationship from medical, molecular, social, and historical perspectives, we argue that aging is neither a disease, nor a non-disease. Instead, it combines all age-related diseases and their preclinical forms, in addition to other pathological changes. More Information

Gladyshev VN, Arnér ES, Berry MJ, Brigelius-Flohé R, Bruford EA, Burk RF, Carlson BA, Castellano S, Chavatte L, Conrad M, Copeland PR, Diamond AM, Driscoll DM, Ferreiro A, Flohé L, Green FR, Guigó R, Handy DE, Hatfield DL, Hesketh J, Hoffmann PR, Holmgren A, Hondal RJ, Howard MT, Huang K, Kim HY, Kim IY, Köhrle J, Krol A, Kryukov GV, Lee BJ, Lee BC, Lei XG, Liu Q, Lescure A, Lobanov AV, Loscalzo J, Maiorino M, Mariotti M, Prabhu KS, Rayman MP, Rozovsky S, Salinas G, Schmidt EE, Schomburg L, Schweizer U, Simonović M, Sunde RA, Tsuji PA, Tweedie S, Ursini F, Whanger PD, Zhang Y. (2016) Selenoprotein Gene Nomenclature. J Biol Chem. 291, 24036-24040.

AbstractThe human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4 and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine-R-sulfoxide reductase 1) and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15 kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV) and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates. More Information

Cox AG, Tsomides A, Kim AJ, Saunders D, Hwang KL, Evason KJ, Heidel J, Brown KK, Yuan M, Lien EC, Lee BC, Nissim S, Dickinson B, Chhangawala S, Chang CJ, Asara JM, Houvras Y, Gladyshev VN, Goessling W. (2016) Selenoprotein H is an essential regulator of redox homeostasis that cooperates with p53 in development and tumorigenesis. Proc Natl Acad Sci USA. 113, E5562-E5571.

AbstractSelenium, an essential micronutrient known for its cancer prevention properties, is incorporated into a class of selenocysteine-containing proteins (selenoproteins). Selenoprotein H (SepH) is a recently identified nucleolar oxidoreductase whose function is not well understood. Here we report that seph is an essential gene regulating organ development in zebrafish. Metabolite profiling by targeted LC-MS/MS demonstrated that SepH deficiency impairs redox balance by reducing the levels of ascorbate and methionine, while increasing methionine sulfoxide. Transcriptome analysis revealed that SepH deficiency induces an inflammatory response and activates the p53 pathway. Consequently, loss of seph renders larvae susceptible to oxidative stress and DNA damage. Finally, we demonstrate that seph interacts with p53 deficiency in adulthood to accelerate gastrointestinal tumor development. Overall, our findings establish that seph regulates redox homeostasis and suppresses DNA damage. We hypothesize that SepH deficiency may contribute to the increased cancer risk observed in cohorts with low selenium levels. More Information

Zhou X, Meng X, Liu Z, Chang J, Wang B, Li M, Orozco-terWengel P, Tian S, Wen C, Wang Z, Garber PA, Pan H, Ye X, Xiang Z, Bruford MW, Edwards SV, Cao Y, Yu S, Gao L, Cao Z, Liu G, Ren B, Shi F, Peterfi Z, Li D, Li B, Jiang Z, Li J, Gladyshev VN, Li R, Li M. (2016) Population genomics reveals low genetic diversity and adaptation to hypoxia in snub-nosed monkeys. Mol Biol Evol. 33, 2670-2681.

AbstractSnub-nosed monkeys (genus Rhinopithecus) are a group of endangered colobines endemic to South Asia. Here, we re-sequenced the whole genomes of 38 snub-nosed monkeys representing four species within this genus. By conducting population genomic analyses, we observed an similar load of deleterious variation in snub-nosed monkeys living in both smaller and larger populations and found that genomic diversity was lower than that reported in other primates. Reconstruction of Rhinopithecus evolutionary history suggested that episodes of climatic variation over the past 2 million years, associated with glacial advances and retreats and population isolation, have shaped snub-nosed monkey demography and evolution. We further identified several hypoxia-related genes under selection in R. bieti (black snub-nosed monkey), a species that exploits habitats higher than any other nonhuman primate. These results provide the first detailed and comprehensive genomic insights into genetic diversity, demography, genetic burden and adaptation in this radiation of endangered primates. More Information

Podolskiy DI, Gladyshev VN. (2016) Intrinsic Versus Extrinsic Cancer Risk Factors and Aging. Trends Mol Med. 22, 833-834.

AbstractTwo recent stimulating publications have examined the causes of cancer, comparing ‘bad luck’ versus environment as main risk factors for cancer incidence. However, bringing aging into the picture might question the entire debate. More Information

Heaphy SM, Mariotti M, Gladyshev VN, Atkins JF, Baranov PV. (2016) Novel ciliate genetic code variants including the reassignment of all three stop codons to sense codons in C. magnum. Mol Biol Evol. 33, 2885-2889.

AbstractmRNA translation in many ciliates utilises variant genetic codes where stop codons are reassigned to specify amino acids. To characterise the repertoire of ciliate genetic codes we analysed ciliate transcriptomes from marine environments. Using codon substitution frequencies in ciliate protein-coding genes and their orthologs we inferred the genetic codes of 24 ciliate species. 9 did not match genetic code tables currently assigned by NCBI. Surprisingly, we identified a novel genetic code where all three standard stop codons (TAA, TAG, TGA) specify amino acids in Condylostoma magnum We provide evidence suggesting that the functions of these codons in C. magnum depends on their location within mRNA. They are decoded as amino acids at internal positions, but specify translation termination when are in close proximity to an mRNA 3′ end. The frequency of stop codons in protein coding sequences of closely related Climacostomum virens suggest that it may represent a transitory state. More Information

Gladyshev VN. (2016) Aging: progressive decline in fitness due to the rising deleteriome adjusted by genetic, environmental, and stochastic processes. Aging Cell 15, 594-602.

AbstractDifferent theories posit that aging is caused by molecular damage, genetic programs, continued development, hyperfunction, antagonistic pleiotropy alleles, mutations, trade-offs, incomplete repair, etc. Here, I discuss that these ideas can be conceptually unified as they capture particular facets of aging, while being incomplete. Their respective deleterious effects impact fitness at different levels of biological organization, adjusting progression through aging, rather than causing it. Living is associated with a myriad of deleterious processes, both random and deterministic, which are caused by imperfectness, exhibit cumulative properties, and represent the indirect effects of biological functions at all levels, from simple molecules to systems. From this, I derive the deleteriome, which encompasses cumulative deleterious age-related changes and represents the biological age. The organismal deleteriome consists of the deleteriomes of cells, organs, and systems, which change along roughly synchronized trajectories and may be assessed through biomarkers of aging. Aging is then a progressive decline in fitness due to the increasing deleteriome, adjusted by genetic, environmental, and stochastic processes. This model allows integration of diverse aging concepts, provides insights into the nature of aging, and suggests how lifespan may be adjusted during evolution and in experimental models. More Information

Mariotti M, Lobanov AV, Manta B, Santesmasses D, Bofill A, Guigó R, Gabaldón T, Gladyshev VN. (2016) Lokiarchaeota Marks the Transition between the Archaeal and Eukaryotic Selenocysteine Encoding Systems. Mol Biol Evol. 33, 2441-2453.

AbstractSelenocysteine (Sec) is the 21st amino acid in the genetic code, inserted in response to UGA codons with the help of RNA structures, the SEC Insertion Sequence (SECIS) elements. The three domains of life feature distinct strategies for Sec insertion in proteins and its utilization. While bacteria and archaea possess similar sets of selenoproteins, Sec biosynthesis is more similar among archaea and eukaryotes. However, SECIS elements are completely different in the three domains of life. Here, we analyze the archaeon Lokiarchaeota that resolves the relationships among Sec insertion systems. This organism has selenoproteins representing five protein families, three of which have multiple Sec residues. Remarkably, these archaeal selenoprotein genes possess conserved RNA structures that strongly resemble the eukaryotic SECIS element, including key eukaryotic protein-binding sites. These structures also share similarity with the SECIS element in archaeal selenoprotein VhuD, suggesting a relation of direct descent. These results identify Lokiarchaeota as an intermediate form between the archaeal and eukaryotic Sec-encoding systems and clarify the evolution of the Sec insertion system. More Information

Peterfi Z, Tarrago L, Gladyshev VN. (2016) Practical guide for dynamic monitoring of protein oxidation using genetically encoded ratiometric fluorescent bioensors of methionine sulfoxide. Methods 109, 149-157.

AbstractIn cells, physiological and pathophysiological conditions may lead to the formation of methionine sulfoxide (MetO). This oxidative modification of methionine exists in the form of two diastereomers, R and S, and may occur in both free amino acid and proteins. MetO is reduced back to methionine by methionine sulfoxide reductases (MSRs). Methionine oxidation was thought to be a nonspecific modification affecting protein functions and methionine availability. However, recent findings suggest that cyclic methionine oxidation and reduction is a posttranslational modification that actively regulates protein function akin to redox regulation by cysteine oxidation and phosphorylation. Methionine oxidation is thus an important mechanism that could play out in various physiological contexts. However, detecting MetO generation and MSR functions remains challenging because of the lack of tools and reagents to detect and quantify this protein modification. We recently developed two genetically encoded diasterospecific fluorescent sensors, MetSOx and MetROx, to dynamically monitor MetO in living cells. Here, we provide a detailed procedure for their use in bacterial and mammalian cells using fluorimetric and fluorescent imaging approaches. This method can be adapted to dynamically monitor methionine oxidation in various cell types and under various conditions. More Information

Carlson BA, Tobe R, Yefremova E, Tsuji PA, Hoffmann VJ, Schweizer U, Gladyshev VN, Hatfield DL, Conrad M. (2016) Glutathione peroxidase 4 and vitamin E cooperatively prevent hepatocellular degeneration. Redox Biol. 9, 22-31.

AbstractThe selenoenzyme glutathione peroxidase 4 (Gpx4) is an essential mammalian glutathione peroxidase, which protects cells against detrimental lipid peroxidation and governs a novel form of regulated necrotic cell death, called ferroptosis. To study the relevance of Gpx4 and of another vitally important selenoprotein, cytosolic thioredoxin reductase (Txnrd1), for liver function, mice with conditional deletion of Gpx4 in hepatocytes were studied, along with those lacking Txnrd1 and selenocysteine (Sec) tRNA (Trsp) in hepatocytes. Unlike Txnrd1- and Trsp-deficient mice, Gpx4-/- mice died shortly after birth and presented extensive hepatocyte degeneration. Similar to Txnrd1-deficient livers, Gpx4-/- livers manifested upregulation of nuclear factor (erythroid-derived)-like 2 (Nrf2) response genes. Remarkably, Gpx4-/- pups born from mothers fed a vitamin E-enriched diet survived, yet this protection was reversible as subsequent vitamin E deprivation caused death of Gpx4-deficient mice ~4 weeks thereafter. Abrogation of selenoprotein expression in Gpx4-/- mice did not result in viable mice, indicating that the combined deficiency aggravated the loss of Gpx4 in liver. By contrast, combined Trsp/Txnrd1-deficient mice were born, but had significantly shorter lifespans than either single knockout, suggesting that Txnrd1 plays an important role in supporting liver function of mice lacking Trsp. In sum our study demonstrates that the ferroptosis regulator Gpx4 is critical for hepatocyte survival and proper liver function, and that vitamin E can compensate for its loss by protecting cells against deleterious lipid peroxidation. More Information

Tobe R, Carlson BA, Huh JH, Castro NP, Xu XM, Tsuji PA, Lee SG, Bang J, Na JW, Kong YY, Beaglehole D, Southon E, Seifried H, Tessarollo L, Salomon DS, Schweizer U, Gladyshev VN, Hatfield DL, Lee BJ. (2016) Selenophosphate Synthetase 1 is an Essential Protein with Roles in Regulation of Redox Homeostasis in Mammals. Biochem J. 473, 2141-2154.

AbstractSelenophosphate synthetase (SPS) was initially detected in bacteria and was shown to synthesize selenophosphate, the active selenium donor. However, mammals have two SPS paralogs, which are designated SPS1 and SPS2. Although it is known that SPS2 catalyzes the synthesis of selenophosphate, the function of SPS1 remains largely unclear. To examine the role of SPS1 in mammals, we generated a Sps1 knockout mouse and found that systemic SPS1 deficiency led to embryos that were clearly underdeveloped by E8.5 and virtually resorbed by E14.5. The knockout of Sps1 in the liver preserved viability, but significantly affected the expression of a large number of mRNAs involved in cancer, embryonic development, and the glutathione system. Particularly notable was the extreme deficiency of glutaredoxin 1 (GLRX1) and glutathione-S-transferase omega 1. To assess these phenotypes at the cellular level, we targeted the removal of SPS1 in F9 cells, a mouse embryonal carcinoma cell line, which affected the glutathione system proteins and accordingly led to the accumulation of hydrogen peroxide in the cell. Further, we found that several malignant characteristics of SPS1-deficient F9 cells were reversed, suggesting that SPS1 played a role in supporting and/or sustaining cancer. In addition, the overexpression of mouse or human GLRX1 led to a reversal of observed increases in reactive oxygen species (ROS) in the F9 SPS1/GLRX1-deficient cells and resulted in levels that were similar to those in F9 SPS1-sufficient cells. The results suggested that SPS1 is an essential mammalian enzyme with roles in regulating redox homeostasis and controlling cell growth. More Information

Patrick A, Seluanov M, Hwang C, Tam J, Khan T, Morgenstern A, Wiener L, Vazquez JM, Zafar H, Wen R, Muratkalyeva M, Doerig K, Zagorulya M, Cole L, Catalano S, Ladd AA, Coppi AA, Coşkun Y, Tian X, Ablaeva J, Nevo E, Gladyshev VN, Zhang ZD, Vijg J, Seluanov A, Gorbunova V. (2016) Sensitivity of primary fibroblasts in culture to atmospheric oxygen does not correlate with species lifespan. Aging (Albany NY) 8, 841-847.

AbstractDifferences in the way human and mouse fibroblasts experience senescence in culture had long puzzled researchers. While senescence of human cells is mediated by telomere shortening, Parrinello et al. demonstrated that senescence of mouse cells is caused by extreme oxygen sensitivity. It was hypothesized that the striking difference in oxygen sensitivity between mouse and human cells explains their different rates of aging. To test if this hypothesis is broadly applicable, we cultured cells from 16 rodent species with diverse lifespans in 3% and 21% oxygen and compared their growth rates. Unexpectedly, fibroblasts derived from laboratory mouse strains were the only cells demonstrating extreme sensitivity to oxygen. Cells from hamster, muskrat, woodchuck, capybara, blind mole rat, paca, squirrel, beaver, naked mole rat and wild-caught mice were mildly sensitive to oxygen, while cells from rat, gerbil, deer mouse, chipmunk, guinea pig and chinchilla showed no difference in the growth rate between 3% and 21% oxygen. We conclude that, although the growth of primary fibroblasts is generally improved by maintaining cells in 3% oxygen, the extreme oxygen sensitivity is a peculiarity of laboratory mouse strains, possibly related to their very long telomeres, and fibroblast oxygen sensitivity does not directly correlate with species’ lifespan. More Information

Lee BC, Kaya A, Gladyshev VN. (2016) Methionine restriction and life-span control. Ann NY Acad Sci. 1363, 116-124.

AbstractDietary restriction (DR) without malnutrition is associated with longevity in various organisms. However, it has also been shown that reduced calorie intake is often ineffective in extending life span. Selecting optimal dietary regimens for DR studies is complicated, as the same regimen may lead to different outcomes depending on genotype and environmental factors. Recent studies suggested that interventions such as moderate protein restriction with or without adequate nutrition (e.g., particular amino acids or carbohydrates) may have additional beneficial effects mediated by certain metabolic and hormonal factors implicated in the biology of aging, regardless of total calorie intake. In particular, it was shown that restriction of a single amino acid, methionine, can mimic the effects of DR and extend life span in various model organisms. We discuss the beneficial effects of a methionine-restricted diet, the molecular pathways involved, and the use of this regimen in longevity interventions. More Information

Bisio H, Bonilla M, Manta B, Graña M, Salzman V, Aguilar P, Gladyshev VN, Comini MA, Salinas G. (2016) A new class of thioredoxin-related protein able to bind iron-sulfur clusters. Antioxid Redox Signal. In Press.

AbstractAIMS: Members of the thioredoxin (Trx) protein family participate mainly in redox pathways and have not been associated with Fe/S binding, in contrast to some closely related glutaredoxins (Grxs). Cestode parasites possess an unusual diversity of Trxs and Trx-related proteins with non-explored functions. Here we addressed the biochemical characterization of a new class of Trx-related protein (IsTRP) and a classical monothiol Grx (EgGrx5) from the human pathogen Echinococcus granulosus.
RESULTS: The dimeric form of IsTRP coordinates Fe2S2 in a glutathione-independent manner; instead, Fe/S binding relies on the CXXC motif conserved among Trxs. This novel binding mechanism allows holo-IsTRP to be highly resistant to oxidation. IsTRP lacks canonical reductase activities. Mitochondrially targeted IsTRP aids growth of a Grx5 null yeast strain. Similar complementation assays performed with EgGrx5 revealed functional conservation for class II Grxs despite the presence of non-conserved structural elements. IsTRP is a cestode-lineage specific protein highly expressed in the gravid adult worm, which releases the infective stage critical for dissemination.
INNOVATION: IsTRP is the first member from the thioredoxin family to be reported to bind Fe/S. We disclose a novel mechanism of Fe/S coordination within the Trx folding unit, which renders the cluster highly resistant to oxidation-mediated disassembly.
CONCLUSION: We demonstrate that IsTRP defines a new protein family within the thioredoxin superfamily, confirm the conservation of function for class II glutaredoxin from non-phylogenetically related species and highlight the versatility of the Trx folding unit to acquire Fe/S binding as a recurrent emergent function. More Information

2015 Articles

MacRae SL, Croken MM, Calder RB, Aliper A, Milholland B, White RR, Zhavoronkov A, Gladyshev VN, Seluanov A, Gorbunova V, Zhang ZD, Vijg J. (2015) DNA repair in species with extreme lifespan differences. Aging (Albany NY) 7, 1171-1184.

AbstractDifferences in DNA repair capacity have been hypothesized to underlie the great range of maximum lifespans among mammals. However, measurements of individual DNA repair activities in cells and animals have not substantiated such a relationship because utilization of repair pathways among animals-depending on habitats, anatomical characteristics, and life styles-varies greatly between mammalian species. Recent advances in high-throughput genomics, in combination with increased knowledge of the genetic pathways involved in genome maintenance, now enable a comprehensive comparison of DNA repair transcriptomes in animal species with extreme lifespan differences. Here we compare transcriptomes of liver, an organ with high oxidative metabolism and abundant spontaneous DNA damage, from humans, naked mole rats, and mice, with maximum lifespans of ~120, 30, and 3 years, respectively, with a focus on genes involved in DNA repair. The results show that the longer-lived species, human and naked mole rat, share higher expression of DNA repair genes, including core genes in several DNA repair pathways. A more systematic approach of signaling pathway analysis indicates statistically significant upregulation of several DNA repair signaling pathways in human and naked mole rat compared with mouse. The results of this present work indicate, for the first time, that DNA repair is upregulated in a major metabolic organ in long-lived humans and naked mole rats compared with short-lived mice. These results strongly suggest that DNA repair can be considered a genuine longevity assurance system. More Information

Tobe R, Carlson BA, Tsuji PA, Lee BJ, Gladyshev VN, Hatfield DL. (2015) Differences in Redox Regulatory Systems in Human Lung and Liver Tumors Suggest Different Avenues for Therapy. Cancers (Basel) 7, 2262-2276.

AbstractA common characteristic of many cancer cells is that they suffer from oxidative stress. They, therefore, require effective redox regulatory systems to combat the higher levels of reactive oxygen species that accompany accelerated growth compared to the normal cells of origin. An elevated dependence on these systems in cancers suggests that targeting these systems may provide an avenue for retarding the malignancy process. Herein, we examined the redox regulatory systems in human liver and lung cancers by comparing human lung adenocarcinoma and liver carcinoma to their respective surrounding normal tissues. Significant differences were found in the two major redox systems, the thioredoxin and glutathione systems. Thioredoxin reductase 1 levels were elevated in both malignancies, but thioredoxin was highly upregulated in lung tumor and only slightly upregulated in liver tumor, while peroxiredoxin 1 was highly elevated in lung tumor, but downregulated in liver tumor. There were also major differences within the glutathione system between the malignancies and their normal tissues. The data suggest a greater dependence of liver on either the thioredoxin or glutathione system to drive the malignancy, while lung cancer appeared to depend primarily on the thioredoxin system. More Information

Zhou X, Seim I, Gladyshev VN. (2015) Convergent evolution of marine mammals is associated with distinct substitutions in common genes. Sci Rep. 5, 16550.

AbstractPhenotypic convergence is thought to be driven by parallel substitutions coupled with natural selection at the sequence level. Multiple independent evolutionary transitions of mammals to an aquatic environment offer an opportunity to test this thesis. Here, whole genome alignment of coding sequences identified widespread parallel amino acid substitutions in marine mammals; however, the majority of these changes were not unique to these animals. Conversely, we report that candidate aquatic adaptation genes, identified by signatures of likelihood convergence and/or elevated ratio of nonsynonymous to synonymous nucleotide substitution rate, are characterized by very few parallel substitutions and exhibit distinct sequence changes in each group. Moreover, no significant positive correlation was found between likelihood convergence and positive selection in all three marine lineages. These results suggest that convergence in protein coding genes associated with aquatic lifestyle is mainly characterized by independent substitutions and relaxed negative selection. More Information

Ma S, Lee SG, Kim EB, Park TJ, Seluanov A, Gorbunova V, Buffenstein R, Seravalli J, Gladyshev VN. (2015) Organization of the Mammalian Ionome According to Organ Origin, Lineage Specialization, and Longevity. Cell Rep. 13, 1319-1326.

AbstractTrace elements are essential to all mammals, but their distribution and utilization across species and organs remains unclear. Here, we examined 18 elements in the brain, heart, kidney, and liver of 26 mammalian species and report the elemental composition of these organs, the patterns of utilization across the species, and their correlation with body mass and longevity. Across the organs, we observed distinct distribution patterns for abundant elements, transition metals, and toxic elements. Some elements showed lineage-specific patterns, including reduced selenium utilization in African mole rats, and positive correlation between the number of selenocysteine residues in selenoprotein P and the selenium levels in liver and kidney across mammals. Body mass was linked positively to zinc levels, whereas species lifespan correlated positively with cadmium and negatively with selenium. This study provides insights into the variation of mammalian ionome by organ physiology, lineage specialization, body mass, and longevity. More Information

Kaya A, Gerashchenko MV, Seim I, Labarre J, Toledano MB, Gladyshev VN. (2015) Adaptive aneuploidy protects against thiol peroxidase deficiency by increasing respiration via key mitochondrial proteins. Proc Natl Acad Sci USA 112, 10685-10690.

AbstractAerobic respiration is a fundamental energy-generating process; however, there is cost associated with living in an oxygen-rich environment, because partially reduced oxygen species can damage cellular components. Organisms evolved enzymes that alleviate this damage and protect the intracellular milieu, most notably thiol peroxidases, which are abundant and conserved enzymes that mediate hydrogen peroxide signaling and act as the first line of defense against oxidants in nearly all living organisms. Deletion of all eight thiol peroxidase genes in yeast (∆8 strain) is not lethal, but results in slow growth and a high mutation rate. Here we characterized mechanisms that allow yeast cells to survive under conditions of thiol peroxidase deficiency. Two independent ∆8 strains increased mitochondrial content, altered mitochondrial distribution, and became dependent on respiration for growth but they were not hypersensitive to H2O2. In addition, both strains independently acquired a second copy of chromosome XI and increased expression of genes encoded by it. Survival of ∆8 cells was dependent on mitochondrial cytochrome-c peroxidase (CCP1) and UTH1, present on chromosome XI. Coexpression of these genes in ∆8 cells led to the elimination of the extra copy of chromosome XI and improved cell growth, whereas deletion of either gene was lethal. Thus, thiol peroxidase deficiency requires dosage compensation of CCP1 and UTH1 via chromosome XI aneuploidy, wherein these proteins support hydroperoxide removal with the reducing equivalents generated by the electron transport chain. To our knowledge, this is the first evidence of adaptive aneuploidy counteracting oxidative stress. More Information

Ma S, Yim SH, Lee SG, Kim EB, Lee SR, Chang KT, Buffenstein R, Lewis KN, Park TJ, Miller RA, Clish CB, Gladyshev VN. (2015) Organization of the mammalian metabolome according to organ function, lineage specialization, and longevity. Cell Metabolism. 22, 332-343.

AbstractBiological diversity among mammals is remarkable. Mammalian body weights range seven orders of magnitude and lifespans differ more than 100-fold among species. While genetic, dietary, and pharmacological interventions can be used to modulate these traits in model organisms, it is unknown how they are determined by natural selection. By profiling metabolites in brain, heart, kidney, and liver tissues of 26 mammalian species representing ten taxonomical orders, we report metabolite patterns characteristic of organs, lineages, and species longevity. Our data suggest different rates of metabolite divergence across organs and reveal patterns representing organ-specific functions and lineage-specific physiologies. We identified metabolites that correlated with species lifespan, some of which were previously implicated in longevity control. We also compared the results with metabolite changes in five long-lived mouse models and observed some similar patterns. Overall, this study describes adjustments of the mammalian metabolome according to lifespan, phylogeny, and organ and lineage specialization. More Information

Turanov AA, Everley RA, Hybsier S, Renko K, Schomburg L, Gygi SP, Hatfield DL, Gladyshev VN. (2015) Regulation of Selenocysteine Content of Human Selenoprotein P by Dietary Selenium and Insertion of Cysteine in Place of Selenocysteine. PLoS One 10, e0140353.

AbstractSelenoproteins are a unique group of proteins that contain selenium in the form of selenocysteine (Sec) co-translationally inserted in response to a UGA codon with the help of cis- and trans-acting factors. Mammalian selenoproteins contain single Sec residues, with the exception of selenoprotein P (SelP) that has 7-15 Sec residues depending on species. Assessing an individual’s selenium status is important under various pathological conditions, which requires a reliable selenium biomarker. Due to a key role in organismal selenium homeostasis, high Sec content, regulation by dietary selenium, and availability of robust assays in human plasma, SelP has emerged as a major biomarker of selenium status. Here, we found that Cys is present in various Sec positions in human SelP. Treatment of cells expressing SelP with thiophosphate, an analog of the selenium donor for Sec synthesis, led to a nearly complete replacement of Sec with Cys, whereas supplementation of cells with selenium supported Sec insertion. SelP isolated directly from human plasma had up to 8% Cys inserted in place of Sec, depending on the Sec position. These findings suggest that a change in selenium status may be reflected in both SelP concentration and its Sec content, and that availability of the SelP-derived selenium for selenoprotein synthesis may be overestimated under conditions of low selenium status due to replacement of Sec with Cys. More Information

Kaya A, Ma S, Wasko B, Lee M, Kaeberiein M, Gladyshev VN. (2015) Defining molecular basis for longevity traits in natural yeast isolates. npj Aging and Mechanisms of Disease 112, 10685-10690.

AbstractBackground: The budding yeast has served as a useful model organism in aging studies, leading to the identification of genetic determinants of longevity, many of which are conserved in higher eukaryotes. However, factors that promote longevity in a laboratory setting often have severe fitness disadvantages in the wild.
Aims and Methods: To obtain an unbiased view on longevity regulation, we analyzed how a replicative lifespan is shaped by transcriptional, translational, metabolic, and morphological factors across 22 wild-type Saccharomyces cerevisiae isolates.
Results: We observed significant differences in lifespan across these strains and found that their longevity is strongly associated with up-regulation of oxidative phosphorylation and respiration and down-regulation of amino- acid and nitrogen compound biosynthesis.
Conclusions: As calorie restriction and TOR signaling also extend the lifespan by adjusting many of the identified pathways, the data suggest that the natural plasticity of yeast lifespan is shaped by the processes that not only do not impose cost on fitness, but also are amenable to dietary intervention. More Information

Tarrago L, Péterfi Z, Lee BC, Michel T, Gladyshev VN. (2015) Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors. Nat Chem Biol. 11, 332-338.

AbstractMethionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological and pathophysiological conditions, but its use as a redox marker suffers from the lack of tools to detect and quantify MetO within cells. In this work, we created a pair of complementary stereospecific genetically encoded mechanism-based ratiometric fluorescent sensors of MetO by inserting a circularly permuted yellow fluorescent protein between yeast methionine sulfoxide reductases and thioredoxins. The two sensors, respectively named MetSOx and MetROx for their ability to detect S and R forms of MetO, were used for targeted analysis of protein oxidation, regulation and repair as well as for monitoring MetO in bacterial and mammalian cells, analyzing compartment-specific changes in MetO and examining responses to physiological stimuli. More Information

Mariotti M, Santesmasses D, Capella-Gutierrez S, Mateo A, Arnan C, Johnson R, D’Aniello S, Yim SH, Gladyshev VN, Serras F, Corominas M, Gabaldon T, Guigo R. (2015) Evolution of selenophosphate synthetases: emergence and relocation of function through independent duplications and recurrent subfunctionalization. Genome Res. 25, 1256-1267.

AbstractSelenoproteins are proteins that incorporate selenocysteine (Sec), a non-standard amino acid that is encoded by UGA, normally a stop codon. The synthesis of Sec requires the enzyme Selenophosphate synthetase (SPS or SelD), conserved in all prokaryotic and eukaryotic genomes encoding selenoproteins. Here we study the evolutionary history of SPS genes, providing a map of selenoprotein function spanning the whole tree of life. SPS is itself a selenoprotein in many species, though functionally equivalent homologues that replace the Sec site with cysteine (Cys) are common. Many metazoans, however, possess SPS genes with substitutions other than Sec or Cys (collectively referred to as SPS1). Using complementation assays in fly mutants, we show that these genes share a common function, which appears to be distinct from the synthesis of selenophosphate carried out by the Sec- and Cys- SPS genes (termed SPS2), and unrelated to Sec synthesis. Even though sharing the same function, we show here that SPS1 genes originated through a number of independent gene duplications from an ancestral metazoan selenoprotein SPS2 gene, that most likely already carried the SPS1 function. Thus, in SPS genes, parallel duplications and subsequent convergent subfunctionalization have resulted in the segregation to different loci of functions initially carried by a single gene. A key actor in the evolution of SPS genes is a stem-loop RNA structure enhancing the readthrough of the Sec-UGA codon, whose origin may be traced back to prokaryotes. The evolutionary history of SPS constitutes a remarkable example of emergence and evolution of gene function, which we have been able to trace with unusual detail thanks to the singular features of SPS genes, wherein the amino acid at a single site determines unequivocally protein function and is intertwined to the evolutionary fate of the entire selenoproteome. More Information

Kaya A, Lee BC, Gladyshev VN. (2015) Regulation of protein function by reversible methionine oxidation and the role of selenoprotein MsrB1. Antioxid Redox Signal. 23, 814-822.

AbstractSIGNIFICANCE: Protein structure and function can be regulated via post-translational modifications by numerous enzymatic and nonenzymatic mechanisms. Regulation involving oxidation of sulfur-containing residues emerged as a key mechanism of redox control. Unraveling the participants and principles of such regulation is necessary for understanding the biological significance of redox control of cellular processes. Recent Advances: Reversible oxidation of methionine residues by monooxygenases of the Mical family and subsequent reduction of methionine sulfoxides by a selenocysteine-containing methionine sulfoxide reductase B1 (MsrB1) was found to control the assembly and disassembly of actin in mammals, and the Mical/MsrB pair similarly regulates actin in fruit flies. This finding has opened up new avenues for understanding the use of stereospecific methionine oxidation in regulating cellular processes and the roles of MsrB1 and Micals in regulation of actin dynamics.
CRITICAL ISSUES: So far, Micals have been the only known partners of MsrB1, and actin is the only target. It is important to identify additional substrates of Micals and characterize other Mical-like enzymes.
FUTURE DIRECTIONS: Oxidation of methionine, reviewed here, is an emerging but not well-established mechanism. Studies suggest that methionine oxidation is a form of oxidative damage of proteins, a modification that alters protein structure or function, a tool in redox signaling, and a mechanism that controls protein function. Understanding the functional impact of reversible oxidation of methionine will require identification of targets, substrates, and regulators of Micals and Msrs. Linking the biological processes, in which these proteins participate, might also lead to insights into disease conditions, which involve regulation of actin by Micals and Msrs. Antioxid. Redox Signal. 00, 000-000. More Information

Gould NS, Evans P, Martínez-Acedo P, Marino SM, Gladyshev VN, Carroll KS, Ischiropoulos H.(2015) Site-Specific proteomic mapping identifies selectively modified regulatory cysteine residues in functionally distinct protein networks. Chem Biol. 22, 965-975.

AbstractS-Acylation, S-glutathionylation, S-nitrosylation, and S-sulfenylation are prominent, chemically distinct modifications that regulate protein function, redox sensing, and trafficking. Although the biological significance of these modifications is increasingly appreciated, their integration in the proteome remains unknown. Novel mass spectrometry-based technologies identified 2,596 predominately unique sites in 1,319 mouse liver proteins under physiological conditions. Structural analysis localized the modifications in unique, evolutionary conserved protein segments, outside commonly annotated functional regions. Contrary to expectations, propensity for modification did not correlate with biophysical properties that regulate cysteine reactivity. However, the in vivo chemical reactivity is fine-tuned for specificity, demonstrated by the nominal complementation between the four modifications and quantitative proteomics which showed that a reduction in S-nitrosylation is not correlated with increased S-glutathionylation. A comprehensive survey uncovered clustering of modifications within biologically related protein networks. The data provide the first evidence for the occurrence of distinct, endogenous protein networks that undergo redox signaling through specific cysteine modifications. More Information

Kaya A, Lobanov AV, Gladyshev VN. (2015) Evidence that mutation accumulation does not cause aging in Saccharomyces cerevisiae. Aging Cell 14, 366-371.

AbstractThe concept that mutations cause aging phenotypes could not be directly tested previously due to inability to identify age-related mutations in somatic cells and determine their impact on organismal aging. Here, we subjected Saccharomyces cerevisiae to multiple rounds of replicative aging and assessed de novo mutations in daughters of mothers of different age. Mutations did increase with age, but their low numbers, < 1 per lifespan, excluded their causal role in aging. Structural genome changes also had no role. A mutant lacking thiol peroxidases had the mutation rate well above that of wild-type cells, but this did not correspond to the aging pattern, as old wild-type cells with few or no mutations were dying, whereas young mutant cells with many more mutations continued dividing. In addition, wild-type cells lost mitochondrial DNA during aging, whereas shorter-lived mutant cells preserved it, excluding a causal role of mitochondrial mutations in aging. Thus, DNA mutations do not cause aging in yeast. These findings may apply to other damage types, suggesting a causal role of cumulative damage, as opposed to individual damage types, in organismal aging. More Information

Fushan AA, Turanov AA, Lee SG, Kim EB, Lobanov AV, Yim SH, Buffenstein R, Lee SR, Chang KT, Rhee H, Kim JS, Yang KS, Gladyshev VN. (2015) Gene expression defines natural changes in mammalian lifespan. Aging Cell 14, 352-365.

AbstractMammals differ more than 100-fold in maximum lifespan, which can be altered in either direction during evolution, but the molecular basis for natural changes in longevity is not understood. Divergent evolution of mammals also led to extensive changes in gene expression within and between lineages. To understand the relationship between lifespan and variation in gene expression, we carried out RNA-seq-based gene expression analyses of liver, kidney, and brain of 33 diverse species of mammals. Our analysis uncovered parallel evolution of gene expression and lifespan, as well as the associated life-history traits, and identified the processes and pathways involved. These findings provide direct insights into how nature reversibly adjusts lifespan and other traits during adaptive radiation of lineages. More Information

Kim MJ, Lee BC, Hwang KY, Gladyshev VN, Kim HY. (2015) Selenium utilization in thioredoxin and catalytic advantage provided by selenocysteine. Biochem Biophys Res Commun. 461, 648-652.

AbstractThioredoxin (Trx) is a major thiol-disulfide reductase that plays a role in many biological processes, including DNA replication and redox signaling. Although selenocysteine (Sec)-containing Trxs have been identified in certain bacteria, their enzymatic properties have not been characterized. In this study, we expressed a selenoprotein Trx from Treponema denticola, an oral spirochete, in Escherichia coli and characterized this selenoenzyme and its natural cysteine (Cys) homologue using E. coli Trx1 as a positive control. 75Se metabolic labeling and mutation analyses showed that the SECIS (Sec insertion sequence) of T. denticola selenoprotein Trx is functional in the E. coli Sec insertion system with specific selenium incorporation into the Sec residue. The selenoprotein Trx exhibited approximately 10-fold higher catalytic activity than the Sec-to-Cys version and natural Cys homologue and E. coli Trx1, suggesting that Sec confers higher catalytic activity on this thiol-disulfide reductase. Kinetic analysis also showed that the selenoprotein Trx had a 10-fold higher Km than Cys-containing homologues, suggesting that this selenoenzyme is adapted to work efficiently with high concentrations of substrate. Collectively, the results of this study support the hypothesis that selenium utilization in oxidoreductase systems is primarily due to the catalytic advantage provided by the rare amino acid, Sec. More Information

Tsuji PA, Carlson BA, Yoo MH, Naranjo-Suarez S, Xu XM, He Y, Asaki E, Seifried HE, Reinhold WC, Davis CD, Gladyshev VN, Hatfield DL. (2015) The 15kDa Selenoprotein and Thioredoxin Reductase 1 Promote Colon Cancer by Different Pathways. PLoS One 10, e0124487.

AbstractSelenoproteins mediate much of the cancer-preventive properties of the essential nutrient selenium, but some of these proteins have been shown to also have cancer-promoting effects. We examined the contributions of the 15kDa selenoprotein (Sep15) and thioredoxin reductase 1 (TR1) to cancer development. Targeted down-regulation of either gene inhibited anchorage-dependent and anchorage-independent growth and formation of experimental metastases of mouse colon carcinoma CT26 cells. Surprisingly, combined deficiency of Sep15 and TR1 reversed the anti-cancer effects observed with down-regulation of each single gene. We found that inflammation-related genes regulated by Stat-1, especially interferon-γ-regulated guanylate-binding proteins, were highly elevated in Sep15-deficient, but not in TR1-deficient cells. Interestingly, components of the Wnt/β-catenin signaling pathway were up-regulated in cells lacking both TR1 and Sep15. These results suggest that Sep15 and TR1 participate in interfering regulatory pathways in colon cancer cells. Considering the variable expression levels of Sep15 and TR1 found within the human population, our results provide insights into new roles of selenoproteins in cancer. More Information

Bang J, Huh JH, Na JW, Lu Q, Carlson BA, Tobe R, Tsuji PA, Gladyshev VN, Hatfield DL, Lee BJ. (2015) Cell Proliferation and Motility are Inhibited by G1 Phase Arrest in 15-kDa Selenoprotein-Deficient Chang Liver Cells. Mol Cells 38, 457-465.

AbstractThe 15-kDa selenoprotein (Sep15) is a selenoprotein residing in the lumen of the endoplasmic reticulum (ER) and implicated in quality control of protein folding. Herein, we established an inducible RNAi cell line that targets Sep15 mRNA in Chang liver cells. RNAi-induced Sep15 deficiency led to inhibition of cell proliferation, whereas cell growth was resumed after removal of the knockdown inducer. Sep15-deficient cells were arrested at the G1 phase by upregulating p21 and p27, and these cells were also characterized by ER stress. In addition, Sep15 deficiency led to the relocation of focal adhesions to the periphery of the cell basement and to the decrease of the migratory and invasive ability. All these changes were reversible depending on Sep15 status. Rescuing the knockdown state by expressing a silent mutant Sep15 mRNA that is resistant to siRNA also reversed the phenotypic changes. Our results suggest that SEP15 plays important roles in the regulation of the G1 phase during the cell cycle as well as in cell motility in Chang liver cells, and that this selenoprotein offers a novel functional link between the cell cycle and cell motility. More Information

MacRae SL, Zhang Q, Lemetre C, Seim I, Calder RB, Hoeijmakers J, Suh Y, Gladyshev VN, Seluanov A, Gorbunova V, Vijg J, Zhang ZD. (2015) Comparative analysis of genome maintenance genes in naked mole rat, mouse, and human. Aging Cell 14, 288-291.

AbstractGenome maintenance (GM) is an essential defense system against aging and cancer, as both are characterized by increased genome instability. Here, we compared the copy number variation and mutation rate of 518 GM-associated genes in the naked mole rat (NMR), mouse, and human genomes. GM genes appeared to be strongly conserved, with copy number variation in only four genes. Interestingly, we found NMR to have a higher copy number of CEBPG, a regulator of DNA repair, and TINF2, a protector of telomere integrity. NMR, as well as human, was also found to have a lower rate of germline nucleotide substitution than the mouse. Together, the data suggest that the long-lived NMR, as well as human, has more robust GM than mouse and identifies new targets for the analysis of the exceptional longevity of the NMR. More Information

Nakao LS, Everley RA, Marino SM, Lo SM, de Souza LE, Gygi SP, Gladyshev VN. (2015) Mechanism-based proteomic screening identifies targets of thioredoxin-like proteins. J Biol Chem. 290, 5685-5695.

AbstractThioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best-characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CxxC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nxn1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIFM1 and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nxn1 could be detected, suggesting that their CxxC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CxxC motifs, providing important insights into thiol-based redox control of cellular processes. Copyright © 2015, The American Society for Biochemistry and Molecular Biology. More Information

Tian X, Azpurua J, Ke Z, Augereau A, Zhang ZD, Vijg J, Gladyshev VN, Gorbunova V, Seluanov A. (2015) INK4 locus of the tumor-resistant rodent, the naked mole rat, expresses a functional p15/p16 hybrid isoform. Proc Natl Acad Sci USA 112, 1053-1058.

AbstractThe naked mole rat (Heterocephalus glaber) is a long-lived and tumor-resistant rodent. Tumor resistance in the naked mole rat is mediated by the extracellular matrix component hyaluronan of very high molecular weight (HMW-HA). HMW-HA triggers hypersensitivity of naked mole rat cells to contact inhibition, which is associated with induction of the INK4 (inhibitors of cyclin dependent kinase 4) locus leading to cell-cycle arrest. The INK4a/b locus is among the most frequently mutated in human cancer. This locus encodes three distinct tumor suppressors: p15INK4b, p16INK4a, and ARF (alternate reading frame). Although p15INK4b has its own ORF, p16INK4a and ARF share common second and third exons with alternative reading frames. Here, we show that, in the naked mole rat, the INK4a/b locus encodes an additional product that consists of p15INK4b exon 1 joined to p16INK4a exons 2 and 3. We have named this isoform pALTINK4a/b (for alternative splicing). We show that pALTINK4a/b is present in both cultured cells and naked mole rat tissues but is absent in human and mouse cells. Additionally, we demonstrate that pALTINK4a/b expression is induced during early contact inhibition and upon a variety of stresses such as UV, gamma irradiation-induced senescence, loss of substrate attachment, and expression of oncogenes. When overexpressed in naked mole rat or human cells, pALTINK4a/b has stronger ability to induce cell-cycle arrest than either p15INK4b or p16INK4a. We hypothesize that the presence of the fourth product, pALTINK4a/b of the INK4a/b locus in the naked mole rat, contributes to the increased resistance to tumorigenesis of this species. More Information

Hine C, Harputlugil E, Zhang Y, Ruckenstuhl C, Lee BC, Brace L, Longchamp A, Treviño-Villarreal JH, Mejia P, Ozaki CK, Wang R, Gladyshev VN, Madeo F, Mair WB, Mitchell JR. (2015) Endogenous Hydrogen Sulfide Production Is Essential for Dietary Restriction Benefits. Cell 160, 132-144.

AbstractDietary restriction (DR) without malnutrition encompasses numerous regimens with overlapping benefits including longevity and stress resistance, but unifying nutritional and molecular mechanisms remain elusive. In a mouse model of DR-mediated stress resistance, we found that sulfur amino acid (SAA) restriction increased expression of the transsulfuration pathway (TSP) enzyme cystathionine γ-lyase (CGL), resulting in increased hydrogen sulfide (H2S) production and protection from hepatic ischemia reperfusion injury. SAA supplementation, mTORC1 activation, or chemical/genetic CGL inhibition reduced H2S production and blocked DR-mediated stress resistance. In vitro, the mitochondrial protein SQR was required for H2S-mediated protection during nutrient/oxygen deprivation. Finally, TSP-dependent H2S production was observed in yeast, worm, fruit fly, and rodent models of DR-mediated longevity. Together, these data are consistent with evolutionary conservation of TSP-mediated H2S as a mediator of DR benefits with broad implications for clinical translation. More Information

Bang J, Jang M, Huh JH, Na J, Shim M, Carlson BA, Tobe R, Tsuji PA, Gladyshev VN, Hatfield DL, Lee BJ. (2015) Deficiency of the 15-kDa selenoprotein led to cytoskeleton remodeling and non-apoptotic membrane blebbing through a RhoA/ROCK pathway. Biochem Biophys Res Commun. 456, 884-890.

AbstractThe 15-kDa selenoprotein (Sep15) has been implicated in etiology of some types of cancer. Herein, inducible RNAi cell lines were established and cell morphology and motility were analyzed. The majority of Sep15-deficient cells (>95%) formed membrane blebs in a dynamic manner. Blebbing cells transformed cell morphology from a normal flat spindle shape to a spherical morphology. In blebbing cells, actin fibers moved to the cell periphery, covering and obscuring visualization of α-tubulin. Bleb formation was suppressed by the inhibitors of Rho-associated protein kinase (ROCK), RhoA or myosin light chain (MLC), restoring blebbing cells to wild-type morphology. RhoA activation and phosphorylation of myosin phosphatase target subunit 1 was induced by Sep15 knockdown. Sep15-deficient cells were non-apoptotic, and displayed a distinct relative localization of F-actin and α-tubulin from typical apoptotic blebbing cells. Our data suggest that Sep15 in Chang liver cells regulates the pathway that antagonizes RhoA/ROCK/MLC-dependent non-apoptotic bleb formation. More Information

Cornelis MC, Fornage M, Foy M, Xun P, Gladyshev VN, Morris S, Chasman DI, Hu FB, Rimm EB, Kraft P, Jordan JM, Mozaffarian D, He K. (2015) Genome-wide association study of selenium concentrations. Hum Mol Genet. 24, 1469-1477.

AbstractSelenium (Se) is an essential trace element in human nutrition, but its role in certain health conditions, particularly among Se sufficient populations, is controversial. A genome-wide association study (GWAS) of blood Se concentrations previously identified a locus at 5q14 near BHMT. We performed a GW meta-analysis of toenail Se concentrations, which reflect a longer duration of exposure than blood Se concentrations, including 4162 European descendants from four US cohorts. Toenail Se was measured using neutron activation analysis. We identified a GW-significant locus at 5q14 (P < 1 × 10(-16)), the same locus identified in the published GWAS of blood Se based on independent cohorts. The lead single-nucleotide polymorphism (SNP) explained ∼1% of the variance in toenail Se concentrations. Using GW-summary statistics from both toenail and blood Se, we observed statistical evidence of polygenic overlap (P < 0.001) and meta-analysis of results from studies of either trait (n = 9639) yielded a second GW-significant locus at 21q22.3, harboring CBS (P < 4 × 10(-8)). Proteins encoded by genes at 5q14 and 21q22.3 function in homocysteine (Hcy) metabolism, and index SNPs for each have previously been associated with betaine and Hcy levels in GWAS. Our findings show evidence of a genetic link between Se and Hcy pathways, both involved in cardiometabolic disease. More Information

2014 Articles

Seim I, Ma S, Zhou X, Gerashchenko MV, Lee SG, Suydam R, George JC, Bickham JW, Gladyshev VN. (2014) The transcriptome of the bowhead whale Balaena mysticetus reveals adaptations of the longest-lived mammal. Aging (Albany NY) 6, 879-899.

AbstractMammals vary dramatically in lifespan, by at least two-orders of magnitude, but the molecular basis for this difference remains largely unknown. The bowhead whaleBalaena mysticetus is the longest-lived mammal known, with an estimated maximal lifespan in excess of two hundred years. It is also one of the two largest animals and the most cold-adapted baleen whale species. Here, we report the first genome-wide gene expression analyses of the bowhead whale, based on the de novo assembly of its transcriptome. Bowhead whale or cetacean-specific changes in gene expression were identified in the liver, kidney and heart, and complemented with analyses of positively selected genes. Changes associated with altered insulin signaling and other gene expression patterns could help explain the remarkable longevity of bowhead whales as well as their adaptation to a lipid-rich diet. The data also reveal parallels in candidate longevity adaptations of the bowhead whale, naked mole rat and Brandt’s bat. The bowhead whale transcriptome is a valuable resource for the study of this remarkable animal, including the evolution of longevity and its important correlates such as resistance to cancer and other diseases. More Information

Fang X, Seim I, Huang Z, Gerashchenko MV, Xiong Z, Turanov AA, Zhu Y, Lobanov AV, Fan D, Yim SH, Yao X, Ma S, Yang L, Lee SG, Kim EB, Bronson RT, Sumbera R, Buffenstein R, Zhou X, Krogh A, Park TJ, Zhang G, Wang J, Gladyshev VN.(2014) Adaptations to a subterranean environment and longevity revealed by the analysis of mole rat genomes. Cell Rep. 8, 1354-1364.

AbstractSubterranean mammals spend their lives in dark, unventilated environments that are rich in carbon dioxide and ammonia and low in oxygen. Many of these animals are also long-lived and exhibit reduced aging-associated diseases, such as neurodegenerative disorders and cancer. We sequenced the genome of the Damaraland mole rat (DMR, Fukomys damarensis) and improved the genome assembly of the naked mole rat (NMR, Heterocephalus glaber). Comparative genome analyses, along with the transcriptomes of related subterranean rodents, revealed candidate molecular adaptations for subterranean life and longevity, including a divergent insulin peptide, expression of oxygen-carrying globins in the brain, prevention of high CO2-induced pain perception, and enhanced ammonia detoxification. Juxtaposition of the genomes of DMR and other more conventional animals with the genome of NMR revealed several truly exceptional NMR features: unusual thermogenesis, an aberrant melatonin system, pain insensitivity, and unique processing of 28S rRNA. Together, these genomes and transcriptomes extend our understanding of subterranean adaptations, stress resistance, and longevity. More Information

Kaya A, Lobanov AV, Gerashchenko MV, Koren A, Fomenko DE, Koc A, Gladyshev VN. (2014) Thiol Peroxidase Deficiency Leads to Increased Mutational Load and Decreased Fitness in Saccharomyces cerevisiae. Genetics 198, 905-917.

AbstractThiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has 8 thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (∆8) is viable. In this study, we employed two independent ∆8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and ∆8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. ∆8 lines showed a significant increase in non-recurrent point mutations and indels. The original ∆8 cells exhibited reduced growth rate and decreased lifespan, which were further reduced in all ∆8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of ∆8 cells. This study shows how deficiency in non-essential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness. More Information

Gerashchenko MV, Gladyshev VN. (2014) Translation inhibitors cause abnormalities in ribosome profiling experiments. Nucleic Acids Res. 42, e134.

AbstractRibosome profiling and high-throughput sequencing provide unprecedented opportunities for the analysis of mRNA translation. Using this novel method, several studies have demonstrated the widespread role of short upstream reading frames in translational control as well as slower elongation at the beginning of open reading frames in response to stress. Based on the initial studies, the importance of adding or omitting translation inhibitors, such as cycloheximide, was noted as it markedly affected ribosome coverage profiles. For that reason, many recent studies omitted translation inhibitors in the culture medium. Here, we investigate the influence of ranging cycloheximide concentrations on ribosome profiles in Saccharomyces cerevisiae and demonstrate that increasing the drug concentration can overcome some of the artifacts. We subjected cells to various manipulations and show that neither oxidative stress nor heat shock nor amino acid starvation affect translation elongation. Instead, the observations in the initial studies are the result of cycloheximide-inflicted artifacts. Likewise, we find little support for short upstream reading frames to be involved in widespread protein synthesis regulation under stress conditions. Our study highlights the need for better standardization of ribosome profiling methods. More Information

Guo C, Chen X, Song H, Maynard MA, Zhou Y, Lobanov AV, Gladyshev VN, Ganis JJ, Wiley D, Jugo RH, Lee NY, Castroneves LA, Zon LI, Scanlan TS, Feldman HA, Huang SA. (2014) Intrinsic Expression of a Multiexon Type 3 Deiodinase Gene Controls Zebrafish Embryo Size. Endocrinology 155, 4069-4080.

AbstractThyroid hormone is a master regulator of differentiation and growth, and its action is terminated by the enzymatic removal of an inner-ring iodine catalyzed by the selenoenzyme type 3 deiodinase (dio3). Our studies of the zebrafish reveal that the dio3 gene is duplicated in this species and that embryonic deiodination is an important determinant of embryo size. Although both dio3 paralogs encode enzymatically active proteins with high affinity for thyroid hormones, their anatomic patterns of expression are markedly divergent and only embryos with knockdown of dio3b, a biallelically expressed selenoenzyme expressed in the developing central nervous system, manifest severe thyroid hormone-dependent growth restriction at 72 hours post fertilization. This indicates that the embryonic deficiency of dio3, once considered only a placental enzyme, causes microsomia independently of placental physiology and raises the intriguing possibility that fetal abnormalities in human deiodination may present as intrauterine growth retardation. By mapping the gene structures and enzymatic properties of all four zebrafish deiodinases, we also identify dio3b as the first multiexon dio3 gene, containing a large intron separating its open reading frame from its selenocysteine insertion sequence (SECIS) element. More Information

Labunskyy VM, Hatfield DL, Gladyshev VN. (2014) Selenoproteins: Molecular Pathways and Physiological Roles. Physiol Rev. 94, 739-777.

AbstractSelenium is an essential micronutrient with important functions in human health and relevance to several pathophysiological conditions. The biological effects of selenium are largely mediated by selenium-containing proteins (selenoproteins) that are present in all three domains of life. Although selenoproteins represent diverse molecular pathways and biological functions, all these proteins contain at least one selenocysteine (Sec), a selenium-containing amino acid, and most serve oxidoreductase functions. Sec is cotranslationally inserted into nascent polypeptide chains in response to the UGA codon, whose normal function is to terminate translation. To decode UGA as Sec, organisms evolved the Sec insertion machinery that allows incorporation of this amino acid at specific UGA codons in a process requiring a cis-acting Sec insertion sequence (SECIS) element. Although the basic mechanisms of Sec synthesis and insertion into proteins in both prokaryotes and eukaryotes have been studied in great detail, the identity and functions of many selenoproteins remain largely unknown. In the last decade, there has been significant progress in characterizing selenoproteins and selenoproteomes and understanding their physiological functions. We discuss current knowledge about how these unique proteins perform their functions at the molecular level and highlight new insights into the roles that selenoproteins play in human health. More Information

Gorbunova V, Seluanov A, Zhang Z, Gladyshev VN, Vijg J. (2014) Comparative genetics of longevity and cancer: insights from long-lived rodents. Nat Rev Genet. 15, 531-540.

AbstractMammals have evolved a remarkable diversity of ageing rates. Within the single order of Rodentia, maximum lifespans range from 4 years in mice to 32 years in naked mole rats. Cancer rates also differ substantially between cancer-prone mice and almost cancer-proof naked mole rats and blind mole rats. Recent progress in rodent comparative biology, together with the emergence of whole-genome sequence information, has opened opportunities for the discovery of genetic factors that control longevity and cancer susceptibility. More Information

Turanov AA, Shchedrina VA, Everley RA, Lobanov AV, Yim SH, Marino SM, Gygi SP, Hatfield DL, Gladyshev VN. (2014) Selenoprotein S is Involved in Maintenance and Transport of Multiprotein Complexes. Biochem J. 462, 555-565.

AbstractSelenoprotein S (SelS) is a selenocysteine-containing protein with roles in endoplasmic reticulum (ER) function and inflammation. It has been implicated in ER-associated protein degradation (ERAD), and clinical studies revealed an association of its promoter polymorphism with cytokine levels and human diseases. However, the pathways and interacting proteins that could shed light on pathogenesis of SelS-associated diseases have not been studied systematically. We performed a large-scale affinity isolation of human SelS and its mutant forms and analyzed proteins that interact with them. All previously known SelS targets and nearly two hundred additional proteins were identified, which were remarkably enriched for various multiprotein complexes. Subsequent chemical cross-linking experiments identified the specific interacting sites in SelS and its several targets. Most of these interactions involved coiled coil domains. The data suggest that SelS participates in intracellular membrane transport and maintenance of protein complexes by anchoring them to the ER membrane. More Information

Ables GP, Brown-Borg HM, Buffenstein R, Church CD, Elshorbagy AK, Gladyshev VN, Huang TH, Miller RA, Mitchell JR, Richie JP, Rogina B, Stipanuk MH, Orentreich DS, Orentreich N. (2014) The first international mini-symposium on methionine restriction and lifespan. Front Genet. 5, 122.

AbstractIt has been 20 years since the Orentreich Foundation for the Advancement of Science, under the leadership Dr. Norman Orentreich, first reported that low methionine (Met) ingestion by rats extends lifespan (Orentreich et al., 1993). Since then, several studies have replicated the effects of dietary methionine restricted (MR) in delaying age-related diseases (Richie et al., 1994; Miller et al., 2005; Ables et al., 2012; Sanchez-Roman and Barja, 2013). We report the abstracts from the First International Mini-Symposium on Methionine Restriction and Lifespan held in Tarrytown, NY, September 2013. The goals were (1) to gather researchers with an interest in MR and lifespan, (2) to exchange knowledge, (3) to generate ideas for future investigations, and (4) to strengthen relationships within this community. The presentations highlighted the importance of research on cysteine, growth hormone (GH), and ATF4 in the paradigm of aging. In addition, the effects of dietary restriction or MR in the kidneys, liver, bones, and the adipose tissue were discussed. The symposium also emphasized the value of other species, e.g., the naked mole rat, Brandt’s bat, and Drosophila, in aging research. Overall, the symposium consolidated scientists with similar research interests and provided opportunities to conduct future collaborative studies (Figure 3). More Information

Avanesov AS, Ma S, Pierce KA, Yim SH, Lee BC, Clish CB, Gladyshev VN. (2014) Age- and diet-associated metabolome remodeling characterizes the aging process driven by damage accumulation. eLife 3, e02077.

AbstractAging is thought to be associated with increased molecular damage, but representative markers vary across conditions and organisms, making it difficult to assess properties of cumulative damage throughout lifespan. We used nontargeted metabolite profiling to follow age-associated trajectories of >15,000 metabolites in Drosophila subjected to control and lifespan-extending diets. We find that aging is associated with increased metabolite diversity and low-abundance molecules, suggesting they include cumulative damage. Remarkably, the number of detected compounds leveled-off in late-life, and this pattern associated with survivorship. Fourteen percent of metabolites showed age-associated changes, which decelerated in late-life and long-lived flies. In contrast, known metabolites changed in abundance similarly to nontargeted metabolites and transcripts, but did not increase in diversity. Targeted profiling also revealed slower metabolism and accumulation of lifespan-limiting molecules. Thus, aging is characterized by gradual metabolome remodeling, and condition- and advanced age-associated deceleration of this remodeling is linked to mortality and molecular damage. More Information

Otero L, Romanelli-Cedrez L, Turanov AA, Gladyshev VN, Miranda-Vizuete A, Salinas G. (2014) Adjustments, extinction, and remains of selenocysteine incorporation machinery in the nematode lineage. RNA 20, 1023-1034.

AbstractSelenocysteine (Sec) is encoded by an UGA codon with the help of a SECIS element present in selenoprotein mRNAs. SECIS-binding protein (SBP2/SCBP-2) mediates Sec insertion, but the roles of its domains and the impact of its deficiency on Sec insertion are not fully understood. We used Caenorhabditis elegans to examine SBP2 function since it possesses a single selenoprotein, thioredoxin reductase-1 (TRXR-1). All SBP2 described so far have an RNA-binding domain (RBD) and a Sec-incorporation domain (SID). Surprisingly, C. elegans SBP2 lacks SID and consists only of an RBD. An sbp2 deletion mutant strain ablated Sec incorporation demonstrating SBP2 essentiality for Sec incorporation. Further in silico analyses of nematode genomes revealed conservation of SBP2 lacking SID and maintenance of Sec incorporation linked to TRXR-1. Remarkably, parasitic plant nematodes lost the ability to incorporate Sec, but retained SecP43, a gene associated with Sec incorporation. Interestingly, both selenophosphate synthetase (SPS) genes are absent in plant parasitic nematodes, while only Cys-containing SPS2 is present in Sec-incorporating nematodes. Our results indicate that C. elegans and the nematode lineage provide key insights into Sec incorporation and the evolution of Sec utilization trait, selenoproteomes, selenoproteins, and Sec residues. Finally, our study provides evidence of noncanonical translation initiation in C. elegans, not previously known for this well-established animal model. More Information

Labunskyy VM, Suzuki Y, Hanly TJ, Murao A, Roth FP, Gladyshev VN. (2014) The Insertion Green Monster (iGM) Method for Expression of Multiple Exogenous Genes in Yeast. G3 (Bethesda) 4, 183-191.

AbstractBeing a simple eukaryotic organism, Saccharomyces cerevisiae provides numerous advantages for expression and functional characterization of proteins from higher eukaryotes, including humans. However, studies of complex exogenous pathways using yeast as a host have been hampered by the lack of tools to engineer strains overexpressing a large number of genetic components. In addition to inserting multiple genes, it is often desirable to knock out or replace multiple endogenous genes that might interfere with the processes studied. Here, we describe the ‘insertion Green Monster’ (iGM) set of expression vectors that enable precise insertion of many heterologous genes into the yeast genome in a rapid and reproducible manner and permit simultaneous replacement of selected yeast genes. As a proof-of-principle, we have used the iGM method to replace components of the yeast pathway for methionine sulfoxide reduction with genes encoding the human selenoprotein biosynthesis machinery, and generated a single yeast strain carrying 11 exogenous components of the selenoprotein biosynthetic pathway in precisely engineered loci.
KEYWORDS: Saccharomyces cerevisiae, flow cytometry, green fluorescent protein, multi-gene insertions, synthetic biology More Information

Han SJ, Lee BC, Yim SH, Gladyshev VN, Lee SR. (2014) Characterization of Mammalian selenoprotein o: a redox-active mitochondrial protein. PLoS One 9, e95518.

AbstractSelenoproteins exhibit diverse biological functions, most of which are associated with redox control. However, the functions of approximately half of mammalian selenoproteins are not known. One such protein is Selenoprotein O (SelO), the largest mammalian selenoprotein with orthologs found in a wide range of organisms, including bacteria and yeast. Here, we report characterization of mammalian SelO. Expression of this protein could be verified in HEK 293T cells by metabolic labeling of cells with 75Se, and it was abolished when selenocysteine was replaced with serine. A CxxU motif was identified in the C-terminal region of SelO. This protein was reversibly oxidized in a time- and concentration-dependent manner in HEK 293T cells when cells were treated with hydrogen peroxide. This treatment led to the formation of a transient 88 kDa SelO-containing complex. The formation of this complex was enhanced by replacing the CxxU motif with SxxC, but abolished when it was replaced with SxxS, suggesting a redox interaction of SelO with another protein through its Sec residue. SelO was localized to mitochondria and expressed across mouse tissues. Its expression was little affected by selenium deficiency, suggesting it has a high priority for selenium supply. Taken together, these results show that SelO is a redox-active mitochondrial selenoprotein. More Information

Barroso M, Florindo C, Kalwa H, Silva Z, Turanov AA, Carlson BA, Tavares de Almeida I, Blom HJ, Gladyshev VN, Hatfield DL, Michel T, Castro R, Loscalzo J, Handy DE. (2014) Inhibition of Cellular Methyltransferases Promotes Endothelial Cell Activation by Suppressing Glutathione Peroxidase-1 Expression. J Biol Chem. 289, 15350-15362.

AbstractS-adenosylhomocysteine (SAH) is a negative regulator of most methyltransferases and the precursor for the cardiovascular risk factor homocysteine. We have previously identified a link between the homocysteine-induced suppression of the selenoprotein glutathione peroxidase-1 (GPx-1) and endothelial dysfunction. Here, we demonstrate a specific mechanism by which hypomethylation, promoted by the accumulation of the homocysteine precursor SAH, suppresses GPx-1 expression and leads to inflammatory activation of endothelial cells. The expression of GPx-1 and a subset of other selenoproteins is dependent on the methylation of the tRNASec to the Um34 form; the formation of methylated tRNASec facilitates translational incorporation of selenocysteine at a UGA codon. Our findings demonstrate that SAH accumulation in endothelial cells suppresses the expression of GPx-1 to promote oxidative stress. Hypomethylation stress, caused by SAH accumulation, inhibits the formation of the methylated isoform of the tRNASec and reduces GPx-1 expression. In contrast, under these conditions, the expression and activity of thioredoxin reductase-1, another selenoprotein, is increased. Furthermore, SAH-induced oxidative stress creates a pro-inflammatory activation of endothelial cells characterized by upregulation of adhesion molecules and an augmented capacity to bind leukocytes. Taken together, these data suggest that SAH accumulation in endothelial cells can induce tRNASec hypomethylation which alters the expression of selenoproteins, such as GPx-1, to contribute to a pro-atherogenic endothelial phenotype. More Information

Lee BC, Kaya A, Ma S, Kim G, Gerashchenko MV, Yim SH, Hu Z, Harshman LG, Gladyshev VN. (2014) Methionine restriction extends lifespan of Drosophila melanogaster under conditions of low amino-acid status. Nat Commun. 5, 3592.

AbstractReduced methionine (Met) intake can extend lifespan of rodents; however, whether this regimen represents a general strategy for regulating aging has been controversial. Here we report that Met restriction extends lifespan in both fruit flies and yeast, and that this effect requires low amino-acid status. Met restriction in Drosophila mimicks the effect of dietary restriction and is associated with decreased reproduction. However, under conditions of high amino-acid status, Met restriction is ineffective and the trade-off between longevity and reproduction is not observed. Overexpression of InRDN or Tsc2 inhibits lifespan extension by Met restriction, suggesting the role of TOR signalling in the Met control of longevity. Overall, this study defines the specific roles of Met and amino-acid imbalance in aging and suggests that Met restiction is a general strategy for lifespan extension. More Information

Malinouski M, Hasan NM, Zhang Y, Seravalli J, Lin J, Avanesov A, Lutsenko S, Gladyshev VN. (2014) Genome-wide RNAi ionomics screen reveals new genes and regulation of human trace element metabolism. Nat Commun. 5, 3301.

AbstractTrace elements are essential for human metabolism and dysregulation of their homoeostasis is associated with numerous disorders. Here we characterize mechanisms that regulate trace elements in human cells by designing and performing a genome-wide high-throughput siRNA/ionomics screen, and examining top hits in cellular and biochemical assays. The screen reveals high stability of the ionomes, especially the zinc ionome, and yields known regulators and novel candidates. We further uncover fundamental differences in the regulation of different trace elements. Specifically, selenium levels are controlled through the selenocysteine machinery and expression of abundant selenoproteins; copper balance is affected by lipid metabolism and requires machinery involved in protein trafficking and post-translational modifications; and the iron levels are influenced by iron import and expression of the iron/haeme-containing enzymes. Our approach can be applied to a variety of disease models and/or nutritional conditions, and the generated data set opens new directions for studies of human trace element metabolism. More Information

Hatfield DL, Tsuji PA, Carlson BA, Gladyshev VN. (2014) Selenium and selenocysteine: roles in cancer, health, and development. Trends Biochem Sci. 39, 112-120.

AbstractThe many biological and biomedical effects of selenium are relatively unknown outside the selenium field. This fascinating element, initially described as a toxin, was subsequently shown to be essential for health and development. By the mid-1990s selenium emerged as one of the most promising cancer chemopreventive agents, but subsequent human clinical trials yielded contradictory results. However, basic research on selenium continued to move at a rapid pace, elucidating its many roles in health, development, and in cancer prevention and promotion. Dietary selenium acts principally through selenoproteins, most of which are oxidoreductases involved in diverse cellular functions. More Information

Labunskyy VM, Gerashchenko MV, Delaney JR, Kaya A, Kennedy BK, Kaeberlein M, Gladyshev VN. (2014) Lifespan extension conferred by endoplasmic reticulum secretory pathway deficiency requires induction of the unfolded protein response. PLoS Genet. 10, e1004019.

AbstractCells respond to accumulation of misfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR) signaling pathway. The UPR restores ER homeostasis by degrading misfolded proteins, inhibiting translation, and increasing expression of chaperones that enhance ER protein folding capacity. Although ER stress and protein aggregation have been implicated in aging, the role of UPR signaling in regulating lifespan remains unknown. Here we show that deletion of several UPR target genes significantly increases replicative lifespan in yeast. This extended lifespan depends on a functional ER stress sensor protein, Ire1p, and is associated with constitutive activation of upstream UPR signaling. We applied ribosome profiling coupled with next generation sequencing to quantitatively examine translational changes associated with increased UPR activity and identified a set of stress response factors up-regulated in the long-lived mutants. Besides known UPR targets, we uncovered up-regulation of components of the cell wall and genes involved in cell wall biogenesis that confer resistance to multiple stresses. These findings demonstrate that the UPR is an important determinant of lifespan that governs ER stress and identify a signaling network that couples stress resistance to longevity. More Information

Romagné F, Santesmasses D, White L, Sarangi GK, Mariotti M, Hübler R, Weihmann A, Parra G, Gladyshev VN, Guigó R, Castellano S. (2014) SelenoDB 2.0: annotation of selenoprotein genes in animals and their genetic diversity in humans. Nucleic Acids Res. 42, D437-D443.

AbstractSelenoDB (http://www.selenodb.org) aims to provide high-quality annotations of selenoprotein genes, proteins and SECIS elements. Selenoproteins are proteins that contain the amino acid selenocysteine (Sec) and the first release of the database included annotations for eight species. Since the release of SelenoDB 1.0 many new animal genomes have been sequenced. The annotations of selenoproteins in new genomes usually contain many errors in major databases. For this reason, we have now fully annotated selenoprotein genes in 58 animal genomes. We provide manually curated annotations for human selenoproteins, whereas we use an automatic annotation pipeline to annotate selenoprotein genes in other animal genomes. In addition, we annotate the homologous genes containing cysteine (Cys) instead of Sec. Finally, we have surveyed genetic variation in the annotated genes in humans. We use exon capture and resequencing approaches to identify single-nucleotide polymorphisms in more than 50 human populations around the world. We thus present a detailed view of the genetic divergence of Sec- and Cys-containing genes in animals and their diversity in humans. The addition of these datasets into the second release of the database provides a valuable resource for addressing medical and evolutionary questions in selenium biology. More Information

Gladyshev VN. (2014) The free radical theory of aging is dead. Long live the damage theory! Antioxid Redox Signal. 20, 727-731.

AbstractThe free radical theory of aging posits that aging is caused by accumulation of damage inflicted by reactive oxygen species (ROS). Although this concept has been very useful in defining the contribution of oxidative damage to the aging process, an increasing number of studies contradict it. The idea that oxidative damage represents only one of many causes of aging also has limitations, as it does not explain causal relationships and inevitability of damage accumulation. Here, it is discussed that infidelity, heterogeneity and imperfectness of each and every biological process may be responsible for the inevitable accumulation of by-products and other damage forms. Although ROS are prototypical by-products, their contribution to aging is governed by the metabolic organization of the cell, its protective systems, and genotype. These factors are controlled by natural selection and, like dietary and genetic interventions that extend lifespan, change the composition of cumulative damage and the rates of accumulation of its various forms. Oxidative damage, like other specific damage types viewed in isolation or in combination, does not represent the cause of aging. Instead, biological imperfectness, which leads to inevitable accumulation of damage in the form of mildly deleterious molecular species, may help define the true root of aging. Free radical and other specialized damage theories served their purpose in the understanding of the aging process, but in the current form they limit further progress in this area. More Information

2013 Articles

Yoo MH, Carlson BA, Gladyshev VN, Hatfield DL. (2013) Abrogated Thioredoxin System Causes Increased Sensitivity to TNF-α-Induced Apoptosis via Enrichment of p-ERK 1/2 in the Nucleus. PLoS One 8, e71427.

AbstractThioredoxin (Trx) and thioredoxin reductase 1 (TR1) are among the major redox regulators in mammalian cells and have a wide variety of roles, including removal of intracellular reactive oxygen species (ROS) and prevention of cell death. Tumor necrosis factor-α (TNF-α) induces cancer cell death. Although ROS have been proposed to participate in this process, the role of the thioredoxin system in TNF-α stimulated cell death remains unclear. We investigated the possibility that the thioredoxin system protects against TNF-α-induced cancer cell death by examining whether TR1/Trx1 status controls TNF-α-induced apoptosis in EMT6 murine breast cancer cells. TR1-deficient cells were more sensitive to TNF-α than control cells. Increased sensitivity to TNF-α was most pronounced in Trx1-deficient cells. TNF-α-induced nuclear localization of phosphorylated ERK 1/2 (p-ERK 1/2) correlated with increased apoptosis in TR1- and Trx1-deficient cells, suggesting a pro-apoptotic role for nuclear p-ERK 1/2 in TNF-α-induced apoptosis. In addition, phosphoinositide 3-kinase (PI3K) inhibition dramatically reduced TNF-α-stimulated apoptosis and nuclear localization of p-ERK 1/2. In contrast, inhibition of ROS, MEK, JNK, or p38 did not significantly alter p-ERK 1/2 localization or apoptosis in TR1- and Trx1-deficient cells compared to control cells. Further, NF-κB p65 localization was not changed in TR1- and Trx1-deficient cells in response to TNF-α relative to control cells. Our data suggest that the thioredoxin system plays a critical role in protecting against TNF-α-induced apoptosis by regulating the levels of nuclear p-ERK 1/2 in a PI3K-dependent manner. More Information

Seim I, Fang X, Xiong Z, Lobanov AV, Huang Z, Ma S, Feng Y, Turanov AA, Zhu Y, Lenz TL, Gerashchenko MV, Fan D, Yim SH, Yao X, Jordan D, Xiong Y, Ma Y, Lyapunov AN, Chen G, Kulakova OI, Sun Y, Lee SG, Bronson RT, Moskalev AA, Sunyaev SR, Zhang G, Krogh A, Wang J, Gladyshev VN. (2013) Genome analysis reveals insights into physiology and longevity of the Brandt’s bat Myotis brandtii. Nature Communications 4, 2212.

AbstractBats account for one-fifth of mammalian species, are the only mammals with powered flight, and are among the few animals that echolocate. The insect-eating Brandt’s bat (Myotis brandtii) is the longest-lived bat species known to date (lifespan exceeds 40 years) and, at 4–8 g adult body weight, is the most extreme mammal with regard to disparity between body mass and longevity. Here we report sequencing and analysis of the Brandt’s bat genome and transcriptome, which suggest adaptations consistent with echolocation and hibernation, as well as altered metabolism, reproduction and visual function. Unique sequence changes in growth hormone and insulin-like growth factor 1 receptors are also observed. The data suggest that an altered growth hormone/insulin-like growth factor 1 axis, which may be common to other long-lived bat species, together with adaptations such as hibernation and low reproductive rate, contribute to the exceptional lifespan of the Brandt’s bat. More Information

Naranjo-Suarez S, Carlson BA, Tobe R, Yoo MH, Tsuji PA, Gladyshev VN, Hatfield DL. (2013) Regulation of HIF-1α activity by overexpression of thioredoxin is independent of thioredoxin reductase status. Mol Cell 36, 151-157.

AbstractUnder hypoxic conditions, cells activate a transcriptional response mainly driven by hypoxia-inducible factors (HIFs). HIF-1α stabilization and activity are known to be regulated by thioredoxin 1 (Txn1), but how the thioredoxin system regulates the hypoxic response is unknown. By examining the effects of Txn1 overexpression on HIF-1α function in HeLa, HT-29, MCF-7 and EMT6 cell lines, we found that this oxidoreductase did not stabilize HIF-1α, yet could increase its activity. These effects were dependent on the redox function of Txn1. However, Txn1 deficiency did not affect HIF-1α hypoxic-stabilization and activity, and overexpression of thioredoxin reductase 1 (TR1), the natural Txn1 reductase, had no influence on HIF-1α activity. Moreover, overexpression of Txn1 in TR1 deficient HeLa and EMT6 cells was still able to increase HIF-1α hypoxic activity. These results indicate that Txn1 is not essential for HIF-1α hypoxic stabilization or activity, that its overexpression can increase HIF-1α hypoxic activity, and that this effect is observed regardless of TR1 status. Thus, regulation of HIF-1α by the thioredoxin system depends on the specific levels of this system’s major components. More Information

Lee BC, Péterfi Z, Hoffmann FW, Moore RE, Kaya A, Avanesov A, Tarrago L, Zhou Y, Weerapana E, Fomenko DE, Hoffmann PR, Gladyshev VN. (2013) MsrB1 and MICALs Regulate Actin Assembly and Macrophage Function via Reversible Stereoselective Methionine Oxidation. Mol Cell 51, 397-404.

AbstractRedox control of protein function involves oxidation and reduction of amino acid residues, but the mechanisms and regulators involved are insufficiently understood. Here, we report that in conjunction with Mical proteins, methionine-R-sulfoxide reductase B1 (MsrB1) regulates mammalian actin assembly via stereoselective methionine oxidation and reduction in a reversible, site-specific manner. Two methionine residues in actin are specifically converted to methionine-R-sulfoxide by Mical1 and Mical2 and reduced back to methionine by selenoprotein MsrB1, supporting actin disassembly and assembly, respectively. Macrophages utilize this redox control during cellular activation by stimulating MsrB1 expression and activity as a part of innate immunity. We identified the regulatory role of MsrB1 as a Mical antagonist in orchestrating actin dynamics and macrophage function. More generally, our study shows that proteins can be regulated by reversible site-specific methionine-R-sulfoxidation. More Information

Mariotti M, Lobanov AV, Guigo R, Gladyshev VN. (2013) SECISearch3 and Seblastian: new tools for prediction of SECIS elements and selenoproteins. Nucleic Acids Res. 41, e149.

AbstractSelenoproteins are proteins containing an uncommon amino acid selenocysteine (Sec). Sec is inserted by a specific translational machinery that recognizes a stem-loop structure, the SECIS element, at the 3′ UTR of selenoprotein genes and recodes a UGA codon within the coding sequence. As UGA is normally a translational stop signal, selenoproteins are generally misannotated and designated tools have to be developed for this class of proteins. Here, we present two new computational methods for selenoprotein identification and analysis, which we provide publicly through the web servers at http://gladyshevlab.org/SelenoproteinPredictionServer or http://seblastian.crg.es. SECISearch3 replaces its predecessor SECISearch as a tool for prediction of eukaryotic SECIS elements. Seblastian is a new method for selenoprotein gene detection that uses SECISearch3 and then predicts selenoprotein sequences encoded upstream of SECIS elements. Seblastian is able to both identify known selenoproteins and predict new selenoproteins. By applying these tools to diverse eukaryotic genomes, we provide a ranked list of newly predicted selenoproteins together with their annotated cysteine-containing homologues. An analysis of a representative candidate belonging to the AhpC family shows how the use of Sec in this protein evolved in bacterial and eukaryotic lineages. More Information

Le DT, Tarrago L, Watanabe Y, Kaya A, Lee BC, Tran U, Nishiyama R, Fomenko DE, Gladyshev VN, Tran LS. (2013) Diversity of plant methionine sulfoxide reductases B and evolution of a form specific for free methionine sulfoxide. PLoS One 8, e65637.

AbstractMethionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological conditions. Organisms evolved two distinct methionine sulfoxide reductase families (MSRA & MSRB) to repair oxidized methionine residues. We found that 5 MSRB genes exist in the soybean genome, including GmMSRB1 and two segmentally duplicated gene pairs (GmMSRB2 and GmMSRB5, GmMSRB3 and GmMSRB4). GmMSRB2 and GmMSRB4 proteins showed MSRB activity toward protein-based MetO with either DTT or thioredoxin (TRX) as reductants, whereas GmMSRB1 was active only with DTT. GmMSRB2 had a typical MSRB mechanism with Cys121 and Cys 68 as catalytic and resolving residues, respectively. Surprisingly, this enzyme also possessed the MSRB activity toward free Met-R-O with kinetic parameters similar to those reported for fRMSR from Escherichia coli, an enzyme specific for free Met-R-O. Overexpression of GmMSRB2 or GmMSRB4 in the yeast cytosol supported the growth of the triple MSRA/MSRB/fRMSR (Δ3MSRs) mutant on MetO and protected cells against H2O2-induced stress. Taken together, our data reveal an unexpected diversity of MSRBs in plants and indicate that, in contrast to mammals that cannot reduce free Met-R-O and microorganisms that use fRMSR for this purpose, plants evolved MSRBs for the reduction of both free and protein-based MetO. More Information

Gladyshev VN. (2013) The origin of aging: imperfectness-driven non-random damage defines the aging process and control of lifespan. Trends Genet. 29, 506-512.

AbstractPhysicochemical properties preclude ideal biomolecules and perfect biological functions. This inherent imperfectness leads to the generation of damage by every biological process, at all levels, from small molecules to cells. The damage is too numerous to be repaired, is partially invisible to natural selection, and manifests as aging. I propose that the inherent imperfectness of biological systems is the true root of the aging process. Because each biomolecule generates specific forms of damage, the cumulative damage is largely non-random and is indirectly encoded in the genome. I consider this concept in light of other proposed theories of aging and integrate these disparate ideas into a single model. I also discuss the evolutionary significance of damage accumulation and strategies for reducing damage. Finally, I suggest ways to test this integrated model of aging. More Information

Read BA, Kegel J, Klute MJ, Kuo A, Lefebvre SC, Maumus F, Mayer C, Miller J, Monier A, Salamov A, Young J, Aguilar M, Claverie JM, Frickenhaus S, Gonzalez K, Herman EK, Lin YC, Napier J, Ogata H, Sarno AF, Shmutz J, Schroeder D, de Vargas C, Verret F, von Dassow P, Valentin K, Van de Peer Y, Wheeler G; Emiliania huxleyi Annotation Consortium, Allen AE, Bidle K, Borodovsky M, Bowler C, Brownlee C, Mark Cock J, Elias M, Gladyshev VN, Groth M, Guda C, Hadaegh A, Debora Iglesias-Rodriguez M, Jenkins J, Jones BM, Lawson T, Leese F, Lindquist E, Lobanov A, Lomsadze A, Malik SB, Marsh ME, Mackinder L, Mock T, Mueller-Roeber B, Pagarete A, Parker M, Probert I, Quesneville H, Raines C, Rensing SA, Riaño-Pachón DM, Richier S, Rokitta S, Shiraiwa Y, Soanes DM, van der Giezen M, Wahlund TM, Williams B, Wilson W, Wolfe G, Wurch LL, Dacks JB, Delwiche CF, Dyhrman ST, Glöckner G, John U, Richards T, Worden AZ, Zhang X, Grigoriev IV. (2013) Pan genome of the phytoplankton Emiliania underpins its global distribution. Nature 499, 209-213.

AbstractCoccolithophores have influenced the global climate for over 200 million years. These marine phytoplankton can account for 20 per cent of total carbon fixation in some systems. They form blooms that can occupy hundreds of thousands of square kilometres and are distinguished by their elegantly sculpted calcium carbonate exoskeletons (coccoliths), rendering them visible from space. Although coccolithophores export carbon in the form of organic matter and calcite to the sea floor, they also release CO2 in the calcification process. Hence, they have a complex influence on the carbon cycle, driving either CO2 production or uptake, sequestration and export to the deep ocean. Here we report the first haptophyte reference genome, from the coccolithophore Emiliania huxleyi strain CCMP1516, and sequences from 13 additional isolates. Our analyses reveal a pan genome (core genes plus genes distributed variably between strains) probably supported by an atypical complement of repetitive sequence in the genome. Comparisons across strains demonstrate that E. huxleyi, which has long been considered a single species, harbours extensive genome variability reflected in different metabolic repertoires. Genome variability within this species complex seems to underpin its capacity both to thrive in habitats ranging from the equator to the subarctic and to form large-scale episodic blooms under a wide variety of environmental conditions. More Information

Turanov AA, Lobanov AV, Hatfield DL, Gladyshev VN. (2013) UGA codon position-dependent incorporation of selenocysteine into mammalian selenoproteins. Nucleic Acids Res. 41, 6952-6959.

AbstractIt is thought that the SelenoCysteine Insertion Sequence (SECIS) element and UGA codon are sufficient for selenocysteine (Sec) insertion. However, we found that UGA supported Sec insertion only at its natural position or in its close proximity in mammalian thioredoxin reductase 1 (TR1). In contrast, Sec could be inserted at any tested position in mammalian TR3. Replacement of the 3′-UTR of TR3 with the corresponding segment of a Euplotes crassus TR restricted Sec insertion into the C-terminal region, whereas the 3′-UTR of TR3 conferred unrestricted Sec insertion into E. crassus TR, in which Sec insertion is normally limited to the C-terminal region. Exchanges of 3′-UTRs between mammalian TR1 and E. crassus TR had no effect, as both proteins restricted Sec insertion. We further found that these effects could be explained by the use of selenoprotein-specific SECIS elements. Examination of Sec insertion into other selenoproteins was consistent with this model. The data indicate that mammals evolved the ability to limit Sec insertion into natural positions within selenoproteins, but do so in a selenoprotein-specific manner, and that this process is controlled by the SECIS element in the 3′-UTR. More Information

Lobanov AV, Gladyshev VN. (2013) Selenoproteome of Kinetoplastids. Drug Dis in Infect Dis. 4, 237-242.

AbstractSelenocysteine (Sec) is a naturally occurring twenty first amino acid that is present in the active sites of several oxidoreductases. Proteins containing Sec, selenoproteins, occur in all three domains of life; however, the use of Sec in lower eukaryotes, and in particular in parasitic protists, is variable, because many organisms have lost the ability to utilize Sec. The genomes of flagellated protozoa Trypanosoma and Leishmania encode three selenoproteins. Two of these proteins are distant homologs of mammalian SelK and SelT. The third selenoprotein is a novel multidomain selenoprotein designated SelTryp. This protein appears to be a Kinetoplastida-specific protein and has neither Sec- nor cysteine-containing homologs in the human host. In all three selenoproteins, Sec is present within predicted redox motifs. The use of selenium for protein synthesis was verified by metabolically labeling Trypanosoma cells with 75Se. In addition, a complete set of genes coding for components of the Sec insertion machinery was identified in the Kinetoplastida genomes. Further studies revealed that the selenoproteome of Trypanosoma is dispensable, but relevant for long-term protection. Finally, it was found that T. b. brucei cells are sensitive to auranofin, the drug that targets selenoproteins. More Information

Gladyshev VN, Zhang Y. (2013) Comparative genomics analysis of the metallomes. Met Ions Life Sci. 12, 529-580.

AbstractBiological trace metals are needed in small quantities, but used by all living organisms. They are employed in key cellular functions in a variety of biological processes, resulting in the various degree of dependence of organisms on metals. Most effort in the field has been placed on experimental studies of metal utilization pathways and metal-dependent proteins. On the other hand, systemic level analyses of metalloproteomes (or metallomes) have been limited for most metals. In this chapter, we focus on the recent advances in comparative genomics, which provides many insights into evolution and function of metal utilization. These studies suggested that iron and zinc are widely used in biology (presumably by all organisms), whereas some other metals such as copper, molybdenum, nickel, and cobalt, show scattered occurrence in various groups of organisms. For these metals, most user proteins are well characterized and their dependence on a specific element is evolutionarily conserved. We also discuss evolutionary dynamics of the dependence of user proteins on different metals. Overall, comparative genomics analysis of metallomes provides a foundation for the systemic level understanding of metal utilization as well as for investigating the general features, functions, and evolutionary dynamics of metal use in the three domains of life. More Information

Tobe R, Naranjo-Suarez S, Everley RA, Carlson BA, Turanov AA, Tsuji PA, Yoo MH, Gygi SP, Gladyshev VN, Hatfield DL. (2013) High error rates in selenocysteine insertion in mammalian cells treated with the antibiotics, doxycycline, chloramphenicol or geneticin. J Biol Chem. 288, 14709-14715.

AbstractAntibiotics cause errors in bacterial translation, but their effects on translation in mammalian cells are less well characterized. We found that doxycycline (Dox), chloramphenicol (Cp) and geneticin (G418) interfered with insertion of selenocysteine (Sec), which is encoded by the stop codon, UGA, into selenoproteins in murine EMT6 cells. Treatment of EMT6 cells with these antibiotics reduced enzymatic activities and Sec insertion into thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1), but differentially affected the levels of these proteins due to varying errors in Sec insertion at UGA. In the presence of Dox, Cp or G418, the Sec-containing form of TR1 decreased, whereas the arginine-containing and truncated forms of this protein increased. We also detected selenoprotein- and antibiotic-specific misinsertion of cysteine and tryptophan. Furthermore, misinsertion of arginine in place of Sec was commonly observed in GPx1 and glutathione peroxidase 4. TR1 was the most and GPx1 the least affected by these translation errors, consistent with the differential use of two Sec tRNA isoforms and their distinct roles in supporting accuracy of Sec insertion into selenoproteins. The data reveal widespread errors in inserting Sec into proteins and in dysregulation of selenoprotein expression and function upon antibiotic treatment. More Information

Gobler CJ, Lobanov AV, Tang YZ, Turanov AA, Zhang Y, Doblin M, Taylor GT, Sañudo-Wilhelmy SA, Grigoriev IV, Gladyshev VN. (2013) The central role of selenium in the biochemistry and ecology of the harmful pelagophyte, Aureococcus anophagefferens. ISME J. 7, 1333-1343.

AbstractThe trace element selenium (Se) is required for the biosynthesis of selenocysteine (Sec), the 21st amino acid in the genetic code, but its role in the ecology of harmful algal blooms (HABs) is unknown. Here, we examined the role of Se in the biology and ecology of the harmful pelagophyte, Aureococcus anophagefferens, through cell culture, genomic analyses, and ecosystem studies. This organism has the largest and the most diverse selenoproteome identified to date that consists of at least 59 selenoproteins, including known eukaryotic selenoproteins, selenoproteins previously only detected in bacteria, and novel selenoproteins. The A. anophagefferens selenoproteome was dominated by the thioredoxin fold proteins and oxidoreductase functions were assigned to the majority of detected selenoproteins. Insertion of Sec in these proteins was supported by a unique Sec insertion sequence. Se was required for the growth of A. anophagefferens as cultures grew maximally at nanomolar Se concentrations. In a coastal ecosystem, dissolved Se concentrations were elevated before and after A. anophagefferens blooms, but were reduced by >95% during the peak of blooms to 0.05 nM. Consistent with this pattern, enrichment of seawater with selenite before and after a bloom did not affect the growth of A. anophagefferens, but enrichment during the peak of the bloom significantly increased population growth rates. These findings demonstrate that Se inventories, which can be anthropogenically enriched, can support proliferation of HABs, such as A. anophagefferens through its synthesis of a large arsenal of Se-dependent oxidoreductases that fine-tune cellular redox homeostasis. More Information

Moustafa ME, Carlson BA, Anver MR, Bobe G, Zhong N, Ward JM, Perella CM, Hoffmann VJ, Rogers K, Combs GF Jr, Schweizer U, Merlino G, Gladyshev VN, Hatfield DL. (2013) Selenium and Selenoprotein Deficiencies Induce Widespread Pyogranuloma Formation in Mice, while High Levels of Dietary Selenium Decrease Liver Tumor Size Driven by TGFα. PLoS One 8, e57389.

AbstractChanges in dietary selenium and selenoprotein status may influence both anti- and pro-cancer pathways, making the outcome of interventions different from one study to another. To characterize such outcomes in a defined setting, we undertook a controlled hepatocarcinogenesis study involving varying levels of dietary selenium and altered selenoprotein status using mice carrying a mutant (A37G) selenocysteine tRNA transgene ( ) and/or a cancer driver transgene. The use of altered selenoprotein expression in a selenoprotein and tissue specific manner and, at sufficient dietary selenium levels, separate the effect of diet and selenoprotein status. Mice were maintained on diets deficient in selenium (0.02 ppm selenium) or supplemented with 0.1, 0.4 or 2.25 ppm selenium or 30 ppm triphenylselenonium chloride (TPSC), a non-metabolized selenium compound. transgenic and / bi-transgenic mice subjected to selenium-deficient or TPSC diets developed a neurological phenotype associated with early morbidity and mortality prior to hepatocarcinoma development. Pathology analyses revealed widespread disseminated pyogranulomatous inflammation. Pyogranulomas occurred in liver, lungs, heart, spleen, small and large intestine, and mesenteric lymph nodes in these transgenic and bi-transgenic mice. The incidence of liver tumors was significantly increased in mice carrying the transgene, while dietary selenium and selenoprotein status did not affect tumor number and multiplicity. However, adenoma and carcinoma size and area were smaller in transgenic mice that were fed 0.4 and 2.25 versus 0.1 ppm of selenium. Thus, selenium and selenoprotein deficiencies led to widespread pyogranuloma formation, while high selenium levels inhibited the size of -induced liver tumors. More Information

Kasaikina MV, Turanov AA, Avanesov A, Schweizer U, Seeher S, Bronson RT, Novoselov SN, Carlson BA, Hatfield DL, Gladyshev VN. (2013) Contrasting roles of dietary selenium and selenoproteins in chemically induced hepatocarcinogenesis. Carcinogenesis 34, 1089-1095.

AbstractSelenium (Se) has long been known for its cancer prevention properties, but the molecular basis remains unclear. The principal questions in assessing the effect of dietary Se in cancer are whether selenoproteins, small molecule selenocompounds, or both, are involved, and under which conditions and genotypes Se may be protective. In this study, we examined diethylnitrosamine-induced hepatocarcinogenesis in mice lacking a subset of selenoproteins due to expression of a mutant selenocysteine tRNA gene (Trsp(A37G) mice). To uncouple the effects of selenocompounds and selenoproteins, these animals were examined at several levels of dietary Se. Our analysis revealed that tumorigenesis in Trsp(A37G) mice maintained on the adequate Se diet was increased. However, in the control, wild-type mice, both Se deficiency and high Se levels protected against tumorigenesis. We further found that the Se-deficient diet induced severe neurological phenotypes in TrspA37G mice. Surprisingly, a similar phenotype could be induced in these mice at high dietary Se intake. Overall, our results show a complex role of Se in chemically induced hepatocarcinogenesis, which involves interaction among selenoproteins, selenocompounds and toxins, and depends on genotype and background of the animals. More Information

Hondal RJ, Marino SM, Gladyshev VN. (2013) Selenocysteine in Thiol/Disulfide-Like Exchange Reactions. Antioxid Redox Signal. 18, 1675-1689.

AbstractAbstract Significance: Among trace elements used as cofactors in enzymes, selenium is unique in that it is incorporated into proteins co-translationally in the form of an amino acid, selenocysteine (Sec). Sec differs from cysteine (Cys) by only one atom (selenium versus sulfur), yet this switch dramatically influences important aspects of enzyme reactivity. Recent Advances: The main focus of this review is an updated and critical discussion on how Sec might be used to accelerate thiol/disulfide-like exchange reactions in natural selenoenzymes, compared with their Cys-containing homologs. Critical Issues: We discuss in detail three major aspects associated with thiol/disulfide exchange reactions: (i) nucleophilicity of the attacking thiolate (or selenolate); (ii) electrophilicity of the center sulfur (or selenium) atom; and (iii) stability of the leaving group (sulfur or selenium). In all these cases, we analyze the benefits that selenium might provide in these types of reactions. Future Directions: It is the biological thiol oxidoreductase-like function that benefits from the use of Sec, since Sec functions to chemically accelerate the rate of these reactions. We review various hypotheses that could help explain why Sec is used in enzymes, particularly with regard to competitive chemical advantages provided by the presence of the selenium atom in enzymes. Ultimately, these chemical advantages must be connected to biological functions of Sec. Antioxid. Redox Signal. 00, 000-000. More Information

Williams DL, Bonilla M, Gladyshev VN, Salinas G. (2013) Thioredoxin glutathione reductase-dependent redox networks in platyhelminth parasites. Antioxid Redox Signal. 9, 735-745.

AbstractSignificance. Platyhelminth parasites cause chronic infections that are a major cause of disability, mortality and economic losses in developing countries. Maintaining redox homeostasis is a major adaptive problem faced by parasites and its disruption can shift the biochemical balance towards the host. Platyhelminth parasites possess a streamlined thiol-based redox system in which a single enzyme, thioredoxin glutathione reductase (TGR), a fusion of a glutaredoxin domain to canonical thioredoxin reductase domains, supplies electrons to oxidized glutathione and thioredoxin. TGR has been validated as a drug target for schistosomiasis. Recent Advances. In addition to glutathione and thioredoxin reduction, TGR supports glutathione-independent deglutathionylase activity conferring an additional advantage to the TGR redox array. Biochemical and structural studies have shown that the thioredoxin reductase activity does not require the glutaredoxin domain, while the glutathione reduction and deglutathionylase activities depend on the glutaredoxin domain, which receives electrons from the thioredoxin reductase domains. The search for TGR inhibitors has identified promising drug leads, notably oxadiazole N-oxides. Critical Issues. A conspicuous feature of platyhelminth TGRs is that their glutaredoxin-dependent activities are temporarily inhibited at high oxidized glutathione concentrations. The mechanism underlying the phenomenon and its biological relevance are not completely understood. Future Directions. The functional diversity of thioredoxins and glutaredoxins encoded in platyhelminth genomes remains to be further assessed to thoroughly understand the TGR-dependent redox network. Optimization of TGR inhibitors and identification of compounds targeting other parasite redox enzymes are good options to develop clinically relevant drugs for these neglected, but important diseases. More Information

Labunskyy VM, Gladyshev VN. (2013) Role of Reactive Oxygen Species-mediated Signaling in Aging. Antioxid Redox Signal. 19, 1362-1372.

AbstractSignificance: Redox biology is a rapidly developing area of research due to the recent evidence for general importance of redox control for numerous cellular functions under both physiological and pathophysiological conditions. Understanding of redox homeostasis is particularly relevant to the understanding of the aging process. The link between reactive oxygen species (ROS) and accumulation of age-associated oxidative damage to macromolecules is well established, but remains controversial and applies only to a subset of experimental models. In addition, recent studies show that ROS may function as signaling molecules and that dysregulation of this process may also be linked to aging. Recent Advances: Many protein factors and pathways that control ROS production and scavenging as well as those that regulate cellular redox homeostasis have been identified. However, much less is known about the mechanisms by which redox signaling pathways influence longevity. In this review, we discuss recent advances in the understanding of the molecular basis for the role of redox signaling in aging. Critical Issues: Recent studies allowed identification of previously uncharacterized redox components and revealed complexity of redox signaling pathways. It would be important to identify functions of these components and elucidate how distinct redox pathways are integrated with each other to maintain homeostatic balance. Future Directions: Further characterization of processes that coordinate redox signaling, redox homeostasis and stress response pathways should allow researchers to dissect how their dysregulation contributes to aging and pathogenesis of various age-related diseases, such as diabetes, cancer and neurodegeneration. More Information

2012 Articles

Dobrovolska O, Shumilina E, Gladyshev VN, Dikiy A.. (2012) Structural Analysis of Glutaredoxin Domain of Mus musculus Thioredoxin Glutathione Reductase. PLoS One 7, e52914.

AbstractThioredoxin glutathione reductase (TGR) is a member of the mammalian thioredoxin reductase family that has a monothiol glutaredoxin (Grx) domain attached to the thioredoxin reductase module. Here, we report a structure of the Grx domain of mouse TGR, determined through high resolution NMR spectroscopy to the final backbone RMSD value of 0.48±0.10 Å. The structure represents a sandwich-like molecule composed of a four stranded β-sheet flanked by five α-helixes, with the CxxS active motif located on the catalytic loop. We structurally characterized the glutathione-binding site in the protein and describe sequence and structural relationships of the domain with glutaredoxins. The structure illuminates a key functional center that evolved in mammalian TGRs to act in thiol-disulfide reactions. Our study allows us to hypothesize that Cys105 might be functionally relevant for TGR catalysis. In addition, the data suggest that the N-terminus of Grx acts as a possible regulatory signal also protecting the protein active site from unwanted interactions in cellular cytosol. More Information

Tsuji PA, Carlson BA, Naranjo-Suarez S, Yoo MH, Xu XM, Fomenko DE, Gladyshev VN, Hatfield DL, Davis CD. (2012) Knockout of the 15 kDa Selenoprotein Protects against Chemically-Induced Aberrant Crypt Formation in Mice. PLoS One 7, e50574.

AbstractEvidence suggests that selenium has cancer preventive properties that are largely mediated through selenoproteins. Our previous observations demonstrated that targeted down-regulation of the 15 kDa selenoprotein (Sep15) in murine colon cancer cells resulted in the reversal of the cancer phenotype. The present study investigated the effect of Sep15 knockout in mice using a chemically-induced colon cancer model. Homozygous Sep15 knockout mice, and wild type littermate controls were given four weekly subcutaneous injections of azoxymethane (10 mg/kg). Sep15 knockout mice developed significantly (p<0.001) fewer aberrant crypt foci than controls demonstrating that loss of Sep15 protects against aberrant crypt foci formation. Dietary selenium above adequate levels did not significantly affect aberrant crypt foci formation in Sep15 knockout mice. To investigate molecular targets affected by loss of Sep15, gene expression patterns in colonic mucosal cells of knockout and wild type mice were examined using microarray analysis. Subsequent analyses verified that guanylate binding protein-1 (GBP-1) mRNA and protein expression were strongly upregulated in Sep15 knockout mice. GBP-1, which is expressed in response to interferon-γ, is considered to be an activation marker during inflammatory diseases, and up-regulation of GBP-1 in humans has been associated with a highly significant, increased five-year survival rate in colorectal cancer patients. In agreement with these studies, we observed a higher level of interferon-γ in plasma of Sep15 knockout mice. Overall, our results demonstrate for the first time, that Sep15 knockout mice are protected against chemically-induced aberrant crypt foci formation and that Sep15 appears to have oncogenic properties in colon carcinogenesis in vivo. More Information

Tarrago L, Gladyshev VN. (2012) Recharging Oxidative Protein Repair: Catalysis by Methionine Sulfoxide Reductases towards Their Amino Acid, Protein, and Model Substrates. Biochemistry (Mosc) 77, 1097-1107.

AbstractThe sulfur-containing amino acid methionine (Met) in its free and amino acid residue forms can be readily oxidized to the R and S diastereomers of methionine sulfoxide (MetO). Methionine sulfoxide reductases A (MSRA) and B (MSRB) reduce MetO back to Met in a stereospecific manner, acting on the S and R forms, respectively. A third MSR type, fRMSR, reduces the R form of free MetO. MSRA and MSRB are spread across the three domains of life, whereas fRMSR is restricted to bacteria and unicellular eukaryotes. These enzymes protect against abiotic and biotic stresses and regulate lifespan. MSRs are thiol oxidoreductases containing catalytic redox-active cysteine or selenocysteine residues, which become oxidized by the substrate, requiring regeneration for the next catalytic cycle. These enzymes can be classified according to the number of redox-active cysteines (selenocysteines) and the strategies to regenerate their active forms by thioredoxin and glutaredoxin systems. For each MSR type, we review catalytic parameters for the reduction of free MetO, low molecular weight MetO-containing compounds, and oxidized proteins. Analysis of these data reinforces the concept that MSRAs reduce various types of MetO-containing substrates with similar efficiency, whereas MSRBs are specialized for the reduction of MetO in proteins. More Information

Liang X, Kaya A, Zhang Y, Le DT, Hua D, Gladyshev VN. (2012) Characterization of methionine oxidation and methionine sulfoxide reduction using methionine-rich cysteine-free proteins. BMC Biochem. 13, 21.

AbstractBACKGROUND: Methionine (Met) residues in proteins can be readily oxidized by reactive oxygen species to Met sulfoxide (MetO). MetO is a promising physiological marker of oxidative stress and its inefficient repair by MetO reductases (Msrs) has been linked to neurodegeneration and aging. Conventional methods of assaying MetO formation and reduction rely on chromatographic or mass spectrometry procedures, but the use of Met-rich proteins (MRPs) may offer a more streamlined alternative.
RESULTS: We carried out a computational search of completely sequenced genomes for MRPs deficient in cysteine (Cys) residues and identified several proteins containing 20% or more Met residues. We used these MRPs to examine Met oxidation and MetO reduction by in-gel shift assays and immunoblot assays with antibodies generated against various oxidized MRPs. The oxidation of Cys-free MRPs by hydrogen peroxide could be conveniently monitored by SDS-PAGE and was specific for Met, as evidenced by quantitative reduction of these proteins with Msrs in DTT- and thioredoxin-dependent assays. We found that hypochlorite was especially efficient in oxidizing MRPs. Finally, we further developed a procedure wherein antibodies made against oxidized MRPs were isolated on affinity resins containing same or other oxidized or reduced MRPs. This procedure yielded reagents specific for MetO in these proteins, but proved to be ineffective in developing antibodies with broad MetO specificity.
CONCLUSION: Our data show that MRPs provide a convenient tool for characterization of Met oxidation, MetO reduction and Msr activities, and could be used for various aspects of redox biology involving reversible Met oxidation.
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Gerashchenko MV, Lobanov AV, Gladyshev VN. (2012) Genome-wide ribosome profiling reveals complex translational regulation in response to oxidative stress. Proc. Natl. Acad. Sci. USA 109, 17394-17399.

AbstractInformation on unique and coordinated regulation of transcription and translation in response to stress is central to the understanding of cellular homeostasis. Here we used ribosome profiling coupled with next-generation sequencing to examine the interplay between transcription and translation under conditions of hydrogen peroxide treatment in Saccharomyces cerevisiae. Hydrogen peroxide treatment led to a massive and rapid increase in ribosome occupancy of short upstream ORFs, including those with non-AUG translational starts, and of the N-terminal regions of ORFs that preceded the transcriptional response. In addition, this treatment induced the synthesis of N-terminally extended proteins and elevated stop codon read-through and frameshift events. It also increased ribosome occupancy at the beginning of ORFs and potentially the duration of the elongation step. We identified proteins whose synthesis was regulated rapidly by hydrogen peroxide posttranscriptionally; however, for the majority of genes increased protein synthesis followed transcriptional regulation. These data define the landscape of genome-wide regulation of translation in response to hydrogen peroxide and suggest that potentiation (coregulation of the transcript level and translation) is a feature of oxidative stress.
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Gladyshev VN. (2012) On the cause of aging and control of lifespan: Heterogeneity leads to inevitable damage accumulation, causing aging; Control of damage composition and rate of accumulation define lifespan. Bioessays 34, 925-928.

AbstractWhat the causes of aging are and which factors define lifespan are key questions in the understanding of aging. Here, it is argued that cellular life involves (i) inevitable accumulation of damage resulting from imperfectness and heterogeneity of every cellular process, and (ii) dilution of damage when cells divide. While severe damage is cleared by protective systems, milder damage can only be diluted. This is due to the high cost of accuracy, the greater number of damage forms compared to protective systems, and the constraints on cellular life inherited from the prokaryotic world. This strategy also applies to cancer cells, which are particularly dependent on damage dilution. Imposing restriction on cell division necessarily leads to aging. Interventions that extend lifespan act through metabolic reprogramming, thereby changing both damage composition and the rate of damage accumulation. Thus, heterogeneity leading to myriad mild damage forms represents the cause of aging, whereas the processes that affect the damage landscape and damage accumulation are lifespan regulators. More Information

Anisimov VN, Bartke A, Barzilai N, Batin MA, Blagosklonny MV, Brown-Borg H, Budovskaya Y, Campisi J, Friguet B, Fraifeld V, Franceschi C, Gems D, Gladyshev V, Gorbunova V, Gudkov AV, Kennedy B, Konovalenko M, Kraemer B, Moskalev A, Petropoulos I, Pasyukova E, Rattan S, Rogina B, Seluanov A, Shaposhnikov M, Shmookler Reis R, Tavernarakis N, Vijg J, Yashin A, Zimniak P. (2012) The second international conference “genetics of aging and longevity”. Aging (Albany NY) 4, 305-317.

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Carlson BA, Yoo MH, Tobe R, Mueller C, Naranjo-Suarez S, Hoffmann VJ, Gladyshev VN, Hatfield DL. (2012) Thioredoxin reductase 1 protects against chemically induced hepatocarcinogenesis via control of cellular redox homeostasis. Carcinogenesis 33, 1806-1813.

AbstractThioredoxin reductase 1 (TR1) controls the redox state of protein thiols in mammalian cells and has been shown to have roles in both preventing and promoting cancer. To define the role of this selenoenzyme in hepatocellular carcinoma development, we examined tumor incidence in the liver of mice with tissue-specific knockout of mouse TR1 subjected to the liver carcinogen, diethylnitrosamine (DEN). TR1-deficient livers manifested ~90% tumor incidence compared with ~16% in control livers. The TR1-dependent effect was observed independent of sex, and, in control mice, tumorigenesis did not affect the expression of TR1. On the other hand, we observed upregulation of another selenoenzyme, glutathione peroxidase 2 (GPx2), and components of the glutathione (GSH) system, including those that generate reduced GSH. Overall, this study shows that TR1 protects against chemically induced hepatocarcinogenesis via the control of the cellular redox state, whereas its role in promoting this type of cancer is minimal.
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Vinogradov DV, Tsoĭ OV, Zaika AV, Lobanov AV, Turanov AA, Gladyshev VN, Gel’fand MS. (2012) [Draft macronuclear genome of a ciliate Euplotes crassus]. Mol. Biol. (Mosk, Russia) 46, 361-6366.

AbstractBasic bioinformatical analysis of the draft Euplotes crassus macronuclear genome and transcriptome suggests that more than a quarter of E. crassus genes contain several exons. A large fraction of all introns is formed by “tiny” introns having length 20-30 bp. Analysis of the transcriptome revealed 63 possible cases of alternative splicing, and also 14 introns with non-standard splicing sites. About 2000 hypothetical genes do not have homologs in other ciliates, and since most of them have the closest homologs in bacterial genomes, they are likely an artifact of the sample preparation. Comparison of the E. crassus genome to the genomes of other ciliates showed an expansion of the same gene families, responsible for the free-living heterotrophic lifestyle.
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Tarrago L, Kaya A, Weerapana E, Marino SM, Gladyshev VN. (2012) Methionine sulfoxide reductases preferentially reduce unfolded oxidized proteins and protect cells from oxidative protein unfolding. J Biol Chem. 287, 24448-24459.

AbstractReduction of methionine sulfoxide (MetO) residues in proteins is catalyzed by methionine sulfoxide reductases A (MSRA) and B (MSRB), which act in a stereospecific manner. Catalytic properties of these enzymes were previously established mostly using low molecular weight MetO-containing compounds, whereas little is known about the catalysis of MetO reduction in proteins, the physiological substrates of MSRA and MSRB. In this work, we exploited an NADPH-dependent thioredoxin system and determined the kinetics parameters of yeast MSRA and MSRB using three different MetO-containing proteins. Both enzymes showed Michaelis-Menten kinetics with the KM lower for protein than for small MetO-containing substrates. MSRA reduced both oxidized proteins and low molecular weight MetO-containing compounds with similar catalytic efficiencies, whereas MSRB was specialized for the reduction of MetO in proteins. Using oxidized glutathione-S-transferase as a model substrate, we showed that both MSR types were more efficient in reducing MetO in unfolded than in folded proteins, and that their activities increased with the unfolding state. Biochemical quantification and identification of MetO reduced in the substrates by mass spectrometry revealed that the increased activity was due to a better access to oxidized MetO in unfolded proteins; it also showed that MSRA was intrinsically more active with unfolded proteins regardless of MetO availability. Moreover, MSRs most efficiently protected cells from oxidative stress that was accompanied by protein unfolding. Overall, this study indicates that MSRs serve a critical function in the folding process by repairing oxidatively damaged nascent polypeptides and unfolded proteins. More Information

Tobe R, Yoo MH, Fradejas N, Carlson BA, Calvo S, Gladyshev VN, Hatfield DL. (2012) Thioredoxin reductase 1 deficiency enhances selenite toxicity in cancer cells via a thioredoxin-independent mechanism. Biochem J. 445, 423-430.

AbstractSelenium is an essential trace element in mammals, but is toxic at high levels. It is best known for its cancer prevention activity, but cancer cells are more sensitive to selenite toxicity than normal cells. Since selenite treatment leads to oxidative stress, and the thioredoxin system is a major antioxidative system, we examined the interplay between thioredoxin reductase 1 (TR1) and thioredoxin 1 (Trx1) deficiencies and selenite toxicity in DT cells, a malignant mouse cell line, and the corresponding parental NIH3T3 cells. TR1 deficient cells were far more sensitive to selenite toxicity than Trx1-deficient or control cells. In contrast, this effect was not seen in cells treated with hydrogen peroxide, suggesting that the increased sensitivity of TR1 deficiency to selenite was not due to oxidative stress caused by this compound. Further analyses revealed that only TR1-deficient cells manifested strongly enhanced production and secretion of glutathione, which was associated with increased sensitivity of the cells to selenite. The data uncover a new role of TR1 in cancer that is independent of Trx reduction and compensated for by the glutathione system. The data also suggest that the enhanced selenite toxicity of cancer cells and simultaneous inhibition of TR1 can provide a new avenue for cancer therapy. More Information

Ross F, Hernández P, Porcal W, López GV, Cerecetto H, González M, Basika T, Carmona C, Fló M, Maggioli G, Bonilla M, Gladyshev VN, Boiani M, Salinas G. (2012) Identification of thioredoxin glutathione reductase inhibitors that kill cestode and trematode parasites. PLoS One 7, e35033.

AbstractParasitic flatworms are responsible for serious infectious diseases that affect humans as well as livestock animals in vast regions of the world. Yet, the drug armamentarium available for treatment of these infections is limited: praziquantel is the single drug currently available for 200 million people infected with Schistosoma spp. and there is justified concern about emergence of drug resistance. Thioredoxin glutathione reductase (TGR) is an essential core enzyme for redox homeostasis in flatworm parasites. In this work, we searched for flatworm TGR inhibitors testing compounds belonging to various families known to inhibit thioredoxin reductase or TGR and also additional electrophilic compounds. Several furoxans and one thiadiazole potently inhibited TGRs from both classes of parasitic flatworms: cestoda (tapeworms) and trematoda (flukes), while several benzofuroxans and a quinoxaline moderately inhibited TGRs. Remarkably, five active compounds from diverse families possessed a phenylsulfonyl group, strongly suggesting that this moiety is a new pharmacophore. The most active inhibitors were further characterized and displayed slow and nearly irreversible binding to TGR. These compounds efficiently killed Echinococcus granulosus larval worms and Fasciola hepatica newly excysted juveniles in vitro at a 20 µM concentration. Our results support the concept that the redox metabolism of flatworm parasites is precarious and particularly susceptible to destabilization, show that furoxans can be used to target both flukes and tapeworms, and identified phenylsulfonyl as a new drug-hit moiety for both classes of flatworm parasites.
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Mariotti M, Ridge PG, Zhang Y, Lobanov AV, Pringle TH, Guigo R, Hatfield DL, Gladyshev VN. (2012) Composition and evolution of the vertebrate and Mammalian selenoproteomes. PLoS One 7, e33066.

AbstractBACKGROUND: Selenium is an essential trace element in mammals due to its presence in proteins in the form of selenocysteine (Sec). Human genome codes for 25 Sec-containing protein genes, and mouse and rat genomes for 24.
METHODOLOGY/PRINCIPAL FINDINGS: We characterized the selenoproteomes of 44 sequenced vertebrates by applying gene prediction and phylogenetic reconstruction methods, supplemented with the analyses of gene structures, alternative splicing isoforms, untranslated regions, SECIS elements, and pseudogenes. In total, we detected 45 selenoprotein subfamilies. 28 of them were found in mammals, and 41 in bony fishes. We define the ancestral vertebrate (28 proteins) and mammalian (25 proteins) selenoproteomes, and describe how they evolved along lineages through gene duplication (20 events), gene loss (10 events) and replacement of Sec with cysteine (12 events). We show that an intronless selenophosphate synthetase 2 gene evolved in early mammals and replaced functionally the original multiexon gene in placental mammals, whereas both genes remain in marsupials. Mammalian thioredoxin reductase 1 and thioredoxin-glutathione reductase evolved from an ancestral glutaredoxin-domain containing enzyme, still present in fish. Selenoprotein V and GPx6 evolved specifically in placental mammals from duplications of SelW and GPx3, respectively, and GPx6 lost Sec several times independently. Bony fishes were characterized by duplications of several selenoprotein families (GPx1, GPx3, GPx4, Dio3, MsrB1, SelJ, SelO, SelT, SelU1, and SelW2). Finally, we report identification of new isoforms for several selenoproteins and describe unusually conserved selenoprotein pseudogenes.
CONCLUSIONS/SIGNIFICANCE: This analysis represents the first comprehensive survey of the vertebrate and mammal selenoproteomes, and depicts their evolution along lineages. It also provides a wealth of information on these selenoproteins and their forms.
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Shumilina E, Soldà A, Gerashchenko M, Gladyshev VN, Dikiy A. (2012) (1)H, (13)C, and (15)N NMR resonance assignments of reduced full length and shortened forms of the Grx domain of Mus musculus TGR. Biomol NMR Assign. 6,103-107.

AbstractTwo forms of the glutaredoxin (Grx) domain (full length Grx domain and short Grx lacking the N-terminal region) of Mus musculus thioredoxin glutathione reductase (TGR) were isotopically labelled with (15)N and (13)C isotopes, expressed and purified to homogeneity. We report here the (1)H, (13)C and (15)N NMR assignment for both Grx forms of this mouse TGR. This investigation represents the first NMR analysis of a mammalian TGR. More Information

Kasaikina MV, Hatfield DL, Gladyshev VN. (2012) Understanding selenoprotein function and regulation through the use of rodent models. Biochim Biophys Acta. 1823, 1633-1642.

AbstractSelenium (Se) is an essential micronutrient. Its biological functions are associated with selenoproteins, which contain this trace element in the form of the 21st amino acid, selenocysteine. Genetic defects in selenocysteine insertion into proteins are associated with severe health issues. The consequences of selenoprotein deficiency are more variable, with several selenoproteins being essential, and several showing no clear phenotypes. Much of these functional studies benefited from the use of rodent models and diets employing variable levels of Se. This review summarizes the data obtained with these models, focusing on mouse models with targeted expression of individual selenoproteins and removal of individual, subsets or all selenoproteins in a systemic or organ-specific manner. This article is part of a Special Issue entitled: Cell Biology of Metals. More Information

Marino SM, Gladyshev VN. (2012) Analysis and functional prediction of reactive cysteine residues. J Biol Chem. 287,4419-4425.

AbstractCys is much different from other common amino acids in proteins. Being one of the least abundant residues, Cys is often observed in functional sites in proteins. This residue is reactive, polarizable, and redox-active; has high affinity for metals; and is particularly responsive to the local environment. A better understanding of the basic properties of Cys is essential for interpretation of high-throughput data sets and for prediction and classification of functional Cys residues. We provide an overview of approaches used to study Cys residues, from methods for investigation of their basic properties, such as exposure and pK(a), to algorithms for functional prediction of different types of Cys in proteins. More Information

Fomenko DE, Gladyshev VN. (2012) Comparative genomics of thiol oxidoreductases reveals widespread and essential functions of thiol-based redox control of cellular processes. Antioxid Redox Signal. 16,193-201.

AbstractAIMS: Redox regulation of cellular processes is an important mechanism that operates in organisms from bacteria to mammals. Much of the redox control is provided by thiol oxidoreductases: proteins that employ cysteine residues for redox catalysis. We wanted to identify thiol oxidoreductases on a genome-wide scale and use this information to obtain insights into the general principles of thiol-based redox control. RESULTS: Thiol oxidoreductases were identified by three independent methods that took advantage of the occurrence of selenocysteine homologs of these proteins and functional linkages among thiol oxidoreductases revealed by comparative genomics. Based on these searches, we describe thioredoxomes, which are sets of thiol oxidoreductases in organisms. Their analyses revealed that these proteins are present in all living organisms, generally account for 0.5%-1% of the proteome and that their use correlates with proteome size, distinguishing these proteins from those involved in core metabolic functions. We further describe thioredoxomes of Saccharomyces cerevisiae and humans, including proteins which have not been characterized previously. Thiol oxidoreductases occur in various cellular compartments and are enriched in the endoplasmic reticulum and cytosol. INNOVATION: We developed bioinformatics methods and used them to characterize thioredoxomes on a genome-wide scale, which in turn revealed properties of thioredoxomes. CONCLUSION: These data provide information about organization and properties of thiol-based redox control, whose use is increased with the increase in complexity of organisms. Our data also show an essential combined function of a set of thiol oxidoreductases, and of thiol-based redox regulation in general, in all living organisms.
More Information

Malinouski M, Kehr S, Finney L, Vogt S, Carlson BA, Seravalli J, Jin R, Handy DE, Park TJ, Loscalzo J, Hatfield DL, Gladyshev VN. (2012) High-resolution imaging of selenium in kidneys: a localized selenium pool associated with glutathione peroxidase 3. Antioxid Redox Signal. 16, 185-192.

AbstractAIM: Recent advances in quantitative methods and sensitive imaging techniques of trace elements provide opportunities to uncover and explain their biological roles. In particular, the distribution of selenium in tissues and cells under both physiological and pathological conditions remains unknown. In this work, we applied high-resolution synchrotron X-ray fluorescence microscopy (XFM) to map selenium distribution in mouse liver and kidney. RESULTS: Liver showed a uniform selenium distribution that was dependent on selenocysteine tRNA([Ser]Sec) and dietary selenium. In contrast, kidney selenium had both uniformly distributed and highly localized components, the latter visualized as thin circular structures surrounding proximal tubules. Other parts of the kidney, such as glomeruli and distal tubules, only manifested the uniformly distributed selenium pattern that co-localized with sulfur. We found that proximal tubule selenium localized to the basement membrane. It was preserved in Selenoprotein P knockout mice, but was completely eliminated in glutathione peroxidase 3 (GPx3) knockout mice, indicating that this selenium represented GPx3. We further imaged kidneys of another model organism, the naked mole rat, which showed a diminished uniformly distributed selenium pool, but preserved the circular proximal tubule signal. INNOVATION: We applied XFM to image selenium in mammalian tissues and identified a highly localized pool of this trace element at the basement membrane of kidneys that was associated with GPx3. CONCLUSION: XFM allowed us to define and explain the tissue topography of selenium in mammalian kidneys at submicron resolution. More Information

Naranjo-Suarez S, Carlson BA, Tsuji PA, Yoo MH, Gladyshev VN, Hatfield DL. (2012) HIF-Independent Regulation of Thioredoxin Reductase 1 Contributes to the High Levels of Reactive Oxygen Species Induced by Hypoxia. PLoS One 7, e40370.

AbstractCellular adaptation to hypoxic conditions mainly involves transcriptional changes in which hypoxia inducible factors (HIFs) play a critical role. Under hypoxic conditions, HIF protein is stabilized due to inhibition of the activity of prolyl hydroxylases (EGLNs). Because the reaction carried out by these enzymes uses oxygen as a co-substrate it is generally accepted that the hypoxic inhibition of EGLNs is due to the reduction in oxygen levels. However, several studies have reported that hypoxic generation of mitochondrial reactive oxygen species (ROS) is required for HIF stabilization. Here, we show that hypoxia downregulates thioredoxin reductase 1 (TR1) mRNA and protein levels. This hypoxic TR1 regulation is HIF independent, as HIF stabilization by EGLNs inhibitors does not affect TR1 expression and HIF deficiency does not block TR1 hypoxic-regulation, and it has an effect on TR1 function, as hypoxic conditions also reduce TR1 activity. We found that, when cultured under hypoxic conditions, TR1 deficient cells showed a larger accumulation of ROS compared to control cells, whereas TR1 over-expression was able to block the hypoxic generation of ROS. Furthermore, the changes in ROS levels observed in TR1 deficient or TR1 over-expressing cells did not affect HIF stabilization or function. These results indicate that hypoxic TR1 down-regulation is important in maintaining high levels of ROS under hypoxic conditions and that HIF stabilization and activity do not require hypoxic generation of ROS. More Information

2011 Articles

Turanov AA, Xu XM, Carlson BA, Yoo MH, Gladyshev VN, Hatfield DL. (2011) Biosynthesis of selenocysteine, the 21st amino Acid in the genetic code, and a novel pathway for cysteine biosynthesis. Adv Nutr. 2, 122-128.

AbstractThe biosynthetic pathway for selenocysteine (Sec), the 21st amino acid in the genetic code whose codeword is UGA, was recently determined in eukaryotes and archaea. Sec tRNA, designated tRNA([Ser]Sec), is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase to form O-phosphoseryl-tRNA([Ser]Sec). Sec synthase (SecS) then uses O-phosphoseryl-tRNA([Ser]Sec) and the active donor of selenium, selenophosphate, to form Sec-tRNA([Ser]Sec). Selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase 2 (SPS2). Sec was the last protein amino acid in eukaryotes whose biosynthesis had not been established and the only known amino acid in eukaryotes whose biosynthesis occurs on its tRNA. Interestingly, sulfide can replace selenide to form thiophosphate in the SPS2-catalyzed reaction that can then react with O-phosphoseryl-tRNA([Ser]Sec) in the presence of SecS to form cysteine-(Cys-)tRNA([Ser]Sec). This novel pathway of Cys biosynthesis results in Cys being decoded by UGA and replacing Sec in normally selenium-containing proteins (selenoproteins). The selenoprotein, thioredoxin reductase 1 (TR1), was isolated from cells in culture and from mouse liver for analysis of Cys/Sec replacement by MS. The level of Cys/Sec replacement in TR1 was proportional to the level of selenium in the diet of the mice. Elucidation of the biosynthesis of Sec and Sec/Cys replacement provides novel ways of regulating selenoprotein functions and ultimately better understanding of the biological roles of dietary selenium. More Information

Gladyshev VN, Zhang G, Wang J. (2011) The naked mole rat genome: understanding aging through genome analysis. Aging (Albany NY) 3, 1124.

AbstractNow that the cost of sequencing decreased dramatically, groups of related organisms with different lifespans can be sequenced and evaluated for differences in genome organization, genes, pathways and systems. It is also clear, however, that there are no easy ways to interpret these differences, so many genomes will need to be examined, and this activity should involve a broader research community. We are now entering an exciting time when aging can be understood through genome analyses. More Information

Uluisik I, Kaya A, Fomenko DE, Karakaya HC, Carlson BA, Gladyshev VN, Koc A. (2011) Boron stress activates the general amino Acid control mechanism and inhibits protein synthesis. PLoS One 6, e27772.

AbstractBoron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS) of Gcn2 is necessary for the phosphorylation of eIF2α in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance. More Information

Shchedrina VA, Everley RA, Zhang Y, Gygi SP, Hatfield DL, Gladyshev VN. (2011) Selenoprotein K binds multi-protein complexes and is involved in the regulation of ER homeostasis. J. Biol. Chem. 286, 42937-42948.

AbstractSelenoprotein K (SelK) is an 11 kDa endoplasmic reticulum (ER) protein of unknown function. Herein, we defined a new eukaryotic protein family that includes SelK, SelS and distantly related proteins. Comparative genomics analyses indicate that this family is the most widespread eukaryotic selenoprotein family. A biochemical search for proteins that interact with SelK revealed ER-associated degradation (ERAD) components (p97 ATPase, Derlins and SelS). In this complex, SelK showed higher affinity for Derlin-1, whereas SelS had affinity for Derlin-2, suggesting that these selenoproteins could determine the nature of the substrate translocated through the Derlin channel. SelK co-precipitated with soluble glycosylated ERAD substrates and was involved in their degradation. Its gene contained a functional ER stress response element and its expression was up-regulated by conditions that induce the accumulation of misfolded proteins in the ER. Components of the oligosaccharyltransferase complex (ribophorins, OST48, STT3A) and an ER chaperone, calnexin, were found to bind SelK. A glycosylated form of SelK was also detected, reflecting its association with the OST complex. These data suggest that SelK is involved in the Derlin-dependent ERAD of glycosylated misfolded proteins and that the function defined by the prototypic SelK is the widespread function of selenium in eukaryotes. More Information

Aachmann FL, Kwak GH, Del Conte R, Kim HY, Gladyshev VN, Dikiy A. (2011) Structural and biochemical analysis of mammalian methionine sulfoxide reductase B2. Proteins 79, 3123-3131.

AbstractMethionine sulfoxide reductases are antioxidant enzymes that repair oxidatively damaged methionine residues in proteins. Mammals have three members of the methionine-R-sulfoxide reductase family, including cytosolic MsrB1, mitochondrial MsrB2, and endoplasmic reticulum MsrB3. Here, we report the solution structure of reduced Mus musculus MsrB2 using high resolution nuclear magnetic resonance (NMR) spectroscopy. MsrB2 is a β-strand rich globular protein consisting of eight antiparallel β-strands and three N-terminal α-helical segments. The latter secondary structure elements represent the main structural difference between mammalian MsrB2 and MsrB1. Structural comparison of mammalian and bacterial MsrB structures indicates that the general topology of this MsrB family is maintained and that MsrB2 more resembles bacterial MsrBs than MsrB1. Structural and biochemical analysis supports the catalytic mechanism of MsrB2 that, in contrast to MsrB1, does not involve a resolving cysteine (Cys). pH dependence of catalytically relevant residues in MsrB2 was accessed by NMR spectroscopy and the pK(a) of the catalytic Cys162 was determined to be 8.3. In addition, the pH-dependence of MsrB2 activity showed a maximum at pH 9.0, suggesting that deprotonation of the catalytic Cys is a critical step for the reaction. Further mobility analysis showed a well-structured N-terminal region, which contrasted with the high flexibility of this region in MsrB1. Our study highlights important structural and functional aspects of mammalian MsrB2 and provides a unifying picture for structure-function relationships within the MsrB protein family. More Information

Kim EB, Fang X, Fushan AA, Huang Z, Lobanov AV, Han L, Marino SM, Sun X, Turanov AA, Yang P, Yim SH, Zhao X, Kasaikina MV, Stoletzki N, Peng C, Polak P, Xiong Z, Kiezun A, Zhu Y, Chen Y, Kryukov GV, Zhang Q, Peshkin L, Yang L, Bronson RT, Buffenstein R, Wang B, Han C, Li Q, Chen L, Zhao W, Sunyaev SR, Park TJ, Zhang G, Wang J, Gladyshev VN. (2011) Genome sequencing reveals insights into physiology and longevity of the naked mole rat. Nature 479, 223-227.

AbstractThe naked mole rat (Heterocephalus glaber) is a strictly subterranean, extraordinarily long-lived eusocial mammal. Although it is the size of a mouse, its maximum lifespan exceeds 30 years, making this animal the longest-living rodent. Naked mole rats show negligible senescence, no age-related increase in mortality, and high fecundity until death. In addition to delayed ageing, they are resistant to both spontaneous cancer and experimentally induced tumorigenesis. Naked mole rats pose a challenge to the theories that link ageing, cancer and redox homeostasis. Although characterized by significant oxidative stress, the naked mole rat proteome does not show age-related susceptibility to oxidative damage or increased ubiquitination. Naked mole rats naturally reside in large colonies with a single breeding female, the ‘queen’, who suppresses the sexual maturity of her subordinates. They also live in full darkness, at low oxygen and high carbon dioxide concentrations8, and are unable to sustain thermogenesis nor feel certain types of pain. Here we report the sequencing and analysis of the naked mole rat genome, which reveals unique genome features and molecular adaptations consistent with cancer resistance, poikilothermy, hairlessness and insensitivity to low oxygen, and altered visual function, circadian rythms and taste sensing. This information provides insights into the naked mole rat’s exceptional longevity and ability to live in hostile conditions, in the dark and at low oxygen. The extreme traits of the naked mole rat, together with the reported genome and transcriptome information, offer opportunities for understanding ageing and advancing other areas of biological and biomedical research. More Information

Carlson BA, Yoo MH, Conrad M, Gladyshev VN, Hatfield DL, Park JM. (2011) Protein kinase-regulated expression and immune function of thioredoxin reductase 1 in mouse macrophages. Mol. Immunol. 49, 311-316.

AbstractMacrophages exposed to lipopolysaccharide (LPS) exhibit radical changes in mRNA and protein profiles. This shift in gene expression is geared not only to activate immune effector and regulatory mechanisms, but also to adjust the immune cell’s metabolism to new physiological demands. However, it remains largely unknown whether immune function and metabolic state are mutually regulatory and, if so, how they are mechanistically interrelated in macrophages. Selenium, a dietary trace element exerting pleiotropic effects on immune homeostasis, and selenium-containing proteins (selenoproteins) may play a role in such coordination. We examined the incorporation of radiolabeled selenium into protein during LPS stimulation, and identified thioredoxin reductase 1 (TR1) as the only LPS-inducible selenoprotein in macrophages. TR1 induction occurred at the transcriptional level and depended on the intracellular signaling pathways mediated by p38 MAP kinase and IκB kinase. Macrophage-specific ablation of TR1 in mice resulted in a drastic decrease in the expression of VSIG4, a B7 family protein known to suppress T cell activation. These results reveal TR1 as both a regulator and a regulated target in the macrophage gene expression network, and suggest a link between selenium metabolism and immune signaling. More Information

Fomenko DE, Gladyshev VN. (2011) Comparative Genomics of Thiol Oxidoreductases Reveals Widespread and Essential Functions of Thiol-based Redox Control of Cellular Processes. Antioxid. Redox Signal. 16, 193-201.

AbstractAims Redox regulation of cellular processes is an important mechanism that operates in organisms from bacteria to mammals. Much of the redox control is provided by thiol oxidoreductases: proteins that employ cysteine residues for redox catalysis. We wanted to identify thiol oxidoreductases on a genome-wide scale and use this information to obtain insights into the general principles of thiol-based redox control. Results Thiol oxidoreductases were identified by three independent methods that took advantage of the occurrence of selenocysteine homologs of these proteins and functional linkages among thiol oxidoreductases revealed by comparative genomics. Based on these searches, we describe thioredoxomes, which are sets of thiol oxidoreductases in organisms. Their analyses revealed that these proteins are present in all living organisms, generally account for 0.5-1% of the proteome and that their use correlates with proteome size, distinguishing these proteins from those involved in core metabolic functions. We further describe thioredoxomes of Saccharomyces cerevisiae and humans, including proteins which have not been characterized previously. Thiol oxidoreductases occur in various cellular compartments and are enriched in the endoplasmic reticulum and cytosol. Innovation We developed bioinformatics methods and used them to characterize thioredoxomes on a genome-wide scale, which in turn revealed properties of thioredoxomes. Conclusion These data provide information about organization and properties of thiol-based redox control, whose use is increased with the increase in complexity of organisms. Our data also show an essential combined function of a set of thiol oxidoreductases, and of thiol-based redox regulation in general, in all living organisms. More Information

Malinouski M, Kehr S, Finney L, Vogt S, Carlson BA, Seravalli J, Jin R, Handy DE, Park TJ, Loscalzo J, Hatfield DL, Gladyshev VN. (2011) High-Resolution Imaging of Selenium in Kidneys: a Localized Selenium Pool Associated with Glutathione Peroxidase 3. Antioxid. Redox Signal. 16, 185-192.

AbstractAim: Recent advances in quantitative methods and sensitive imaging techniques of trace elements provide opportunities to uncover and explain their biological roles. In particular, the distribution of selenium in tissues and cells under both physiological and pathological conditions remains unknown. In this work, we applied high-resolution synchrotron X-ray fluorescence microscopy (XFM) to map selenium distribution in mouse liver and kidney. Results: Liver showed a uniform selenium distribution that was dependent on selenocysteine tRNA[Ser]Sec and dietary selenium. In contrast, kidney selenium had both uniformly distributed and highly localized components, the latter visualized as thin circular structures surrounding proximal tubules. Other parts of the kidney, such as glomeruli and distal tubules, only manifested the uniformly distributed selenium pattern that co-localized with sulfur. We found that proximal tubule selenium localized to the basement membrane. It was preserved in Selenoprotein P knockout mice, but was completely eliminated in glutathione peroxidase 3 (GPx3) knockout mice, indicating that this selenium represented GPx3. We further imaged kidneys of another model organism, the naked mole rat, which showed a diminished uniformly distributed selenium pool, but preserved the circular proximal tubule signal. Innovation: We applied XFM to image selenium in mammalian tissues and identified a highly localized pool of this trace element at the basement membrane of kidneys that was associated with GPx3. Conclusion: XFM allowed us to define and explain the tissue topography of selenium in mammalian kidneys at submicron resolution. More Information

Yim SH, Kim YJ, Oh SY, Fujii J, Zhang Y, Gladyshev VN, Rhee SG. (2011) Identification and characterization of an alternatively transcribed form of peroxiredoxin IV that is specifically expressed in spermatids of the postpubertal mouse testis. J. Biol. Chem. 286, 39002-39012.

Abstract2-cysteine (Cys) peroxiredoxins (Prxs), which include mammalian Prx I to IV, possess two conserved Cys residues that are readily oxidized by H2O2 to form a disulfide. In the case of Prx I to III, the disulfide is reduced by thioredoxin, thus enabling these proteins to function as peroxidases. Prx IV was previously shown to be synthesized as a 31-kDa polypeptide with an NH2-terminal signal peptide that is subsequently cleaved to generate a 27-kDa form of the protein that is localized to the endoplasmic reticulum. A form of Prx IV larger than 27-kDa revealed by immunoblot analysis was suggested to represent the unprocessed, 31-kDa form, but this larger form was detected only in spermatids of the post pubertal testis. We now show that the larger form of Prx IV (here designated Prx IV-L) detected in the testis is actually a product of alternative transcription of the Prx IV gene that is encoded by a newly identified exon 1A together with exons 2 to 7 that are shared with the 27-kDa form (designated Prx IV-S). Prx IV-L was detected in spermatids but not in mature sperm, it was found to exist mostly as a disulfide-linked dimer in the testis, and it appears not to function as a peroxidase. Phylogenetic analysis showed that the Prx IV-S gene is present in all vertebrates examined, whereas the Prx IV-L gene was detected only in placental mammals. We suggest that Prx IV-L functions as an H2O2 sensor that mediates protein thiol oxidation required for the maturation of spermatozoa in placental mammals. More Information

Lee BC, Fomenko DE, Gladyshev VN. (2011) Selective Reduction of Methylsulfinyl-containing Compounds by mammalian MsrA Suggests a Strategy for Improved Drug Efficacy. ACS Chem Biol., 6, 1029-1035.

AbstractIdentification of pathways of drug metabolism provides critical information regarding efficacy and safety of these compounds. Particularly challenging cases involve stereospecific processes. We found that broad classes of compounds containing methylsulfinyl groups are reduced to methylsulfides specifically by methionine sulfoxide reductase A, which acts on the S-stereomers of methionine sulfoxides, whereas the R-stereomers of these compounds could not be efficiently reduced by any methionine sulfoxide reductase in mammals. The findings of efficient reduction of S-methylsulfinyls and deficiency in the reduction of R-methylsulfinyls by methionine sulfoxide reductases suggest strategies for improved efficacy and decreased toxicity of drugs and natural compounds containing methylsulfinyls through targeted use of their enantiomers. More Information

Goponenko AV, Boyle BJ, Jahan KI, Gerashchenko MV, Fomenko DE, Gladyshev VN, Dzenis YA. (2011) Use of environmental scanning electron microscopy for in situ observation of interaction of cells with micro- and nanoprobes. Micro Nano Lett. 8, 603-608.

AbstractPrecision intracellular sensing, probing and manipulation offer unprecedented opportunities for advances in biological sciences. Next-generation ultra-fine probes will be capable of targeting individual cell organelles. Development of such probes as well as probes capable of penetrating through tough cell walls requires detailed knowledge of cell-probe interaction. This Letter evaluated the applicability of environmental scanning electron microscopy (ESEM) for cell and cell-probe interaction imaging. Several types of cells (plant and yeast cells as well as mouse spermatozoa) were successfully imaged in their natural state, with mouse spermatoza observed by ESEM for the first time. Computerised stage applied to image was tough plant cell walls interactions with several probes. Substantial damage to the cell walls was observed as a result of microprobe penetration. The damage persisted after the probe withdrawal and there was residue of cellular content on the withdrawn probes. Several mechanisms of probe failure were observed in situ global buckling, localised bending followed by the tip break-off, and plastic deformation with permanent bending in the case of ultra-fine metal nanoprobe. The results demonstrate applicability of ESEM for high-resolution in situ imaging of cells. Observed mechanisms of cell damage and probe failure provide guidance for future development of probes for minimally-invasive intercellular probing.

Kasaikina MV, Fomenko DE, Labunskyy VM, Lachke SA, Qiu W, Moncaster JA, Zhang J, Wojnarowicz MW Jr, Natarajan SK, Malinouski M, Schweizer U, Tsuji PA, Carlson BA, Maas RL, Lou MF, Goldstein LE, Hatfield DL, Gladyshev VN. (2011) Roles of the 15-kDa Selenoprotein (Sep15) in Redox Homeostasis and Cataract Development Revealed by the Analysis of Sep 15 Knockout Mice. J. Biol. Chem. 286, 33203-33212.

AbstractThe 15 kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum (ER)-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase (UGT). Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In the current study, we developed and characterized Sep15 knockout (KO) mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteine-rich UGT-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable and fertile, showed normal brain morphology and did not activate ER stress pathways. However, parameters of oxidative stress were elevated in the livers of these mice. We found that Sep15 mRNA was enriched during lens development. Further phenotypic characterization of Sep15 KO mice revealed a prominent nuclear cataract that developed at an early age. These cataracts did not appear to be associated with severe oxidative stress or glucose dysregulation. We suggest that the cataracts resulted from improper folding status of lens proteins caused by Sep15 deficiency. More Information

Kim JY, Carlson BA, Xu XM, Zeng Y, Chen S, Gladyshev VN, Lee BJ, Hatfield DL. (2011) Inhibition of selenocysteine tRNA([Ser]Sec) aminoacylation provides evidence that aminoacylation is required for regulatory methylation of this tRNA. Biochem. Biophys. Res. Commun. 409, 814-819.

AbstractThere are two isoforms of selenocysteine (Sec) tRNA([Ser]Sec) that differ by a single methyl group, Um34. The non-Um34 isoform supports the synthesis of a subclass of selenoproteins, designated housekeeping, while the Um34 isoform supports the expression of another subclass, designated stress-related selenoproteins. Herein, we investigated the relationship between tRNA([Ser]Sec) aminoacylation and Um34 synthesis which is the last step in the maturation of this tRNA. Mutation of the discriminator base at position 73 in tRNA([Ser]Sec) dramatically reduced aminoacylation with serine, as did an inhibitor of seryl-tRNA synthetase, SB-217452. Although both the mutation and the inhibitor prevented Um34 synthesis, neither precluded the synthesis of any other of the known base modifications on tRNA([Ser]Sec) following microinjection and incubation of the mutant tRNA([Ser]Sec) transcript, or the wild type transcript along with inhibitor, in Xenopus oocytes. The data demonstrate that Sec tRNA([Ser]Sec) must be aminoacylated for Um34 addition. The fact that selenium is required for Um34 methylation suggests that Sec must be attached to its tRNA for Um34 methylation. This would explain why selenium is essential for the function of Um34 methylase and provides further insights into the hierarchy of selenoprotein expression. More Information

Shchedrina VA, Kabil H, Vorbruggen G, Lee BC, Turanov AA, Hirosawa-Takamori M, Kim HY, Harshman LG, Hatfield DL, Gladyshev VN. (2011) Analyses of fruit flies that do not express selenoproteins or express a mouse selenoprotein, methionine sulfoxide reductase B1, reveal a role of selenoproteins in stress resistance.
J. Biol. Chem. 286, 29449-29461.

AbstractSelenoproteins are essential in vertebrates because of their crucial role in cellular redox homeostasis, but some invertebrates that lack selenoproteins have recently been identified. Genetic disruption of selenoprotein biosynthesis had no effect on lifespan and oxidative stress resistance of Drosophila melanogaster. In the current study, fruit flies with knockout of selenocysteine-specific elongation factor were metabolically labeled with 75Se; they did not incorporate selenium into proteins and had the same lifespan on a chemically defined diet with or without selenium supplementation. These flies were, however, more susceptible to starvation than controls, and this effect could be ascribed to the function of Selenoprotein K. We further expressed mouse methionine sulfoxide reductase B1 (MsrB1), a selenoenzyme that catalyzes the reduction of oxidized methionine residues and has protein repair function, in the whole body or the nervous system of fruit flies. This exogenous selenoprotein could only be expressed when the Drosophila selenocysteine insertion sequence element was used, whereas the corresponding mouse element did not support selenoprotein synthesis. Ectopic expression of MsrB1 in the nervous system led to an increase in the resistance against oxidative stress and starvation, but did not affect lifespan and reproduction, whereas ubiquitous MsrB1 expression had no effect. Dietary selenium did not influence lifespan of MsrB1-expressing flies. Thus, in contrast to vertebrates, fruit flies preserve only three selenoproteins, which are not essential and play a role only under certain stress conditions, thereby limiting the use of the micronutrient selenium by these organisms. More Information

Zhang Y, Rump S, Gladyshev VN. (2011) Comparative genomics and evolution of molybdenum utilization. Coord. Chem. Rev. 255, 1206-1217.

AbstractThe trace element molybdenum (Mo) is the catalytic component of important enzymes involved in global nitrogen, sulfur, and carbon metabolism in both prokaryotes and eukaryotes. With the exception of nitrogenase, Mo is complexed by a pterin compound thus forming the biologically active molybdenum cofactor (Moco) at the catalytic sites of molybdoenzymes. The physiological roles and biochemical functions of many molybdoenzymes have been characterized. However, our understanding of the occurrence and evolution of Mo utilization is limited. This article focuses on recent advances in comparative genomics of Mo utilization in the three domains of life. We begin with a brief introduction of Mo transport systems, the Moco biosynthesis pathway, the role of posttranslational modifications, and enzymes that utilize Mo. Then, we proceed to recent computational and comparative genomics studies of Mo utilization, including a discussion on novel Moco-binding proteins that contain the C-terminal domain of the Moco sulfurase and that are suggested to represent a new family of molybdoenzymes. As most molybdoenzymes need additional cofactors for their catalytic activity, we also discuss interactions between Mo metabolism and other trace elements and finish with an analysis of factors that may influence evolution of Mo utilization. More Information

Zhang Y, Gladyshev VN. (2011) Comparative genomics of trace element dependence in biology. J. Biol. Chem. 286, 23623-23629.

AbstractBiological trace elements are needed in small quantities but are used by all living organisms. A growing list of trace element-dependent proteins and trace element utilization pathways highlights importance of these elements for life. In this review, we focus on recent advances in comparative genomics of trace elements and explore evolutionary dynamics of the dependence of user proteins on these elements. Many zinc protein families evolved representatives that lack this metal, whereas selenocysteine in proteins is dynamically exchanged with cysteine. Several other elements, such as molybdenum and nickel, have a limited number of user protein families but they are strictly dependent on these metals. Comparative genomics of trace elements provides a foundation for investigating fundamental properties, functions and evolutionary dynamics of trace element dependence in biology. More Information

Kasaikina MV, Kravtsova MA, Lee BC, Seravalli J, Peterson DA, Walter J, Legge R, Benson AK, Hatfield DL, Gladyshev VN. (2011) Dietary selenium affects host selenoproteome expression by influencing the gut microbiota. FASEB J. 25, 2492-2499.

AbstractColonization of the gastrointestinal tract and composition of the microbiota may be influenced by components of the diet, including trace elements. To understand how selenium regulates the intestinal microflora, we used high-throughput sequencing to examine the composition of gut microbiota of mice maintained on selenium-deficient, selenium-sufficient, and selenium-enriched diets. The microbiota diversity increased as a result of selenium in the diet. Specific phylotypes showed differential effects of selenium, even within a genus, implying that selenium had unique effects across microbial taxa. Conventionalized germ-free mice subjected to selenium diets gave similar results and showed an increased diversity of the bacterial population in animals fed with higher levels of selenium. Germ-free mice fed selenium diets modified their selenoproteome expression similar to control mice but showed higher levels and activity of glutathione peroxidase 1 and methionine-R-sulfoxide reductase 1 in the liver, suggesting partial sequestration of selenium by the gut microorganisms, limiting its availability for the host. These changes in the selenium status were independent of the levels of other trace elements. The data show that dietary selenium affects both composition of the intestinal microflora and colonization of the gastrointestinal tract, which, in turn, influence the host selenium status and selenoproteome expression. More Information

Wu C, Parrott AM, Fu C, Liu T, Marino SM, Gladyshev VN, Jain MR, Baykal AT, Li Q, Oka S, Sadoshima J, Beuve A, Simmons WJ, Li H. (2011) Thioredoxin 1-Mediated Post-Translational Modifications: Reduction, Transnitrosylation, Denitrosylation and Related Proteomics Methodologies. Antioxid. Redox Signal. 15, 2565-2604.

AbstractDespite the significance of redox post-translational modifications (PTMs) in regulating diverse signal transduction pathways, the enzymatic systems that catalyze reversible and specific oxidative or reductive modifications have yet to be firmly established. Thioredoxin 1 (Trx1) is a conserved antioxidant protein that is well-known for its disulfide reductase activity. Interestingly, Trx1 is also able to transnitrosylate or denitrosylate (defined as processes to transfer or remove a nitric oxide entity to/from substrates) specific proteins. An intricate redox regulatory mechanism has recently been uncovered that accounts for the ability of Trx1 to catalyze these different redox PTMs. In this review, we will summarize the available evidence in support of Trx1 as a specific disulfide reductase, and denitrosylation and transnitrosylation agent, as well as the biological significance of the diverse array of Trx1-regulated pathways and processes under different physiological contexts. The dramatic progress in redox proteomics techniques has enabled the identification of an increasing number of proteins, including peroxiredoxin 1, whose disulfide bond formation and nitrosylation status are regulated by Trx1. This review will also summarize the advancements of redox proteomics techniques for the identification of the protein targets of Trx1-mediated PTMs. Collectively, these studies have shed light on the mechanisms that regulate Trx1-mediated reduction, transnitrosylation and denitrosylation of specific target proteins, solidifying the role of Trx1 as a master regulator of redox signal transduction. More Information

Lee BC, Lobanov AV, Marino SM, Kaya A, Seravalli J, Hatfield DL, Gladyshev VN. (2011) A 4-Selenocysteine, 2-Selenocysteine Insertion Sequence (SECIS) Element Methionine Sulfoxide Reductase from Metridium senile Reveals a Non-catalytic Function of Selenocysteines. J. Biol. Chem. 286, 18747-18755.

AbstractSelenocysteine (Sec) residues occur in thiol oxidoreductase families, and functionally characterized selenoenzymes typically have a single Sec residue used directly for redox catalysis. But how new Sec residues evolve and if non-catalytic Sec residues exist in proteins is not known. Here, we computationally identified several genes with multiple Sec insertion sequence (SECIS) elements, one of which was a methionine-R-sulfoxide reductase (MsrB) homolog from Metridium senile that has four in-frame UGA codons and two nearly identical SECIS elements. One of UGA codons corresponded to the conserved catalytic Sec or Cys in MsrBs, whereas three other UGA codons evolved recently and had no homologs with Sec or Cys in these positions. Metabolic 75Se labeling showed that all four in-frame UGA codons supported Sec insertion and that both SECIS elements were functional and collaborated in Sec insertion at each UGA codon. Interestingly, recombinant M. senile MsrB bound iron, and further analyses suggested the possibility of binding an iron-sulfur cluster by the protein. These data show that Sec residues may appear transiently in genes containing SECIS elements and be adapted for non-catalytic functions. More Information

Kasaikina MV, Lobanov AV, Malinouski MY, Lee BC, Seravalli J, Fomenko DE, Turanov AA, Finney L, Vogt S, Park TJ, Miller RA, Hatfield DL, Gladyshev VN. (2011) Reduced utilization of selenium by naked mole rats due to a specific defect in GPx1 expression.J. Biol. Chem. 286, 17005-17014.

AbstractNaked mole rat (MR) Heterocephalus glaber is a rodent model of delayed aging because of its unusually long lifespan (>28 years). It is also not known to develop cancer. In the current work, tissue imaging by X-ray fluorescence microscopy and direct analyses of trace elements revealed low levels of selenium in the MR liver and kidney, whereas MR and mouse brains had similar selenium levels. This effect was not explained by uniform selenium deficiency as methionine sulfoxide reductase activities were similar in mice and MR. However, glutathione peroxidase activity was an order of magnitude lower in MR liver and kidney than in mouse tissues. In addition, metabolic labeling of MR cells with 75Se revealed a loss of the abundant glutathione peroxidase 1 (GPx1) band, whereas other selenoproteins were preserved. To characterize the MR selenoproteome, we sequenced its liver transcriptome. Gene reconstruction revealed standard selenoprotein sequences except for GPx1, which had an early stop codon, and SelP, which had low selenocysteine content. When expressed in HEK 293 cells, MR GPx1 was present in low levels and its expression could be rescued neither by removing the early stop codon nor by replacing its SECIS element. In addition, GPx1 mRNA was present in lower levels in MR liver than in mouse liver. To determine if GPx1 deficiency could account for the reduced selenium content, we analyzed GPx1 knockout mice and found reduced selenium levels in their livers and kidneys. Thus, MR is characterized by the reduced utilization of selenium due to a specific defect in GPx1 expression. More Information

Gobler CJ, Berry DL, Dyhrman ST, Wilhelm SW, Salamov A, Lobanov AV, Zhang Y, Collier JL, Wurch LL, Kustka AB, Dill BD, Shah M, VerBerkmoes NC, Kuo A, Terry A, Pangilinan J, Lindquist E, Lucas S, Paulsen I, Hattenrath TK, Talmage SC, Walker EA, Koch F, Burson AM, Alejandra Marcoval M, Tang YZ, LeCleir GR, Coyne KJ, Mine Berg G, Bertrand EM, Saito MA, Gladyshev VN, Grigoriev IV. (2011) Niche of harmful alga Aureococcus anophagefferens revealed through ecogenomics. Proc. Natl. Acad. Sci. USA 108, 4352-4357.

AbstractHarmful algal blooms (HABs) cause significant economic and ecological damage worldwide. Despite considerable efforts, a comprehensive understanding of the factors that promote these blooms has been lacking, because the biochemical pathways that facilitate their dominance relative to other phytoplankton within specific environments have not been identified. Here, biogeochemical measurements showed that the harmful alga Aureococcus anophagefferens outcompeted co-occurring phytoplankton in estuaries with elevated levels of dissolved organic matter and turbidity and low levels of dissolved inorganic nitrogen. We subsequently sequenced the genome of A. anophagefferens and compared its gene complement with those of six competing phytoplankton species identified through metaproteomics. Using an ecogenomic approach, we specifically focused on gene sets that may facilitate dominance within the environmental conditions present during blooms. A. anophagefferens possesses a larger genome (56 Mbp) and has more genes involved in light harvesting, organic carbon and nitrogen use, and encoding selenium- and metal-requiring enzymes than competing phytoplankton. Genes for the synthesis of microbial deterrents likely permit the proliferation of this species, with reduced mortality losses during blooms. Collectively, these findings suggest that anthropogenic activities resulting in elevated levels of turbidity, organic matter, and metals have opened a niche within coastal ecosystems that ideally suits the unique genetic capacity of A. anophagefferens and thus, has facilitated the proliferation of this and potentially other HABs. More Information

Bonilla M, Denicola A, Marino SM, Gladyshev VN, Salinas G. (2011) Linked thioredoxin-glutathione systems in platyhelminth parasites: Alternative pathways for glutathione reduction and deglutathionylation. J. Biol. Chem. 286, 4959-4967.

AbstractIn most organisms, thioredoxin (Trx) and/or glutathione (GSH) systems are essential for redox homeostasis and deoxyribonucleotide synthesis. Platyhelminth parasites have a unique and simplified thiol-based redox system, in which the selenoprotein thioredoxin-glutathione reductase (TGR), a fusion of a glutaredoxin (Grx) domain to canonical thioredoxin reductase domains, is the sole enzyme supplying electrons to oxidized glutathione (GSSG) and Trx. This enzyme has recently been validated as a key drug target for flatworm infections. In this study, we show that TGR possesses GSH-independent deglutathionylase activity on a glutathionylated peptide. Furthermore, we demonstrate that deglutathionylation and GSSG reduction are mediated by the Grx domain by a monothiolic mechanism and that the glutathionylated TGR intermediate is resolved by selenocysteine. Deglutathionylation and GSSG reduction via Grx domain, but not Trx reduction, are inhibited at high [GSSG]/[GSH] ratios. We found that Trxs (cytosolic and mitochondrial) provide alternative pathways for deglutathionylation and GSSG reduction. These pathways are operative at high [GSSG]/[GSH] and function in a complementary manner to the Grx domain-dependent one. Despite the existence of alternative pathways, the thioredoxin reductase domains of TGR are an obligate electron route for both the Grx domain- and the Trx-dependent pathways. Overall, our results provide an explanation for the unique array of thiol-dependent redox pathways present in parasitic platyhelminths. Finally, we found that TGR is inhibited by 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7), giving further evidence for NO donation as a mechanism of action for oxadiazole N-oxide TGR inhibitors. Thus, NO donors aimed at TGR could disrupt the entire redox homeostasis of parasitic flatworms. More Information

Marino SM, Gladyshev VN. (2011) Proteomics: Mapping reactive cysteines. Nature Chem. Biol. 7, 72-73.

AbstractA new quantitative proteomic approach can identify reactive cysteine residues in native proteins and distinguish them on the basis of reactivity. This resource-rich study offers a useful new technology and is a significant step toward understanding the reactivity and functions of cysteines in cells. More Information

Suzuki Y, St. Onge RP, Mani R, King OD, Heilbut A, Labunskyy VM, Chen W, Pham L, Zhang LV, Tong AHY, Nislow C, Giaever G, Gladyshev VN, Vidal M, Schow P, Lehár J, Roth FP. (2011) Knocking out multi-gene redundancies via cycles of sexual assortment and fluorescence selection. Nat. Methods 8, 159-164.

AbstractPhenotypes that might otherwise reveal a gene’s function can be obscured by genes with overlapping function. This phenomenon is best known within gene families, in which an important shared function may only be revealed by mutating all family members. Here we describe the ‘green monster’ technology that enables precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein reporter gene, is subjected to repeated rounds of mating, meiosis and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci in single cells. The green monster strategy is potentially applicable to assembling other engineered alterations in any species with sex or alternative means of allelic assortment. To test the technology, we generated a single broadly drug-sensitive strain of Saccharomyces cerevisiae bearing precise deletions of all 16 ATP-binding cassette transporters within clades associated with multidrug resistance. More Information

Fomenko DE, Koc A, Agisheva N, Jacobsen M, Kaya A, Malinouski M, Rutherford JC, Siu KL, Jin DY, Winge DR, Gladyshev VN. (2011) Thiol peroxidases mediate specific genome-wide regulation of gene expression in response to hydrogen peroxide. Proc. Natl. Acad. Sci. USA 108, 2729-2734.

AbstractHydrogen peroxide is thought to regulate cellular processes by direct oxidation of numerous cellular proteins, whereas antioxidants, most notably thiol peroxidases, are thought to reduce peroxides and inhibit H(2)O(2) response. However, thiol peroxidases have also been implicated in activation of transcription factors and signaling. It remains unclear if these enzymes stimulate or inhibit redox regulation and whether this regulation is widespread or limited to a few cellular components. Herein, we found that Saccharomyces cerevisiae cells lacking all eight thiol peroxidases were viable and withstood redox stresses. They transcriptionally responded to various redox treatments, but were unable to activate and repress gene expression in response to H(2)O(2) Further studies involving redox transcription factors suggested that thiol peroxidases are major regulators of global gene expression in response to H(2)O(2) The data suggest that thiol peroxidases sense and transfer oxidative signals to the signaling proteins and regulate transcription, whereas a direct interaction between H(2)O(2) and other cellular proteins plays a secondary role. More Information

Malinouski M, Zhou Y, Belousov VV, Hatfield DL, Gladyshev VN. (2011) Hydrogen peroxide probes directed to different cellular compartments. PloS One 6, e14564, 1-10.

AbstractBACKGROUND: Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells. PRINCIPAL FINDINGS: Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed in each of these compartments, consistent with a low peroxide tone in mammalian cells. In contrast, HyPer was mostly oxidized in the endoplasmic reticulum. Using this system, we characterized control of hydrogen peroxide in various cell systems, such as cells deficient in thioredoxin reductase, sulfhydryl oxidases or subjected to selenium deficiency. Generation of hydrogen peroxide could also be monitored in various compartments following signaling events. CONCLUSIONS: We found that HyPer can be used as a valuable tool to monitor hydrogen peroxide generated in different cellular compartments. The data also show that hydrogen peroxide generated in one compartment could translocate to other compartments. Our data provide information on compartmentalization, dynamics and homeostatic control of hydrogen peroxide in mammalian cells. More Information

Lee BC, Gladyshev VN. (2011) The biological significance of methionine sulfoxide stereochemistry. Free Rad. Biol. Med. 50, 221-227.

AbstractMethionine can be oxidized by reactive oxygen species to a mixture of two diastereomers, methionine-S-sulfoxide and methionine-R-sulfoxide. Both free amino acid and protein-based forms of methionine-S-sulfoxide are stereospecifically reduced by MsrA, whereas the reduction of methionine-R-sulfoxide requires two enzymes, MsrB and fRMsr, which act on its protein-based and free amino acid forms, respectively. However, mammals lack fRMsr and are characterized by deficiency in the reduction of free methionine-R-sulfoxide. The biological significance of such biased reduction of methionine sulfoxide has not been fully explored. MsrA and MsrB activities decrease during aging, leading to accumulation of protein-based and free amino acid forms of methionine sulfoxide. Since methionine is an indispensible amino acid in human nutrition and a key metabolite in sulfur, methylation, and transsulfuration pathways, the consequences of accumulation of its oxidized forms require further studies. Finally, in addition to methionine, methylsulfinyl groups are present in various drugs and natural compounds, and their differential reduction by Msrs may have important therapeutic implications. More Information

Ahmed ZM, Yousaf R, Lee BC, Khan SN, Lee S, Lee K, Husnain T, Rehman AU, Bonneux S, Ansar M, Ahmad W, Leal SM, Gladyshev VN, Belyantseva IA, Van Camp G, Riazuddin S, Friedman TB, Riazuddin S. (2011) Functional null mutations of MSRB3 encoding methionine sulfoxide reductase are associated with human deafness DFNB74. Am. J. Hum. Genet. 88, 19-29.

AbstractThe DFNB74 locus for autosomal-recessive, nonsyndromic deafness segregating in three families was previously mapped to a 5.36 Mb interval on chromosome 12q14.2-q15. Subsequently, we ascertained five additional consanguineous families in which deafness segregated with markers at this locus and refined the critical interval to 2.31 Mb. We then sequenced the protein-coding exons of 18 genes in this interval. The affected individuals of six apparently unrelated families were homozygous for the same transversion (c.265T>G) in MSRB3, which encodes a zinc-containing methionine sulfoxide reductase B3. c.265T>G results in a substitution of glycine for cysteine (p.Cys89Gly), and this substitution cosegregates with deafness in the six DFNB74 families. This cysteine residue of MSRB3 is conserved in orthologs from yeast to humans and is involved in binding structural zinc. In vitro, p.Cys89Gly abolished zinc binding and MSRB3 enzymatic activity, indicating that p.Cys89Gly is a loss-of-function allele. The affected individuals in two other families were homozygous for a transition mutation (c.55T>C), which results in a nonsense mutation (p.Arg19X) in alternatively spliced exon 3, encoding a mitochondrial localization signal. This finding suggests that DFNB74 deafness is due to a mitochondrial dysfunction. In a cohort of 1,040 individuals (aged 53-67 years) of European ancestry, we found no association between 17 tagSNPs for MSRB3 and age-related hearing loss. Mouse Msrb3 is expressed widely. In the inner ear, it is found in the sensory epithelium of the organ of Corti and vestibular end organs as well as in cells of the spiral ganglion. Taken together, MSRB3-catalyzed reduction of methionine sulfoxides to methionine is essential for hearing. More Information

Labunskyy VM, Lee BC, Handy DE, Loscalzo J, Hatfield DL, Gladyshev VN. (2011) Both maximal expression of selenoproteins and selenoprotein deficiency can promote development of type 2 diabetes-like phenotype in mice. Antioxid. Redox Signal. 14, 2327-2336.

AbstractSelenium (Se) is an essential trace element in mammals, which has been shown to exert its function through selenoproteins. Whereas optimal levels of Se in the diet have important health benefits, a recent clinical trial has suggested that supplemental intake of Se above the adequate level potentially may raise the risk of type 2 diabetes mellitus. However, the molecular mechanisms for the effect of dietary Se on the development of this disease are not understood. In the present study, we examined the contribution of selenoproteins to increased risk of developing diabetes using animal models. C57BL/6J mice (n=6-7 per group) were fed either Se-deficient Torula yeast-based diet or diets supplemented with 0.1 and 0.4 ppm Se. Our data show that mice maintained on a Se-supplemented diet develop hyperinsulinemia and have decreased insulin sensitivity. These effects are accompanied by elevated expression of a selective group of selenoproteins. We also observed that reduced synthesis of these selenoproteins caused by overexpression of an i6A- mutant selenocysteine tRNA promotes glucose intolerance and leads to a diabetes-like phenotype. These findings indicate that both high expression of selenoproteins and selenoprotein deficiency may dysregulate glucose homeostasis and suggest a role for selenoproteins in development of diabetes. More Information

Marino SM, Gladyshev VN. (2011) Redox Biology: Computational Approaches to the Investigation of Functional Cysteine Residues.Antioxid. Redox Signal. 15, 135-146.

AbstractCysteine (Cys) residues serve many functions, such as catalysis, stabilization of protein structure through disulfides, metal binding, and regulation of protein function. Cys residues are also subject to numerous posttranslational modifications. In recent years, various computational tools aiming at classifying and predicting different functional categories of Cys have been developed, particularly for structural and catalytic Cys. On the other hand, given complexity of the subject, bioinformatics approaches have been less successful for the investigation of regulatory Cys sites. In this review, we introduce different functional categories of Cys residues. For each category, an overview of state-of-the-art bioinformatics methods and tools is provided, along with examples of successful applications and potential limitations associated with each approach. Finally, we discuss Cys-based redox switches, which modify the view of distinct functional categories of Cys in proteins. More Information

Turanov A, Xu XM, Carlson BA, Yoo MH, Gladyshev VN, Hatfield DL. (2011) Biosynthesis of selenocysteine, the 21st amino acid in the genetic code, and a novel pathway for cysteine biosynthesis. Adv. Nutr. 2, 122-128.

AbstractThe biosynthetic pathway for selenocysteine (Sec), the 21st amino acid in the genetic code whose codeword is UGA, was recently determined in eukaryotes and archaea. Sec tRNA, designated tRNA[Ser]Sec, is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase to form O-phosphoseryl-tRNA[Ser]Sec. Sec synthase (SecS) then uses O-phosphoseryl-tRNA[Ser]Sec and the active donor of selenium, selenophosphate, to form Sec-tRNA[Ser]Sec. Selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase 2 (SPS2). Sec was the last protein amino acid in eukaryotes whose biosynthesis had not been established and the only known amino acid in eukaryotes whose biosynthesis occurs on its tRNA. Interestingly, sulfide can replace selenide to form thiophosphate in the SPS2-catalyzed reaction that can then react with O-phosphoseryl-tRNA[Ser]Sec in the presence of SecS to form cysteine-(Cys-)tRNA[Ser]Sec. This novel pathway of Cys biosynthesis results in Cys being decoded by UGA and replacing Sec in normally selenium-containing proteins (selenoproteins). The selenoprotein, thioredoxin reductase 1 (TR1), was isolated from cells in culture and from mouse liver for analysis of Cys/Sec replacement by MS. The level of Cys/Sec replacement in TR1 was proportional to the level of selenium in the diet of the mice. Elucidation of the biosynthesis of Sec and Sec/Cys replacement provides novel ways of regulating selenoprotein functions and ultimately better understanding of the biological roles of dietary selenium.. More Information

Kim MJ, Lee BC, Jeong J, Lee KJ, Hwang KY, Gladyshev VN, Kim HY. (2011) Tandem use of selenocysteine: adaptation of a selenoprotein glutaredoxin for reduction of selenoprotein methionine sulfoxide reductase. Mol. Microbiol. 79, 1194-1203.

AbstractSeveral engineered selenocysteine (Sec)-containing glutaredoxins (Grxs) and their enzymatic properties have been reported, but natural selenoprotein Grxs have not been previously characterized. We expressed a bacterial selenoprotein Grx from Clostridium sp. (also known as Alkaliphilus oremlandii) OhILAs in Escherichia coli and characterized this selenoenzyme and its natural Cys homologues in Clostridium and E. coli. The selenoprotein Grx had a 200-fold higher activity than its Sec-to-Cys mutant form, suggesting that Sec is essential for catalysis by this thiol-disulfide oxidoreductase. Kinetic analysis also showed that the selenoprotein Grx had a 10-fold lower K(m) than Cys homologues. Interestingly, this selenoenzyme efficiently reduced a Clostridium selenoprotein methionine sulfoxide reductase A (MsrA), suggesting that it is the natural reductant for the protein that is not reducible by thioredoxin, a common reductant for Cys-containing MsrAs. We also found that the selenoprotein Grx could not efficiently reduce a Cys version of Clostridium MsrA, whereas natural Clostridium and E. coli Cys-containing Grxs, which efficiently reduce Cys-containing MsrAs, poorly acted on the selenoprotein MsrA. This specificity for MsrA reduction could explain why Sec is utilized in Clostridium Grx and more generally provides a novel example of the use of Sec in biological systems. More Information

2010 Articles

Gladyshev VN, Hatfield DL. (2010) Selenocysteine Biosynthesis, Selenoproteins, and Selenoproteomes. Nucleic Acids and Mol. Bio. 24, 3-27.

AbstractSelenocysteine (Sec), the 21st amino acid in the genetic code, is encoded by UGA. The pathway of Sec biosynthesis in eukaryotes has only recently been discovered. Sec is constructed on its tRNA that is initially aminoacylated with serine and modified to a phosphoseryl-tRNA intermediate with the help of several dedicated enzymes. More than 50 selenoprotein families are now known with most selenoproteins being oxidoreductases. Development of bioinformatics tools led to the identification of entire sets of selenoproteins in organisms, selenoproteomes, which in turn helped explain biological and biomedical effects of dietary selenium and identify new functions of selenium in biology. Roles of selenium and selenoproteins in health have also been addressed through sophisticated transgenic/knockout models that targeted removal or modulation of Sec tRNA expression. More Information

Marino SM, Gladyshev VN. (2010) Cysteine function governs its conservation and degeneration and restricts its utilization on protein surfaces. J. Mol. Biol. 404, 902-916.

AbstractCysteine (Cys) is an enigmatic amino acid residue. Although one of the least abundant, it often occurs in the functional sites of proteins. Whereas free Cys is a polar amino acid, Cys in proteins is often buried, and its classification on the hydrophobicity scale is ambiguous. We hypothesized that the deviation of Cys residues from the properties of a free amino acid is due to their reactivity and addressed this possibility by examining Cys in large protein structure data sets. Compared to other amino acids, Cys was characterized by the most extreme conservation pattern, with the majority of Cys being either highly conserved or poorly conserved. In addition, clustering of Cys with another Cys residue was associated with high conservation, whereas exposure of Cys on protein surfaces was associated with low conservation. Moreover, although clustered Cys behaved as polar residues, isolated Cys was the most buried residue of all, in disagreement with known chemical properties of Cys. Thus, the anomalous hydrophobic behavior and conservation pattern of Cys can be explained by elimination of isolated Cys from protein surfaces during evolution and by clustering of other Cys residues. These findings indicate that Cys abundance is governed by Cys function in proteins rather than by the sheer chemical-physical properties of free amino acids, and suggest that a high tendency of Cys to be functionally active can considerably limit its abundance on protein surfaces. More Information

Xu XM, Turanov AA, Carlson BA, Yoo MH, Everley RA, Nandakumar R, Sorokina I, Gygi SP, Gladyshev VN, Hatfield DL. (2010) Targeted insertion of cysteine by decoding UGA codons with mammalian selenocysteine machinery. Proc. Natl. Acad. Sci. USA. 107, 21430-21434.

AbstractCysteine (Cys) is inserted into proteins in response to UGC and UGU codons. Herein, we show that supplementation of mammalian cells with thiophosphate led to targeted insertion of Cys at the UGA codon of thioredoxin reductase 1 (TR1). This Cys was synthesized by selenocysteine (Sec) synthase on tRNA[Ser]Sec and its insertion was dependent on the Sec insertion sequence element in the 3′UTR of TR1 mRNA. The substrate for this reaction, thiophosphate, was synthesized by selenophosphate synthetase 2 from ATP and sulfide and reacted with phosphoseryl-tRNA[Ser]Sec to generate Cys-tRNA[Ser]Sec. Cys was inserted in vivo at UGA codons in natural mammalian TRs, and this process was regulated by dietary selenium and availability of thiophosphate. Cys occurred at 10% of the Sec levels in liver TR1 of mice maintained on a diet with normal amounts of selenium and at 50% in liver TR1 of mice maintained on a selenium deficient diet. These data reveal a novel Sec machinery-based mechanism for biosynthesis and insertion of Cys into protein at UGA codons and suggest new biological functions for thiophosphate and sulfide in mammals. More Information

Hacioglu E, Esmer I, Fomenko DE, Gladyshev VN, Koc A. (2010) The roles of thiol oxidoreductases in yeast replicative aging. Mech. Ageing Dev. 131, 692-699.

Abstract Thiol-based redox reactions are involved in the regulation of a variety of biological functions, such as protection against oxidative stress, signal transduction and protein folding. Some proteins involved in redox regulation have been shown to modulate life span in organisms from yeast to mammals. To assess the role of thiol oxidoreductases in aging on a genome-wide scale, we analyzed the replicative life span of yeast cells lacking known and candidate thiol oxidoreductases. The data suggest the role of several pathways in controlling yeast replicative life span, including thioredoxin reduction, protein folding and degradation, peroxide reduction, PIP3 signaling, and ATP synthesis. More Information

Sengupta A, Lichti UF, Carlson BA, Ryscavage AO, Gladyshev VN, Yuspa SH, Hatfield DL. (2010) Selenoproteins Are Essential for Proper Keratinocyte Function and Skin Development. PLoS 5, e12249, 1-15.

AbstractDietary selenium is known to protect skin against UV-induced damage and cancer and its topical application improves skin surface parameters in humans, while selenium deficiency compromises protective antioxidant enzymes in skin. Furthermore, skin and hair abnormalities in humans and rodents may be caused by selenium deficiency, which are overcome by dietary selenium supplementation. Most important biological functions of selenium are attributed to selenoproteins, proteins containing selenium in the form of the amino acid, selenocysteine (Sec). Sec insertion into proteins depends on Sec tRNA; thus, knocking out the Sec tRNA gene (Trsp) ablates selenoprotein expression. We generated mice with targeted removal of selenoproteins in keratin 14 (K14) expressing cells and their differentiated descendents. The knockout progeny had a runt phenotype, developed skin abnormalities and experienced premature death. Lack of selenoproteins in epidermal cells led to the development of hyperplastic epidermis and aberrant hair follicle morphogenesis, accompanied by progressive alopecia after birth. Further analyses revealed that selenoproteins are essential antioxidants in skin and unveiled their role in keratinocyte growth and viability. This study links severe selenoprotein deficiency to abnormalities in skin and hair and provides genetic evidence for the role of these proteins in keratinocyte function and cutaneous development. More Information

Kaya A, Koc A, Lee BC, Fomenko DE, Rederstorff M, Krol A, Lescure A, Gladyshev VN. (2010) Compartmentalization and Regulation of Mitochondrial Function by Methionine Sulfoxide Reductases in Yeast. Biochemistry 49, 8618-8625.

AbstractElevated levels of reactive oxygen species can damage proteins. Sulfur-containing amino acid residues, cysteine and methionine, are particularly susceptible to such damage. Various enzymes evolved to protect proteins or repair oxidized residues, including methionine sulfoxide reductases MsrA and MsrB, which reduce methionine-S-sulfoxide (Met-SO), and methionine-R-sulfoxide (Met-RO) residues, respectively, back to methionine. Here, we show that MsrA and MsrB are involved in the regulation of mitochondrial function. Saccharomyces cerevisiae mutant cells lacking MsrA, MsrB or both proteins, had normal levels of mitochondria, but lower levels of cytochrome c and fewer respiration-competent mitochondria. The growth of single MsrA or MsrB mutants on respiratory carbon sources was inhibited, and that of the double mutant was severely compromised, indicating impairment of mitochondrial function. Although MsrA and MsrB are thought to have similar roles in oxidative protein repair each targeting a diastereomer of methionine sulfoxide, their deletion resulted in different phenotypes. GFP fusions of MsrA and MsrB showed different localization patterns and primarily localized to cytoplasm and mitochondria, respectively. This finding agreed with compartment-specific enrichment of MsrA and MsrB activities. These results show that oxidative stress contributes to mitochondrial dysfunction through oxidation of methionine residues in proteins located in different cellular compartments. More Information

Marino SM, Li Y, Fomenko DE, Agisheva N, Cerny RL, Gladyshev VN. (2010) Characterization of Surface-Exposed Reactive Cysteine Residues in Saccharomyces cerevisiae. Biochemistry 49, 7709-7721.

AbstractNumerous cellular processes are subject to redox regulation, and thiol-dependent redox control, acting through reactive cysteine (Cys) residues, is among the major mechanisms of redox regulation. However, information on the sets of proteins that provide thiol-based redox regulation or are affected by it is limited. Here, we describe proteomic approaches to characterize proteins that contain reactive thiols and methods to identify redox Cys in these proteins. Using Saccharomyces cerevisiae as a eukaryotic model organism, we identified 284 proteins with exposed reactive Cys and determined the identities of 185 of these residues. We then characterized subsets of these proteins as in vitro targets of major cellular thiol oxidoreductases, thioredoxin and glutaredoxin, and found that these enzymes can control the redox state of a significant number of thiols in target proteins. We further examined common features of exposed reactive Cys and compared them with an unbiased control set of Cys using computational approaches. This analysis (i) validated the efficacy of targeting exposed Cys in proteins in their native, folded state, (ii) quantified the proportion of targets that can be redox regulated via thiol oxidoreductase systems, and (iii) revealed the theoretical range of the experimental approach with regard to protein abundance and physicochemical properties of reactive Cys. From these analyses, we estimate that approximately one-fourth of exposed Cys in the yeast proteome can be regarded as functional sites, either subject to regulation by thiol oxidoreductases or involved in structural disulfides and metal binding. More Information

Liang X, Fomenko DE, Hua D, Kaya A, Gladyshev VN. (2010) Diversity of protein and mRNA forms of mammalian methionine sulfoxide reductase B1 due to intronization and protein processing. PLoS One 5, e11497, 1-8.

Abstract Methionine sulfoxide reductases (Msrs) are repair enzymes that protect proteins from oxidative stress by catalyzing stereospecific reduction of oxidized methionine residues. MsrB1 is a selenocysteine-containing cytosolic/nuclear Msr with high expression in liver and kidney. PRINCIPAL FINDINGS: Here, we identified differences in MsrB1 gene structure among mammals. Human MsrB1 gene consists of four, whereas the corresponding mouse gene of five exons, due to occurrence of an additional intron that flanks the stop signal and covers a large part of the 3-UTR. This intron evolved in a subset of rodents through intronization of exonic sequences, whereas the human gene structure represents the ancestral form. In mice, both splice forms were detected in liver, kidney, brain and heart with the five-exon form being the major form. We found that both mRNA forms were translated and supported efficient selenocysteine insertion into MsrB1. In addition, MsrB1 occurs in two protein forms that migrate as 14 and 5 kDa proteins. We found that each mRNA splice form generated both protein forms. The abundance of the 5 kDa form was not influenced by protease inhibitors, replacement of selenocysteine in the active site or mutation of amino acids in the cleavage site. However, mutation of cysteines that coordinate a structural zinc decreased the levels of 5 and 14 kDa forms, suggesting importance of protein structure for biosynthesis and/stability of these forms. CONCLUSIONS: This study characterized unexpected diversity of protein and mRNA forms of mammalian selenoprotein MsrB1. More Information

Yoo MH, Carlson BA, Tsuji P, Irons R, Gladyshev VN, Hatfield DL. (2010) Alteration of thioredoxin reductase 1 levels in elucidating cancer etiology. Methods Enzymol. 474, 255-275.

AbstractThioredoxin reductase 1 (TR1) is a major antioxidant and redox regulator in mammalian cells and appears to function as a double-edged sword in that it has roles in preventing and promoting/sustaining cancer. TR1 is overexpressed in many cancer cells and targeting its removal often leads to a reversal in numerous malignant characteristics which has marked this selenoenzyme as a prime target for cancer therapy. Since alterations in TR1 activity may lead to a better understanding of the etiology of cancer and new avenues for providing better therapeutic procedures, we have described herein techniques for removing and reexpressing TR1 employing RNAi technology and for assessing the catalytic activity of this enzyme. More Information

Turanov AA, Hatfield DL, Gladyshev VN. (2010) Characterization of protein targets of mammalian thioredoxin reductases. Methods Enzymol. 474, 245-254.

AbstractMammalian thioredoxin reductases (TRs) are members of the pyridine nucleotide-disulfide oxidoreductase family. The main function of these enzymes is to maintain thioredoxins (Trxs) in the reduced state. The accessibility and high reactivity of selenocysteine in the C-terminal tetrapeptide allows mammalian TRs to couple to a range of substrates from proteins, such as Trx, to small molecules, such as selenite and hydroperoxides. However, identification of physiological substrates of TRs remains a challenge, with new targets identified primarily by testing random candidates in in vitro assays. The reaction mechanism of TRs supports a procedure that could trap substrates in a covalent nonproductive complex with TRs. Accordingly, attachment of TRs to affinity matrices allows isolation and identification of these targets. Application of this method revealed that Trxs are the major targets of TRs and supported efficient isolation of Trx substrates on TR affinity columns. We suggest that this procedure may be used as a general method of affinity isolation of Trxs and other TR substrates. More Information

Aachmann FL, Sal LS, Kim HY, Marino SM, Gladyshev VN, Dikiy A. (2010) Insights into function, catalytic mechanism and fold evolution of selenoprotein methionine sulfoxide reductase B1 through structural analysis. J. Biol. Chem. 285, 33315-33323.

AbstractMethionine sulfoxide reductases protect cells by repairing oxidatively damaged methionine residues in proteins. Here, we report the first three-dimensional structure of the mammalian selenoprotein methionine sulfoxide reductase B1 (MsrB1), determined by high-resolution NMR spectroscopy. Heteronuclear multidimensional spectra yielded NMR spectral assignments for the reduced form of MsrB1 in which catalytic selenocysteine (Sec) was replaced with cysteine (Cys). MsrB1 consists of a central structured core of two b-sheets and a highly flexible, disordered N-terminal region. Analysis of pH dependence of NMR signals of catalytically relevant residues, comparison with the data for bacterial MsrBs, and NMR-based structural analysis of methionine sulfoxide (substrate) and methionine sulfone (inhibitor) binding to MsrB1 at the atomic level reveal a mechanism involving catalytic Sec95 and resolving Cys4 residues in catalysis. The MsrB1 structure differs from the structures of Cys-containing MsrBs in the use of distal selenenylsulfide, residues needed for catalysis and the mode in which the active form of the enzyme is regenerated. In addition, this is the first structure of a zinc-containing MsrB, which highlights the structural role of this metal ion bound to four conserved Cys. We integrated this information into a structural model of evolution of MsrB superfamily. More Information

Carlson BA, Yoo MH, Shrimali RK, Irons R, Gladyshev VN, Hatfield DL, Park JM. (2010) Role of selenium-containing proteins in T-cell and macrophage function. Proc. Nutr. Soc. 69, 300-310.

AbstractSelenium (Se) has been known for many years to have played a role in boosting the immune function, but the manner in which this element acts at the molecular level in host defence and inflammatory diseases is poorly understood. To elucidate the role of Se-containing proteins in the immune function, we knocked out the expression of this protein class in T-cells or macrophages of mice by targeting the removal of the selenocysteine tRNA gene using loxP-Cre technology. Mice with selenoprotein-less T-cells manifested reduced pools of mature and functional T-cells in lymphoid tissues and an impairment in T-cell-dependent antibody responses. Furthermore, selenoprotein deficiency in T-cells led to an inability of these cells to suppress reactive oxygen species production, which in turn affected their ability to proliferate in response to T-cell receptor stimulation. Selenoprotein-less macrophages, on the other hand, manifested mostly normal inflammatory responses, but this deficiency resulted in an altered regulation in extracellular matrix-related gene expression and a diminished migration of macrophages in a protein gel matrix. These observations provided novel insights into the role of selenoproteins in the immune function and tissue homeostasis. More Information

Turanov AA, Kehr S, Marino SM, Yoo MH, Carlson BA, Hatfield DL, Gladyshev VN. (2010) Mammalian thioredoxin reductase 1: roles in redox homoeostasis and characterization of cellular targets. Biochem. J. 430, 285-293.

AbstractThe classical Trx (thioredoxin) system, composed of TR (Trx reductase), Trx and NADPH, defines a major pathway of cellular thiol-based redox regulation. Three TRs have been identified in mammals: (i) cytosolic TR1, (ii) mitochondrial TR3 and (iii) testes-specific TGR (Trx-glutathione reductase). All three are selenocysteine-containing enzymes with broad substrate specificity in in vitro assays, but which protein substrates are targeted by TRs in vivo is not well understood. In the present study, we used a mechanism-based approach to characterize the molecular targets of TR1. Cytosolic Trx1 was the major target identified in rat and mouse liver, as well as in rat brain and mouse serum. The results suggest that the main function of TR1 is to reduce Trx1. We also found that TR1-based affinity resins provide a convenient tool for specific isolation of Trxs from a variety of biological samples. To better assess the role of TRs in redox homoeostasis, we comparatively analysed TR1- and TR3-knockdown cells. Although cells deficient in TR1 were particularly sensitive to diamide, TR3-knockdown cells were more sensitive to hydrogen peroxide. To further examine the TR1-Trx1 redox pair, we used mice with a liver-specific knockout of selenocysteine tRNA. In this model, selenocysteine insertion into TR1 was blocked, but the truncated form of this protein was not detected. Instead, TR1 and TR3 levels were decreased in the knockout samples. Diminished hepatic TR1 function was associated with elevated Trx1 levels, but this protein was mostly in the oxidized state. Overall, this study provides evidence for the key role of the TR1-Trx1 pair in redox homoeostasis. More Information

Lobanov AV, Turanov AA, Hatfield DL, Gladyshev VN. (2010) Dual functions of codons in the genetic code. Critical Reviews in Biochemistry and Molecular Biology 45, 257-265.

AbstractThe discovery of the genetic code provided one of the basic foundations of modern molecular biology. Most organisms use the same genetic language, but there are also well-documented variations representing codon reassignments within specific groups of organisms (such as ciliates and yeast) or organelles (such as plastids and mitochondria). In addition, duality in codon function is known in the use of AUG in translation initiation and methionine insertion into internal protein positions as well as in the case of selenocysteine and pyrrolysine insertion (encoded by UGA and UAG, respectively) in competition with translation termination. Ambiguous meaning of CUG in coding for serine and leucine is also known. However, a recent study revealed that codons in any position within the open reading frame can serve a dual function and that a change in codon meaning can be achieved by availability of a specific type of RNA stem-loop structure in the 3?-untranslated region. Thus, duality of codon function is a more widely used feature of the genetic code than previously known, and this observation raises the possibility that additional recoding events and additional novel features have evolved in the genetic code. More Information

Irons R, Tsuji PA, Carlson BA, Ouyang P, Yoo MH, Xu XM, Hatfield DL, Gladyshev VN, Davis CD. (2010) Deficiency in the 15-kDa Selenoprotein Inhibits Tumorigenicity and Metastasis of Colon Cancer Cells. Cancer Prev. Res. (Phila Pa) 3, 630-639.

AbstractSelenium has cancer-preventive activity that is mediated, in part, through selenoproteins. The role of the 15-kDa selenoprotein (Sep15) in colon cancer was assessed by preparing and using mouse colon CT26 cells stably transfected with short hairpin RNA constructs targeting Sep15. Metabolic (75)Se labeling and Northern and Western blot analyses revealed that >90% of Sep15 was downregulated. Growth of the resulting Sep15-deficient CT26 cells was reduced (P < 0.01), and cells formed significantly (P < 0.001) fewer colonies in soft agar compared with control CT26 cells. Whereas most (14 of 15) BALB/c mice injected with control cells developed tumors, few (3 of 30) mice injected with Sep15-deficient cells developed tumors (P < 0.0001). The ability to form pulmonary metastases had similar results. Mice injected with the plasmid-transfected control cells had >250 lung metastases per mouse; however, mice injected with cells with downregulation of Sep15 only had 7.8 +/- 5.4 metastases. To investigate molecular targets affected by Sep15 status, gene expression patterns between control and knockdown CT26 cells were compared. Ingenuity Pathways Analysis was used to analyze the 1,045 genes that were significantly (P < 0.001) affected by Sep15 deficiency. The highest-scored biological functions were cancer and cellular growth and proliferation. Consistent with these observations, subsequent analyses revealed a G(2)-M cell cycle arrest in cells with targeted downregulation of Sep15. In contrast to CT26 cells, Sep15-targeted downregulation in Lewis lung carcinoma (LLC1) cells did not affect anchorage-dependent or anchorage-independent cell growth. These data suggest tissue specificity in the cancer-protective effects of Sep15 downregulation, which are mediated, at least in part, by influencing the cell cycle. More Information

Otero L, Bonilla M, Protasio AV, Fernandez C, Gladyshev VN, Salinas G. (2010) Thioredoxin and glutathione systems differ in parasitic and free-living platyhelminths. BMC Genomics 11, 237.

Abstract The thioredoxin and/or glutathione pathways occur in all organisms. They provide electrons for deoxyribonucleotide synthesis, function as antioxidant defenses, in detoxification, Fe/S biogenesis and participate in a variety of cellular processes. In contrast to their mammalian hosts, platyhelminth (flatworm) parasites studied so far, lack conventional thioredoxin and glutathione systems. Instead, they possess a linked thioredoxin-glutathione system with a selenocysteine-containing enzyme thioredoxin glutathione reductase (TGR) as the single redox hub that controls the overall redox homeostasis. TGR has been recently validated as a drug target for schistosomiasis and new drug leads targeting TGR have recently been identified for these platyhelminth infections that affect more than 200 million people and for which a single drug is currently available. Little is known regarding the genomic structure of flatworm TGRs, the expression of TGR variants and whether the absence of conventional thioredoxin and glutathione systems is a signature of the entire platyhelminth phylum. RESULTS: We examine platyhelminth genomes and transcriptomes and find that all platyhelminth parasites (from classes Cestoda and Trematoda) conform to a biochemical scenario involving, exclusively, a selenium-dependent linked thioredoxin-glutathione system having TGR as a central redox hub. In contrast, the free-living platyhelminth Schmidtea mediterranea (Class Turbellaria) possesses conventional and linked thioredoxin and glutathione systems. We identify TGR variants in Schistosoma spp. derived from a single gene, and demonstrate their expression. We also provide experimental evidence that alternative initiation of transcription and alternative transcript processing contribute to the generation of TGR variants in platyhelminth parasites. CONCLUSIONS: Our results indicate that thioredoxin and glutathione pathways differ in parasitic and free-living flatworms and that canonical enzymes were specifically lost in the parasitic lineage. Platyhelminth parasites possess a unique and simplified redox system for diverse essential processes, and thus TGR is an excellent drug target for platyhelminth infections. Inhibition of the central redox wire hub would lead to overall disruption of redox homeostasis and disable DNA synthesis. More Information

Yoo MH, Gu X, Xu XM, Kim JY, Carlson BA, Patterson AD, Cai H, Gladyshev VN, Hatfield DL. (2010) Delineating the role of glutathione peroxidase 4 in protecting cells against lipid hydroperoxide damage and in Alzheimer’s disease. Antioxid. Redox Signal. 12, 819-827.

AbstractNumerous studies characterizing the function of glutathione peroxidase 4 (GPx4) have demonstrated that this selenoenzyme is protective against oxidative stress. Herein, we characterized the function of this protein by targeting GPx4 down-regulation using RNA interference. Partial knockdown of GPx4 levels resulted in growth retardation and morphological changes. Surprisingly, GPx4 knockdown cells showed virtually unchanged levels of intracellular ROS, yet highly increased levels of oxidized lipid by-products. GPx1, another glutathione peroxidase and a major cellular peroxide scavenging enzyme, did not rescue GPx4 deficient cells and did not reduce lipid peroxide levels. The data established an essential role of GPx4 in protecting cells against lipid hydroperoxide damage, yet a limited role as a general antioxidant enzyme. As oxidized lipid hydroperoxides are a characteristic of neurodegenerative diseases, we analyzed brain tissues of mice suffering from a model of Alzheimers disease and found that oxidized lipid by-products were enriched and expression of both GPx4 and guanine-rich sequence-binding factor, which is known to control GPx4 synthesis, was down-regulated. Brain tissue from an Alzheimers diseased human also manifested enhanced levels of one of the oxidized lipid by-products, 4-hydroxynonenal. These data suggest a role of GPx4 in neurodegenerative diseases through its function in removal of lipid hydroperoxides. More Information

Novoselov SV, Kim HY, Hua D, Lee BC, Astle CM, Harrison DE, Friguet B, Moustafa MM, Carlson BA, Hatfield DL, Gladyshev V. (2010) Regulation of Selenoproteins and Methionine Sulfoxide Reductases A and B1 by Age, Calorie Restriction, and Dietary Selenium in Mice. Antioxid. Redox Signal. 12, 829-838.

AbstractMethionine residues are susceptible to oxidation, but this damage may be reversed by methionine sulfoxide reductases MsrA and MsrB. Mammals contain one MsrA and three MsrBs, including a selenoprotein MsrB1. Here, we show that MsrB1 is the major methionine sulfoxide reductase in liver of mice and it is among the proteins that are most easily regulated by dietary selenium. MsrB1, but not MsrA activities, were reduced with age and the selenium regulation of MsrB1 was preserved in the aging liver, suggesting that MsrB1 could account for the impaired methionine sulfoxide reduction in aging animals. We also examined regulation of Msr and selenoprotein expression by a combination of dietary selenium and calorie restriction and found that, under calorie restriction conditions, selenium regulation was preserved. In addition, mice overexpressing a mutant form of selenocysteine tRNA reduced MsrB1 activity to the level observed in selenium deficiency, whereas MsrA activity was elevated in these animals. Finally, we show that selenium regulation in inbred mouse strains is preserved in an outbred aging model. Taken together, these findings better define dietary regulation of methionine sulfoxide reduction and selenoprotein expression in mice with regard to age, calorie restriction, dietary Se and a combination of these factors. More Information

Shchedrina VA, Zhang Y, Labunskyy VM, Hatfield DL, Gladyshev V. (2010) Structure-function relationships, physiological roles and evolution of mammalian ER-resident selenoproteins. Antioxid. Redox Signal. 12, 839-848.

AbstractSelenium is an essential trace element in mammals. The major biological form of this micronutrient is the amino acid selenocysteine that is present in the active sites of selenoenzymes. Seven out of 25 mammalian selenoproteins have been identified as residents of the endoplasmic reticulum, including the 15-kDa selenoprotein, type 2 iodothyronine deiodinase and selenoproteins K, M, N, S, and T. Most of these proteins are poorly characterized. However, recent studies implicate some of them in quality control of protein folding in the ER, retrotranslocation of misfolded proteins from the ER to cytosol, metabolism of thyroid hormone and regulation of calcium homeostasis. In addition, some of these proteins are involved in regulation of glucose metabolism and inflammation. This review discusses evolution and structure-function relationships of the ER-resident selenoproteins and summarizes recent findings on these proteins, which reveal the emerging important role of selenium and selenoproteins in ER function. More Information

Zhang Y, Gladyshev VN. (2010) dbTEU: a protein database of trace element utilization. Bioinformatics 26, 700-702.

Abstract Biological trace elements are required for numerous biological processes and by all organisms. We describe a database, dbTEU (DataBase of Trace Element Utilization), that features known transporters and user proteins for five trace elements (copper, molybdenum, nickel, cobalt and selenium) and represents sequenced organisms from the three domains of life. The manually curated dbTEU currently includes approximately 16,500 proteins from more than seven hundred organisms, and offers interactive trace element, protein, organism and sequence search and browse tools. Availability and Implementation: dbTEU is freely available at http://gladyshevlab.bwh.harvard.edu/trace_element/. CONTACT: vgladyshev@rics.bwh.harvard.edu. More Information

Gerashchenko MV, Su D, Gladyshev VN. (2010) CUG start codon generates thioredoxin/glutathione reductase isoforms in mouse testes. J. Biol. Chem. 285, 4595-4602.

AbstractMammalian cytosolic and mitochondrial thioredoxin reductases are essential selenocysteine-containing enzymes that control thioredoxin functions. Thioredoxin/glutathione reductase (TGR) is a third member of this enzyme family. It has an additional glutaredoxin domain and shows highest expression in testes. Herein, we found that human and several other mammalian TGR genes lack any AUG codons that could function in translation initiation. Although mouse and rat TGRs have such codons, we detected protein sequences upstream of them by immunoblot assays and direct proteomic analyses. Further gene engineering and expression analyses demonstrated that a CUG codon, located upstream of the sequences previously thought to initiate translation, is the actual start codon in mouse TGR. The use of this codon relies on the Kozak consensus sequence and ribosome scanning mechanism. However, CUG serves as an inefficient start codon that allows downstream initiation, thus generating two isoforms of the enzyme in vivo and in vitro. The use of CUG evolved in mammalian TGRs, and, in some of these organisms, GUG is used instead. The newly discovered longer TGR form shows cytosolic localization in cultured cells and is expressed in spermatids in mouse testes. This study shows that CUG codon is used as an inefficient start codon to generate protein isoforms in mouse. More Information

Zhang Y, Gladyshev VN. (2010) General trends in trace element utilization revealed by comparative genomic analyses of Co, Cu, Mo, Ni and Se. J. Biol. Chem. 285, 3393-3405.

AbstractTrace elements are used by all organisms and provide proteins with unique coordination, catalytic, and electron transfer properties. Although many trace element-containing proteins are well characterized, little is known about the general trends in trace element utilization. We carried out comparative genomic analyses of copper, molybdenum, nickel, cobalt (in the form of vitamin B12) and selenium (in the form of selenocysteine) in 747 sequenced organisms at the level of (i) transporters and transport-related proteins, (ii) cofactor biosynthesis traits, and (iii) trace element-dependent proteins. Few organisms were found to utilize all five trace elements whereas many symbionts, parasites and yeasts used only one or none of these elements. Investigation of metalloproteomes and selenoproteomes revealed examples of increased utilization of proteins that use copper in land plants, cobalt in Dehalococcoides and Dictyostelium, and selenium in fish and algae, whereas nematodes were found to have great diversity of copper transporters. These analyses also characterized trace element metabolism in common model organisms and suggested new model organisms for experimental studies of individual trace elements. Mismatches in the occurrence of user proteins and corresponding transport systems revealed deficiencies in our understanding of trace element biology. Biological interactions among some trace elements were observed; however, such links were limited, and trace elements generally had unique utilization patterns. Finally, environmental factors, such as oxygen requirement and habitat, correlated with the utilization of certain trace elements. These data provide insights into the general features of utilization and evolution of trace elements in the three domains of life. More Information

Marino SM, Gladyshev VN. (2010) Structural analysis of cysteine S-nitrosylation: A modified acid-based motif and the emerging role of trans-nitrosylation. J. Mol. Biol. 395, 844-859.

AbstractS-nitrosylation, the selective and reversible addition of nitric oxide (NO) moiety to cysteine (Cys) sulfur in proteins, regulates numerous cellular processes. In recent years, proteomic approaches have been developed that are capable of identifying nitrosylated Cys residues. However, the features underlying specificity of Cys modification with NO remain poorly defined. Previous studies suggested that S-nitrosylated Cys may be flanked by an acid-base motif or hydrophobic areas, and show high reactivity, low pKa and high sulfur atom exposure. In the current study, we prepared an extensive, manually curated dataset of proteins with S-nitrosothiols, accounting for a variety of biochemical functions, organisms of origin and physiological responses to NO. Analysis of this generic NO-Cys dataset revealed that proximal acid-base motif, Cys pKa, sulfur atom exposure, Cys conservation or hydrophobicity in the vicinity of the modified Cys do not define the specificity of S-nitrosylation. Instead, this analysis revealed a revised acid-base motif, which is located more distantly to the Cys and has its charged groups exposed. We hypothesize that, rather than being strictly employed for direct activation of Cys, the modified acid-base motif is engaged in protein-protein interactions whereby contributing to trans-nitrosylation as an important and widespread mechanism for reversible modification of Cys with NO moiety. For proteins lacking the revised motif, we discuss alternative mechanisms including a potential role of nitrosoglutathione as a trans-acting agent. More Information

Bekaert M, Firth AE, Zhang Y, Gladyshev VN, Atkins JF, Baranov PV. (2010) Recode-2: new design, new search tools, and many more genes. Nucleic Acids Res. 38, D69-74.

AbstractRecoding is a term used to describe non-standard read-out of the genetic code, and encompasses such phenomena as programmed ribosomal frameshifting, stop codon readthrough, selenocysteine insertion and translational bypassing. Although only a small proportion of genes utilize recoding in protein synthesis, accurate annotation of recoded genes lags far behind annotation of standard genes. In order to address this issue, provide a service to researchers in the field, and offer training data for developers of gene-annotation software, we have gathered together known cases of recoding within the Recode database. Recode-2 is an improved and updated version of the database. It provides access to detailed information on genes known to utilize translational recoding and allows complex search queries, browsing of recoding data and enhanced visualization of annotated sequence elements. At present, the Recode-2 database stores information on approximately 1500 genes that are known to utilize recoding in their expression-a factor of approximately three increase over the previous version of the database. Recode-2 is available at http://recode.ucc.ie. More Information

2009 Articles

Sengupta A, Carlson BA, Labunskyy VM, Gladyshev VN, Hatfield DL. (2009) Selenoprotein T deficiency alters cell adhesion and elevates selenoprotein W expression in murine fibroblast cells. Biochem. Cell Biol. 87, 953-961.

AbstractMammalian selenoproteins have diverse functions, cellular locations, and evolutionary histories, but all use the amino acid selenocysteine (Sec), often present in the enzyme active site. Only about half of mammalian selenoproteins have been functionally characterized, with most being oxidoreductases. The cellular role of selenoprotein T (SelT), manifesting a CxxU motif in a thioredoxin-like fold and localized to Golgi and the endoplasmic reticulum, is not known. To examine its biological function, we knocked down SelT expression in mouse fibroblast cells and found that SelT deficiency alters cell adhesion and enhances the expression of several oxidoreductase genes, while decreasing the expression of genes involved in cell structure organization, suggesting the involvement of SelT in redox regulation and cell anchorage. Furthermore, we found that the loss of SelT elevates expression of another selenoprotein, selenoprotein W (SepW1). SelT and SepW1 belong to the same protein family, suggesting that SepW1 may functionally compensate for SelT. More Information

Carlson BA, Yoo MH, Sano Y, Sengupta A, Kim JY, Irons R, Gladyshev VN, Hatfield DL, Park JM. (2009) Selenoproteins regulate macrophage invasiveness and extracellular matrix-related gene expression. BMC Immunol. 10, 57.

AbstractBACKGROUND: Selenium, a micronutrient whose deficiency in diet causes immune dysfunction and inflammatory disorders, is thought to exert its physiological effects mostly in the form of selenium-containing proteins (selenoproteins). Incorporation of selenium into the amino acid selenocysteine (Sec), and subsequently into selenoproteins is mediated by Sec tRNA[Ser]Sec. RESULTS: To define macrophage-specific selenoprotein functions, we generated mice with the Sec tRNA[Ser]Sec gene specifically deleted in myeloid cells. These mutant mice were devoid of the selenoproteome in macrophages, yet exhibited largely normal inflammatory responses. However, selenoprotein deficiency led to aberrant expression of extracellular matrix-related genes, and diminished migration of macrophages in a protein gel matrix. CONCLUSIONS: Selenium status may affect immune defense and tissue homeostasis through its effect on selenoprotein expression and the trafficking of tissue macrophages. More Information

Jin BY, Sartoretto JL, Gladyshev VN, Michel T. (2009) Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells. Proc. Natl. Acad. Sci. USA 106, 17343-17348.

AbstractHydrogen peroxide and other reactive oxygen species are intimately involved in endothelial cell signaling. In many cell types, the AMP-activated protein kinase (AMPK) has been implicated in the control of metabolic responses, but the role of endothelial cell redox signaling in the modulation of AMPK remains to be completely defined. We used RNA interference and pharmacological methods to establish that H(2)O(2) is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAECs). H(2)O(2) treatment of BAECs rapidly and significantly increases the phosphorylation of AMPK. The EC(50) for H(2)O(2)-promoted phosphorylation of AMPK is 65 +/- 15 muM, within the physiological range of cellular H(2)O(2) concentrations. The Ca(2+)/calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) inhibitor STO-609 abolishes H(2)O(2)-dependent AMPK activation, whereas eNOS inhibitors enhance AMPK activation. Similarly, siRNA-mediated knockdown of CaMKKbeta abrogates AMPK activation, whereas siRNA-mediated knockdown of eNOS leads to a striking increase in AMPK phosphorylation. Cellular imaging studies using the H(2)O(2) biosensor HyPer show that siRNA-mediated eNOS knockdown leads to a marked increase in intracellular H(2)O(2) generation, which is blocked by PEG-catalase. eNOS(-/-) mice show a marked increase in AMPK phosphorylation in liver and lung compared to wild-type mice. Lung endothelial cells from eNOS(-/-) mice also show a significant increase in AMPK phosphorylation. Taken together, these results establish that CaMKKbeta is critically involved in mediating the phosphorylation of AMPK promoted by H(2)O(2) in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation and intracellular H(2)O(2) generation in endothelial cells. More Information

Zhang Y, Gladyshev VN. (2009) Comparative Genomics of Trace Elements: Emerging Dynamic View of Trace Element Utilization and Function. Chem. Rev. 109, 4828-4861.

AbstractNo abstract available. More Information

Carlson BA, Yoo MH, Tsuji PA, Gladyshev VN, Hatfield DL.(2009) Mouse models targeting selenocysteine tRNA expression for elucidating the role of selenoproteins in health and development. Molecules 14, 3509-3527.

AbstractSelenium (Se) deficiency has been known for many years to be associated with disease, impaired growth and a variety of other metabolic disorders in mammals. Only recently has the major role that Se-containing proteins, designated selenoproteins, play in many aspects of health and development begun to emerge. Se is incorporated into protein by way of the Se-containing amino acid, selenocysteine (Sec). The synthesis of selenoproteins is dependent on Sec tRNA for insertion of Sec, the 21(st) amino acid in the genetic code, into protein. We have taken advantage of this dependency to modulate the expression of Sec tRNA that in turn modulates the expression of selenoproteins by generating transgenic, conditional knockout, transgenic/standard knockout and transgenic/conditional knockout mouse models, all of which involve the Sec tRNA gene, to elucidate the intracellular roles of this protein class. More Information

Labunskyy VM, Yoo MH, Hatfield DL, Gladyshev VN. (2009) Sep15, a thioredoxin-like selenoprotein, is involved in the unfolded protein response and differentially regulated by adaptive and acute ER stresses. Biochemistry 48, 8458-8465.

AbstractThe accumulation of misfolded proteins in the endoplasmic reticulum (ER) results in activation of signaling pathways collectively known as the unfolded protein response (UPR). The UPR promotes adaptation of cells to ER stress by transient inhibition of protein translation and transcriptional up-regulation of genes encoding chaperones, oxidoreductases, and ER-associated degradation components. However, it may also trigger apoptosis in response to persistent ER stress. Recently, a novel selenocysteine-containing oxidoreductase, Sep15, has been reported to reside in the ER lumen. It has been proposed that this oxidoreductase may assist oxidative folding and structural maturation of N-glycosylated proteins targeted by UDP-glucose:glycoprotein glucosyltransferase, a chaperone implicated in quality control in the ER that forms a 1:1 complex with Sep15. To address the role of Sep15 in protein folding, we analyzed changes in Sep15 expression in murine fibroblast NIH3T3 cells in response to tunicamycin, brefeldin A (brefA), thapsigargin, and DTT that lead to accumulation of unfolded proteins within the ER. We show that expression of this protein is transcriptionally up-regulated in response to adaptive UPR caused by tunicamycin and brefA, whereas acute ER stress caused by DTT and thapsigargin leads to rapid and specific degradation of Sep15 by proteasomes. However, Sep15 deficiency did not result in detectable ER stress, consistent with the idea that Sep15 assists in the maturation of a restricted group of N-glycosylated proteins and/or that its function may be compensated by other mechanisms. More Information

Shchedrina VA, Vorbruggen G, Lee BC, Kim HY, Kabil H, Harshman LG, Gladyshev VN. (2009) Overexpression of methionine-R-sulfoxide reductases has no influence on fruit fly aging. Mech. Ageing Dev. 130, 429-443.

AbstractMethionine sulfoxide reductases (Msrs) are enzymes that repair oxidized methionine residues in proteins. This function implicated Msrs in antioxidant defense and the regulation of aging. There are two known Msr types in animals: MsrA specific for the reduction of methionine-S-sulfoxide, and MsrB that catalyzes the reduction of methionine-R-sulfoxide. In a previous study, overexpression of MsrA in the nervous system of Drosophila was found to extend lifespan by 70%. Overexpression of MsrA in yeast also extended lifespan, whereas MsrB overexpression did so only under calorie restriction conditions. The effect of MsrB overexpression on lifespan has not yet been characterized in any animal model systems. Here, the GAL4-UAS binary system was used to drive overexpression of cytosolic Drosophila MsrB and mitochondrial mouse MsrB2 in whole body, fatbody, and the nervous system of flies. In contrast to MsrA, MsrB overexpression had no consistent effect on the lifespan of fruit flies on both corn meal and sugar yeast diets. Physical activity, fecundity, and stress resistance were also similar in MsrB-overexpressing and control flies. Thus, MsrA and MsrB, the two proteins with identical function in antioxidant protein repair, have different effects on aging in fruit flies. More Information

Kaya A, Karakaya HC, Fomenko DE, Gladyshev VN, Koc A. (2009) Identification of a novel system for boron transport: Atr1 is a main boron exporter in yeast. Mol. Cell Biol. 29, 3665-3674.

AbstractBoron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to this element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated and they all contained the ATR1 (YML116w) gene. Atr1 is a member of multidrug resistance transport proteins belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1Delta mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron-resistant. In addition, atr1Delta cells accumulated boron, whereas ATR1 overexpressing cells had low intracellular levels of this element. Furthermore, atr1Delta cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance. More Information

Kehr S, Malinouski M, Finney L, Vogt S, Labunskyy VM, Kasaikina MV, Carlson BA, Zhou Y, Hatfield DL, Gladyshev VN. (2009) X-ray fluorescence microscopy reveals the role of selenium in spermatogenesis. J. Mol. Biol. 389, 808-18.

AbstractSelenium (Se) is a trace element with important roles in human health. Several selenoproteins have essential functions in development. However, the cellular and tissue distribution of Se remains largely unknown because of the lack of analytical techniques that image this element with sufficient sensitivity and resolution. Herein, wIn this worke report that X-ray fluorescence microscopy (XFM) can be used to visualize and quantify the tissue, cellular and subcellular topography of Se. We applied this technique to characterize the role of Se in spermatogenesis and identified a dramatic Se enrichment specifically in late spermatids, a pattern that was not seen in any other elemental maps. This enrichment was due to elevated levels of the mitochondrial form of glutathione peroxidase 4 and was fully dependent on the supplies of Se by Selenoprotein P. High-resolution scans revealed that Se concentrated near the lumen side of elongating spermatids, where structural components of sperm are formed. During spermatogenesis, maximal Se associated with decreased phosphorus, whereas Zn did not change. In sperm, Se was primarily in the midpiece and co-localized with Cu and Fe. XFM allowed quantification of Se in the midpiece (0.8 fg) and head (0.14 fg) of individual sperm cells, revealing the ability of sperm cells to handle the amounts of this element well above its toxic levels. Overall, the use of XFM allowed visualization of tissue and cellular Se and provided important insights in the role of this and other trace elements in spermatogenesis. More Information

Lobanov AV, Hatfield DL, Gladyshev VN. (2009) Eukaryotic Selenoproteins and Selenoproteomes. Biochim. Biophys. Acta. 1790, 1424-1428.

AbstractSelenium is an essential trace element for which both beneficial and toxic effects in human health have been described. It is now clear that the importance of having adequate amounts of this micronutrient in the diet is primarily due to the fact that selenium is required for biosynthesis of selenocysteine, the twenty first naturally occurring amino acid in protein. In this review, we provide an overview of eukaryotic selenoproteins and selenoproteomes, which are sets of selenoproteins in these organisms. In eukaryotes, selenoproteins show a mosaic occurrence, with some organisms, such as vertebrates and algae, having dozens of these proteins, while other organisms, such as higher plants and fungi, having lost all selenoproteins during evolution. We also discuss selenoprotein functions and evolutionary trends in the use of these proteins in eukaryotes. Functional analysis of selenoproteins is critical for better understanding of the role of selenium in human health and disease. More Information

Lee BC, Dikiy A, Kim HY, Gladyshev VN. (2009) Functions and evolution of selenoprotein methionine sulfoxide reductases. Biochim. Biophys. Acta. 1790, 1471-1477.

AbstractMethionine sulfoxide reductases (Msrs) are thiol-dependent enzymes which catalyze conversion of methionine sulfoxide to methionine. Three Msr families, MsrA, MsrB, and fRMsr, are known. MsrA and MsrBs are responsible for the reduction of methionine-S-sulfoxide and methionine-R-sulfoxide residues in proteins, respectively, whereas fRMsr reduces free methionine-R-sulfoxide. Besides acting on proteins, MsrA can additionally reduce free methionine-S-sulfoxide. Some MsrAs and MsrBs evolved to utilize catalytic selenocysteine. This includes MsrB1, which is a major MsrB in cytosol and nucleus in mammalian cells. Specialized machinery is used for insertion of selenocysteine into MsrB1 and other selenoproteins at in-frame UGA codons. Selenocysteine offers catalytic advantage to the protein repair function of Msrs, but also makes these proteins dependent on the supply of selenium and requires adjustments in their strategies for regeneration of active enzymes. Msrs have roles in protecting cellular proteins from oxidative stress and through this function they may regulate lifespan in several model organisms. More Information

Marino SM, Gladyshev VN. (2009) A structure-based approach for detection of thiol oxidoreductases and their catalytic redox-active cysteine residues. PLoS Comput. Biol. 5, e1000383, 1-13.

AbstractCysteine (Cys) residues often play critical roles in proteins, for example, in the formation of structural disulfide bonds, metal binding, targeting proteins to the membranes, and various catalytic functions. However, the structural determinants for various Cys functions are not clear. Thiol oxidoreductases, which are enzymes containing catalytic redox-active Cys residues, have been extensively studied, but even for these proteins there is little understanding of what distinguishes their catalytic redox Cys from other Cys functions. Herein, we characterized thiol oxidoreductases at a structural level and developed an algorithm that can recognize these enzymes by (i) analyzing amino acid and secondary structure composition of the active site and its similarity to known active sites containing redox Cys and (ii) calculating accessibility, active site location, and reactivity of Cys. For proteins with known or modeled structures, this method can identify proteins with catalytic Cys residues and distinguish thiol oxidoreductases from the enzymes containing other catalytic Cys types. Furthermore, by applying this procedure to Saccharomyces cerevisiae proteins containing conserved Cys, we could identify the majority of known yeast thiol oxidoreductases. This study provides insights into the structural properties of catalytic redox-active Cys and should further help to recognize thiol oxidoreductases in protein sequence and structure databases. More Information

Hatfield DL, Yoo MH, Carlson BA, Gladyshev VN. (2009) Selenoproteins that function in cancer prevention and promotion. Biochim. Biophys. Acta, 1790, 1541-1545.

AbstractOf the many health benefits attributed to selenium, the one that has received the most attention is its role in cancer prevention. Selenium-containing proteins (selenoproteins) have been shown in recent years to have roles in cancer prevention. However, selenoproteins have diverse functions and their view as antioxidants is oversimplified. Some selenoproteins appear to have a split personality in having roles both in preventing and promoting cancer. The contrasting roles of one selenoprotein, thioredoxin reductase 1, in cancer are discussed in detail, but as also noted, at least one other selenoprotein may also have such a dual function. In addition, we discuss examples of inhibition of cancer development by selenoprotein deficiency in mouse models. These studies highlight the complex nature of selenium in relation to cancer. More Information

Kim HY,Zhang Y, Lee BC, Kim JR, Gladyshev VN. (2009) The selenoproteome of Clostridium sp. OhlLAs: characterization of anaerobic bacterial selenoprotein methionine sulfoxide reductase A. Proteins 74, 1008-1017.

AbstractSelenocysteine (Sec) is incorporated into proteins in response to UGA codons. This residue is frequently found at the catalytic sites of oxidoreductases. In this study, we characterized the selenoproteome of an anaerobic bacterium, Clostridium sp. (also known as Alkaliphilus oremlandii) OhILA, and identified 13 selenoprotein genes, five of which have not been previously described. One of the detected selenoproteins was methionine sulfoxide reductase A (MsrA), an antioxidant enzyme that repairs oxidatively damaged methionines in a stereospecific manner. To date, little is known about MsrA from anaerobes. We characterized this selenoprotein MsrA which had a single Sec residue at the catalytic site but no cysteine (Cys) residues in the protein sequence. Its SECIS (Sec insertion sequence) element did not resemble those in Escherichia coli. Although with low translational efficiency, the expression of the Clostridium selenoprotein msrA gene in E. coli could be demonstrated by (75)Se metabolic labeling, immunoblot analyses, and enzyme assays, indicating that its SECIS element was recognized by the E. coli Sec insertion machinery. We found that the Sec-containing MsrA exhibited at least a 20-fold higher activity than its Cys mutant form, indicating a critical role of Sec in the catalytic activity of the enzyme. Furthermore, our data revealed that the Clostridium MsrA was inefficiently reducible by thioredoxin, which is a typical reducing agent for MsrA, suggesting the use of alternative electron donors in this anaerobic bacterium that directly act on the selenenic acid intermediate and do not require resolving Cys residues. More Information

Fomenko DE, Novoselov SV, Natarajan SK, Lee BC, Koc A, Carlson BA, Lee TH, Kim HY, Hatfield DL, Gladyshev VN. (2009) MsrB1 (Methionine-R-sulfoxide reductase 1) knockout mice: roles of MsrB1 in redox regulation and identification of a novel selenoprotein form. J. Biol. Chem. 284, 5986-5993.

AbstractProtein oxidation has been linked to accelerated aging and is a contributing factor to many diseases. Methionine residues are particularly susceptible to oxidation, but the resulting mixture of methionine-R-sulfoxide (Met-RO) and methionine-S-sulfoxide (Met-SO) can be repaired by thioredoxin-dependent enzymes MsrB and MsrA, respectively. Here, we describe a knockout mouse deficient in selenoprotein MsrB1, the main mammalian MsrB located in the cytosol and nucleus. In these mice, in addition to the deletion of 14 kDa MsrB1, a 5 kDa selenoprotein form was specifically removed. Further studies revealed that the 5 kDa protein occurred in both mouse tissues and human HEK 293 cells, was downregulated by MsrB1 siRNA, selenium deficiency and selenocysteine tRNA mutations, and was immunoprecipitated and recognized by MsrB1 antibodies. Specific labeling with 75Se and mass-spectrometry analyses revealed that the 5 kDa selenoprotein corresponded to the C-terminal sequence of MsrB1. The MsrB1 knockout mice lacked both 5 and 14 kDa MsrB1 forms and showed reduced MsrB activity with the strongest effect seen in liver and kidney. In addition, MsrA activity was decreased by MsrB1 deficiency. Liver and kidney of the MsrB1 knockout mice also showed increased levels of malondialdehyde, protein carbonyls, protein methionine sulfoxide and oxidized glutathione, as well as reduced levels of free and protein thiols, whereas these parameters were little changed in other organs examined. Overall, this study established an important contribution of MsrB1 to the redox control in mouse liver and kidney and identified a novel form of this protein. More Information

Carlson BA, Schweizer U, Perella C, Shrimali RK, Feigenbaum L, Shen L, Speransky S, Floss T, Jeong SJ, Watts J, Hoffmann V, Combs Jr GF, Gladyshev VN, Hatfield DL. (2009) The selenocysteine tRNA STAF-binding region is essential for adequate selenocysteine tRNA status, selenoprotein expression and early age survival of mice. Biochem. J. 418, 61-71.

AbstractSTAF is a transcription activating factor for a number of RNA Pol III- and RNA Pol II-dependent genes including the selenocysteine (Sec) tRNA gene, which in turn controls the expression of all selenoproteins. Here, the role of STAF in regulating expression of Sec tRNA and selenoproteins was examined. We generated transgenic mice expressing the Trsp transgene lacking the STAF binding site and made these mice dependent on the transgene for survival by removing the wild type Sec tRNA gene. The level of Sec tRNA was unaffected or slightly elevated in heart and testis, but reduced ~60% in liver and kidney, ~70% in lung and spleen, and ~80% in brain and muscle compared to the corresponding organs in control mice. Moreover, the ratio of the two isoforms of Sec tRNA that differ by methylation at position 34 (Um34) was altered significantly, and the Um34-containing form was substantially reduced in all tissues examined. Selenoprotein expression in these animals was most affected in tissues in which the Sec tRNA levels were most severely reduced. Importantly, mice had a neurological phenotype strikingly similar to that of mice in which the selenoprotein P gene had been removed and their lifespan was substantially reduced. The data indicate that STAF influences selenoprotein expression by enhancing Trsp synthesis in an organ-specific manner and by controlling Sec tRNA modification in each tissue examined. More Information

Le DT, Lee BC, Marino SM, Zhang Y, Fomenko DE, Kaya A, Hacioglu E, Kwak GH, Koc A, Kim HY, Gladyshev VN. (2009) Functional analysis of free methionine-R-sulfoxide reductase from Saccharomyces cerevisiae. J. Biol. Chem. 284, 4354-4364.

AbstractMethionine sulfoxide reductases (Msrs) are oxidoreductases that catalyze thiol-dependent reduction of oxidized methionines. MsrA and MsrB are the best known Msrs that repair methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO) residues in proteins, respectively. In addition, an Escherichia coli enzyme specific for free Met-R-SO, designated fRMsr, was recently discovered. In this work, we carried out comparative genomic and experimental analyses to examine occurrence, evolution and function of fRMsr. This protein is present in single copies and two mutually exclusive subtypes in about half of prokaryotes and unicellular eukaryotes, but is missing in higher plants and animals. A Saccharomyces cerevisiae fRMsr homolog was found to reduce free Met-R-SO, but not free Met-S-SO or dabsyl-Met-R-SO. fRMsr was responsible for growth of yeast cells on Met-R-SO, and the double fRMsr/MsrA mutant could not grow on a mixture of methionine sulfoxides. However, in the presence of methionine, even the triple fRMsr/MsrA/MsrB mutant was viable. In addition, fRMsr deletion strain showed an increased sensitivity to oxidative stress and a decreased lifespan, whereas overexpression of fRMsr conferred higher resistance to oxidants. Molecular modeling and cysteine residue targeting by thioredoxin pointed to Cys101 as catalytic and Cys125 as resolving residues in yeast fRMsr. These residues as well as the third Cys, resolving Cys91, clustered in the structure, and each was required for the catalytic activity of the enzyme. The data show that fRMsr is the main enzyme responsible for the reduction of free Met-R-SO in S. cerevisiae. More Information

Zhang Y, Rodionov DA, Gelfand MS, Gladyshev VN. (2009) Comparative genomic analyses of nickel, cobalt and vitamin B12 utilization. BMC Genomics 10, Feb 10;78.

AbstractNickel (Ni) and cobalt (Co) are trace elements required for a variety of biological processes. Ni is directly coordinated by proteins, whereas Co is mainly used as a component of vitamin B12. Although a number of Ni and Co-dependent enzymes have been characterized, systematic evolutionary analyses of utilization of these metals are limited. RESULTS: We carried out comparative genomic analyses to examine occurrence and evolutionary dynamics of the use of Ni and Co at the level of (i) transport systems, and (ii) metalloproteomes. Our data show that both metals are widely used in bacteria and archaea. Cbi/NikMNQO is the most common prokaryotic Ni/Co transporter, while Ni-dependent urease and Ni-Fe hydrogenase, and B12-dependent methionine synthase (MetH), ribonucleotide reductase and methylmalonyl-CoA mutase, are the most widespread metalloproteins for Ni and Co, respectively. Occurrence of other metalloenzymes showed a mosaic distribution and a new B12-dependent protein family was predicted. Deltaproteobacteria and Methanosarcina generally have larger Ni- and Co-dependent proteomes. On the other hand, utilization of these two metals is limited in eukaryotes, and very few of these organisms utilize both of them. The Ni-utilizing eukaryotes are mostly fungi (except saccharomycotina) and plants, whereas most B12-utilizing organisms are animals. The NiCoT transporter family is the most widespread eukaryotic Ni transporter, and eukaryotic urease and MetH are the most common Ni- and B12-dependent enzymes, respectively. Finally, investigation of environmental and other conditions and identity of organisms that show dependence on Ni or Co revealed that host-associated organisms (particularly obligate intracellular parasites and endosymbionts) have a tendency for loss of Ni/Co utilization. CONCLUSIONS: Our data provide information on the evolutionary dynamics of Ni and Co utilization and highlight widespread use of these metals in the three domains of life, yet only a limited number of user proteins. More Information

Hatfield DL, Gladyshev VN. (2009) The Outcome of Selenium and Vitamin E Cancer Prevention Trial (SELECT) Reveals the Need for Better Understanding of Selenium Biology. Mol. Interv. 9, 18-21.

AbstractThe recently completed Selenium and Vitamin E Cancer Prevention Trial (SELECT) was one of the largest human cancer prevention trials ever undertaken. Its purpose was to assess the role of selenium and vitamin E in prostate cancer prevention, but SELECT found no decline in prostate cancer. Comparison of this study to other clinical trials involving selenium and to the results of animal studies suggests that the source of the selenium supplement, L-selenomethionine, and the relatively high initial levels of selenium in the enrolled men may have contributed to this outcome. Further analysis of the clinical and animal data highlights the need for mechanistic studies to better understand selenium biology in order to target dietary selenium to appropriate subsets of the human population: those individuals most likely to benefit from this micronutrient. More Information

Turanov AA, Lobanov AV, Fomenko DE, Morrison HG, Sogin ML, Klobutcher LA, Hatfield DL, Gladyshev VN. (2009) Genetic code supports targeted insertion of two amino acids by one codon. Science 323, 259-261.

AbstractStrict one-to-one correspondence between codons and amino acids is thought to be an essential feature of the genetic code. However, we report that one codon can code for two different amino acids with the choice of the inserted amino acid determined by a specific 3′ untranslated region structure and location of the dual-function codon within the messenger RNA (mRNA). We found that the codon UGA specifies insertion of selenocysteine and cysteine in the ciliate Euplotes crassus, that the dual use of this codon can occur even within the same gene, and that the structural arrangements of Euplotes mRNA preserve location-dependent dual function of UGA when expressed in mammalian cells. Thus, the genetic code supports the use of one codon to code for multiple amino acids. More Information

Hirosawa-Takamori M, Ossipov D, Novoselov SV, Turanov AA, Zhang Y, Gladyshev VN, Krol A, Vorbruggen G, Jackle H. (2009) A novel stem loop control element-dependent UGA read-through system without translational selenocysteine incorporation in Drosophila. FASEB J. 23, 107-113.

AbstractTranslational read-through of the UGA stop codon is an evolutionarily conserved feature that most prominently represents the basis of selenoprotein biosynthesis. It requires a specific cis-acting stem loop control element, termed SECIS, which is located in the 3;-untranslated region of eukaryotic selenoprotein mRNAs. In a search for novel factors underlying the SECIS-directed UGA read-through process, we identified an evolutionary conserved GTPase-activating protein, termed GAPsec. We show that the activity of the Drosophila GAPsec (dGAPsec) is necessary to support SECIS-dependent UGA read-through activity in flies and the mouse homolog mGAPsec in mice tissue culture cells. However, selenoprotein biosynthesis is not impaired in flies that lack dGAPsec activity. The results indicate that GAPsec is part of a novel SECIS-dependent translational read-through system that does not involve selenocysteine incorporation.-Hirosawa-Takamori, M, Ossipov, D, Novoselov, SV, Turanov, AA, Zhang, Y, Gladyshev, VN, Krol, A, Vorbruggen, G, Jackle, H. A novel stem loop control element-dependent UGA read-through system without translational selenocysteine incorporation in Drosophila. More Information

Xu XM, Yoo MH, Carlson BA, Gladyshev VN, Hatfield DL. (2009) Simultaneous knockdown of the expression of two genes using multiple shRNAs and subsequent knock-in of their expression. Nat. Protoc. 4, 1338-1348.

AbstractSmall hairpin RNA (shRNA) is a powerful tool for inhibiting gene expression. One limitation has been that this technique has been used primarily to target a single gene. This protocol expands upon previous methods by describing a knockdown vector that facilitates cloning of multiple shRNAs; this allows targeted knockdown of more than one gene or of a single gene that may otherwise be difficult to knockdown using a single shRNA. The targeted gene(s) can be readily re-expressed by transfecting knockdown cells with a knock-in vector, containing an shRNA-refractive cDNA that will express the protein-of-interest even in the presence of shRNAs. The constructed knockdown and knock-in vectors can be easily used concurrently to assess possible interrelationships between genes, the effects of gene loss on cell function and/or their restoration by replacing targeted genes one at a time. The entire knockdown or knock-in procedure can be completed in approximately 3-4 months. More Information

2008 Articles

Papp LV, Wang J, Kennedy D, Boucher D, Zhang Y, Gladyshev VN, Singh RN, Khanna KK. (2008) Functional characterization of alternatively spliced human SECISBP2 transcript variants. Nucleic Acids Res. 36, 7192-7206.

AbstractSynthesis of selenoproteins depends on decoding of the UGA stop codon as the amino acid selenocysteine (Sec). This process requires the presence of a Sec insertion sequence element (SECIS) in the 3-untranslated region of selenoprotein mRNAs and its interaction with the SECIS binding protein 2 (SBP2). In humans, mutations in the SBP2-encoding gene Sec insertion sequence binding protein 2 (SECISBP2) that alter the amino acid sequence or cause splicing defects lead to abnormal thyroid hormone metabolism. Herein, we present the first in silico and in vivo functional characterization of alternative splicing of SECISBP2. We report a complex splicing pattern in the 5-region of human SECISBP2, wherein at least eight splice variants encode five isoforms with varying N-terminal sequence. One of the isoforms, mtSBP2, contains a mitochondrial targeting sequence and localizes to mitochondria. Using a minigene-based in vivo splicing assay we characterized the splicing efficiency of several alternative transcripts, and show that the splicing event that creates mtSBP2 can be modulated by antisense oligonucleotides. Moreover, we show that full-length SBP2 and some alternatively spliced variants are subject to a coordinated transcriptional and translational regulation in response to ultraviolet type A irradiation-induced stress. Overall, our data broadens the functional scope of a housekeeping protein essential to selenium metabolism. More Information

Lee BC, Le DT, Gladyshev VN. (2008) Mammals reduce methionine-S-sulfoxide with MsrA, are unable to reduce methionine-R-sulfoxide, and this function can be restored with a yeast reductase. J. Biol. Chem. 283, 28361-28369.

AbstractMethionine is an essential amino acid in mammals at the junction of methylation, protein synthesis and sulfur pathways. However, this amino acid is highly susceptible to oxidation, resulting in a mixture of methionine-S-sulfoxide and methionine-R-sulfoxide. Whether methionine is quantitatively regenerated from these compounds is unknown. Here we report that SK-Hep1 hepatocytes grew on methionine-S-sulfoxide and consumed this compound by import and methionine-S-sulfoxide reductase (MsrA)-dependent reduction, but methionine-R-sulfoxide reductases (MsrBs) were not involved in this process and methionine-R-sulfoxide could not be used by the cells. However, SK-Hep1 cells expressing a yeast free methionine-R-sulfoxide reductase (fRMsr) proliferated in the presence of either sulfoxide, reduced them and showed increased resistance to oxidative stress. Only methionine-R-sulfoxide was detected in plasma of wild-type mice, but both sulfoxides in plasma of MsrA knockout mice. These results show that mammals can support methionine metabolism by reduction of methionine-S-sulfoxide, that this process is dependent on MsrA, that mammals are inherently deficient in the reduction of methionine-R-sulfoxide, and that expression of yeast fRMsr can fully compensate for this deficiency. More Information

Fomenko DE, Marino SM, Gladyshev VN. (2008) Functional Diversity of Cysteine Residues in Proteins and Unique Features of Catalytic Redox-Active Cysteines in Thiol Oxidoreductases. Mol. Cells 26, 228-235.

AbstractThiol-dependent redox systems are involved in regulation of diverse biological processes, such as response to stress, signal transduction, and protein folding. The thiol-based redox control is provided by mechanistically similar, but structurally distinct families of enzymes known as thiol oxidoreductases. Many such enzymes have been characterized, but identities and functions of the entire sets of thiol oxidoreductases in organisms are not known. Extreme sequence and structural divergence makes identification of these proteins difficult. All thiol oxidoreductases contain a redox-active cysteine residue, or its functional analog selenocysteine, in their active sites. Here, we describe computational methods for in silico prediction of thiol oxidoreductases in nucleotide and protein sequence databases and identification of their redox-active cysteines. We discuss different functional categories of cysteine residues, describe methods for discrimination between catalytic and noncatalytic and between redox and non-redox cysteine residues and highlight unique properties of the redox-active cysteines based on evolutionary conservation, secondary and three-dimensional structures, and sporadic replacement of cysteines with catalytically superior selenocysteine residues. More Information

Shrimali RK, Irons RD, Carlson BA, Sano Y, Gladyshev VN, Park JM, Hatfield DL. (2008) Selenoproteins mediate T cell immunity through an antioxidant mechanism. J. Biol. Chem. 283, 20181-20185.

AbstractSelenium is an essential dietary element with antioxidant roles in immune regulation, but there is little understanding of how this element acts at the molecular level in host defense and inflammatory disease. Selenium is incorporated into the amino acid selenocysteine (Sec), which in turn is inserted into selenoproteins in a manner dependent on Sec tRNA([Ser]Sec). To investigate the molecular mechanism that links selenium to T cell immunity, we generated mice with selenoprotein-less T cells by cell type-specific ablation of the Sec tRNA([Ser]Sec) gene (trsp). Herein, we show that these mutant mice exhibit decreased pools of mature T cells and a defect in T cell-dependent antibody responses. We also demonstrate that selenoprotein deficiency leads to oxidant hyperproduction in T cells and thereby suppresses T cell proliferation in response to T cell receptor stimulation. These findings offer novel insights into immune function of selenium and physiological antioxidants. More Information

Sengupta A, Carlson BA, Weaver JA, Novoselov SV, Fomenko DE, Gladyshev VN, Hatfield DL. (2008) A functional link between housekeeping selenoproteins and phase II enzymes. Biochem. J. 413, 151-161.

AbstractSec (selenocysteine) is biosynthesized on its tRNA and incorporated into selenium-containing proteins (selenoproteins) as the 21st amino acid residue. Selenoprotein synthesis is dependent on Sec tRNA and the expression of this class of proteins can be modulated by altering Sec tRNA expression. The gene encoding Sec tRNA (Trsp) is a single-copy gene and its targeted removal in liver demonstrated that selenoproteins are essential for proper function wherein their absence leads to necrosis and hepatocellular degeneration. In the present study, we found that the complete loss of selenoproteins in liver was compensated for by an enhanced expression of several phase II response genes and their corresponding gene products. The replacement of selenoprotein synthesis in mice carrying mutant Trsp transgenes, wherein housekeeping, but not stress-related selenoproteins are expressed, led to normal expression of phase II response genes. Thus the present study provides evidence for a functional link between housekeeping selenoproteins and phase II enzymes. More Information

Bonilla M, Denicola A, Novoselov SV, Turanov AA, Protasio A, Izmendi D, Gladyshev VN, Salinas G. (2008) Platyhelminth mitochondrial and cytosolic redox homeostasis is controlled by a single thioredoxin glutathione reductase and dependent on selenium and glutathione. J. Biol. Chem. 283, 17898-17907.

AbstractPlatyhelminth parasites are a major health problem in developing countries. In contrast to their mammalian hosts, platyhelminth thiol-disulfide redox homeostasis relies on linked thioredoxin-glutathione systems, which are fully dependent on thioredoxin-glutathione reductase (TGR), a promising drug target. TGR is a homodimeric enzyme comprising a glutaredoxin domain and thioredoxin reductase (TR) domains with a C-terminal redox center containing selenocysteine (Sec). In this study, we demonstrate the existence of functional linked thioredoxin-glutathione systems in the cytosolic and mitochondrial compartments of Echinococcus granulosus, the platyhelminth responsible for hydatid disease. The glutathione reductase (GR) activity of TGR exhibited hysteretic behavior regulated by the [GSSG]/[GSH] ratio. This behavior was associated with glutathionylation by GSSG and abolished by deglutathionylation. The K(m) and k(cat) values for mitochondrial and cytosolic thioredoxins (9.5 microm and 131 s(-1), 34 microm and 197 s(-1), respectively) were higher than those reported for mammalian TRs. Analysis of TGR mutants revealed that the glutaredoxin domain is required for the GR activity but did not affect the TR activity. In contrast, both GR and TR activities were dependent on the Sec-containing redox center. The activity loss caused by the Sec-to-Cys mutation could be partially compensated by a Cys-to-Sec mutation of the neighboring residue, indicating that Sec can support catalysis at this alternative position. Consistent with the essential role of TGR in redox control, 2.5 microm auranofin, a known TGR inhibitor, killed larval worms in vitro. These studies establish the selenium- and glutathione-dependent regulation of cytosolic and mitochondrial redox homeostasis through a single TGR enzyme in platyhelminths. More Information

Zhang Y, Gladyshev VN. (2008) Molybdoproteomes and evolution of molybdenum utilization. J. Mol. Biol. 379, 881-899.

AbstractThe trace element molybdenum (Mo) is utilized in many life forms, and it is a key component of several enzymes involved in nitrogen, sulfur, and carbon metabolism. With the exception of nitrogenase, Mo is bound in proteins to a pterin, thus forming the molybdenum cofactor (Moco) at the catalytic sites of molybdoenzymes. Although a number of molybdoenzymes are well characterized structurally and functionally, evolutionary analyses of Mo utilization are limited. Here, we carried out comparative genomic and phylogenetic analyses to examine the occurrence and evolution of Mo utilization in bacteria, archaea and eukaryotes at the level of (i) Mo transport and Moco utilization trait, and (ii) Mo-dependent enzymes. Our results revealed that most prokaryotes and all higher eukaryotes utilize Mo whereas many unicellular eukaryotes including parasites and most yeasts lost the ability to use this metal. In addition, eukaryotes have fewer molybdoenzyme families than prokaryotes. Dimethylsulfoxide reductase (DMSOR) and sulfite oxidase (SO) families were the most widespread molybdoenzymes in prokaryotes and eukaryotes, respectively. A distant group of the ModABC transport system, was predicted in the hyperthermophilic archaeon Pyrobaculum. ModE-type regulation of Mo uptake occurred in less than 30% of Moco-utilizing organisms. A link between Mo and selenocysteine utilization in prokaryotes was also identified wherein the selenocysteine trait was largely a subset of the Mo trait, presumably due to formate dehydrogenase, a Mo- and selenium-containing protein. Finally, analysis of environmental conditions and organisms that do or do not depend on Mo revealed that host-associated organisms and organisms with low G+C content tend to reduce their Mo utilization. Overall, our data provide new insights into Mo utilization and show its wide occurrence, yet limited use of this metal in individual organisms in all three domains of life. More Information

Zhang Y, Gladyshev VN. (2008) Trends in selenium utilization in marine microbial world revealed through the analysis of the global ocean sampling (GOS) project. PLoS Genet. 4, e1000095, 1-20.

AbstractSelenium is an important trace element that occurs in proteins in the form of selenocysteine (Sec) and in tRNAs in the form of selenouridine. Recent large-scale metagenomics projects provide an opportunity for understanding global trends in trace element utilization. Herein, we characterized the selenoproteome of the microbial marine community derived from the Global Ocean Sampling (GOS) expedition. More than 3,600 selenoprotein gene sequences belonging to 58 protein families were detected, including sequences representing 7 newly identified selenoprotein families, such as homologs of ferredoxin-thioredoxin reductase and serine protease. In addition, a new eukaryotic selenoprotein family, thiol reductase GILT, was identified. Most GOS selenoprotein families originated from Cys-containing thiol oxidoreductases. In both Pacific and Atlantic microbial communities, SelW-like and SelD were the most widespread selenoproteins. Geographic location had little influence on Sec utilization as measured by selenoprotein variety and the number of selenoprotein genes detected; however, both higher temperature and marine (as opposed to freshwater and other aquatic) environment were associated with increased use of this amino acid. Selenoproteins were also detected with preference for either environment. We identified novel fusion forms of several selenoproteins that highlight redox activities of these proteins. Almost half of Cys-containing SelDs were fused with NADH dehydrogenase, whereas such SelD forms were rare in terrestrial organisms. The selenouridine utilization trait was also analyzed and showed an independent evolutionary relationship with Sec utilization. Overall, our study provides insights into global trends in microbial selenium utilization in marine environments. More Information

See commentary in: Eisen JA. (2008) Open Metagenomics: Selenium in the Oceans. Commentary

Zhang Y, Turanov AA, Hatfield DL, Gladyshev VN. (2008) In silico identification of genes involved in selenium metabolism: evidence for a third selenium utilization trait. BMC Genomics 9, 251.

AbstractBACKGROUND: Selenium (Se) is a trace element that occurs in proteins in the form of selenocysteine (Sec) and in tRNAs in the form of selenouridine (SeU). Selenophosphate synthetase (SelD) is required for both utilization traits. However, previous research also revealed SelDs in two organisms lacking Sec and SeU, suggesting a possible additional use of Se that is dependent on SelD. RESULTS: In this study, we conducted comparative genomics and phylogenetic analyses to characterize genes involved in Se utilization. Candidate genes identified included SelA/SelB and YbbB that define Sec and SeU pathways, respectively, and NADH oxidoreductase that is predicted to generate a SelD substrate. In addition, among 227 organisms containing SelD, 10 prokaryotes were identified that lacked SelA/SelB and YbbB. Investigation of selD neighboring genes in these organisms revealed a SirA-like protein and two hypothetical proteins HP1 and HP2 that were strongly linked to a novel Se utilization. With these new signature proteins, 32 bacteria and archaea were found that utilized these proteins, likely as part of the new Se utilization trait. Metabolic labeling of one organism containing an orphan SelD, Enterococcus faecalis, with 75Se revealed a protein containing labile Se species that could be released by treatment with reducing agents, suggesting non-Sec utilization of Se in this organism. CONCLUSION: These studies suggest the occurrence of a third Se utilization trait in bacteria and archaea. More Information

Le DT, Liang X, Fomenko DE, Raza AS, Chong CK, Carlson BA, Hatfield DL, Gladyshev VN. (2008) Analysis of Methionine/Selenomethionine Oxidation and Methionine Sulfoxide Reductase Function Using Methionine-Rich Proteins and Antibodies against Their Oxidized Forms. Biochemistry 47, 6685-6694.

AbstractMethionine (Met) residues are present in most proteins. However, this sulfur-containing amino acid is highly susceptible to oxidation. In cells, the resulting Met sulfoxides are reduced back to Met by stereospecific reductases MsrA and MsrB. Reversible Met oxidation occurs even in the absence of stress, is elevated during aging and disease, but is notoriously difficult to monitor. In this work, we computationally identified natural Met-rich proteins (MRPs) and characterized three such proteins containing 21-33% Met residues. Oxidation of multiple Met residues in MRPs with H 2O 2 and reduction of Met sulfoxides with MsrA/MsrB dramatically influenced the mobility of these proteins on polyacrylamide gels and could be monitored by simple SDS-PAGE. We further prepared antibodies enriched for reduced and Met sulfoxide forms of these proteins and used them to monitor Met oxidation and reduction by immunoblot assays. We describe applications of these reagents for the analysis of MsrA and MsrB functions, as well as the development of the assay for high-throughput analysis of their activities. We also show that all Met sulfoxide residues in an MRP can be reduced by MsrA and MsrB. Furthermore, we prepared a selenomethionine form of an MRP and found that selenomethionine selenoxide residues can be efficiently reduced nonenzymatically by glutathione and other thiol compounds. Selenomethionine selenoxide residues were not recognized by antibodies specific for the Met sulfoxide form of an MRP. These findings, reagents, assays, and approaches should facilitate research and applications in the area of Met sulfoxide reduction, oxidative stress, and aging. More Information

Ganichkin OM, Xu XM, Carlson BA, Mix H, Hatfield DL, Gladyshev VN, Wahl MC. (2008) Structure and catalytic mechanism of eukaryotic selenocysteine synthase. J. Biol. Chem. 283, 5849-5865.

AbstractIn eukaryotes and Archaea, selenocysteine synthase (SecS) converts O-phospho-L-seryl-tRNA [Ser]Sec into selenocysteyl-tRNA [Ser]Sec using selenophosphate as the selenium donor compound. The molecular mechanisms underlying SecS activity are presently unknown. We have delineated a 450-residue core of mouse SecS, which retained full selenocysteyl-tRNA [Ser]Sec synthesis activity, and determined its crystal structure at 1.65 A resolution. SecS exhibits three domains that place it in the fold type I family of pyridoxal phosphate (PLP)-dependent enzymes. Two SecS monomers interact intimately and together build up two identical active sites around PLP in a Schiff-base linkage with lysine 284. Two SecS dimers further associate to form a homotetramer. The N terminus, which mediates tetramer formation, and a large insertion that remodels the active site set SecS aside from other members of the family. The active site insertion contributes to PLP binding and positions a glutamate next to the PLP, where it could repel substrates with a free alpha-carboxyl group, suggesting why SecS does not act on free O-phospho-l-serine. Upon soaking crystals in phosphate buffer, a previously disordered loop within the active site insertion contracted to form a phosphate binding site. Residues that are strictly conserved in SecS orthologs but variant in related enzymes coordinate the phosphate and upon mutation corrupt SecS activity. Modeling suggested that the phosphate loop accommodates the gamma-phosphate moiety of O-phospho-l-seryl-tRNA [Ser]Sec and, after phosphate elimination, binds selenophosphate to initiate attack on the proposed aminoacrylyl-tRNA [Ser]Sec intermediate. Based on these results and on the activity profiles of mechanism-based inhibitors, we offer a detailed reaction mechanism for the enzyme. More Information

Zhang Y, Zhou Y, Schweizer U, Savaskan NE, Hua D, Kipnis J, Hatfield DL, Gladyshev VN. (2008) Comparative analysis of selenocysteine machinery and selenoproteome gene expression in mouse brain identifies neurons as key functional sites of selenium in mammals. J. Biol. Chem. 283, 2427-2438.

AbstractAlthough dietary selenium (Se) deficiency results in phenotypes associated with selenoprotein depletion in various organs, the brain is protected from Se loss. To address the basis for the critical role of Se in brain function, we carried out comparative gene expression analyses for the complete selenoproteome and associated biosynthetic factors. Using the Allen Brain Atlas, we evaluated 159 regions of adult mouse brain and provided experimental analyses of selected selenoproteins. All 24 selenoprotein mRNAs were expressed in the mouse brain. Most strikingly, neurons in olfactory bulb, hippocampus, cerebral cortex, and cerebellar cortex were exceptionally rich in selenoprotein gene expression, in particular in GPx4, SelK, SelM, SelW, and Sep15. Over half of the selenoprotein genes were also expressed in the choroid plexus. A unique expression pattern was observed for one of the highly expressed selenoprotein genes, SelP, which we suggest to provide neurons with Se. Cluster analysis of the expression data linked certain selenoproteins and selenocysteine machinery genes and suggested functional linkages among selenoproteins, such as that between SelM and Sep15. Overall, this study suggests that the main functions of selenium in mammals are confined to certain neurons in the brain. More Information

Sengupta A, Carlson BA, Hoffmann VJ, Gladyshev VN, Hatfield DL. (2008) Loss of housekeeping selenoprotein expression in mouse liver modulates lipoprotein metabolism. Biochem. Biophys. Res. Commun. 365, 446-452.

AbstractSelenium is incorporated into proteins as selenocysteine (Sec), which is dependent on its specific tRNA, designated tRNA([Ser]Sec). Targeted removal of the tRNA([Ser]Sec) gene (Trsp) in mouse hepatocytes previously demonstrated the importance of selenoproteins in liver function. Herein, analysis of plasma proteins in this Trsp knockout mouse revealed increases in apolipoprotein E (ApoE) that was accompanied by elevated plasma cholesterol levels. The expression of genes involved in cholesterol biosynthesis, metabolism and transport were also altered in knockout mice. Additionally, in two transgenic Trsp mutant mouse lines (wherein only housekeeping selenoprotein synthesis was restored), the expression of ApoE, as well as genes involved in cholesterol biosynthesis, metabolism and transport were similar to those observed in wild type mice. These data correlate with reports that selenium deficiency results in increased levels of ApoE, indicating for the first time that housekeeping selenoproteins have a role in regulating lipoprotein biosynthesis and metabolism. More Information

Ridge PG, Zhang Y, Gladyshev VN. (2008) Comparative Genomic Analyses of Copper Transporters and Cuproproteomes Reveal Evolutionary Dynamics of Copper Utilization and Its Link to Oxygen. PLOS ONE 3, e1378, 1-9.

AbstractCopper is an essential trace element in many organisms and is utilized in all domains of life. It is often used as a cofactor of redox proteins, but is also a toxic metal ion. Intracellular copper must be carefully handled to prevent the formation of reactive oxygen species which pose a threat to DNA, lipids, and proteins. In this work, we examined patterns of copper utilization in prokaryotes by analyzing the occurrence of copper transporters and copper-containing proteins. Many organisms, including those that lack copper-dependent proteins, had copper exporters, likely to protect against copper ions that inadvertently enter the cell. We found that copper use is widespread among prokaryotes, but also identified several phyla that lack cuproproteins. This is in contrast to the use of other trace elements, such as selenium, which shows more scattered and reduced usage, yet larger selenoproteomes. Copper transporters had different patterns of occurrence than cuproproteins, suggesting that the pathways of copper utilization and copper detoxification are independent of each other. We present evidence that organisms living in oxygen-rich environments utilize copper, whereas the majority of anaerobic organisms do not. In addition, among copper users, cuproproteomes of aerobic organisms were larger than those of anaerobic organisms. Prokaryotic cuproproteomes were small and dominated by a single protein, cytochrome c oxidase. The data are consistent with the idea that proteins evolved to utilize copper following the oxygenation of the Earth. More Information

Lobanov AV, Hatfield DL, Gladyshev VN. (2008) Reduced reliance on the trace element selenium during evolution of mammals. Genome Biol. 9, R62.

AbstractBACKGROUND: Selenium (Se) is an essential trace element that occurs in proteins in the form of selenocysteine (Sec). It is transported throughout the body in the form of Sec residues in Selenoprotein P (SelP), a plasma protein of unclear origin recently proposed as an experimental marker of dietary Se status. RESULTS: Here, we report that the amino-terminal domain of SelP is distantly related to ancestral bacterial thiol oxidoreductases of the thioredoxin superfamily, and that its carboxy-terminal Se transport domain may have originated in early metazoan evolution by de novo accumulation of Sec residues. Reconstruction of evolutionary changes in the Se transport domain indicates a decrease in Sec content of SelP specifically in the mammalian lineage via replacement of Sec with cysteine (Cys). Sec content of mammalian SelPs varies more than two-fold and is lowest in rodents and primates. Compared to mammals, fish show higher Sec content of SelP, larger selenoproteomes, elevated SelP gene expression, and higher levels of tissue Se. In addition, mammals replaced Sec with Cys in several proteins and lost several selenoproteins altogether, whereas such events are not found in fish. CONCLUSION: These data suggest that evolution from fish to mammals was accompanied by decreased use of Sec and that analyses of SelP, selenoproteomes and Sec/Cys transitions provide a genetic marker of utilization of this trace element in vertebrates. The evolved reduced reliance on Se raises questions regarding the need to maximize selenoprotein expression by Se dietary supplements in situations when pathology is not imminent, a currently accepted practice. More Information

Castellano S, Gladyshev VN, Guigó R, Berry MJ. (2008) SelenoDB 1.0 : a database of selenoprotein genes, proteins and SECIS elements. Nucleic Acids Res. 36, D332-338.

AbstractSelenoproteins are a diverse group of proteins usually misidentified and misannotated in sequence databases. The presence of an in-frame UGA (stop) codon in the coding sequence of selenoprotein genes precludes their identification and correct annotation. The in-frame UGA codons are recoded to cotranslationally incorporate selenocysteine, a rare selenium-containing amino acid. The development of ad hoc experimental and, more recently, computational approaches have allowed the efficient identification and characterization of the selenoproteomes of a growing number of species. Today, dozens of selenoprotein families have been described and more are being discovered in recently sequenced species, but the correct genomic annotation is not available for the majority of these genes. SelenoDB is a long-term project that aims to provide, through the collaborative effort of experimental and computational researchers, automatic and manually curated annotations of selenoprotein genes, proteins and SECIS elements. Version 1.0 of the database includes an initial set of eukaryotic genomic annotations, with special emphasis on the human selenoproteome, for immediate inspection by selenium researchers or incorporation into more general databases. SelenoDB is freely available at http://www.selenodb.org. More Information

Lobanov AV, Hatfield DL, Gladyshev VN. (2008) Selenoproteinless animals: Selenophosphate synthetase SPS1 functions in a pathway unrelated to selenocysteine biosynthesis. Protein Sci. 17, 176-182.

AbstractProteins containing the 21st amino acid, selenocysteine (Sec), have been described in all three domains of life, but the composition of selenoproteomes in organisms varies significantly. Here, we report that aquatic arthropods possess many selenoproteins also detected in other animals and unicellular eukaryotes, and that most of these proteins were either lost or replaced with cysteine-containing homologs in insects. As a result of this selective selenoproteome reduction, fruit flies and mosquitoes have three known selenoproteins, and the honeybee, Apis mellifera, a single detected candidate selenoprotein. Moreover, we identified the red flour beetle, Tribolium castaneum, and the silkworm, Bombyx mori, as the first animals that lack any Sec-containing proteins. These insects also lost the Sec biosynthesis and insertion machinery, but selenophosphate synthetase 1 (SPS1), an enzyme previously implicated in Sec biosynthesis, is present in all insects, including T. castaneum and B. mori. These data indicate that SPS1 functions in a pathway unrelated to selenoprotein synthesis. Since SPS1 evolved from a protein that utilizes selenium for Sec biosynthesis, an attractive possibility is that SPS1 may define a new pathway of selenium utilization in animals. More Information

Glass RS, Berry MJ, Block E, Boakye HT, Carlson BA, Gailer J, George GN, Gladyshev VN, Hatfield DL, Jacobsen NE, Johnson S, Kahakachchi C, Kaminski R, Manley SA, Mix H, Pickering IJ, Prenner EJ, Saira K, Skowronska A, Tyson JF, Uden PC, Wu Q, Xu X-M, Yamdagni R, Zhang Y. (2008) Insights into the Chemical Biology of Selenium. Phosphorus, Sulfur, and Silicon and the Related Elements 183, 924-930.

AbstractThe long-sought pathway by which selenocysteyl-tRNA[Ser]Sec is synthesized in eukaryotes has been revealed. Seryl-tRNA[Ser]Sec is O-phosphorylated and SecS, a pyridoxal phosphate-dependent protein, catalyzes the reaction of O-phosphoseryltRNA[Ser]Sec with monoselenophosphate to give selenocysteyl-tRNA[Ser]Sec . 1H-{77Se} HMQC-TOCSY NMR spectroscopy has been developed to detect the selenium-containing amino acids present in selenized yeast after protease XIV digestion. An archived selenized yeast sample is found to contain the novel amino acid S-(methylseleno)cysteine in addition to selenomethionine. Arsenite and selenite react with GSH to form (GS)2AsSe-. The structure of this compound has been determined by EXAFS, 77Se NMR and Raman spectroscopic and chromatographic studies. Its formation under biological conditions has been demonstrated. More Information

2007 Articles

Yoo MH, Xu XM, Carlson BA, Patterson AD, Gladyshev VN, Hatfield DL. (2007) Targeting thioredoxin reductase 1 reduction in cancer cells inhibits self-sufficient growth and DNA replication. PLoS ONE 2, e1112, 1-7.

AbstractThioredoxin reductase 1 (TR1) is a major redox regulator in mammalian cells. As an important antioxidant selenoprotein, TR1 is thought to participate in cancer prevention, but is also known to be over-expressed in many cancer cells. Numerous cancer drugs inhibit TR1, and this protein has been proposed as a target for cancer therapy. We previously reported that reduction of TR1 levels in cancer cells reversed many malignant characteristics suggesting that deficiency in TR1 function is antitumorigenic. The molecular basis for TR1’s role in cancer development, however, is not understood. Herein, we found that, among selenoproteins, TR1 is uniquely overexpressed in cancer cells and its knockdown in a mouse cancer cell line driven by oncogenic k-ras resulted in morphological changes characteristic of parental (normal) cells, without significant effect on cell growth under normal growth conditions. When grown in serum-deficient medium, TR1 deficient cancer cells lose self-sufficiency of growth, manifest a defective progression in their S phase and a decreased expression of DNA polymerase alpha, an enzyme important in DNA replication. These observations provide evidence that TR1 is critical for self-sufficiency in growth signals of malignant cells, that TR1 acts largely as a pro-cancer protein and it is indeed a primary target in cancer therapy. More Information

Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Marechal-Drouard L, Marshall WF, Qu LH, Nelson DR, Sanderfoot AA, Spalding MH, Kapitonov VV, Ren Q, Ferris P, Lindquist E, Shapiro H, Lucas SM, Grimwood J, Schmutz J, Cardol P, Cerutti H, Chanfreau G, Chen CL, Cognat V, Croft MT, Dent R, Dutcher S, Fernandez E, Fukuzawa H, Gonzalez-Ballester D, Gonzalez-Halphen D, Hallmann A, Hanikenne M, Hippler M, Inwood W, Jabbari K, Kalanon M, Kuras R, Lefebvre PA, Lemaire SD, Lobanov AV., Lohr M, Manuell A, Meier I, Mets L, Mittag M, Mittelmeier T, Moroney JV, Moseley J, Napoli C, Nedelcu AM, Niyogi K, Novoselov SV., Paulsen IT, Pazour G, Purton S, Ral JP, Riano-Pachon DM, Riekhof W, Rymarquis L, Schroda M, Stern D, Umen J, Willows R, Wilson N, Zimmer SL, Allmer J, Balk J, Bisova K, Chen CJ, Elias M, Gendler K, Hauser C, Lamb MR, Ledford H, Long JC, Minagawa J, Page MD, Pan J, Pootakham W, Roje S, Rose A, Stahlberg E, Terauchi AM, Yang P, Ball S, Bowler C, Dieckmann CL, Gladyshev VN., Green P, Jorgensen R, Mayfield S, Mueller-Roeber B, Rajamani S, Sayre RT, Brokstein P, Dubchak I, Goodstein D, Hornick L, Huang YW, Jhaveri J, Luo Y, Martinez D, Ngau WC, Otillar B, Poliakov A, Porter A, Szajkowski L, Werner G, Zhou K, Grigoriev IV, Rokhsar DS, Grossman AR. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318, 245-250.

AbstractChlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella. More Information

Gladyshev VN. (2007) Methionine Sulfoxide Reductases. In Redox Biochemistry. Ed., Banerjee R. (ed., Gladyshev VN, Ragsdale SW, Becker DF, Dickman MB), John Wiley & Sons, pp. 84-87.
More Information

Gladyshev VN. (2007) Selenoproteins. In Redox Biochemistry. Ed., Banerjee R. (ed., Gladyshev VN, Ragsdale SW, Becker DF, Dickman MB), John Wiley & Sons, pp. 127-131.
More Information

Fomenko DE, Gladyshev VN. (2007) Bioinformatics Methods to Study Thiol-Based Oxidoreductases. In Redox Biochemistry. Ed., Banerjee R. (ed., Gladyshev VN, Ragsdale SW, Becker DF, Dickman MB), John Wiley & Sons, pp. 251-256.
More Information

Aachmann FL, Fomenko DE, Soragni A, Gladyshev VN, Dikiy A. (2007) Structural analysis of selenoprotein W and NMR analysis of its interaction with 14-3-3 proteins. J. Biol. Chem. 282, 37036-37044.

AbstractSelenium is a trace element with significant biomedical potential. It is essential in mammals due to its occurrence in several proteins in the form of selenocysteine (Sec). One of the most abundant mammalian Sec-containing proteins is selenoprotein W (SelW). This protein of unknown function has a broad expression pattern and contains a candidate CXXU (where U represents Sec) redox motif. Here, we report the solution structure of the Sec13–>Cys variant of mouse SelW determined through high resolution NMR spectroscopy. The protein has a thioredoxin-like fold with the CXXU motif located in an exposed loop similarly to the redox-active site in thioredoxin. Protein dynamics studies revealed the rigidity of the protein backbone and mobility of two external loops and suggested a role of these loops in interaction with SelW partners. Molecular modeling of structures of other members of the Rdx family based on the SelW structure identified new conserved features in these proteins, including an aromatic cluster and interacting loops. Our previous study suggested an interaction between SelW and 14-3-3 proteins. In the present work, with the aid of NMR spectroscopy, we demonstrated specificity of this interaction and identified mobile loops in SelW as interacting surfaces. This finding suggests that 14-3-3 are redox-regulated proteins. More Information

Kim HY, Gladyshev VN. (2007) Methionine sulfoxide reductases: selenoprotein forms and roles in antioxidant protein repair in mammals. Biochem. J. 407, 321-329.

AbstractMsrs (methionine sulfoxide reductases), MsrA and MsrB, are repair enzymes that reduce methionine sulfoxide residues in oxidatively damaged proteins to methionine residues in a stereospecific manner. These enzymes protect cells from oxidative stress and have been implicated in delaying the aging process and progression of neurodegenerative diseases. In recent years, significant efforts have been made to explore the catalytic properties and physiological functions of these enzymes. In the current review, we present recent progress in this area, with the focus on mammalian MsrA and MsrBs including their roles in disease, evolution and function of selenoprotein forms of MsrA and MsrB, and the biochemistry of these enzymes. More Information

Lobanov AV, Fomenko DE, Zhang Y, Sengupta A, Hatfield DL, Gladyshev VN. (2007) Evolutionary dynamics of eukaryotic selenoproteomes: large selenoproteomes may associate with aquatic and small with terrestrial life. Genome Biol. 8, R198.

AbstractBACKGROUND: Selenocysteine (Sec) is a selenium-containing amino acid that is co-translationally inserted into nascent polypeptides by recoding UGA codons. Selenoproteins occur in both eukaryotes and prokaryotes, but the selenoprotein content of organisms (selenoproteome) is highly variable and some organisms do not utilize Sec at all. RESULTS: We analyzed the selenoproteomes of several model eukaryotes and detected 26 and 29 selenoprotein genes in the green algae Ostreococcus tauri and Ostreococcus lucimarinus, respectively, five in the social amoebae Dictyostelium discoideum, three in the fly Drosophila pseudoobscura, and 16 in the diatom Thalassiosira pseudonana, including several new selenoproteins. Distinct selenoprotein patterns were verified by metabolic labeling of O. tauri and D. discoideum with 75Se. More than half of the selenoprotein families were shared by unicellular eukaryotes and mammals, consistent with their ancient origin. Further analyses identified massive, independent selenoprotein losses in land plants, fungi, nematodes, insects and some protists. Comparative analyses of selenoprotein-rich and -deficient organisms revealed that aquatic organisms generally have large selenoproteomes, whereas several groups of terrestrial organisms reduced their selenoproteomes through loss of selenoprotein genes and replacement of Sec with cysteine. CONCLUSION: Our data suggest many selenoproteins originated at the base of the eukaryotic domain and show that the environment plays an important role in selenoproteome evolution. In particular, aquatic organisms apparently retained and sometimes expanded their selenoproteomes, whereas the selenoproteomes of some terrestrial organisms were reduced or completely lost. These findings suggest a hypothesis that, with the exception of vertebrates, aquatic life supports selenium utilization, whereas terrestrial habitats lead to reduced use of this trace element due to an unknown environmental factor. More Information

Carlson BA, Moustafa ME, Sengupta A, Schweizer U, Shrimali R, Rao M, Zhong N, Wang S, Feigenbaum L, Lee BJ, Gladyshev VN, Hatfield DL. (2007) Selective restoration of the selenoprotein population in a mouse hepatocyte selenoproteinless background with different mutant selenocysteine tRNAs lacking Um34. J. Biol. Chem. 282, 32591-32602.

AbstractNovel mouse models were developed in which the hepatic selenoprotein population was targeted for removal by disrupting the selenocysteine (Sec) tRNA([Ser]Sec) gene (trsp), and selenoprotein expression was then restored by introducing wild type or mutant trsp transgenes. The selenoprotein population was partially replaced in liver with mutant transgenes encoding mutations at either position 34 (34T–>A) or 37 (37A–>G) in tRNA([Ser]Sec). The A34 transgene product lacked the highly modified 5-methoxycarbonylmethyl-2′-O-methyluridine, and its mutant base A was converted to I34. The G37 transgene product lacked the highly modified N(6)-isopentenyladenosine. Both mutant tRNAs lacked the 2′-methylribose at position 34 (Um34), and both supported expression of housekeeping selenoproteins (e.g. thioredoxin reductase 1) in liver but not stress-related proteins (e.g. glutathione peroxidase 1). Thus, Um34 is responsible for synthesis of a select group of selenoproteins rather than the entire selenoprotein population. The ICA anticodon in the A34 mutant tRNA decoded Cys codons, UGU and UGC, as well as the Sec codon, UGA. However, metabolic labeling of A34 transgenic mice with (75)Se revealed that selenoproteins incorporated the label from the A34 mutant tRNA, whereas other proteins did not. These results suggest that the A34 mutant tRNA did not randomly insert Sec in place of Cys, but specifically targeted selected selenoproteins. High copy numbers of A34 transgene, but not G37 transgene, were not tolerated in the absence of wild type trsp, further suggesting insertion of Sec in place of Cys in selenoproteins. More Information

Xu XM, Carlson BA, Zhang Y, Mix H, Kryukov GV, Glass RS, Berry MJ, Gladyshev VN, Hatfield DL. (2007) New developments in selenium biochemistry: selenocysteine biosynthesis in eukaryotes and archaea. Biol. Trace Elem. Res. 119, 234-241.

AbstractWe used comparative genomics and experimental analyses to show that (1) eukaryotes and archaea, which possess the selenocysteine (Sec) protein insertion machinery contain an enzyme, O-phosphoseryl-transfer RNA (tRNA) [Ser]Sec kinase (designated PSTK), which phosphorylates seryl-tRNA [Ser]Sec to form O-phosphoseryl-tRNA [Ser]Sec and (2) the Sec synthase (SecS) in mammals is a pyridoxal phosphate-containing protein previously described as the soluble liver antigen (SLA). SecS uses the product of PSTK, O-phosphoseryl-tRNA[Ser]Sec, and selenophosphate as substrates to generate selenocysteyl-tRNA [Ser]Sec. Sec could be synthesized on tRNA [Ser]Sec from selenide, adenosine triphosphate (ATP), and serine using tRNA[Ser]Sec, seryl-tRNA synthetase, PSTK, selenophosphate synthetase, and SecS. The enzyme that synthesizes monoselenophosphate is a previously identified selenoprotein, selenophosphate synthetase 2 (SPS2), whereas the previously identified mammalian selenophosphate synthetase 1 did not serve this function. Monoselenophosphate also served directly in the reaction replacing ATP, selenide, and SPS2, demonstrating that this compound was the active selenium donor. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that contain selenoproteins. More Information

Shchedrina VA, Novoselov SV, Malinouski MY, Gladyshev VN. (2007) Identification and characterization of a selenoprotein family containing a diselenide bond in a redox motif. Proc. Natl. Acad. Sci. USA 104, 13919-13924.

AbstractSelenocysteine (Sec, U) insertion into proteins is directed by translational recoding of specific UGA codons located upstream of a stem-loop structure known as Sec insertion sequence (SECIS) element. Selenoproteins with known functions are oxidoreductases containing a single redox-active Sec in their active sites. In this work, we identified a family of selenoproteins, designated SelL, containing two Sec separated by two other residues to form a UxxU motif. SelL proteins show an unusual occurrence, being present in diverse aquatic organisms, including fish, invertebrates, and marine bacteria. Both eukaryotic and bacterial SelL genes use single SECIS elements for insertion of two Sec. In eukaryotes, the SECIS is located in the 3′ UTR, whereas the bacterial SelL SECIS is within a coding region and positioned at a distance that supports the insertion of either of the two Sec or both of these residues. SelL proteins possess a thioredoxin-like fold wherein the UxxU motif corresponds to the catalytic CxxC motif in thioredoxins, suggesting a redox function of SelL proteins. Distantly related SelL-like proteins were also identified in a variety of organisms that had either one or both Sec replaced with Cys. Danio rerio SelL, transiently expressed in mammalian cells, incorporated two Sec and localized to the cytosol. In these cells, it occurred in an oxidized form and was not reducible by DTT. In a bacterial expression system, we directly demonstrated the formation of a diselenide bond between the two Sec, establishing it as the first diselenide bond found in a natural protein. More Information

See commentary in: Drahl C. (2007) Selenium Doubles Up In Proteins. Chemical & Engineering News 85, 14. Commentary

Zhang Y, Gladyshev VN. (2007) High content of proteins containing 21st and 22nd amino acids, selenocysteine and pyrrolysine, in a symbiotic deltaproteobacterium of gutless worm Olavius algarvensis. Nucleic Acids Res. 35, 4952-4963.

AbstractSelenocysteine (Sec) and pyrrolysine (Pyl) are rare amino acids that are cotranslationally inserted into proteins and known as the 21st and 22nd amino acids in the genetic code. Sec and Pyl are encoded by UGA and UAG codons, respectively, which normally serve as stop signals. Herein, we report on unusually large selenoproteomes and pyrroproteomes in a symbiont metagenomic dataset of a marine gutless worm, Olavius algarvensis. We identified 99 selenoprotein genes that clustered into 30 families, including 17 new selenoprotein genes that belong to six families. In addition, several Pyl-containing proteins were identified in this dataset. Most selenoproteins and Pyl-containing proteins were present in a single deltaproteobacterium, delta1 symbiont, which contained the largest number of both selenoproteins and Pyl-containing proteins of any organism reported to date. Our data contrast with the previous observations that symbionts and host-associated bacteria either lose Sec utilization or possess a limited number of selenoproteins, and suggest that the environment in the gutless worm promotes Sec and Pyl utilization. Anaerobic conditions and consistent selenium supply might be the factors that support the use of amino acids that extend the genetic code. More Information

See commentary in: Atkins JF. Baranov PV. (2007) Duality in the genetic code. Nature 448, 1004-1005. Commentary

Sal LS, Aachmann FL, Kim H, Gladyshev VN, Dikiy A. (2007) NMR assignments of 1H, 13C and 15N spectra of methionine sulfoxide reductase B1 from Mus musculus. Biomol NMR Assign, 1, 131-133.

AbstractIsotopically labeled, 15N and 15N/13C forms of recombinant methionine-r-sulfoxide reductase 1 (MsrB1, SelR) from Mus musculus were produced, in which catalytic selenocysteine was replaced with cysteine. We report here the 1H, 15N and 13C NMR assignment of the reduced form of this mammalian protein. More Information

Su D, Berndt C, Fomenko DE, Holmgren A, Gladyshev VN. (2007) A Conserved cis-Proline Precludes Metal Binding by the Active Site Thiolates in Members of the Thioredoxin Family of Proteins. Biochemistry 46, 6903-6910.

AbstractMany thioredoxin-fold proteins possess a conserved cis-proline located in their C-terminal portions. This residue, as well as catalytic and resolving cysteines, is a key functional group in the active sites of these thiol-disulfide oxidoreductases. However, the specific function of the proline is poorly understood, and some thioredoxin-fold proteins lack this residue. Herein, we found that mutation of a cis-proline, Pro75, in human thioredoxin to serine, threonine, or alanine leads to the formation of an Fe2-S2 cluster in this protein. Further mutagenesis studies revealed that the first cysteine in the CxxC motif and a cysteine in the C-terminal region of the protein were responsible for metal binding. Replacement of Pro75 with arginine, a residue that occurs in place of Pro in peroxiredoxins, also led to the formation of the cluster in the thioredoxin. In addition, we found that mutation of the TxxC active site in a peroxiredoxin to the CxxC form could lead to coordination of an Fe2-S2 cluster in these proteins in vitro. Sco1, a distantly related thioredoxin-fold protein, has histidine in place of the cis-proline, and this residue binds copper. The Pro75His mutation led to increased copper binding by human thioredoxin when cells were grown in the presence of this trace element. Taken together, our data suggest that an important function of Pro75 in human thioredoxin, and likely other members of this superfamily, is to prevent metal binding by the reactive thiolate-based active site. More Information

Dikiy A, Novoselov SV, Fomenko DE, Sengupta A, Carlson BA, Cerny RL, Ginalski K, Grishin NV, Hatfield DL, Gladyshev VN. (2007) SelT, SelW, SelH, and Rdx12: Genomics and Molecular Insights into the Functions of Selenoproteins of a Novel Thioredoxin-like Family. Biochemistry 46, 6871-6882.

AbstractSelenium is an essential trace element in many life forms due to its occurrence as a selenocysteine (Sec) residue in selenoproteins. The majority of mammalian selenoproteins, however, have no known function. Herein, we performed extensive sequence similarity searches to define and characterize a new protein family, designated Rdx, that includes mammalian selenoproteins SelW, SelV, SelT and SelH, bacterial SelW-like proteins and cysteine-containing proteins of unknown function in all three domains of life. An additional member of this family is a mammalian cysteine-containing protein, designated Rdx12, and its fish selenoprotein orthologue. Rdx proteins are proposed to possess a thioredoxin-like fold and a conserved CxxC or CxxU (U is Sec) motif, suggesting a redox function. We cloned and characterized three mammalian members of this family, which showed distinct expression patterns in mouse tissues and different localization patterns in cells transfected with the corresponding GFP fusion proteins. By analogy to thioredoxin, Rdx proteins can use catalytic cysteine (or Sec) to form transient mixed disulfides with substrate proteins. We employed this property to identify cellular targets of Rdx proteins using affinity columns containing mutant versions of these proteins. Rdx12 was found to interact with glutathione peroxidase 1, whereas 14-3-3 protein was identified as one of the targets of mammalian SelW, suggesting a mechanism for redox regulation of the 14-3-3 family of proteins. More Information

Novoselov SV, Lobanov AV, Hua D, Kasaikina MV, Hatfield DL, Gladyshev VN. (2007) A highly efficient form of the selenocysteine insertion sequence element in protozoan parasites and its use in mammalian cells. Proc. Natl. Acad. Sci. USA 104, 7857-7862.

AbstractSelenoproteins are an elite group of proteins containing a rare amino acid, selenocysteine (Sec), encoded by the codon, UGA. In eukaryotes, incorporation of Sec requires a Sec insertion sequence (SECIS) element, a stem-loop structure located in the 3′-untranslated regions of selenoprotein mRNAs. Here we report identification of a noncanonical form of SECIS element in Toxoplasma gondii and Neospora canine, single-celled apicomplexan parasites of humans and domestic animals. This SECIS has a GGGA sequence in the SBP2-binding site in place of AUGA previously considered invariant. Using a combination of computational and molecular techniques, we show that Toxoplasma and Neospora possess both canonical and noncanonical SECIS elements. The GGGA-type SECIS element supported Sec insertion in mammalian HEK 293 and NIH 3T3 cells and did so more efficiently than the natural mammalian SECIS elements tested. In addition, mammalian type I and type II SECIS elements mutated into the GGGA forms were functional but manifested decreased Sec insertion efficiency. We carried out computational searches for both AUGA and GGGA forms of SECIS elements in Toxoplasma and detected five selenoprotein genes, including one coding for a previously undescribed selenoprotein, designated SelQ, and two containing the GGGA form of the SECIS element. In contrast, the GGGA-type SECIS elements were not detected in mammals and nematodes. As a practical outcome of the study, we developed pSelExpress1, a vector for convenient expression of selenoproteins in mammalian cells. It contains an SBP2 gene and the most efficient tested SECIS element: an AUGA mutant of the GGGA-type Toxoplasma SelT structure. More Information

Yoo MH, Xu XM, Turanov AA, Carlson BA, Gladyshev VN, Hatfield DL. (2007) A new strategy for assessing selenoprotein function: siRNA knockdown/knock-in targeting the 3′-UTR. RNA 13, 921-929.

AbstractSelenocysteine insertion into protein in mammalian cells requires RNA elements in the 3′-untranslated regions (3′-UTRs) of selenoprotein genes. The occurrence of these conserved sequences should make selenoproteins particularly amenable for knockdown/knock-in strategies to examine selenoprotein functions. Herein, we utilized the 3′-UTR of various selenoproteins to knock down their expression using siRNAs and then knock in expression using constructs containing mutations within the target region. Thioredoxin reductase 1 (TR1) knockdown in a mouse kidney cell line resulted in the cells growing about 10% more slowly, being more sensitive to UV radiation, and having increased apoptosis in response to UV than control cells. The knockdown cells transfected with a construct encoding the wild-type TR1 gene and having mutations in the sequences targeted by siRNA restored TR1 expression and catalytic activity, rendered the knockdown cells less sensitive to UV, and protected the cells against apoptosis. We also applied this technique to other selenoproteins, selenophosphate synthetase 2 and glutathione peroxidase 1, and found that mRNA and protein levels were restored following transfection of knockdown cells with the corresponding knock-in constructs. In addition to important new insights into the functions of key mammalian selenoproteins, the data suggest that the RNAi-based knock-in technology could distinguish phenotypes due to off-targeting and provide a new method for examining many of the subtleties of selenoprotein function not available using RNAi technology alone. More Information

Koc A, Gladyshev VN. (2007) Methionine sulfoxide reduction and the aging process. Ann. NY Acad. Sci. 1100, 383-386.

AbstractAging has been described for multicellular and asymmetrically dividing organisms, but the mechanisms are poorly understood. Oxidation of proteins is considered to be one of the major factors that leads to aging. Oxidative damage to proteins results in the oxidation of certain amino acid residues, among which oxidation of sulfur-containing amino acids, methionine and cysteine, is notable because of the susceptibility of these residues to damage, and occurrence of repair mechanisms. Methionine sulfoxide reductases, MsrA and MsrB, are thioredoxin-dependent oxidoreductases that reduce oxidized forms of methionine, methionine sulfoxides, in a stereospecific manner. These enzymes are present in all cell types and have shown to be regulating life spans in mammals, insects, and yeast. Here, their roles in modulating yeast life span are discussed. More Information

Labunskyy VM, Hatfield DL, Gladyshev VN. (2007) The Sep15 protein family: Roles in disulfide bond formation and quality control in the endoplasmic reticulum. IUBMB Life 59, 1-5.

AbstractDisulfide bonds play an important role in the structure and function of membrane and secretory proteins. The formation of disulfide bonds in the endoplasmic reticulum (ER) of eukaryotic cells is catalyzed by a complex network of thiol-disulfide oxidoreductases. Whereas a number of ER-resident oxidoreductases have been identified, the function of only a few of them is firmly established. Recently, a selenocysteine-containing oxidoreductase, Sep15, has been implicated in disulfide bond assisted protein folding, and a role in quality control for this selenoprotein has been proposed. This review summarizes up-to-date information on the Sep15 family proteins and highlights new insights into their physiological function. More Information

Xu XM, Carlson BA, Irons R, Mix H, Zhong N, Gladyshev VN, Hatfield DL. (2007) Selenophosphate synthetase 2 is essential for selenoprotein biosynthesis. Biochem. J. 404, 115-20.

AbstractSelenophosphate synthetase (SelD) generates the selenium donor for selenocysteine biosynthesis in eubacteria. One homologue of SelD in eukaryotes is SPS1 (selenophosphate synthetase 1) and a second one, SPS2, was identified as a selenoprotein in mammals. Earlier in vitro studies showed SPS2, but not SPS1, synthesized selenophosphate from selenide, whereas SPS1 may utilize a different substrate. The roles of these enzymes in selenoprotein synthesis in vivo remain unknown. To address their function in vivo, we knocked down SPS2 in NIH3T3 cells using small interfering RNA and found that selenoprotein biosynthesis was severely impaired, whereas knockdown of SPS1 had no effect. Transfection of SPS2 into SPS2 knockdown cells restored selenoprotein biosynthesis, but SPS1 did not, indicating that SPS1 cannot complement SPS2 function. These in vivo studies indicate that SPS2 is essential for generating the selenium donor for selenocysteine biosynthesis in mammals, whereas SPS1 probably has a more specialized, non-essential role in selenoprotein metabolism. More Information

Novoselov SV, Kryukov GV, Xu XM, Carlson BA, Hatfield DL, Gladyshev VN. (2007) Selenoprotein H is a nucleolar thioredoxin-like protein with a unique expression pattern. J. Biol. Chem. 282,11960-11968.

AbstractThe human selenoproteome consists of 25 known selenoproteins, but functions of many of these proteins are not known. Selenoprotein H (SelH) is a recently discovered 14-kDa mammalian protein with no sequence homology to functionally characterized proteins. By sensitive sequence and structure analyses, we identified SelH as a thioredoxin fold-like protein in which a conserved CXXU motif (cysteine separated by two other residues from selenocysteine) corresponds to the CXXC motif in thioredoxins. These data suggest a redox function of SelH. Indeed, a recombinant SelH shows significant glutathione peroxidase activity. In addition, SelH has a conserved RKRK motif in the N-terminal sequence. We cloned wild-type and cysteine mutant forms of SelH either upstream or downstream of green fluorescent protein (GFP) and localized this fusion protein to the nucleus in transfected mammalian cells, whereas mutations in the RKRK motif resulted in the cytosolic protein. Interestingly, the full-length SelH-GFP fusion protein localized specifically to nucleoli, whereas the N-terminal sequence of SelH fused to GFP had a diffuse nucleoplasm location. Northern blot analyses revealed low expression levels of SelH mRNA in various mouse tissues, but it was elevated in the early stages of embryonic development. In addition, SelH mRNA was overexpressed in human prostate cancer LNCaP and mouse lung cancer LCC1 cells. Down-regulation of SelH by RNA interference made LCC1 cells more sensitive to hydrogen peroxide but not to other peroxides tested. Overall, these data establish SelH as a novel nucleolar oxidoreductase and suggest that some functions in this compartment are regulated by redox and dependent on the trace element selenium. More Information

Grossman AR, Croft M, Gladyshev VN, Merchant SS, Posewitz MC, Prochnik S, Spalding MH. (2007) Novel metabolism in Chlamydomonas through the lens of genomics. Curr. Opin. Plant Biol. 10, 190-198.

AbstractChlamydomonas has traditionally been exploited as an organism that is associated with sophisticated physiological, genetic and molecular analyses, all of which have been used to elucidate several biological processes, especially photosynthesis and flagella function and assembly. Recently, the genomics of Chlamydomonas has been combined with other technologies to unveil new aspects of metabolism, including inorganic carbon utilization, anaerobic fermentation, the suite and functions of selenoproteins, and the regulation of vitamin biosynthesis. These initial findings represent the first glimpse through a genomic window onto the highly complex metabolisms that characterize a unicellular, photosynthetic eukaryote that has maintained both plant-like and animal-like characteristics over evolutionary time. More Information

Fomenko DE, Xing W, Adair BM, Thomas DJ, Gladyshev VN. (2007) High-Throughput Identification of Catalytic Redox-Active Cysteine Residues. Science 315, 387-389.

AbstractCysteine (Cys) residues often play critical roles in proteins; however, identification of their specific functions has been limited to case-by-case experimental approaches. We developed a procedure for high-throughput identification of catalytic redox-active Cys in proteins by searching for sporadic selenocysteine-Cys pairs in sequence databases. This method is independent of protein family, structure, and taxon. We used it to selectively detect the majority of known proteins with redox-active Cys and to make additional predictions, one of which was verified. Rapid accumulation of sequence information from genomic and metagenomic projects should allow detection of many additional oxidoreductase families as well as identification of redox-active Cys in these proteins. More Information

Mix H, Lobanov AV, Gladyshev VN. (2007) SECIS elements in the coding regions of selenoprotein transcripts are functional in higher eukaryotes. Nucleic Acids Res. 35, 414-423.

AbstractExpression of selenocysteine (Sec)-containing proteins requires the presence of a cis-acting mRNA structure, called selenocysteine insertion sequence (SECIS) element. In bacteria, this structure is located in the coding region immediately downstream of the Sec-encoding UGA codon, whereas in eukaryotes a completely different SECIS element has evolved in the 3′-untranslated region. Here, we report that SECIS elements in the coding regions of selenoprotein mRNAs support Sec insertion in higher eukaryotes. Comprehensive computational analysis of all available viral genomes revealed a SECIS element within the ORF of a naturally occurring selenoprotein homolog of glutathione peroxidase 4 in fowlpox virus. The fowlpox SECIS element supported Sec insertion when expressed in mammalian cells as part of the coding region of viral or mammalian selenoproteins. In addition, readthrough at UGA was observed when the viral SECIS element was located upstream of the Sec codon. We also demonstrate successful de novo design of a functional SECIS element in the coding region of a mammalian selenoprotein. Our data provide evidence that the location of the SECIS element in the untranslated region is not a functional necessity but rather is an evolutionary adaptation to enable a more efficient synthesis of selenoproteins. More Information

Shrimali RK, Weaver JA, Miller GF, Starost MF, Carlson BA, Novoselov SV, Kumaraswamy E, Gladyshev VN, Hatfield DL. (2007) Neuromuscul. Disord. 7, 135-142.

AbstractLoxP-Cre technology was used to remove the selenocysteine tRNA gene, trsp, in either endothelial cells or myocytes of skeletal and heart muscle to elucidate the role of selenoproteins in cardiovascular disease. Loss of selenoprotein expression in endothelial cells was embryonic lethal. A 14.5-day-old embryo had numerous abnormalities including necrosis of the central nervous system, subcutaneous hemorrhage and erythrocyte immaturity. Loss of selenoprotein expression in myocytes manifested no apparent phenotype until about day 12 after birth. Affected mice had decreased mobility and an increased respiratory rate, which proceeded rapidly to death. Pathological analysis revealed that mice lacking trsp had moderate to severe myocarditis with inflammation extending into the mediastinitis. Thus, ablation of selenoprotein expression demonstrated an essential role of selenoproteins in endothelial cell development and in proper cardiac muscle function. The data suggest a direct connection between the loss of selenoprotein expression in these cell types and cardiovascular disease. More Information

Xu XM, Carlson BA, Mix H, Zhang Y, Saira K, Glass RS, Berry MJ, Gladyshev VN, Hatfield DL. (2007) Biosynthesis of Selenocysteine on Its tRNA in Eukaryotes. PLoS Biol. 5, e4, 1-9.

AbstractSelenocysteine (Sec) is cotranslationally inserted into protein in response to UGA codons and is the 21st amino acid in the genetic code. However, the means by which Sec is synthesized in eukaryotes is not known. Herein, comparative genomics and experimental analyses revealed that the mammalian Sec synthase (SecS) is the previously identified pyridoxal phosphate-containing protein known as the soluble liver antigen. SecS required selenophosphate and O-phosphoseryl-tRNA([Ser]Sec) as substrates to generate selenocysteyl-tRNA([Ser]Sec). Moreover, it was found that Sec was synthesized on the tRNA scaffold from selenide, ATP, and serine using tRNA([Ser]Sec), seryl-tRNA synthetase, O-phosphoseryl-tRNA([Ser]Sec) kinase, selenophosphate synthetase, and SecS. By identifying the pathway of Sec biosynthesis in mammals, this study not only functionally characterized SecS but also assigned the function of the O-phosphoseryl-tRNA([Ser]Sec) kinase. In addition, we found that selenophosphate synthetase 2 could synthesize monoselenophosphate in vitro but selenophosphate synthetase 1 could not. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that synthesize selenoproteins. More Information

2006 Articles

Kim HY, Fomenko DE, Yoon YE, Gladyshev VN. (2006) Catalytic Advantages Provided by Selenocysteine in Methionine-S-Sulfoxide Reductases. Biochemistry 45, 13697-13704.

AbstractMethionine sulfoxide reductases are key enzymes that repair oxidatively damaged proteins. Two distinct stereospecific enzyme families are responsible for this function: MsrA (methionine-S-sulfoxide reductase) and MsrB (methionine-R-sulfoxide reductase). In the present study, we identified multiple selenoprotein MsrA sequences in organisms from bacteria to animals. We characterized the selenocysteine (Sec)-containing Chlamydomonas MsrA and found that this protein exhibited 10-50-fold higher activity than either its cysteine (Cys) mutant form or the natural mouse Cys-containing MsrA, making this selenoenzyme the most efficient MsrA known. We also generated a selenoprotein form of mouse MsrA and found that the presence of Sec increased the activity of this enzyme when a resolving Cys was mutated in the protein. These data suggest that the presence of Sec improves the reduction of methionine sulfoxide by MsrAs. However, the oxidized selenoprotein could not always be efficiently reduced to regenerate the active enzyme. Overall, this study demonstrates that sporadically evolved Sec-containing forms of methionine sulfoxide reductases reflect catalytic advantages provided by Sec in these and likely other thiol-dependent oxidoreductases. More Information

Fernando MR, Lechner JM, Lofgren S, Gladyshev VN, Lou MF. (2006) Mitochondrial thioltransferase (glutaredoxin 2) has GSH-dependent and thioredoxin reductase-dependent peroxidase activities in vitro and in lens epithelial cells. FASEB J. 20, 2645-2647.

AbstractThioltransferase (or Grx) belongs to the oxidoreductase family and is known to regulate redox homeostasis in cells. Mitochondrial Grx2 is a recent discovery, but its function is largely unknown. In this study we investigate Grx2 function by examining its potential peroxidase activity using lens epithelial cells (LEC). cDNA for human and mouse Grx2 was cloned into pET21d(+) vector and used to produce respective recombinant Grx2 for kinetic studies. cDNA for human Grx2 was transfected into human LEC and used for in vivo studies. Both human and mouse Grx2 showed glutathione (GSH)-dependent and thioredoxin reductase (TR)-dependent peroxidase activity. The catalytic efficiency of human and mouse Grx2 was lower than that of glutathione peroxidases (2.5 and 0.8×10(4) s(-1) M(-1), respectively), but comparable with TR-dependent peroxiredoxins (16.5 and 2.7×10(4) s(-1) M(-1), respectively). TR-dependent peroxidase activity increased 2-fold in the transfected cells and was completely abolished by addition of anti-Grx2 antibody (Ab). Flow cytometry (FACS) analysis and confocal microscopy revealed that cells preloaded with pure Grx2 detoxified peroxides more efficiently. Grx2 over-expression protected cells against H2O2-mediated disruption of mitochondrial transmembrane potential. These results suggest that Grx2 has a novel function as a peroxidase, accepting electrons both from GSH and TR. This unique property may play a role in protecting the mitochondria from oxidative damage. More Information

Carlson, B.A., Xu, X.M., Shrimali, R., Sengupta, A., Yoo, M.H., Irons, R., Zhong, N., Hatfield, D.L., Lee, B.J., Lobanov, A.V., and Gladyshev, V.N. (2006) Mammalian and other eukaryotic selenocysteine tRNAs. In Selenium: Its molecular biology and role in human health (ed., Hatfield, D.L., Berry, M.J., Gladyshev, V.N.), Springer, pp. 31-40.

AbstractSelenocysteine (Sec) tRNA occupies a prominent position in the expression of selenoproteins as it is essential for their synthesis and it provides the means by which selenium is co-translationally inserted into protein as the amino acid, Sec. Thus, Sec tRNA is regarded as the principle constituent in selenoprotein synthesis. Many features unique to this tRNA have been characterized over the years in mammals and other eukaryotes. In the last five years, the major advances have been in an elucidation of the different roles that the two major Sec tRNA isoforms play in selenoprotein biosynthesis and in Sec biosynthesis. One isoform appears to be responsible for the synthesis of selenoproteins that have roles in housekeeping functions and are less dependent on selenium status for their expression. The second isoforrn, that differs by only a single methyl group at the 2′-0-hydroxylribosyl moiety at position 34 (designated Um34), appears to be responsible for the expression of selenoproteins that have roles in stress-related phenomena and are highly dependent on selenium for their expression. Several new observations regarding Sec biosynthesis, which occurs on its tRNA, have also been recently made. Other recent advances involving Sec tRNA have used this molecule as a tool for determining whether eukaryotes outside the animal kingdom contain the machinery dedicated for the insertion of Sec into protein. These recent findings are discussed in this chapter. More Information

Salinas, G., Romero, H., Xu, X.M., Carlson, B.A., Hatfield, D.L. and Gladyshev, V.N. (2006) Evolution of Sec decoding and the key role of selenophosphate synthetase in the pathway of selenium utilization. In Selenium: Its molecular biology and role in human health (ed., Hatfield, D.L., Berry, M.J., Gladyshev, V.N.), Springer, pp. 41-52.

AbstractThe complete sequencing of genomes and the development of in silico methods for identification of genes encoding selenocysteine (Sec)-containing proteins have greatly contributed to shape our view on the evolution of selenium utilization in nature. Current evidence is consistent with the idea that Sec decoding is a late addition to the genetic code and it evolved once, before the separation of archaeal, bacterial and eukaryal domains. Many organisms have lost the Sec decoding trait, but recent evidence has shown that the loss is not irreversible. The distribution of organisms that use UGA as a Sec codon suggests that Sec decoding evolved as a result of speciation, differential gene loss and horizontal gene transfer. Selenium is also used in the synthesis 2-selenouridine, a modified base of unknown function located in the wobble position of certain tRNAs. It has been recently demonstrated that selenouridine and Sec-decoding traits can evolve independently of each other, but both require selenophosphate synthetase. This ATP-dependent enzyme emerged as a key feature of selenium utilization that allows separation of selenium from the pathways of sulfur utilization and non-specific use of selenium. Some animals, including mammals, evolved two selenophosphate synthetases, highlighting an unknown complexity of selenium utilization in nature. More Information

Gladyshev, V.N. (2006) Selenoproteins and selenoproteomes. In Selenium: Its molecular biology and role in human health (ed., Hatfield, D.L., Berry, M.J., Gladyshev, V.N.), Springer, pp. 101-112.

AbstractIn the past several years, progress in genome sequencing and development of specialized bioinformatics tools allowed efficient identification of selenocysteine-containing proteins encoded in completely sequenced genomes. Information is currently available on selenoproteomes from a variety of organisms, including humans, which contain 25 known selenoprotein genes. This review provides basic information about mammalian selenoproteins and other known selenoprotein families. Analysis of full sets of selenoproteins in organisms provides exciting avenues for examining selenoprotein evolution and dependence of organisms on the trace element selenium and allows linking selenoproteins with specific biological and biomedical effects of dietary selenium. More Information

Kim, H.Y., and Gladyshev, V.N. (2006) Selenium and methionine sulfoxide reduction. In Selenium: Its molecular biology and role in human health (ed., Hatfield, D.L., Berry, M.J., Gladyshev, V.N.), Springer, pp. 125-136.

AbstractMethionine residues in proteins can be readily oxidized to a diastereomeric mixture of methionine sulfoxides by reactive oxygen species. In most organisms, methionine sulfoxides are reversibly and stereospecifically reduced back to methionine by two distinct classes of repair enzymes, methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). Methionine sulfoxide reduction is thought to be an essential pathway that protects cells from oxidative stress and regulates protein function. This pathway is also implicated in delaying the aging process in organisms from yeast to mammals. The first selenoprotein identified using bioinformatics methods, SelR (also known as SelX or MsrB1), was recently found to be a selenocysteine-containing MsrB. In mammals, selenoprotein MsrB1 is a major MsrB, while MsrB2 and MsrB3 contain cysteine in place of selenocysteine. It has been found that selenocysteine- and cysteine-containing MsrBs employ different catalytic mechanisms. Interestingly, a selenocysteine-containing form of MsrA was also described, but so far was only detected in green algae. More Information

Labunskyy, V.M., Gladyshev, V.N., and Hatfield, D.L. (2006) The 15-kDa selenoprotein (Sep15): functional analysis and role in cancer. In Selenium: Its molecular biology and role in human health (ed., Hatfield, D.L., Berry, M.J., Gladyshev, V.N.), Springer, pp. 143-150.

AbstractThe 15-kDa selenoprotein (Sep15) was identified several years ago as a protein of unknown function. In recent years, several lines of evidence implicated Sepl5 in the effect of dietary selenium in cancer prevention. These lines of evidence include: 1) protein expression patterns in normal and malignant cells; 2) identification of polymorphic sites that regulate Sep15 levels and differentially respond to selenium supplementation; 3) location of the Sep15 gene in the human genome; and 4) correlation between Sep15 haplotype and susceptibility to cancer. Functional analyses revealed a specific interaction between Sep15 and a protein folding sensor in the endoplasmic reticulum of mammalian cells and identified Sep15 as a novel thioredoxin-like fold redox regulator. Sep15 defines a new protein family that occurs in several organisms from green algae to mammals and also contains selenoprotein M (SelM) and a recently identified fish-specific selenoprotein Fep15. More Information

Carlson, B.A., Xu, X.M., Shrimali, R., Sengupta, A., Yoo, M.H., Zhong, N., Hatfield, D.L., Irons, R., Davis, C., Lee , B.J., Novoselov, S.V., and Gladyshev, V.N. (2006) Mouse models for assessing the role of selenoproteins in health and development. In Selenium: Its molecular biology and role in human health (ed., Hatfield, D.L., Berry, M.J., Gladyshev, V.N.), Springer, pp.337-346.

AbstractMouse models have been generated to assess the roles of selenoproteins involved with housekeeping tasks and/or stress-related phenomena in development and health. Each mouse model has taken advantage of the fact that the synthesis of all selenoproteins is dependent on the expression of two selenocysteine (Sec) tRNA[Ser]Sec isoforms that differ fiom each other by a single methyl group on the ribosyl moiety at position 34. The endogenous (Sec) tRNA[Ser]Sec population was selectively altered by generating mouse models involving 1) transgenic animals carrying mutant or wild type (Sec) tRNA[Ser]Sec transgenes, 2) conditional knockout animals carrying a floxed (Sec) tRNA[Ser]Sec gene that was targeted for removal in specific tissues and organs using loxP-Cre technology and 3) transgenic/standard knockout animals carrying mutant or wild type transgenes and a knockout of the (Sec) tRNA[Ser]Sec gene wherein the animal’s survival is dependent on the transgene. These mouse models perturbed selenoprotein expression, often in a protein- and tissue-specific manner, permitting us to better assess their function in health and development. More Information

Salinas, G., Lobanov, A.V., and Gladyshev, V.N. (2006) Selenium in parasites. In Selenium: Its molecular biology and role in human health (ed., Hatfield, D.L., Berry, M.J., Gladyshev, V.N.), Springer, pp. 359-370.

AbstractParasites, which cause an enormous burden in the population of the third world, are a diverse group of organisms, many of which are sensitive to oxidative stress imposed by their hosts. In recent years, several selenoprotein families, some with antioxidant properties, have been described and characterized in metazoan parasites. Glutathione peroxidase and thioredoxin glutathione reductase (TGR) appear to be essential selenoproteins in flatworms (phylum Platyhelminthes). TGR is the single enzyme that provides reducing equivalents to both thioredoxin and glutathione pathways, in contrast to hosts, which evolve parallel pathways. In roundworms (phylum Nematoda), selenoproteins have recently been described, revealing species differences in the Sec/Cys protein sets and the presence of an unusual SECIS element. Plasmodium sp, one of the most important protozoan parasites that affect humans, also decode Sec. The selenoprotein families encoded by Plasmodial genomes have neither Sec nor Cys homologs in their hosts, raising the possibility that targeting their selenoproteomes may provide new treatment strategies. More Information

Zhang Y, Romero H, Salinas G, Gladyshev VN. (2006) Dynamic evolution of selenocysteine utilization in bacteria: a balance between selenoprotein loss and evolution of selenocysteine from redox-active cysteine residues. Genome Biology 7, R94.

AbstractBACKGROUND: Selenocysteine (Sec) is co-translationally inserted into protein in response to UGA codons. It occurs in oxidoreductase active sites and often is catalytically superior to cysteine (Cys). However, Sec is used very selectively in proteins and organisms. The wide distribution of Sec and its restricted use have not been explained. RESULTS: We conducted comparative genomics and phylogenetic analyses to examine dynamics of Sec decoding in bacteria at both selenium utilization trait and selenoproteome levels. These searches revealed that 21.5% of sequenced bacteria utilize Sec, their selenoproteomes have 1 to 31 selenoproteins, and selenoprotein-rich organisms are mostly Deltaproteobacteria or Firmicutes/Clostridia. Evolutionary histories of selenoproteins suggest that Cys-to-Sec replacement is a general trend for most selenoproteins. In contrast, only a small number of Sec-to-Cys replacements were detected, and these were mostly restricted to formate dehydrogenase and selenophosphate synthetase families. In addition, specific selenoprotein gene losses were observed in many sister genomes. Thus, the Sec/Cys replacements were mostly unidirectional, and increased utilization of Sec by existing protein families was counterbalanced by loss of selenoprotein genes or entire selenoproteomes. Lateral transfers of the Sec trait were an additional factor, and we describe the first example of selenoprotein gene transfer between archaea and bacteria. Finally, oxygen requirement and optimal growth temperature were identified as environmental factors that correlate with changes in Sec utilization. CONCLUSION: Our data reveal a dynamic balance between selenoprotein origin and loss, and may account for the discrepancy between catalytic advantages provided by Sec and the observed low number of selenoprotein families and Sec-utilizing organisms. More Information

Lobanov AV, Gromer S, Salinas G, Gladyshev VN. (2006) Selenium metabolism in Trypanosoma: characterization of selenoproteomes and identification of a Kinetoplastida-specific selenoprotein. Nucleic Acids Res. 34, 4012-4024.

AbstractProteins containing the 21st amino acid selenocysteine (Sec) are present in the three domains of life. However, within lower eukaryotes, particularly parasitic protists, the dependence on the trace element selenium is variable as many organisms lost the ability to utilize Sec. Herein, we analyzed the genomes of Trypanosoma and Leishmania for the presence of genes coding for Sec-containing proteins. The selenoproteomes of these flagellated protozoa have three selenoproteins, including distant homologs of mammalian SelK and SelT, and a novel multidomain selenoprotein designated SelTryp. In SelK and SelTryp, Sec is near the C-terminus, and in all three selenoproteins, it is within predicted redox motifs. SelTryp has neither Sec- nor cysteine-containing homologs in the human host and appears to be a Kinetoplastida-specific protein. The use of selenium for protein synthesis was verified by metabolically labeling Trypanosoma cells with 75Se. In addition, genes coding for components of the Sec insertion machinery were identified in the Kinetoplastida genomes. Finally, we found that Trypanosoma brucei brucei cells were highly sensitive to auranofin, a compound that specifically targets selenoproteins. Overall, these data establish that Trypanosoma, Leishmania and likely other Kinetoplastida utilize and depend on the trace element selenium, and this dependence is due to occurrence of selenium in at least three selenoproteins. More Information

Hatfield DL, Carlson BA, Xu XM, Mix H, Gladyshev VN. (2006) Selenocysteine incorporation machinery and the role of selenoproteins in development and health. Prog. Nucleic Acid Res. Mol. Biol. 81, 97-142.

Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16891170&query_hl=1&itool=pubmed_docsum

Turanov AA, Su D, Gladyshev VN. (2006) Mouse mitochondrial thioredoxin reductase: Characterization of alternative cytosolic forms and cellular targets. J. Biol. Chem. 281, 22953-22963.

AbstractThioredoxin reductase (TR) and thioredoxin (Trx) define a major cellular redox system that maintains cysteine residues in numerous proteins in the reduced state. Both cytosolic (TR1 and Trx1) and mitochondrial (TR3 and Trx2) enzymes are essential in mammals, but the function of the mitochondrial system is less understood. In this study, we characterized subcellular localization of three TR3 forms that are generated by alternative first exon splicing and that differ in their N-terminal sequences. Only one of these forms resides in mitochondria, whereas the two other isoforms are cytosolic. Consistent with this finding, TR3 did not have catalytic preferences for mitochondrial Trx2 versus cytosolic Trx1, both of which could serve as TR3 substrates. Similarly, TR1 was equally active with Trx1, Trx2, or a bacterial Trx. We generated recombinant selenoprotein forms of TR1 and TR3 and found that these enzymes were inhibited by zinc, but not by calcium or cobalt ions. We further developed a proteomic method for identification of targets of TRs in mammalian cells utilizing affinity columns containing recombinant TR3 forms differing in C-terminal sequences. Using this procedure, we found that Trx1 was the major target of TR3 in both rat and mouse liver cytosol. The truncated form of TR3 lacking selenocysteine was particularly efficient in binding Trx1, consistent with the previously observed role of truncated TR1 in apoptosis. Overall, these data establish that the function of TR3 is not limited to its role in Trx2 reduction. More Information

Lobanov AV, Kryukov GV, Hatfield DL, Gladyshev VN. (2006) Is there a twenty third amino acid in the genetic code?. Trends Genet. 22, 357-360.

AbstractThe universal genetic code includes 20 common amino acids. In addition, selenocysteine (Sec) and pyrrolysine (Pyl), known as the twenty first and twenty second amino acids, are encoded by UGA and UAG, respectively, which are the codons that usually function as stop signals. The discovery of Sec and Pyl suggested that the genetic code could be further expanded by reprogramming stop codons. To search for the putative twenty third amino acid, we employed various tRNA identification programs that scanned 16 archaeal and 130 bacterial genomes for tRNAs with anticodons corresponding to the three stop signals. Our data suggest that the occurrence of additional amino acids that are widely distributed and genetically encoded is unlikely. More Information

Eckenroth B, Harris K, Turanov AA, Gladyshev VN, Raines RT, Hondal RJ. (2006) Semisynthesis and Characterization of Mammalian Thioredoxin Reductase. Biochemistry 45, 5158-5170.

AbstractThioredoxin reductase and thioredoxin constitute the cellular thioredoxin system, which provides reducing equivalents to numerous intracellular target disulfides. Mammalian thioredoxin reductase contains the rare amino acid selenocysteine. Known as the 21st amino acid, selenocysteine is inserted into proteins by recoding UGA stop codons. Some model eukaryotic organisms lack the ability to insert selenocysteine, and prokaryotes have a recoding apparatus different from that of eukaryotes, thus making heterologous expression of mammalian selenoproteins difficult. Here, we present a semisynthetic method for preparing mammalian thioredoxin reductase. This method produces the first 487 amino acids of mouse thioredoxin reductase-3 as an intein fusion protein in Escherichia coli cells. The missing C-terminal tripeptide containing selenocysteine is then ligated to the thioester-tagged protein by expressed protein ligation. The semisynthetic version of thioredoxin reductase that we produce in this manner has k(cat) values ranging from 1500 to 2220 min(-)(1) toward thioredoxin and has strong peroxidase activity, indicating a functional form of the enzyme. We produced the semisynthetic thioredoxin reductase with a total yield of 24 mg from 6 L of E. coli culture (4 mg/L). This method allows production of a fully functional, semisynthetic selenoenzyme that is amenable to structure-function studies. A second semisynthetic system is also reported that makes use of peptide complementation to produce a partially active enzyme. The results of our peptide complementation studies reveal that a tetrapeptide that cannot ligate to the enzyme (Ac-Gly-Cys-Sec-Gly) can form a noncovalent complex with the truncated enzyme to form a weak complex. This noncovalent peptide-enzyme complex has 350-500-fold lower activity than the semisynthetic enzyme produced by peptide ligation. More Information

Yoo MH, Xu XM, Carlson BA, Gladyshev VN, Hatfield DL. (2006) Thioredoxin reductase 1 deficiency reverses tumor phenotype and tumorigenicity of lung carcinoma cells. J. Biol. Chem. 281, 13005-13008.

AbstractDietary selenium has potent cancer prevention activity. Both low molecular weight selenocompounds and selenoproteins are implicated in this effect. Thioredoxin reductase 1 (TR1) is one of the major antioxidant and redox regulators in mammals that supports p53 function and other tumor suppressor activities. However, this selenium-containing oxidoreductase is also overexpressed in many malignant cells and has been proposed as a target for cancer therapy. To further assess the role of TR1 in the malignancy process, we used RNA interference technology to decrease its expression in mouse lung carcinoma (LLC1) cells. Stable transfection of LLC1 cells with a small interfering RNA construct that specifically targets TR1 removal manifested a reversal in the morphology and anchorage-independent growth properties of these cancer cells that made them similar to those of normal cells. The expression of at least two cancer-related protein mRNAs, Hgf and Opn1, were reduced dramatically in the TR1 knockdown cells. Mice injected with the TR1 knockdown showed a dramatic reduction in tumor progression and metastasis compared with those mice injected with the corresponding control vector. In addition, tumors that arose from injected TR1 knockdown cells lost the targeting construct, suggesting that TR1 is essential for tumor growth in mice. These observations provide direct evidence that the reduction of TR1 levels in malignant cells is antitumorigenic and suggest that the enzyme is a prime target for cancer therapy. More Information

Kim HY, Gladyshev VN. (2006) Alternative first exon splicing regulates subcellular distribution of methionine sulfoxide reductases. BMC Mol. Biol. 7, 11.

AbstractBACKGROUND: Methionine sulfoxide reduction is an important protein repair pathway that protects against oxidative stress, controls protein function and has a role in regulation of aging. There are two enzymes that reduce stereospecifically oxidized methionine residues: MsrA (methionine-S-sulfoxide reductase) and MsrB (methionine-R-sulfoxide reductase). In many organisms, these enzymes are targeted to various cellular compartments. In mammals, a single MsrA gene is known, however, its product is present in cytosol, nucleus, and mitochondria. In contrast, three mammalian MsrB genes have been identified whose products are located in different cellular compartments. RESULTS: In the present study, we identified and characterized alternatively spliced forms of mammalian MsrA. In addition to the previously known variant containing an N-terminal mitochondrial signal peptide and distributed between mitochondria and cytosol, a second mouse and human form was detected in silico. This form, MsrA(S), was generated using an alternative first exon. MsrA(S) was enzymatically active and was present in cytosol and nucleus in transfected cells, but occurred below detection limits in tested mouse tissues. The third alternative form lacked the active site and could not be functional. In addition, we found that mitochondrial and cytosolic forms of both MsrA and MsrB in Drosophila could be generated by alternative first exon splicing. CONCLUSION: Our data suggest conservation of alternative splicing to regulate subcellular distribution of methionine sulfoxide reductases. More Information

Lobanov AV, Delgado C, Rahlfs S, Novoselov SV, Kryukov GV, Gromer S, Hatfield DL, Becker K, Gladyshev VN. (2006) The Plasmodium selenoproteome. Nucleic Acids Res. 34, 496-505.

AbstractThe use of selenocysteine (Sec) as the 21st amino acid in the genetic code has been described in all three major domains of life. However, within eukaryotes, selenoproteins are only known in animals and algae. In this study, we characterized selenoproteomes and Sec insertion systems in protozoan Apicomplexa parasites. We found that among these organisms, Plasmodium and Toxoplasma utilized Sec, whereas Cryptosporidium did not. However, Plasmodium had no homologs of known selenoproteins. By searching computationally for evolutionarily conserved selenocysteine insertion sequence (SECIS) elements, which are RNA structures involved in Sec insertion, we identified four unique Plasmodium falciparum selenoprotein genes. These selenoproteins were incorrectly annotated in PlasmoDB, were conserved in other Plasmodia and had no detectable homologs in other species. We provide evidence that two Plasmodium SECIS elements supported Sec insertion into parasite and endogenous selenoproteins when they were expressed in mammalian cells, demonstrating that the Plasmodium SECIS elements are functional and indicating conservation of Sec insertion between Apicomplexa and animals. Dependence of the plasmodial parasites on selenium suggests possible strategies for antimalarial drug development. More Information

Ferguson AD, Labunskyy VM, Fomenko DE, Arac D, Chelliah Y, Amezcua CA, Rizo J, Gladyshev VN, Deisenhofer J. (2006) NMR structures of the selenoproteins Sep15 and SelM reveal redox activity of new thioredoxin-like family. J. Biol. Chem. 281, 3536-3543.

AbstractSelenium has significant health benefits, including potent cancer prevention activity and roles in immune function and the male reproductive system. Selenium-containing proteins, which incorporate this essential micronutrient as selenocysteine, are proposed to mediate the positive effects of dietary selenium. Presented here are the solution NMR structures of the selenoprotein SelM and an ortholog of the selenoprotein Sep15. These data reveal that Sep15 and SelM are structural homologs that establish a new thioredoxin-like protein family. The location of the active-site redox motifs within the fold together with the observed localized conformational changes after thiol-disulfide exchange and measured redox potential indicate that they have redox activity. In mammals, Sep15 expression is regulated by dietary selenium, and either decreased or increased expression of this selenoprotein alters redox homeostasis. A physiological role for Sep15 and SelM as thiol-disulfide oxidoreductases and their contribution to the quality control pathways of the endoplasmic reticulum are discussed. More Information

Novoselov SV, Hua D, Lobanov AV, Gladyshev VN. (2006) Identification and characterization of Fep15, a new selenocysteine-containing member of the Sep15 protein family. Biochem. J. 394, 575-579.

AbstractSec (selenocysteine) is a rare amino acid in proteins. It is co-translationally inserted into proteins at UGA codons with the help of SECIS (Sec insertion sequence) elements. A full set of selenoproteins within a genome, known as the selenoproteome, is highly variable in different organisms. However, most of the known eukaryotic selenoproteins are represented in the mammalian selenoproteome. In addition, many of these selenoproteins have cysteine orthologues. Here, we describe a new selenoprotein, designated Fep15, which is distantly related to members of the 15 kDa selenoprotein (Sep15) family. Fep15 is absent in mammals, can be detected only in fish and is present in these organisms only in the selenoprotein form. In contrast with other members of the Sep15 family, which contain a putative active site composed of Sec and cysteine, Fep15 has only Sec. When transiently expressed in mammalian cells, Fep15 incorporated Sec in an SECIS- and SBP2 (SECIS-binding protein 2)-dependent manner and was targeted to the endoplasmic reticulum by its N-terminal signal peptide. Phylogenetic analyses of Sep15 family members suggest that Fep15 evolved by gene duplication. More Information

2005 Articles

Kim HY, Gladyshev VN. (2005) Different Catalytic Mechanisms in Mammalian Selenocysteine- and Cysteine-Containing Methionine-R-Sulfoxide Reductases. PLoS Biol. 3, e375, 1-9.

AbstractSelenocysteine (Sec) is found in active sites of several oxidoreductases in which this residue is essential for catalytic activity. However, many selenoproteins have fully functional orthologs, wherein cysteine (Cys) occupies the position of Sec. The reason why some enzymes evolve into selenoproteins if the Cys versions may be sufficient is not understood. Among three mammalian methionine-R-sulfoxide reductases (MsrBs), MsrB1 is a Sec-containing protein, whereas MsrB2 and MsrB3 contain Cys in the active site, making these enzymes an excellent system for addressing the question of why Sec is used in biological systems. In this study, we found that residues, which are uniquely conserved in Cys-containing MsrBs and which are critical for enzyme activity in MsrB2 and MsrB3, were not required for MsrB1, but increased the activity of its Cys mutant. Conversely, selenoprotein MsrB1 had a unique resolving Cys reversibly engaged in the selenenylsulfide bond. However, this Cys was not necessary for activities of either MsrB2, MsrB3, or the Cys mutant of MsrB1. We prepared Sec-containing forms of MsrB2 and MsrB3 and found that they were more than 100-fold more active than the natural Cys forms. However, these selenoproteins could not be reduced by the physiological electron donor, thioredoxin. Yet, insertion of the resolving Cys, which was conserved in MsrB1, into the selenoprotein form of MsrB3 restored the thioredoxin-dependent activity of this enzyme. These data revealed differences in catalytic mechanisms between selenoprotein MsrB1 and non-selenoproteins MsrB2 and MsrB3, and identified catalytic advantages and disadvantages of Sec- and Cys-containing proteins. The data also suggested that Sec- and Cys-containing oxidoreductases require distinct sets of active-site features that maximize their catalytic efficiencies and provide strategies for protein design with improved catalytic properties. More Information

Sun QA, Su D, Novoselov SV, Carlson BA, Hatfield DL, Gladyshev VN. (2005) Reaction mechanism and regulation of Mammalian thioredoxin/glutathione reductase. Biochemistry 44, 14528-14537.

AbstractThioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes. More Information

Castellano S, Lobanov AV, Chapple C, Novoselov SV, Albrecht M, Hua D, Lescure A, Lengauer T, Krol A, Gladyshev VN, Guigo R. (2005) Diversity and functional plasticity of eukaryotic selenoproteins: Identification and characterization of the SelJ family. Proc. Natl. Acad. Sci. 102, 16188-16193.

AbstractSelenoproteins are a diverse group of proteins that contain selenocysteine (Sec), the 21st amino acid. In the genetic code, UGA serves as a termination signal and a Sec codon. This dual role has precluded the automatic annotation of selenoproteins. Recent advances in the computational identification of selenoprotein genes have provided a first glimpse of the size, functions, and phylogenetic diversity of eukaryotic selenoproteomes. Here, we describe the identification of a selenoprotein family named SelJ. In contrast to known selenoproteins, SelJ appears to be restricted to actinopterygian fishes and sea urchin, with Cys homologues only found in cnidarians. SelJ shows significant similarity to the jellyfish J1-crystallins and with them constitutes a distinct subfamily within the large family of ADP-ribosylation enzymes. Consistent with its potential role as a structural crystallin, SelJ has preferential and homogeneous expression in the eye lens in early stages of zebrafish development. A structural role for SelJ would be in contrast to the majority of known selenoenzymes. The unusually highly restricted phylogenetic distribution of SelJ, its specialization, and the comparative analysis of eukaryotic selenoproteomes reveal the diversity and functional plasticity of selenoproteins and point to a mosaic evolution of the use of Sec in proteins. More Information

Xu XM, Mix H, Carlson BA, Grabowski PJ, Gladyshev VN, Berry MJ, Hatfield DL. (2005) Evidence for direct roles of two additional factors, SECp43 and SLA, in the selenoprotein synthesis machinery. J. Biol. Chem. 280, 41568-41575.

AbstractSelenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA([Ser]Sec), but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2′-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA([Ser]Sec), and consequently reduced glutathione peroxidase 1 expression. Double knockdown of SECp43 and SLA resulted in decreased selenoprotein expression. SECp43 formed a complex with Sec tRNA([Ser]Sec) and SLA, and the targeted removal of one of these proteins affected the binding of the other to Sec tRNA([Ser]Sec). SECp43 was located primarily in the nucleus, whereas SLA was found in the cytoplasm. Co-transfection of both proteins resulted in the nuclear translocation of SLA suggesting that SECp43 may also promote shuttling of SLA and Sec tRNA([Ser]Sec) between different cellular compartments. Taken together, these data establish the role of SECp43 and SLA in selenoprotein biosynthesis through interaction with tRNA([Ser]Sec) in a multiprotein complex. The data also reveal a role of SECp43 in regulation of selenoprotein expression by affecting the synthesis of Um34 on tRNA([Ser]Sec) and the intracellular location of SLA. More Information

Biterova EI, Turanov AA, Gladyshev VN, Barycki JJ. (2005) Crystal structures of oxidized and reduced mitochondrial thioredoxin reductase provide molecular details of the reaction mechanism. Proc. Natl. Acad Sci. 102, 15018-15023.

AbstractThioredoxin reductase (TrxR) is an essential enzyme required for the efficient maintenance of the cellular redox homeostasis, particularly in cancer cells that are sensitive to reactive oxygen species. In mammals, distinct isozymes function in the cytosol and mitochondria. Through an intricate mechanism, these enzymes transfer reducing equivalents from NADPH to bound FAD and subsequently to an active-site disulfide. In mammalian TrxRs, the dithiol then reduces a mobile C-terminal selenocysteine-containing tetrapeptide of the opposing subunit of the dimer. Once activated, the C-terminal redox center reduces a disulfide bond within thioredoxin. In this report, we present the structural data on a mitochondrial TrxR, TrxR2 (also known as TR3 and TxnRd2). Mouse TrxR2, in which the essential selenocysteine residue had been replaced with cysteine, was isolated as a FAD-containing holoenzyme and crystallized (2.6 A; R = 22.2%; R(free) = 27.6%). The addition of NADPH to the TrxR2 crystals resulted in a color change, indicating reduction of the active-site disulfide and formation of a species presumed to be the flavin-thiolate charge transfer complex. Examination of the NADP(H)-bound model (3.0 A; R = 24.1%; R(free) = 31.2%) indicates that an active-site tyrosine residue must rotate from its initial position to stack against the nicotinamide ring of NADPH, which is juxtaposed to the isoalloxazine ring of FAD to facilitate hydride transfer. Detailed analysis of the structural data in conjunction with a model of the unusual C-terminal selenenylsulfide suggests molecular details of the reaction mechanism and highlights evolutionary adaptations among reductases. More Information

Novoselov SV, Calvisi DV, Labunskyy VM, Factor VM, Carlson BA, Fomenko DE, Moustafa ME, Hatfield DL. and Gladyshev VN. (2005) Selenoprotein deficiency and high levels of selenium compounds can effectively inhibit hepatocarcinogenesis in transgenic mice. Oncogene 24, 8003-8011.

AbstractThe micronutrient element selenium (Se) has been shown to be effective in reducing the incidence of cancer in animal models and human clinical trials. Selenoproteins and low molecular weight Se compounds were implicated in the chemopreventive effect, but specific mechanisms are not clear. We examined the role of Se and selenoproteins in liver tumor formation in TGFalpha/c-Myc transgenic mice, which are characterized by disrupted redox homeostasis and develop liver cancer by 6 months of age. In these mice, both Se deficiency and high levels of Se compounds suppressed hepatocarcinogenesis. In addition, both treatments induced expression of detoxification genes, increased apoptosis and inhibited cell proliferation. Within low-to-optimal levels of dietary Se, tumor formation correlated with expression of most selenoproteins. These data suggest that changes in selenoprotein expression may either suppress or promote tumorigenesis depending on cell type and genotype. Since dietary Se may have opposing effects on cancer, it is important to identify the subjects who will benefit from Se supplementation as well as those who will not. More Information

Labunskyy VM, Ferguson AD, Fomenko DE, Chelliah Y, Hatfield DL, Gladyshev VN. (2005) A novel cysteine-rich domain of SEP15 mediates the interactions with UDP-glucose: Glycoprotein glucosyltransferase. J. Biol. Chem. 280, 37839-37845.

AbstractSelenium is an essential trace element with potent cancer prevention activity in mammals. The 15-kDa selenoprotein (Sep15) has been implicated in the chemopreventive effect of dietary selenium. Although the precise function of Sep15 remains elusive, Sep15 co-purifies with UDP-glucose:glycoprotein glucosyltransferase (GT), an essential regulator of quality control mechanisms within the endoplasmic reticulum. Recent studies identified two GT and two Sep15 homologues in mammals. We characterize interactions between these protein families in this report. Sep15 and GT form a tight 1:1 complex, and these interactions are conserved between mammals and fruit flies. In mammalian cells, Sep15 co-immunoprecipitates with both GT isozymes. In contrast, a Sep15 homologue, designated selenoprotein M (SelM), does not form a complex with GT. Sequence analysis of members of the Sep15 family identified a novel N-terminal cysteine-rich domain in Sep15 that is absent in SelM. This domain contains six conserved cysteine residues that form two CxxC motifs that do not coordinate metal ions. If this domain is deleted or the cysteines are mutated, Sep15 no longer forms a complex with GT. Conversely, if the cysteine-rich domain of Sep15 is fused to the N-terminus of SelM, the resulting chimera is capable of binding GT. These data indicate that the cysteine-rich domain of Sep15 exclusively mediates protein-protein interactions with GT. More Information

Romero H, Zhang Y, Gladyshev VN, Salinas G. (2005) Evolution of selenium utilization traits. Genome Biol. 6, R66.

AbstractBACKGROUND: The essential trace element selenium is used in a wide variety of biological processes. Selenocysteine (Sec), the 21st amino acid, is co-translationally incorporated into a restricted set of proteins. It is encoded by an UGA codon with the help of tRNASec (SelC), Sec-specific elongation factor (SelB) and a cis-acting mRNA structure (SECIS element). In addition, Sec synthase (SelA) and selenophosphate synthetase (SelD) are involved in the biosynthesis of Sec on the tRNASec. Selenium is also found in the form of 2-selenouridine, a modified base present in the wobble position of certain tRNAs, whose synthesis is catalyzed by YbbB using selenophosphate as a precursor. RESULTS: We analyzed completely sequenced genomes for occurrence of the selA, B, C, D and ybbB genes. We found that selB and selC are gene signatures for the Sec-decoding trait. However, selD is also present in organisms that do not utilize Sec, and shows association with either selA, B, C and/or ybbB. Thus, selD defines the overall selenium utilization. A global species map of Sec-decoding and 2-selenouridine synthesis traits is provided based on the presence/absence pattern of selenium-utilization genes. The phylogenies of these genes were inferred and compared to organismal phylogenies, which identified horizontal gene transfer (HGT) events involving both traits. CONCLUSION: These results provide evidence for the ancient origin of these traits, their independent maintenance, and a highly dynamic evolutionary process that can be explained as the result of speciation, differential gene loss and HGT. The latter demonstrated that the loss of these traits is not irreversible as previously thought. More Information

Kim HY, Gladyshev VN. (2005) Role of structural and functional elements of mouse methionine-s-sulfoxide reductase in its subcellular distribution. Biochemistry 44, 8059-8067.

AbstractOxidized forms of methionine residues in proteins can be repaired by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). In mammals, three MsrBs are present, which are targeted to various subcellular compartments. In contrast, only a single mammalian MsrA gene is known whose products have been detected in both cytosol and mitochondria. Factors that determine the location of the protein in these compartments are not known. Here, we found that MsrA was present in cytosol, nucleus, and mitochondria in mouse cells and tissues and that the major enzyme forms detected in various compartments were generated from a single-translation product rather than by alternative translation initiation. Both cytosolic and mitochondrial forms were processed with respect to the N-terminal signal peptide, and the distribution of the protein occurred post-translationally. Deletion of amino acids 69-108, 69-83, 84-108, or 217-233, which contained elements important for MsrA structure and function, led to exclusive mitochondrial location of MsrA, whereas a region that affected substrate binding but was not part of the overall fold had no influence on the subcellular distribution. The data suggested that proper structure-function organization of MsrA played a role in subcellular distribution of this protein in mouse cells. These findings were recapitulated by expressing various forms of mouse MsrA in Saccharomyces cerevisiae, suggesting conservation of the mechanisms responsible for distribution of the mammalian enzyme among different cellular compartments. More Information

Su D, Novoselov SV, Sun QA, Moustafa ME, Zhou Y, Oko R, Hatfield DL, Gladyshev VN. (2005) Mammalian selenoprotein thioredoxin/glutathione reductase: Roles in disulfide bond formation and sperm maturation. J. Biol. Chem. 280, 26491-26498.

AbstractThioredoxin reductases (TRs) are important redox regulatory enzymes, which control the redox state of thioredoxins. Mammals have cytosolic and mitochondrial TRs, which contain an essential selenocysteine residue and reduce cytosolic and mitochondrial thioredoxins. In addition, thioredoxin/glutathione reductase (TGR) was identified, which is a fusion of an N-terminal glutaredoxin domain and the TR module. Here we show that TGR is expressed at low levels in various tissues but accumulates in testes after puberty. The protein is particularly abundant in elongating spermatids at the site of mitochondrial sheath formation but is absent in mature sperm. We found that TGR can catalyze isomerization of protein and interprotein disulfide bonds and localized this function to its thiol domain. TGR targets include proteins that form structural components of the sperm, including glutathione peroxidase GPx4/PHGPx. Together, TGR and GPx4 can serve as a novel disulfide bond formation system. Both enzymes contain a catalytic selenocysteine consistent with the role of selenium in male reproduction. More Information

Su D, Li Y, Gladyshev VN. (2005) Selenocysteine insertion directed by the 3′-UTR SECIS element in Escherichia coli. Nucleic Acids Res. 33, 2486-2492.

AbstractCo-translational insertion of selenocysteine (Sec) into proteins in response to UGA codons is directed by selenocysteine insertion sequence (SECIS) elements. In known bacterial selenoprotein genes, SECIS elements are located in the coding regions immediately downstream of UGA codons. Here, we report that a distant SECIS element can also function in Sec insertion in bacteria provided that it is spatially close to the UGA codon. We expressed a mammalian phospholipid hydroperoxide glutathione peroxidase in Escherichia coli from a construct in which a natural E.coli SECIS element was located in the 3′-untranslated region (3′-UTR) and adjacent to a sequence complementary to the region downstream of the Sec UGA codon. Although the major readthrough event at the UGA codon was insertion of tryptophan, Sec was also incorporated and its insertion was dependent on the functional SECIS element in the UTR, base-pairing potential of the SECIS flanking region and the Sec UGA codon. These data provide important implications into evolution of SECIS elements and development of a system for heterologous expression of selenoproteins and show that in addition to the primary sequence arrangement between UGA codons and SECIS elements, their proximity within the tertiary structure can support Sec insertion in bacteria. More Information

Taskov K, Chapple C, Kryukov GV, Castellano S, Lobanov AV, Korotkov KV, Guigó R. and Gladyshev VN. (2005) Nematode selenoproteome: the use of the selenocysteine insertion system to decode one codon in an animal genome? Nucleic Acids Res. 33, 2227-2238.

AbstractSelenocysteine (Sec) is co-translationally inserted into selenoproteins in response to codon UGA with the help of the selenocysteine insertion sequence (SECIS) element. The number of selenoproteins in animals varies, with humans having 25 and mice having 24 selenoproteins. To date, however, only one selenoprotein, thioredoxin reductase, has been detected in Caenorhabditis elegans, and this enzyme contains only one Sec. Here, we characterize the selenoproteomes of C.elegans and Caenorhabditis briggsae with three independent algorithms, one searching for pairs of homologous nematode SECIS elements, another searching for Cys- or Sec-containing homologs of potential nematode selenoprotein genes and the third identifying Sec-containing homologs of annotated nematode proteins. These methods suggest that thioredoxin reductase is the only Sec-containing protein in the C.elegans and C.briggsae genomes. In contrast, we identified additional selenoproteins in other nematodes. Assuming that Sec insertion mechanisms are conserved between nematodes and other eukaryotes, the data suggest that nematode selenoproteomes were reduced during evolution, and that in an extreme reduction case Sec insertion systems probably decode only a single UGA codon in C.elegans and C.briggsae genomes. In addition, all detected genes had a rare form of SECIS element containing a guanosine in place of a conserved adenosine present in most other SECIS structures, suggesting that in organisms with small selenoproteomes SECIS elements may change rapidly. More Information

Zhang Y, Fomenko DE, Gladyshev VN. (2005) The microbial selenoproteome of the Sargasso Sea. Genome Biology 6, R37.

AbstractBACKGROUND: Selenocysteine (Sec) is a rare amino acid which occurs in proteins in major domains of life. It is encoded by TGA, which also serves as the signal for termination of translation, precluding identification of selenoprotein genes by available annotation tools. Information on full sets of selenoproteins (selenoproteomes) is essential for understanding the biology of selenium. Herein, we characterized the selenoproteome of the largest microbial sequence dataset, the Sargasso Sea environmental genome project. RESULTS: We identified 310 selenoprotein genes that clustered into 25 families, including 101 new selenoprotein genes that belonged to 15 families. Most of these proteins were predicted redox proteins containing catalytic selenocysteines. Several bacterial selenoproteins previously thought to be restricted to eukaryotes were detected by analyzing eukaryotic and bacterial SECIS elements, suggesting that eukaryotic and bacterial selenoprotein sets partially overlapped. The Sargasso Sea microbial selenoproteome was rich in selenoproteins and its composition was different from that observed in the combined set of completely sequenced genomes, suggesting that these genomes do not accurately represent the microbial selenoproteome. Most detected selenoproteins occurred sporadically compared to the widespread presence of their cysteine homologs, suggesting that many selenoproteins recently evolved from cysteine-containing homologs. CONCLUSIONS: This study yielded the largest selenoprotein dataset to date, doubled the number of prokaryotic selenoprotein families and provided insights into forces that drive selenocysteine evolution. More Information

See commentary in:Copeland PR. (2005) Making sense of nonsense: the evolution of selenocysteine usage in proteins. Genome Biol., 6, 221. Commentary

Zhang Y, Gladyshev VN. (2005) An algorithm for identification of bacterial selenocysteine insertion sequence elements and selenoprotein genes. Bioinformatics 21, 2580-2589.

AbstractMOTIVATION: Incorporation of selenocysteine (Sec) into proteins in response to UGA codons requires a cis-acting RNA structure, Sec insertion sequence (SECIS) element. Whereas SECIS elements in Escherichia coli are well characterized, a bacterial SECIS consensus structure is lacking. RESULTS: We developed a bacterial SECIS consensus model, the key feature of which is a conserved guanosine in a small apical loop of the properly positioned structure. This consensus was used to build a computational tool, bSECISearch, for detection of bacterial SECIS elements and selenoprotein genes in sequence databases. The program identified 96.5% of known selenoprotein genes in completely sequenced bacterial genomes and predicted several new selenoprotein genes. Further analysis revealed that the size of bacterial selenoproteomes varied from 1 to 11 selenoproteins. Formate dehydrogenase was present in most selenoproteomes, often as the only selenoprotein family, whereas the occurrence of other selenoproteins was limited. The availability of the bacterial SECIS consensus and the tool for identification of these structures should help in correct annotation of selenoprotein genes and characterization of bacterial selenoproteomes. More Information

Zhang Y, Baranov PV, Atkins JF, Gladyshev VN. (2005) Pyrrolysine and selenocysteine use dissimilar decoding strategies. J. Biol. Chem. 280, 20740-20751.

AbstractSelenocysteine (Sec) and pyrrolysine (Pyl) are known as the 21st and 22nd amino acids in protein. Both are encoded by codons that normally function as stop signals. Sec specification by UGA codons requires the presence of a cis-acting selenocysteine insertion sequence (SECIS) element. Similarly, it is thought that Pyl is inserted by UAG codons with the help of a putative pyrrolysine insertion sequence (PYLIS) element. Herein, we analyzed the occurrence of Pyl-utilizing organisms, Pyl-associated genes, and Pyl-containing proteins. The Pyl trait is restricted to several microbes, and only one organism has both Pyl and Sec. We found that methanogenic archaea that utilize Pyl have few genes that contain in-frame UAG codons, and many of these are followed with nearby UAA or UGA codons. In addition, unambiguous UAG stop signals could not be identified. This bias was not observed in Sec-utilizing organisms and non-Pyl-utilizing archaea, as well as with other stop codons. These observations as well as analyses of the coding potential of UAG codons, overlapping genes, and release factor sequences suggest that UAG is not a typical stop signal in Pyl-utilizing archaea. On the other hand, searches for conserved Pyl-containing proteins revealed only four protein families, including methylamine methyltransferases and transposases. Only methylamine methyltransferases matched the Pyl trait and had conserved Pyl, suggesting that this amino acid is used primarily by these enzymes. These findings are best explained by a model wherein UAG codons may have ambiguous meaning and Pyl insertion can effectively compete with translation termination for UAG codons obviating the need for a specific PYLIS structure. Thus, Sec and Pyl follow dissimilar decoding and evolutionary strategies. More Information

Shrimali RK, Lobanov AV, Xu XM, Rao M, Carlson BA, Mahadeo DC, Parent CA, Gladyshev VN, Hatfield DL. (2005) Selenocysteine tRNA identification in the model organisms Dictyostelium discoideum and Tetrahymena thermophila. Biochem. Biophys. Res. Commun. 329, 147-151.

AbstractCharacterizing Sec tRNAs that decode UGA provides one of the most direct and easiest means of determining whether an organism possesses the ability to insert selenocysteine (Sec) into protein. Herein, we used a combination of two techniques, computational to identify Sec tRNA genes and RT-PCR to sequence the gene products, to unequivocally demonstrate that two widely studied, model protozoans, Dictyostelium discoideum and Tetrahymena thermophila, encode Sec tRNA in their genomes. The advantage of using both procedures is that computationally we could easily detect potential Sec tRNA genes and then confirm by sequencing that the Sec tRNA was present in the tRNA population, and thus the identified gene was not a pseudogene. Sec tRNAs from both organisms decode UGA. T. thermophila Sec tRNA, like all other sequenced Sec tRNAs, is 90 nucleotides in length, while that from D. discoideum is 91 nucleotides long making it the longest eukaryotic sequenced to date. Evolutionary analyses of known Sec tRNAs reveal the two forms identified herein are the most divergent eukaryotic Sec tRNAs thus far sequenced. More Information

Carlson BA, Xu XM, Gladyshev VN, Hatfield DL. (2005) Selective rescue of selenoprotein expression in mice lacking a highly specialized methyl group in selenocysteine tRNA. J. Biol. Chem. 280, 5542-5548.

AbstractSelenocysteine (Sec) is the 21st amino acid in the genetic code. Its tRNA is variably methylated on the 2′-O-hydroxyl site of the ribosyl moiety at position 34 (Um34). Herein, we identified a role of Um34 in regulating the expression of some, but not all, selenoproteins. A strain of knock-out transgenic mice was generated, wherein the Sec tRNA gene was replaced with either wild type or mutant Sec tRNA transgenes. The mutant transgene yielded a tRNA that lacked two base modifications, N(6)-isopentenyladenosine at position 37 (i(6)A37) and Um34. Several selenoproteins, including glutathione peroxidases 1 and 3, SelR, and SelT, were not detected in mice rescued with the mutant transgene, whereas other selenoproteins, including thioredoxin reductases 1 and 3 and glutathione peroxidase 4, were expressed in normal or reduced levels. Northern blot analysis suggested that other selenoproteins (e.g. SelW) were also poorly expressed. This novel regulation of protein expression occurred at the level of translation and manifested a tissue-specific pattern. The available data suggest that the Um34 modification has greater influence than the i(6)A37 modification in regulating the expression of various mammalian selenoproteins and Um34 is required for synthesis of several members of this protein class. Many proteins that were poorly rescued appear to be involved in responses to stress, and their expression is also highly dependent on selenium in the diet. Furthermore, their mRNA levels are regulated by selenium and are subject to nonsense-mediated decay. Overall, this study described a novel mechanism of regulation of protein expression by tRNA modification that is in turn regulated by levels of the trace element, selenium. More Information

Earlier Articles

Gladyshev VN, Kryukov GV, Fomenko DE, Hatfield DL. (2004) Identification of trace element-containing proteins in genomic databases. Annu. Rev. Nutr. 24, 579-596.

AbstractDevelopment of bioinformatics tools provided researchers with the ability to identify full sets of trace element-containing proteins in organisms for which complete genomic sequences are available. Recently, independent bioinformatics methods were used to identify all, or almost all, genes encoding selenocysteine-containing proteins in human, mouse, and Drosophila genomes, characterizing entire selenoproteomes in these organisms. It also should be possible to search for entire sets of other trace element-associated proteins, such as metal-containing proteins, although methods for their identification are still in development. More Information

Wang C, Scott S, Tao Q, Fomenko DE, Gladyshev VN. (2004) New Techniques for Generation and Analysis of Evolutionary Trees. International Conference on Mathematics and Engineering Techniques in Medicine and Biological Sciences 283-289.

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Su D, Gladyshev VN. (2004) Alternative splicing involving the thioredoxin reductase module in mammals: a glutaredoxin-containing thioredoxin reductase 1. Biochemistry 43, 12177-12188.

AbstractThioredoxin reductase 1 (TR1) is a key component in the thioredoxin system, one of major redox systems in mammals that links NADPH and thiol-dependent processes. Mammalian TR1 genes are known to be regulated by alternative splicing. In this report, comparative genomic analyses were used to identify and characterize species-specific and common alternative forms of mammalian TR1 genes. Six human TR1 isoforms were identified that were derived from a large number of transcripts and differed in their N-terminal sequences. One isoform resulted from exons located 30-70 kb upstream of the previously identified core TR1 promoter and was composed of a basic TR1 module fused to a glutaredoxin (Grx) domain that contained an unusual active site CTRC sequence. This TR1 form occurred in humans, dogs, and chimpanzees but was inactivated in mice and rats. The CTRC motif in the human enzyme made the N-terminal domain inactive in the Grx assays tested. However, when mutated to CPYC, an active site present in most Grxs, the Grx domain was active. In addition, the presence of the Grx domain interfered with the TR1 activity, distinguishing this enzyme from other proteins with Grx and TR fusions. The data suggest that the fusion of the basic TR1 module and variable N-terminal sequences links the pyridine nucleotide thiol/disulfide oxidoreductase pathway to specific cellular redox functions and may control spatial and temporal expression of TR1 transcripts. Our data also suggest that various N-terminal extensions in mammalian TRs are often expressed in testes. More Information

Carlson BA, Xu XM, Kryukov GV, Rao M, Berry MJ, Gladyshev VN, Hatfield,DL. (2004) Identification and characterization of phosphoseryl-tRNA[Ser]Sec kinase. Proc. Natl. Acad. Sci. USA 101, 12848-12853.

AbstractIn 1970, a kinase activity that phosphorylated a minor species of seryl-tRNA to form phosphoseryl-tRNA was found in rooster liver [Maenpaa, P. H. & Bernfield, M. R. (1970) Proc. Natl. Acad. Sci. USA 67, 688-695], and a minor seryl-tRNA that decoded the nonsense UGA was detected in bovine liver. The phosphoseryl-tRNA and the minor UGA-decoding seryl-tRNA were subsequently identified as selenocysteine (Sec) tRNA[Ser]Sec, but the kinase activity remained elusive. Herein, by using a comparative genomics approach that searched completely sequenced archaeal genomes for a kinase-like protein with a pattern of occurrence similar to that of components of Sec insertion machinery, we detected a candidate gene for mammalian phosphoseryl-tRNA[Ser]Sec kinase (pstk). Mouse pstk was cloned, and the gene product (PSTK) was expressed and characterized. PSTK specifically phosphorylated the seryl moiety on seryl-tRNA[Ser]Sec and, in addition, had a requirement for ATP and Mg2+. Proteins with homology to mammalian PSTK occur in Drosophila, Caenorhabditis elegans, Methanopyrus kandleri, and Methanococcus jannaschii, suggesting a conservation of its function across archaea and eukaryotes that synthesize selenoproteins and the absence of this function in bacteria, plants, and yeast. The fact that PSTK has been highly conserved in evolution suggests that it plays an important role in selenoprotein biosynthesis and/or regulation. Copyright 2004 The National Academy of Sciencs of the USA More Information

Kim HY, Gladyshev VN. (2004) Characterization of mouse endoplasmic reticulum methionine-R-sulfoxide reductase. Biochem. Biophys. Res. Commun. 320, 1277-1283.

AbstractMethionine-R-sulfoxide reductases (MsrBs) catalyze a stereospecific reduction of methionine-R-sulfoxides to methionines in proteins. Mammals possess three MsrB genes. MsrB1 (SelR) is a selenoprotein located in the cytosol and nucleus, MsrB2 (CBS-1) is a mitochondrial protein, and MsrB3 is a recently identified protein with an unusual localization pattern. Human MsrB3 occurs in two protein forms, MsrB3A and MsrB3B, which can be targeted to the endoplasmic reticulum (ER) and mitochondria, respectively. These forms are generated by alternative first exon splicing that introduces contrasting N-terminal signal peptides. Herein, we characterized mouse MsrB3 and found no evidence of alternative splicing of its gene. The ER signal was located upstream of the predicted mitochondrial signal sequence in a single coding region, whose product was targeted to the ER. Although the mitochondrial signal could function if placed at the N-terminus, it did not target MsrB3 to mitochondria as part of the entire coding region. In addition, immunoblot assays detected no mitochondrial MsrB3 in examined mouse tissues. The data suggest that, in mice, MsrB3 is largely or exclusively an ER-resident protein, and that the reduction of methionine-R-sulfoxides in different cellular compartments is provided by individual MsrB isozymes. More Information

Koc A, Gasch AP, Rutherford JC, Kim HY, Gladyshev VN. (2004) Methionine sulfoxide reductase regulation of yeast lifespan reveals reactive oxygen species-dependent and -independent components of aging. Proc. Natl. Acad. Sci. USA 101, 7999-8004.

AbstractAging is thought to be caused by the accumulation of damage, primarily from oxidative modifications of cellular components by reactive oxygen species (ROS). Here we used yeast methionine sulfoxide reductases MsrA and MsrB to address this hypothesis. In the presence of oxygen, these antioxidants could increase yeast lifespan and did so independent of the lifespan extension offered by caloric restriction. However, under ROS-deficient, strictly anaerobic conditions, yeast lifespan was shorter, not affected by MsrA or MsrB, and further reduced by caloric restriction. In addition, we identified changes in the global gene expression associated with aging in yeast, and they did not include oxidative stress genes. Our findings suggest how the interplay between ROS, antioxidants, and efficiency of energy production regulates the lifespan. The data also suggest a model wherein factors implicated in aging (for example, ROS) may influence the lifespan yet not be the cause of aging. More Information

Kryukov GV, Gladyshev VN. (2004) The prokaryotic selenoproteome. EMBO Rep. 5, 538-543.

AbstractIn the genetic code, the UGA codon has a dual function as it encodes selenocysteine (Sec) and serves as a stop signal. However, only the translation terminator function is used in gene annotation programs, resulting in misannotation of selenoprotein genes. Here, we applied two independent bioinformatics approaches to characterize a selenoprotein set in prokaryotic genomes. One method searched for selenoprotein genes by identifying RNA stem-loop structures, selenocysteine insertion sequence elements; the second approach identified Sec/Cys pairs in homologous sequences. These analyses identified all or almost all selenoproteins in completely sequenced bacterial and archaeal genomes and provided a view on the distribution and composition of prokaryotic selenoproteomes. In addition, lineage-specific and core selenoproteins were detected, which provided insights into the mechanisms of selenoprotein evolution. Characterization of selenoproteomes allows interpretation of other UGA codons in completed genomes of prokaryotes as terminators, addressing the UGA dual-function problem. More Information

Castellano S, Novoselov SV, Kryukov GV, Lescure A, Blanco E, Krol A, Gladyshev VN, Guigo R. (2004) Reconsidering the evolution of eukaryotic selenoproteins: a novel nonmammalian family with scattered phylogenetic distribution. EMBO Rep. 7, 71-77.

AbstractWhile the genome sequence and gene content are available for an increasing number of organisms, eukaryotic selenoproteins remain poorly characterized. The dual role of the UGA codon confounds the identification of novel selenoprotein genes. Here, we describe a comparative genomics approach that relies on the genome-wide prediction of genes with in-frame TGA codons, and the subsequent comparison of predictions from different genomes, wherein conservation in regions flanking the TGA codon suggests selenocysteine coding function. Application of this method to human and fugu genomes identified a novel selenoprotein family, named SelU, in the puffer fish. The selenocysteine-containing form also occurred in other fish, chicken, sea urchin, green algae and diatoms. In contrast, mammals, worms and land plants contained cysteine homologues. We demonstrated selenium incorporation into chicken SelU and characterized the SelU expression pattern in zebrafish embryos. Our data indicate a scattered evolutionary distribution of selenoproteins in eukaryotes, and suggest that, contrary to the picture emerging from data available so far, other taxa-specific selenoproteins probably exist. More Information

Kim HY, Gladyshev VN. (2004) Methionine sulfoxide reduction in mammals: characterization of methionine-R-sulfoxide reductases. Mol. Biol. Cell 15, 1055-1064.

AbstractMethionine residues in proteins are susceptible to oxidation by reactive oxygen species, but can be repaired via reduction of the resulting methionine sulfoxides by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). However, the identity of all methionine sulfoxide reductases involved, their cellular locations and relative contributions to the overall pathway are poorly understood. Here, we describe a methionine-R-sulfoxide reduction system in mammals, in which two MsrB homologues were previously described. We found that human and mouse genomes possess three MsrB genes and characterized their protein products, designated MsrB1, MsrB2, and MsrB3. MsrB1 (Selenoprotein R) was present in the cytosol and nucleus and exhibited the highest methionine-R-sulfoxide reductase activity because of the presence of selenocysteine (Sec) in its active site. Other mammalian MsrBs contained cysteine in place of Sec and were less catalytically efficient. MsrB2 (CBS-1) resided in mitochondria. It had high affinity for methionine-R-sulfoxide, but was inhibited by higher concentrations of the substrate. The human MsrB3 gene gave rise to two protein forms, MsrB3A and MsrB3B. These were generated by alternative splicing that introduced contrasting N-terminal and C-terminal signals, such that MsrB3A was targeted to the endoplasmic reticulum and MsrB3B to mitochondria. We found that only mitochondrial forms of mammalian MsrBs (MsrB2 and MsrB3B) could compensate for MsrA and MsrB deficiency in yeast. All mammalian MsrBs belonged to a group of zinc-containing proteins. The multiplicity of MsrBs contrasted with the presence of a single mammalian MsrA gene as well as with the occurrence of single MsrA and MsrB genes in yeast, fruit flies, and nematodes. The data suggested that different cellular compartments in mammals maintain a system for repair of oxidized methionine residues and that this function is tuned in enzyme- and stereo-specific manner. More Information

Carlson BA, Novoselov SV, Kumaraswamy E, Lee BJ, Anver MR, Gladyshev VN, Hatfield DL. (2004) Specific excision of the selenocysteine tRNA[Ser]Sec (Trsp) gene in mouse liver demonstrates an essential role of selenoproteins in liver function. J. Biol. Chem. 279, 8011-8017.

AbstractSelenium is essential in mammalian embryonic development. However, in adults, selenoprotein levels in several organs including liver can be substantially reduced by selenium deficiency without any apparent change in phenotype. To address the role of selenoproteins in liver function, mice homozygous for a floxed allele encoding the selenocysteine (Sec) tRNA([Ser]Sec) gene were crossed with transgenic mice carrying the Cre recombinase under the control of the albumin promoter that expresses the recombinase specifically in liver. Recombination was nearly complete in mice 3 weeks of age, whereas liver selenoprotein synthesis was virtually absent, which correlated with the loss of Sec tRNA([Ser]Sec) and activities of major selenoproteins. Total liver selenium was dramatically decreased, whereas levels of low molecular weight selenocompounds were little affected. Plasma selenoprotein P levels were reduced by about 75%, suggesting that selenoprotein P is primarily exported from the liver. Glutathione S-transferase levels were elevated in the selenoprotein-deficient liver, suggesting a compensatory activation of this detoxification program. Mice appeared normal until about 24 h before death. Most animals died between 1 and 3 months of age. Death appeared to be due to severe hepatocellular degeneration and necrosis with concomitant necrosis of peritoneal and retroperitoneal fat. These studies revealed an essential role of selenoproteins in liver function. More Information

Fomenko DE, Gladyshev VN. (2003) Genomics perspective on disulfide bond formation. Antioxid. Redox Signal. 5, 397-402.

AbstractDisulfide bond formation, reduction, and isomerization in substrate proteins are catalyzed by designated pathways composed of thiol-dependent enzymes. Disulfides are generated in oxidizing environments, such as bacterial periplasm and eukaryotic endoplasmic reticulum (ER), but could also be formed in the cytosol. Major contributors to the formation of intramolecular disulfides in proteins are thiol/disulfide oxidoreductases containing a conserved CxxC motif (two cysteines separated by two other residues), which in turn transfer reducing equivalents to adapter or membrane-bound oxidoreductases. Disulfide bond formation is accompanied by disulfide bond reduction and isomerization processes, allowing disulfide repair and quality control. Higher eukaryotes evolved a complex network of thiol/disulfide oxidoreductases that are involved in disulfide bond formation and isomerization and thiol-dependent protein retention. Emerging evidence suggests that these ER functions might be assisted by mammalian selenocysteine-containing oxidoreductases Sep15 and SelM. More Information

Kwon SY, Badenhorst P, Martin-Romero FJ, Carlson BA, Paterson BM, Gladyshev VN, Lee BJ, Hatfield DL. (2003) The Drosophila selenoprotein BthD is required for survival and has a role in salivary gland development. Mol. Cell. Biol. 23, 8495-8504.

AbstractSelenium is implicated in many diseases, including cancer, but its function at the molecular level is poorly understood. BthD is one of three selenoproteins recently identified in Drosophila. To elucidate the function of BthD and the role of selenoproteins in cellular metabolism and health, we analyzed the developmental expression profile of this protein and used inducible RNA interference (RNAi) to ablate function. We find that BthD is dynamically expressed during Drosophila development. bthD mRNA and protein are abundant in the ovaries of female flies and are deposited into the developing oocyte. Maternally contributed protein and RNA persist during early embryonic development but decay by the onset of gastrulation. At later stages of embryogenesis, BthD is expressed highly in the developing salivary gland. We generated transgenic fly lines carrying an inducible gene-silencing construct, in which an inverted bthD genomic-cDNA hybrid is under the control of the Drosophila Gal4 upstream activation sequence system. Duplex RNAi induced from this construct targeted BthD mRNA for destruction and reduced BthD protein levels. We found that loss of BthD compromised salivary gland morphogenesis and reduced animal viability. More Information

Fomenko DE, Gladyshev VN. (2003) Identity and functions of CxxC-derived motifs. Biochemistry 42, 11214-11225.

AbstractTwo cysteines separated by two other residues (the CxxC motif) are employed by many redox proteins for formation, isomerization, and reduction of disulfide bonds and for other redox functions. The place of the C-terminal cysteine in this motif may be occupied by serine (the CxxS motif), modifying the functional repertoire of redox proteins. Here we found that the CxxC motif may also give rise to a motif, in which the C-terminal cysteine is replaced with threonine (the CxxT motif). Moreover, in contrast to a view that the N-terminal cysteine in the CxxC motif always serves as a nucleophilic attacking group, this residue could also be replaced with threonine (the TxxC motif), serine (the SxxC motif), or other residues. In each of these CxxC-derived motifs, the presence of a downstream alpha-helix was strongly favored. A search for conserved CxxC-derived motif/helix patterns in four complete genomes representing bacteria, archaea, and eukaryotes identified known redox proteins and suggested possible redox functions for several additional proteins. Catalytic sites in peroxiredoxins were major representatives of the TxxC motif, whereas those in glutathione peroxidases represented the CxxT motif. Structural assessments indicated that threonines in these enzymes could stabilize catalytic thiolates, suggesting revisions to previously proposed catalytic triads. Each of the CxxC-derived motifs was also observed in natural selenium-containing proteins, in which selenocysteine was present in place of a catalytic cysteine. More Information

Thisse C, Degrave A, Kryukov GV, Gladyshev VN, Obrecht-Pflumio S, Krol A, Thisse B, Lescure A. (2003) Spatial and temporal expression patterns of selenoprotein genes during embryogenesis in zebrafish. Gene Expr. Patterns 3, 525-532.

AbstractSelenium is important for embryogenesis in vertebrates but little is known about the expression patterns and biological functions of most selenoprotein genes. Taking advantage of the zebrafish model, systematic analysis of selenoprotein gene expression was performed by in situ hybridization on whole-mount embryos at different developmental stages. Twenty-one selenoprotein mRNAs were analyzed and all of them exhibited expression patterns restricted to specific tissues. Moreover, we demonstrated that highly similar selenoprotein paralogs were expressed within distinct territories. Therefore, tissue- and development-specific expression patterns provided new information for selenoproteins of unknown function. More Information

Rao M, Carlson BA, Novoselov SV, Weeks DP, Gladyshev VN, Hatfield DL. (2003) Chlamydomonas reinhardtii selenocysteine tRNA[Ser]Sec. RNA 9, 923-930.

AbstractEukaryotic selenocysteine (Sec) protein insertion machinery was thought to be restricted to animals, but the occurrence of both Sec-containing proteins and the Sec insertion system was recently found in Chlamydomonas reinhardtii, a member of the plant kingdom. Herein, we used RT-PCR to determine the sequence of C. reinhardtii Sec tRNA[Ser]Sec, the first non-animal eukaryotic Sec tRNA[Ser]Sec sequence. Like its animal counterpart, it is 90 nucleotides in length, is aminoacylated with serine by seryl-tRNA synthetase, and decodes specifically UGA. Evolutionary analyses of known Sec tRNAs identify the C. reinhardtii form as the most diverged eukaryotic Sec tRNA[Ser]Sec and reveal a common origin for this tRNA in bacteria, archaea, and eukaryotes. More Information

Kryukov GV, Castellano S, Novoselov SV, Lobanov AV, Zehtab O, Guigo R, Gladyshev VN. (2003) Characterization of mammalian selenoproteomes. Science 300, 1439-1443.

AbstractIn the genetic code, UGA serves as a stop signal and a selenocysteine codon, but no computational methods for identifying its coding function are available. Consequently, most selenoprotein genes are misannotated. We identified selenoprotein genes in sequenced mammalian genomes by methods that rely on identification of selenocysteine insertion RNA structures, the coding potential of UGA codons, and the presence of cysteine-containing homologs. The human selenoproteome consists of 25 selenoproteins. More Information

Kumaraswamy E, Carlson BA, Morgan F, Miyoshi K, Robinson GW, Su D, Wan, S, Southo, E, Tessaroll, L, Le, B J, Gladyshe, VN, Hennighausen L, Hatfield DL. (2003) Selective removal of the selenocysteine tRNA [Ser]Sec gene (Trsp) in mouse mammary epithelium. Mol. Cell. Biol. 23, 1477-1488.

AbstractMice homozygous for an allele encoding the selenocysteine (Sec) tRNA [Ser]Sec gene (Trsp) flanked by loxP sites were generated. Cre recombinase-dependent removal of Trsp in these mice was lethal to embryos. To investigate the role of Trsp in mouse mammary epithelium, we deleted this gene by using transgenic mice carrying the Cre recombinase gene under control of the mouse mammary tumor virus (MMTV) long terminal repeat or the whey acidic protein promoter. While both promoters target Cre gene expression to mammary epithelium, MMTV-Cre is also expressed in spleen and skin. Sec tRNA [Ser]Sec amounts were reduced by more than 70% in mammary tissue with either transgene, while in skin and spleen, levels were reduced only with MMTV-Cre. The selenoprotein population was selectively affected with MMTV-Cre in breast and skin but not in the control tissue, kidney. Moreover, within affected tissues, expression of specific selenoproteins was regulated differently and often in a contrasting manner, with levels of Sep15 and the glutathione peroxidases GPx1 and GPx4 being substantially reduced. Expression of the tumor suppressor genes BRCA1 and p53 was also altered in a contrasting manner in MMTV-Cre mice, suggesting greater susceptibility to cancer and/or increased cell apoptosis. Thus, the conditional Trsp knockout mouse allows tissue-specific manipulation of Sec tRNA and selenoprotein expression, suggesting that this approach will provide a useful tool for studying the role of selenoproteins in health. More Information

Novoselov SV, Gladyshev VN. (2003) Non-animal origin of animal thioredoxin reductases: Implications for selenocysteine evolution and evolution of protein function through carboxy-terminal extensions. Protein Sci. 12, 372-378.

AbstractThioredoxin reductase (TR) and thioredoxin constitute a major cellular redox system present in all organisms. In contrast to a single form of thioredoxin, there are two TR types: One (bacterial type or small TR) is present in bacteria, archaea, plants, and most unicellular eukaryotes, whereas the second (animal or large TR) is only found in animals and typically contains a carboxy-terminal penultimate selenocysteine encoded by TGA. Surprisingly, we detected sequences of large TRs in various unicellular eukaryotes. Moreover, green algae Chlamydomonas reinhardtii had both small and large TRs, with the latter being a selenoprotein, but no examples of horizontal gene transfer from animals to the green algae could be detected. In addition, phylogenetic analyses revealed that large TRs formed a subgroup of lower eukaryotic glutathione reductases (GRs). The data suggest that the large TR evolved in a lower eukaryote capable of selenocysteine insertion rather than in an animal. The enzyme appeared to evolve by a carboxy-terminal extension of GR such that the resulting carboxy-terminal glutathionelike peptide became an intramolecular substrate for GR and a reductant for thioredoxin. Subsequently, small TRs were lost in an organism that gave rise to animals, large TRs were lost in plants and fungi, and selenocysteine/cysteine replacements took place in some large TRs. Our data implicate carboxy-terminal extension of proteins as a general mechanism of evolution of new protein function. More Information

Xu XM, Carlson BA, Grimm TA, Kutza J, Berry MJ, Arreola R, Fields, KH, Shanmugam I, Jeang KT, Oroszlan S, Combs GF, Marx PA, Gladyshev VN, Clouse KA, Hatfield DL. (2002) Rhesus monkey simian immunodeficiency virus infection as a model for assessing the role of selenium in AIDS. J. Acquir. Immune. Defic. Syndr. 31, 453-463.

AbstractThe objective of this study was to determine whether simian immunodeficiency virus (SIV) infection of macaques could be used as a model system to assess the role of selenium in AIDS. Plasma and serum selenium levels were determined by standard assays in monkeys before and after inoculation of SIV. SIV-infected cells or cells expressing the HIV Tat protein were labeled with 75Se, and protein extracts were prepared and electrophoresed to analyze selenoprotein expression. Total tRNA was isolated from CEMx174 cells infected with SIV or from KK1 cells infected with HIV, and selenocysteine tRNA isoforms were characterized by reverse phase chromatography. SIV-infected monkeys show a decrease in blood selenium levels similar to that observed in AIDS with development of SAIDS. Cells infected with SIV in vitro exhibit reduced selenoprotein levels and an accumulation of small molecular weight selenium compounds relative to uninfected cells. Examination of the selenocysteine tRNA isoforms in HIV-infected KK1 cells or SIV-infected CEMx174 cells reveals an isoform distribution characteristic of selenium-deficient cells. Furthermore, transfection of Jurkat E6 cells with the Tat gene selectively altered selenoprotein synthesis, with GPX4 and Sep15 being the most inhibited and TR1 the most enhanced. Taken together, the data show that monkeys infected with SIV in vivo and cells infected with SIV in vitro will provide appropriate models for investigating the mechanism(s) responsible for reduced selenium levels that accompany the progression of AIDS in HIV disease. More Information

Fomenko DE, Gladyshev VN. (2002) CxxS: fold-independent redox motif revealed by genome-wide searches for thiol/disulfide oxidoreductase function. Protein Sci. 11, 2285-2296.

AbstractRedox reactions involving thiol groups in proteins are major participants in cellular redox regulation and antioxidant defense. Although mechanistically similar, thiol-dependent redox processes are catalyzed by structurally distinct families of enzymes, which are difficult to identify by available protein function prediction programs. Herein, we identified a functional motif, CxxS (cysteine separated from serine by two other residues), that was often conserved in redox enzymes, but rarely in other proteins. Analyses of complete Escherichia coli, Campylobacter jejuni, Methanococcus jannaschii, and Saccharomyces cerevisiae genomes revealed a high proportion of proteins known to use the CxxS motif for redox function. This allowed us to make predictions in regard to redox function and identity of redox groups for several proteins whose function previously was not known. Many proteins containing the CxxS motif had a thioredoxin fold, but other structural folds were also present, and CxxS was often located in these proteins upstream of an alpha-helix. Thus, a conserved CxxS sequence followed by an alpha-helix is typically indicative of a redox function and corresponds to thiol-dependent redox sites in proteins. The data also indicate a general approach of genome-wide identification of redox proteins by searching for simple conserved motifs within secondary structure patterns. More Information

Kumar RA, Koc A, Cerny RL, Gladyshev VN. (2002) Reaction mechanism, evolutionary analysis, and role of zinc in Drosophila methionine-R-sulfoxide reductase. J. Biol. Chem. 277, 37527-37535.

AbstractMethionine residues in proteins are susceptible to oxidation, and the resulting methionine sulfoxides can be reduced back to methionines by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). Herein, we have identified two MsrB families that differ by the presence of zinc. Evolutionary analyses suggested that the zinc-containing MsrB proteins are prototype enzymes and that the metal was lost in certain MsrB proteins later in evolution. Zinc-containing Drosophila MsrB was further characterized. The enzyme was found to employ a catalytic Cys(124) thiolate, which directly interacted with methionine sulfoxide, resulting in methionine and a Cys(124) sulfenic acid intermediate. A subsequent reaction of this intermediate with Cys(69) generated an intramolecular disulfide. Dithiothreitol could reduce either the sulfenic acid or the disulfide, but the disulfide was a preferred substrate for thioredoxin, a natural electron donor. Interestingly, the C69S mutant could complement MsrA/MsrB deficiency in yeast, and the corresponding natural form of mouse MsrB was active with thioredoxin. These data indicate that MsrB proteins employ alternative mechanisms for sulfenic acid reduction. Four other conserved cysteines in Drosophila MsrB (Cys(51), Cys(54), Cys(101), and Cys(104)) were found to coordinate structural zinc. Mutation of any one or a combination of these residues resulted in complete loss of metal and catalytic activity, demonstrating an essential role of zinc in Drosophila MsrB. In contrast, two conserved histidines were important for thioredoxin-dependent activity, but were not involved in zinc binding. A Drosophila MsrA gene was also cloned, and the recombinant enzyme was found to be metal-free and specific for methionine S-sulfoxide and to employ a similar sulfenic acid/disulfide mechanism. More Information

Novoselov SV, Rao M, Onoshko NV, Zhi H, Kryukov GV, Xiang Y, Weeks DP, Hatfield DL, Gladyshev VN. (2002) Selenoproteins and selenocysteine insertion system in the model plant cell system. Chlamydomonas reinhardtii. EMBO J. 21, 3681-3693.

AbstractKnown eukaryotic selenocysteine (Sec)-containing proteins are animal proteins, whereas selenoproteins have not been found in yeast and plants. Surprisingly, we detected selenoproteins in a member of the plant kingdom, Chlamydomonas reinhardtii, and directly identified two of them as phospholipid hydroperoxide glutathione peroxidase and selenoprotein W homologs. Moreover, a selenocysteyl-tRNA was isolated that recognized specifically the Sec codon UGA. Subsequent gene cloning and bioinformatics analyses identified eight additional selenoproteins, including methionine-S-sulfoxide reductase, a selenoprotein specific to Chlamydomonas: Chlamydomonas selenoprotein genes contained selenocysteine insertion sequence (SECIS) elements that were similar, but not identical, to those of animals. These SECIS elements could direct selenoprotein synthesis in mammalian cells, indicating a common origin of plant and animal Sec insertion systems. We found that selenium is required for optimal growth of Chlamydomonas: Finally, evolutionary analyses suggested that selenoproteins present in Chlamydomonas and animals evolved early, and were independently lost in land plants, yeast and some animals. More Information

Kryukov GV, Kumar RA, Koc A, Sun Z, Gladyshev VN. (2002) Selenoprotein R is a zinc-containing stereo-specific methionine sulfoxide reductase. Proc. Natl. Acad. Sci. USA 99, 4245-4250.

AbstractSelenoprotein R (SelR) is a mammalian selenocysteine-containing protein with no known function. Here we report that cysteine homologs of SelR are present in all organisms except certain parasites and hyperthermophiles, and this pattern of occurrence closely matches that of only one protein, peptide methionine sulfoxide reductase (MsrA). Moreover, in several genomes, SelR and MsrA genes are fused or clustered, and their expression patterns suggest a role of both proteins in protection against oxidative stress. Consistent with these computational screens, growth of Saccharomyces cerevisiae SelR and MsrA mutant strains was inhibited, and the strain lacking both genes could not grow, in the presence of H2O2 and methionine sulfoxide. We found that the cysteine mutant of mouse SelR, as well as the Drosophila SelR homolog, contained zinc and reduced methionine-R-sulfoxide, but not methionine-S-sulfoxide, in in vitro assays, a function that is both distinct and complementary to the stereo-specific activity of MsrA. These findings identify a function of the conserved SelR enzyme family, define a pathway of methionine sulfoxide reduction, reveal a case of convergent evolution of similar function in structurally distinct enzymes, and suggest a previously uncharacterized redox regulatory role of selenium in mammals. More Information

Korotkov KV, Novoselov SV, Hatfield DL, Gladyshev VN. (2002) Mammalian selenoprotein in which selenocysteine (Sec) incorporation is supported by a new form of Sec insertion sequence element. Mol. Cell. Biol. 22, 1402-1411.

AbstractSelenocysteine (Sec), the 21st amino acid in protein, is encoded by UGA. The Sec insertion sequence (SECIS) element, which is the stem-loop structure present in 3′ untranslated regions (UTRs) of eukaryotic selenoprotein-encoding genes, is essential for recognition of UGA as a codon for Sec rather than as a stop signal. We now report the identification of a new eukaryotic selenoprotein, designated selenoprotein M (SelM). The 3-kb human SelM-encoding gene has five exons and is located on chromosome 22 but has not been correctly identified by either Celera or the public Human Genome Project. We characterized human and mouse SelM cDNA sequences and expressed the selenoprotein in various mammalian cell lines. The 3′ UTR of the human, mouse, and rat SelM-encoding genes lacks a canonical SECIS element. Instead, Sec is incorporated in response to a conserved mRNA structure, in which cytidines are present in place of the adenosines previously considered invariant. Substitution of adenosines for cytidines did not alter Sec incorporation; however, other mutant structures did not support selenoprotein synthesis, demonstrating that this new form of SECIS element is functional. SelM is expressed in a variety of tissues, with increased levels in the brain. It is localized to the perinuclear structures, and its N-terminal signal peptide is necessary for protein translocation. More Information

Gladyshev VN. (2002) Thioredoxin and peptide methionine sulfoxide reductase: convergence of similar structure and function in distinct structural folds. Proteins 146, 149-152.

AbstractThioredoxin (Trx) and peptide methionine sulfoxide reductase (PMSR) are small thiol oxidoreductases implicated in antioxidant defense and redox regulation of cellular processes. Here we show that the structures of Trx and PMSR exhibit resemblance in their alphabeta core regions and that the active site cysteines in two proteins occupy equivalent positions downstream of a central beta-strand and at the N-terminus of an alpha-helix. Moreover, we identified a PMSR subfamily that contains an active site CxxC motif (two cysteines separated by two other amino acids) positioned similarly to the catalytic redox active CxxC motif in Trx. However, Trx and PMSR are characterized by distinct ancient folds that differ in both orientation of secondary structures and their patterns. Trx is a member of the Trx-fold superfamily, whereas PMSR has a unique fold not found in other proteins. The data suggest that similar structures and functions of Trx and PMSR were acquired independently during evolution and point to a general strategy of identifying new redox regulatory proteins. Copyright 2001 Wiley-Liss, Inc. More Information

Kumaraswamy E, Korotkov KV, Diamond AM, Gladyshev VN, Hatfield DL. (2002) Genetic and functional analysis of mammalian Sep15 selenoprotein. Methods Enzymol. 347, 187-197.

Kryukov GV, Gladyshev VN. (2002) Mammalian Selenoprotein Gene Signature: identification and functional analysis of selenoprotein genes using bioinformatics methods. Methods Enzymol. 347, 84-100.

Hatfield DL, Gladyshev VN. (2002) How selenium has altered our understanding of the genetic code. Mol. Cell. Biol. 22, 3565-3576.

AbstractSelenium is an essential micronutrient in the diet of many life forms, including humans and other mammals. Significant health benefits have been attributed to this element. It is rapidly becoming recognized as one of the more promising cancer chemopreventive agents (19), and there are strong indications that it has a role in reducing viral expression (4), in preventing heart disease and other cardiovascular and muscle disorders (23), and in delaying the progression of AIDS in human immunodeficiency virus-infected patients (3). Additional evidence suggests that selenium may have a role in mammalian development (51), in immune function (70), in male reproduction (30), and in slowing the aging process (70). More Information

Sun Q, Gladyshev VN. (2002) Redox regulation of cell signaling by thioredoxin reductase. Methods Enzymol. 347, 451-461.

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Gladyshev VN, Liu A, Novoselov SV, Krysan K, Sun QA, Kryukov VM, Kryukov GV, Lou MF (2001) Identification and characterization of a new mammalian glutaredoxin (thioltransferase) Grx2. J. Biol. Chem. 276, 30374-30380.

AbstractA thiol/disulfide oxidoreductase component of the GSH system, glutaredoxin (Grx), is involved in the reduction of GSH-based mixed disulfides and participates in a variety of cellular redox pathways. A single cytosolic Grx (Grx1) was previously described in mammals. We now report identification and characterization of a second mammalian Grx, designated Grx2. Grx2 exhibited 36% identity with Grx1 and had a disulfide active center containing the Cys-Ser-Tyr-Cys motif. Grx2 was encoded in the genomes of mammals and birds and expressed in a variety of cell types. The gene for human Grx2 consisted of four exons and three introns, spanned 10 kilobase pairs, and localized to chromosome 1q31.2-31.3. The coding sequence was present in all exons, with the first exon encoding a mitochondrial signal peptide. The mitochondrial leader sequence was also present in mouse and rat Grx2 sequences and was shown to direct either Grx2 or green fluorescent protein to mitochondria. Alternative splicing forms of mammalian Grx2 mRNAs were identified that differed in sequences upstream of exon 2. To functionally characterize the new protein, human and mouse Grx2 proteins were expressed in Escherichia coli, and the purified proteins were shown to reduce mixed disulfides formed between GSH and S-sulfocysteine, hydroxyethyldisulfide, or cystine. Grx1 and Grx2 were sensitive to inactivation by iodoacetamide and H(2)O(2) and exhibited similar pH dependence of catalytic activity. However, H(2)O(2)-inactivated Grx2 could only be reactivated with 5 mm GSH, whereas Grx1 could also be reactivated with dithiothreitol or thioredoxin/thioredoxin reductase. The Grx2 structural model suggested a common reaction mechanism for this class of proteins. The data provide the first example of a mitochondrial Grx and also indicate the occurrence of a second functional Grx in mammals. More Information

Martin-Romero FJ, Kryukov GK, Lobanov AV, Carlson BA, Lee BJ, Gladyshev VN, Hatfield DL. (2001) Selenium metabolism in Drosophila: selenoproteins, selenoprotein mRNA expression, fertility and mortality. J. Biol. Chem. 276, 29798-29804.

AbstractSelenocysteine is a rare amino acid in protein that is encoded by UGA with the requirement of a downstream mRNA stem-loop structure, the selenocysteine insertion sequence element. To detect selenoproteins in Drosophila, the entire genome was analyzed with a novel program that searches for selenocysteine insertion sequence elements, followed by selenoprotein gene signature analyses. This computational screen and subsequent metabolic labeling with (75)Se and characterization of selenoprotein mRNA expression resulted in identification of three selenoproteins: selenophosphate synthetase 2 and novel G-rich and BthD selenoproteins that had no homology to known proteins. To assess a biological role for these proteins, a simple chemically defined medium that supports growth of adult Drosophila and requires selenium supplementation for optimal survival was devised. Flies survived on this medium supplemented with 10(-8) to 10(-6) m selenium or on the commonly used yeast-based complete medium at about twice the rate as those on a medium without selenium or with >10(-6) m selenium. This effect correlated with changes in selenoprotein mRNA expression. The number of eggs laid by Drosophila was reduced approximately in half in the chemically defined medium compared with the same medium supplemented with selenium. The data provide evidence that dietary selenium deficiency shortens, while supplementation of the diet with selenium normalizes the Drosophila life span by a process that may involve the newly identified selenoproteins. More Information

Moustafa ME, Carlson BA, El-Saadani MA, Kryukov GV, Sun Q.-A, Harney JW, Hill KE, Combs GF, Feigenbaum L, Mansur DB, Burk RF, Berry MJ, Diamond AM, Lee BJ, Gladyshev VN, Hatfield DL. (2001) Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine mutant selenocysteine tRNA transgenes. Mol. Cell. Biol. 21, 3840-3852.

AbstractSelenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins. More Information

Sun Q-A, Kirnarsky L, Sherman S, Gladyshev VN. (2001) Selenoprotein oxidoreductase with specificity for thioredoxin and glutathione systems. Proc. Natl. Acad. Sci. USA 98, 3673-3678.

AbstractThioredoxin (Trx) and glutathione (GSH) systems are considered to be two major redox systems in animal cells. They are reduced by NADPH via Trx reductase (TR) or oxidized GSH (GSSG) reductase and further supply electrons for deoxyribonucleotide synthesis, antioxidant defense, and redox regulation of signal transduction, transcription, cell growth, and apoptosis. We cloned and characterized a pyridine nucleotide disulfide oxidoreductase, Trx and GSSG reductase (TGR), that exhibits specificity for both redox systems. This enzyme contains a selenocysteine residue encoded by the TGA codon. TGR can reduce Trx, GSSG, and a GSH-linked disulfide in in vitro assays. This unusual substrate specificity is achieved by an evolutionary conserved fusion of the TR and glutaredoxin domains. These observations, together with the biochemical probing and molecular modeling of the TGR structure, suggest a mechanism whereby the C-terminal selenotetrapeptide serves a role of a protein-linked GSSG and shuttles electrons from the disulfide center within the TR domain to either the glutaredoxin domain or Trx. More Information

Korotkov KV, Kumaraswamy E, Zhou Y, Hatfield DL, Gladyshev VN. (2001) Association between the 15 kDa selenoprotein and UDP-glucose:glycoprotein glucosyltransferase in the endoplasmic reticulum of mammalian cells. J. Biol. Chem. 276, 15330-15336.

AbstractMammalian selenocysteine-containing proteins characterized with respect to function are involved in redox processes and exhibit distinct expression patterns and cellular locations. A recently identified 15-kDa selenoprotein (Sep15) has no homology to previously characterized proteins, and its function is not known. Here we report the intracellular localization and identification of a binding partner for this selenoprotein which implicate Sep15 in the regulation of protein folding. The native Sep15 isolated from rat prostate and mouse liver occurred in a complex with a 150-kDa protein. The latter protein was identified as UDP-glucose:glycoprotein glucosyltransferase (UGTR), the endoplasmic reticulum (ER)-resident protein, which was previously shown to be involved in the quality control of protein folding. UGTR functions by glucosylating misfolded proteins, retaining them in the ER until they are correctly folded or transferring them to degradation pathways. To determine the intracellular localization of Sep15, we expressed a green fluorescent protein-Sep15 fusion protein in CV-1 cells, and this protein was localized to the ER and possibly other perinuclear compartments. We determined that Sep15 contained the N-terminal signal peptide that was essential for translocation and that it was cleaved in the mature protein. However, C-terminal sequences of Sep15 were not involved in trafficking and retention of Sep15. The data suggest that the association between Sep15 and UGTR is responsible for maintaining the selenoprotein in the ER. This report provides the first example of the ER-resident selenoprotein and suggests a possible role of the trace element selenium in the quality control of protein folding. More Information

Hu YJ, Korotkov KV, Mehta R, Hatfield DL, Rotimi C, Luke A, Prewitt TE, Cooper RS, Stock W, Vokes EE, Dolan ME, Gladyshev VN, Diamond AM. (2001) Distribution and functional consequences of nucleotide polymorphisms in the 3′-untranslated region of the human Sep15 gene. Cancer Res. 61, 2307-2310.

AbstractSelenium has been shown to prevent cancer in a variety of animal model systems. Both epidemiological studies and supplementation trials have supported its efficacy in humans. However, the mechanism by which selenium suppresses tumor development remains unknown. Selenium is present in known human selenoproteins as the amino acid selenocysteine (Sec). Sec is inserted cotranslationally in response to UGA codons within selenoprotein mRNAs in a process requiring a sequence within the 3′-untranslated region (UTR), referred to as a Sec insertion sequence (SECIS) element. Recently, a human Mr 15,000 selenoprotein (Sep15) was identified that contains an in-frame UGA codon and a SECIS element in the 3′-UTR. Examination of the available cDNA sequences for this protein revealed two polymorphisms located at position 811 (C/T) and at position 1125 (G/A) located within the 3′-UTR. Here, we demonstrate significant differences in Sep15 allele frequencies by ethnicity and that the identity of the nucleotides at the polymorphic sites influences SECIS function in a selenium-dependent manner. This, together with genetic data indicating loss of heterozygosity at the Sep15 locus in certain human tumor types, suggests that Sep15 may be involved in cancer development, risk, or both. More Information

Sun Q-A, Zappacosta F, Factor VM, Wirth P, Hatfield DL, Gladyshev VN. (2001) Heterogeneity within animal thioredoxin reductases: evidence for alternative first exon splicing. J. Biol. Chem. 276, 3106-3114.

AbstractAnimal thioredoxin reductases (TRs) are selenocysteine-containing flavoenzymes that utilize NADPH for reduction of thioredoxins and other protein and nonprotein substrates. Three types of mammalian TRs are known, with TR1 being a cytosolic enzyme, and TR3, a mitochondrial enzyme. Previously characterized TR1 and TR3 occurred as homodimers of 55-57-kDa subunits. We report here that TR1 isolated from mouse liver, mouse liver tumor, and a human T-cell line exhibited extensive heterogeneity as detected by electrophoretic, immunoblot, and mass spectrometry analyses. In particular, a 67-kDa band of TR1 was detected. Furthermore, a novel form of mouse TR1 cDNA encoding a 67-kDa selenoprotein subunit with an additional N-terminal sequence was identified. Subsequent homology analyses revealed three distinct isoforms of mouse and rat TR1 mRNA. These forms differed in 5′ sequences that resulted from the alternative use of the first three exons but had common downstream sequences. Similarly, expression of multiple mRNA forms was observed for human TR3 and Drosophila TR. In these genes, alternative first exon splicing resulted in the formation of predicted mitochondrial and cytosolic proteins. In addition, a human TR3 gene overlapped with the gene for catechol-O-methyltransferase (COMT) on a complementary DNA strand, such that mitochondrial TR3 and membrane-bound COMT mRNAs had common first exon sequences; however, transcription start sites for predicted cytosolic TR3 and soluble COMT forms were separated by approximately 30 kilobases. Thus, this study demonstrates a remarkable heterogeneity within TRs, which, at least in part, results from evolutionary conserved genetic mechanisms employing alternative first exon splicing. Multiple transcription start sites within TR genes may be relevant to complex regulation of expression and/or organelle- and cell type-specific location of animal thioredoxin reductases. More Information

Gladyshev VN. (2001) Comparison of selenium-containing molybdoenzymes. In Molybdenum and Tungsten. Their roles in biological systems”. “ Metal Ions in Biological Systems 39, 655-672.

Gladyshev VN, Kryukov GV. (2001) Evolution of selenocysteine-containing proteins: significance of identification and functional characterization of selenoproteins. BioFactors 14, 87-92.

AbstractIn the genetic code, UGA serves as either a signal for termination or a codon for selenocysteine (Sec). Sec rarely occurs in protein and is different from other amino acids in that much of the biosynthetic machinery governing its incorporation into protein is unique to this amino acid. Sec-containing proteins have diverse functions and lack a common amino acid motif or consensus sequence. Sec has previously been considered to be a relic of the primordial genetic code that was counter-selected by the presence of oxygen in the atmosphere. In the present report, it is proposed that Sec was added to the already existing genetic code and its use has accumulated during evolution of eukaryotes culminating in vertebrates. The more recently evolved selenoproteins appear to take advantage of unique redox properties of Sec that are superior to those of Cys for specific biological functions. Further understanding of the evolution of selenoproteins as well as biological properties and biomedical applications of the trace element selenium requires identification and functional characterization of all mammalian selenoproteins. More Information

Gladyshev VN. (2001) Identity, evolution and functions of selenoproteins and selenoprotein genes. In Selenium: its molecular biology and role in human health (ed., Hatfield DL), Kluwer Academic Publishers, pp. 99-113.

Gladyshev VN, Diamond DL, Hatfield DL. (2001) The 15 kDa selenoprotein (Sep15): functional studies and a role in cancer etiology. In Selenium: its molecular biology and role in human health (ed., Hatfield DL), Kluwer Academic Publishers, pp. 147-155.

AbstractThe 15 kDa selenoprotein (Sepl5) is one of several recently identified selenoproteins. It contains a single selenocysteine residue in the middle of a 162-amino acid open reading frame and has no detectable homology to known proteins. The human Sepl5 gene spans 5 1 kb, has 5 exons and is located on chromosome 1 at position p3 1. The gene contains two single nucleotide polymorphisms in the 3\’-untranslated region (3\’-UTR) including one in the SECIS element, that are distributed differently between Caucasians and African Americans. Sep15 localizes to the endoplasmic reticulum where it is tightly bound to UDP-glucose:glycoprotein glucosyltransferase, a protein involved in the quality control of protein folding. Sepl5 may be involved in the chemopreventive effect of dietary selenium. This hypothesis is based on its differential expression in normal and malignant tissues, the distribution and functional consequences of natural polymorphisms within its gene, and the location of the Sepl5 gene in a region that is often altered in a variety of cancers. More Information

Gladyshev VN. (2001) Selenium in biology and human health: controversies and perspectives. In Selenium: its molecular biology and role in human health (ed., Hatfield DL), Kluwer Academic Publishers, pp. 313-317.

AbstractImportant unresolved questions raised by the contributors of this book and addressing roles of selenium in biology and human health are discussed. Resolving major scientific controversies in the field should further highlight a bright future for selenium in fundamental science, biotechnology and medicine. More Information

Gladyshev VN, Hatfield DL. (2001) Analysis of selenocysteine-containing proteins. Curr. Protoc. Protein Sci. 3, 3.8.

AbstractRepresentatives of three primary life domains–bacteria, archaea, and eukaryotes–possess specific selenium-containing proteins. The majority of naturally occurring selenoproteins contain an amino acid, selenocysteine, that is incorporated into protein in response to the code word UGA. The presence of selenium in natural selenoproteins and in proteins in which this element is introduced by chemical or biological manipulations provides additional opportunities for characterizing structure, function, and mechanism of action. This unit provides an overview of known selenocysteine-containing proteins, examples of targeted incorporation of selenium into proteins, and methods specific for selenoprotein identification and characterization. More Information

Kumaraswamy E, Malykh A, Korotkov KV, Kozyavkin S, Hu Y, Moustafa ME, Carlson B, Berry MJ, Lee BJ, Hatfield DL, Diamond AM, Gladyshev VN. (2000) Structure-expression relationships of the 15 kDa selenoprotein gene: possible role of the protein in cancer etiology. J. Biol. Chem. 275, 35540-35547.

AbstractSelenium has been implicated in cancer prevention, but the mechanism and possible involvement of selenoproteins in this process are not understood. To elucidate whether the 15-kDa selenoprotein may play a role in cancer etiology, the complete sequence of the human 15-kDa protein gene was determined, and various characteristics associated with expression of the protein were examined in normal and malignant cells and tissues. The 51-kilobase pair gene for the 15-kDa selenoprotein consisted of five exons and four introns and was localized on chromosome 1p31, a genetic locus commonly mutated or deleted in human cancers. Two stem-loop structures resembling selenocysteine insertion sequence elements were identified in the 3′-untranslated region of the gene, and only one of these was functional. Two alleles in the human 15-kDa protein gene were identified that differed by two single nucleotide polymorphic sites that occurred within the selenocysteine insertion sequence-like structures. These 3′-untranslated region polymorphisms resulted in changes in selenocysteine incorporation into protein and responded differently to selenium supplementation. Human and mouse 15-kDa selenoprotein genes manifested the highest level of expression in prostate, liver, kidney, testis, and brain, and the level of the selenoprotein was reduced substantially in a malignant prostate cell line and in hepatocarcinoma. The expression pattern of the 15-kDa protein in normal and malignant tissues, the occurrence of polymorphisms associated with protein expression, the role of selenium in differential regulation of polymorphisms, and the chromosomal location of the gene may be relevant to a role of this protein in cancer. More Information

Kryukov GV, Gladyshev VN. (2000) Selenium metabolism in zebrafish: multiplicity of selenoprotein genes and expression of a protein containing seventeen selenocysteine residues. Genes Cells 5, 1049-1060.

AbstractBACKGROUND: Fish are an important source of selenium in human nutrition and the zebrafish is a potentially useful model organism for the study of selenium metabolism and its role in biology and medicine. Selenium is present in vertebrate proteins in the form of selenocysteine (Sec), the 21st natural amino acid in protein which is encoded by UGA. RESULTS: We report here the detection of 18 zebrafish genes for Sec-containing proteins. We found two zebrafish orthologs of human SelT, glutathione peroxidase 1 and glutathione peroxidase 4, and single orthologs of several other selenoproteins. In addition, new zebrafish selenoproteins were identified that were distant homologues of SelP, SelT and SelW, but their direct orthologs in other species are not known. This multiplicity of selenoprotein genes appeared to result from gene and genome duplications, followed by the retention of new selenoprotein genes. We found a zebrafish selenoprotein P gene (designated zSelPa) that contained two Sec insertion sequence (SECIS) elements and encoded a protein containing 17 Sec residues, the largest number of Sec residues found in any known protein. In contrast, a second SelP gene (designated zSelPb) was also identified that contained one SECIS element and encoded a protein with a single Sec. We found that zSelPa could be expressed and secreted by mammalian cells. CONCLUSIONS: The occurrence of zSelPa and zSelPb suggested that the function of the N-terminal domain of mammalian SelP proteins may be separated from that of the C-terminal Sec-rich sequence: the N-terminal domain containing the UxxC motif is likely involved in oxidoreduction, whereas the C-terminal portion of the protein may function in selenium transport or storage. Our data also suggest that the utilization of Sec is more common in zebrafish than in previously characterized species, including mammals. More Information

Mansur DB, Hao H, Gladyshev VN, Korotkov K, Hu Y, Moustafa ME, El-Saadani MA. Carlson BA, Hatfield DL, Diamond AM. (2000) Multiple levels of regulation of selenoprotein biosynthesis revealed from the analysis of human glioma cell lines. Biochem. Pharm. 60, 489-497.

AbstractTo gain a better understanding of the biological consequences of the exposure of tumor cells to selenium, we evaluated the selenium-dependent responses of two selenoproteins (glutathione peroxidase and the recently characterized 15-kDa selenoprotein) in three human glioma cell lines. Protein levels, mRNA levels, and the relative distribution of the two selenocysteine tRNA isoacceptors (designated mcm(5)U and mcm(5)Um) were determined for standard as well as selenium-supplemented conditions. The human malignant glioma cell lines D54, U251, and U87 were maintained in normal or selenium-supplemented (30 nM sodium selenite) conditions. Northern blot analysis demonstrated only minor increases in steady-state GSHPx-1 mRNA in response to selenium addition. Baseline glutathione peroxidase activity was 10.7 +/- 0.7, 7.6 +/- 0.7, and 4.3 +/- 0.7 nmol NADPH oxidized/min/mg protein for D54, U251, and U87, respectively, as determined by the standard coupled spectrophotometric assay. Glutathione peroxidase activity increased in a cell line-specific manner to 19.7 +/- 1.4, 15.6 +/- 2.1, and 6. 7 +/- 0.5 nmol NADPH oxidized/min/mg protein, respectively, as did a proportional increase in cellular resistance to H(2)O(2), in response to added selenium. The 15-kDa selenoprotein mRNA levels likewise remained constant despite selenium supplementation. The selenium-dependent change in distribution between the two selenocysteine tRNA isoacceptors also occurred in a cell line-specific manner. The percentage of the methylated isoacceptor, mcm(5)Um, changed from 35.5 to 47.2 for D54, from 38.1 to 47.3 for U251, and from 49.0 to 47.6 for U87. These data represent the first time that selenium-dependent changes in selenoprotein mRNA and protein levels, as well as selenocysteine tRNA distribution, were examined in human glioma cell lines. More Information

Kryukov GV, Kryukov VM, Gladyshev VN. (1999) New Mammalian Selenocysteine-containing Proteins Identified with an Algorithm That Searches for Selenocysteine Insertion Sequence Elements. J. Biol. Chem. 274, 33888-33897.

Sun QA, Wu Y, Zappacosta F, Jeang KT, Lee BJ, Hatfield DL, Gladyshev VN. (1999) Redox regulation of cell signaling by selenocysteine in mammalian thioredoxin reductases. J. Biol. Chem. 274, 24522-24530.

Gladyshev VN, Krause M, Xu XM, Korotkov KV, Kryukov GV, Sun QA, Lee BJ, Wootton JC, Hatfield DL. (1999) Selenocysteine-containing thioredoxin reductase in C. elegans. Biochem. Biophys. Res. Commun. 259, 244-249.

Gladyshev VN, Stadtman TC, Hatfield DL, Jeang KT. (1999) Levels of major selenoproteins in T cells decrease during HIV infection and low molecular mass selenium compounds increase. Proc. Natl. Acad. Sci. USA 96, 835-839.

AbstractIt has been observed previously that plasma selenium and glutathione levels are subnormal in HIV-infected individuals, and plasma glutathione peroxidase activity is decreased. Under these conditions the survival rate of AIDS patients is reduced significantly. In the present study, using 75Se-labeled human Jurkat T cells, we show that the levels of four 75Se-containing proteins are lower in HIV-infected cell populations than in uninfected cells. These major selenoproteins migrated as 57-, 26-, 21-, and 15-kDa species on SDS/PAGE gels. In our earlier studies, the 57-kDa protein was purified from T cells and identified as a subunit of thioredoxin reductase. The 26- and 21-kDa proteins were identified in immunoblot assays as the glutathione peroxidase (cGPX or GPX1) subunit and phospholipid hydroperoxide glutathione peroxidase (PHGPX or GPX4), respectively. We recently purified the 15-kDa protein and characterized it as a selenoprotein of unknown function. In contrast to selenoproteins, low molecular mass [75Se]compounds accumulated during HIV infection and migrated as a diffuse band near the front of SDS/PAGE gels. More Information

Gladyshev VN, Martin-Romero FJ, Xu XM, Kumaraswamy E, Carlson BA, Hatfield DL, Lee BJ. (1999) Molecular biology of selenium and its role in cancer, AIDS and other human diseases. Recent Research Developments in Biochemistry 1, 145-167

Gladyshev VN, Hatfield DL. (1999) Selenocysteine-containing proteins in mammals. J. Biomed. Sci. 6, 151-160.

Hatfield DL, Gladyshev VN, Park J, Park SI, Chittum HS, Baek HJ, Carlson BA, Yang ES, Moustafa ME, Lee BJ. (1999) Biosynthesis of selenocysteine and its incorporation into protein as the 21st amino acid. Comprehensive Natural Products Chemistry 4, 353-380.

Gladyshev VN, Factor VM, Housseau F, Hatfield DL. (1998) Contrasting patterns of regulation of the antioxidant selenoproteins, thioredoxin reductase, and glutathione peroxidase, in cancer cells. Biochem. Biophys. Res. Commun. 251, 488-93.

Gladyshev VN, Jeang KT, Wootton JC, Hatfield DL. (1998) A new human selenium-containing protein: purification, characterization and cDNA sequence. J. Biol. Chem. 273, 8910-8915.

AbstractSelenium which occurs in proteins as the amino acid, selenocysteine, is essential for numerous biological processes and for human health. A prominent 75Se-labeled protein detected in human T-cells migrated as a 15-kDa band by SDS-polyacrylamide gel electrophoresis. This protein subunit was purified and subjected to tryptic digestion and peptide sequence analyses. Sequences of tryptic peptides derived from the protein corresponded to a human placental gene sequence containing an open reading frame of 162 residues and a readthrough in-frame TGA codon. Three different peptide sequences of the 15-kDa protein corresponded to a nucleotide sequence located downstream of this codon, suggesting that the T-cell 15-kDa selenoprotein contains a selenocysteine residue encoded by TGA. Post-translational processing of the N-terminal portion of the predicted gene product to give the 15-kDa protein was suggested on the basis of molecular mass, amino acid analysis, and immunoblot assays of the purified protein. The 3-untranslated region (UTR) of the gene encoding the 15-kDa protein contained a sequence that is very similar to the canonical selenocysteine-inserting sequence element. Computer analysis of transcript map data bases indicated that this gene was located on human chromosome 1. Its coding sequence showed no homology to known protein-encoding genes. The 15-kDa protein gene was expressed as mRNA in a wide range of tissues, with increased levels in the thyroid, parathyroid, and prostate-derived cells as evidenced by searches of partial cDNA sequences in public data bases. Genes corresponding to the 15-kDa selenocysteinecontaining protein were found in mice and rats, while the corresponding genes in Caenorhabditis elegans and Brugia malayi contained a cysteine codon in place of TGA. The discovery of a new human selenoprotein provides an additional example of the role of selenium in mammalian systems. More Information

Khangulov SV, Gladyshev VN, Dismukes C, Stadtman TC. (1998) Selenium-containing formate dehydrogenase from E. coli: molybdopterin enzyme that catalyes formate oxidation without oxygen transfer. Biochemistry 37, 3518-3528.

George GN, Colangelo CM, Dong J, Scott RA, Khangulov SV, Gladyshev VN, Stadtman TC. (1998) X-ray absorbtion spectroscopy of the molybdenum site of Escherichia coli formate dehydrogenase. J. Am. Chem. Soc. 120, 1267-1273.

Boyington JC, Gladyshev VN, Khangulov SV, Stadtman TC, Sun PD. (1997) Crystal structure of formate dehydrogenase H: catalysis involving molybdenum, molybdopterin, selenocysteine and an Fe4S4 cluster. Science 275, 1305-1308.

AbstractFormate dehydrogenase H from Escherichia coli contains selenocysteine (SeCys), molybdenum, two molybdopterin guanine dinucleotide (MGD) cofactors, and an Fe4S4 cluster at the active site and catalyzes the two-electron oxidation of formate to carbon dioxide. The crystal structures of the oxidized [Mo( VI), Fe4S4(ox)] form of formate dehydrogenase H (with and without bound inhibitor) and the reduced [Mo(IV ), Fe4S4(red)] form have been determined, revealing a four-domain ab structure with the molybdenum directly coordinated to selenium and both MGD cofactors. These structures suggest a reaction mechanism that directly involves SeCys140 and His141 in proton abstraction and the molybdenum, molybdopterin, Lys44, and the Fe4S4 cluster in electron transfer. More Information

Baek HJ, Chittum HS, Yang ES, Park JM, Gladyshev VN, Moustafa ME, Carlson BA, Diamond AM, Lee BJ, Hatfield DL. (1997) Response of the selenocysteine tRNA population to selenium in mammals and Xenopus oocytes. Nucl. Acids Res. Symp. Ser. 36, 157-158

Gladyshev VN, Lecchi P. (1996) Identification of molybdopterins in molybdenum- and selenium-containing enzymes. BioFactors 5, 94-97.

Gladyshev VN, Jeang KT, Stadtman TC. (1996) Selenocysteine, identified as the penultimate C- terminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene. Proc. Natl. Acad. Sci. USA 93, 6146-6151.

AbstractThe possible relationship of selenium to immunological function which has been suggested for decades was investigated in studies on selenuim metabolism in human T cells. One of the major 75Se-labeled selenoproteins detected was purified to homogeneity and shown to be a homodimer of 55-kDa subunits. Each subunit contained about 1 FAD and at least 0.74 Se. This protein proved to be thioredoxin reductase (TR) on the basis of its catalytic activities, cross-reactivity with anti-rat liver TR antibodies, and sequence identities of several tryptic peptides with the published deduced sequence of human placental TR. Physicochemical characteristics of T-cell TR were similar to those of a selenocysteine (Secys)- containing TR recently isolated from human lung adenocarcinoma cells. The sequence of a 12-residue 75Se-labeled tryptic peptide from T-cell TR was identical with a C-terminaldeduced sequence of human placental TR except that Secys was present in the position corresponding to TGA, previously thought to be the termination codon, and this was followed by Gly-499, the actual C-terminal amino acid. The presence of the unusual conserved Cys-Secys-Gly sequence at the C terminus of TR in addition to the redox active cysteines of the Cys-Val- Asn-Val-Gly-Cys motif in the FAD-binding region may account for the peroxidase activity and the relatively low substrate specificity of mammalian TRs. The finding that T-cell TR is a selenoenzyme that contains Se in a conserved Cterminal region provides another example of the role of selenium in a major antioxidant enzyme system (i.e., thioredoxin- thioredoxin reductase), in addition to the well-known glutathione peroxidase enzyme system. More Information

Gladyshev VN, Khangulov SV, Stadtman TC. (1996) Properties of selenium- and molybdenum-containing nicotinic acid hydroxylase from Clostridium barkeri. Biochemistry 35, 212-223.

Gladyshev VN, Boyington JC, Khangulov SV, Grahame DA, Stadtman TC, Sun PD. (1996) Characterization of crystalline formate dehydrogenase H from Escherichia Coli: stabilization, EPR spectroscopy and preliminary crystallographic analysis. J. Biol. Chem. 271, 8095-8100.

AbstractThe selenocysteine-containing formate dehydrogenase H (FDH) is an 80-kDa component of the Escherichia coli formate-hydrogen lyase complex. The molybdenumcoordinated selenocysteine is essential for catalytic activity of the native enzyme. FDH in dilute solutions (30 µg/ml) was rapidly inactivated at basic pH or in the presence of formate under anaerobic conditions, but at higher enzyme concentrations ( 3 mg/ml) the enzyme was relatively stable. The formate-reduced enzyme was extremely sensitive to air inactivation under all conditions examined. Active formate-reduced FDH was crystallized under anaerobic conditions in the presence of ammonium sulfate and PEG 400. The crystals diffract to 2.6 � resolution and belong to a space group of P41212 or P43212 with unit cell dimensions a = b = 146.1 � and c = 82.7 �. There is one monomer of FDH per crystallographic asymmetric unit. Similar diffraction quality crystals of oxidized FDH could be obtained by oxidation of crystals of formate-reduced enzyme with benzyl viologen. By EPR spectroscopy, a signal of a single reduced FeS cluster was found in a crystal of reduced FDH, but not in a crystal of oxidized enzyme, whereas Mo(V) signal was not detected in either form of crystalline FDH. This suggests that Mo(IV)- and the reduced FeS cluster-containing form of the enzyme was crystallized and this could be converted into Mo(VI)- and oxidized FeS cluster form upon oxidation. A procedure that combines anaerobic and cryocrystallography has been developed that is generally applicable to crystallographic studies of oxygen-sensitive enzymes. These data provide the first example of crystallization of a substrate-reduced form of a Se- and Mo-containing enzyme. More Information

Tamura T, Gladyshev VN, Liu SY, Stadtman TC. (1996) The mutual sparring effects of selenium and vitamin E in animal nutrition may be further explained by the discovery that mammalian thioredoxin reductase is a selenoenzyme. BioFactors 5, 99-102.

Gladyshev VN, Khangulov SV, Stadtman TC. (1994) Nicotinc acid hydroxylase from Clostridium barkeri: Electron paramagnetic resonance studies show that selenium is coordinated with molybdenum in the catalytically active selenium-dependent enzyme. Proc. Natl. Acad. Sci. USA 91, 232-236.

Gladyshev VN, Khangulov SV, Axley MJ, Stadtman TC. (1994) Coordination of selenium to molybdenum in formate dehydrogenase H from Escherichia coli.. Proc. Natl. Acad. Sci. USA 91, 7708-7711.

Tishkov VI, Galkin AG, Gladyshev VN, Karzanov VV, Egorov AM. (1992) Analysis of gene structure, optimization of expression in E. coli, and properties of recombinant formate dehydrogenase from Pseudomonas sp. 101. Biotechnology 5, 52-59.

Books

Hatfield DL, Schweizer U, Tsuji PA, Gladyshev VN. Editors. (2016) Selenium: Its Molecular Biology and Role in Human Health, 4th Edition. Springer.

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Carlson BA, Lee BJ, Tsuji PA, Tobe R, Park JM, Schweitzer U, Gladyshev VN, Hatfield DL. Selenocysteine tRNA^[Ser]Sec: From Nonsense Suppressor tRNA to the Quintessential Constituent in Selenoprotein Biosynthesis. Springer, 3-12.
Gladyshev VN, Carlson BA, Hatfield DL. Pathways in De Novo Biosynthesis of Selenocysteine and Cysteine in Eukaryotes. Springer, 39-45.
Gladyshev VN. Eukaryotic Selenoproteomes. Springer, 127-139.
Tsuji PA, Carlson BA, Lee BJ, Gladyshev VN, Hatfield DL. Interplay of Selenoproteins and Different Antioxidant Systems in Various Cancers. Springer, 441-449.
Lobanov AV, Gladyshev VN. The Naked Mole Rat and Selenium. Springer, 579-585.

Jakob U, Reichmann D. Editors. (2013) Oxidative Stress and Redox Regulation. Springer.

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Marino SM, Roos G, Gladyshev VN. Computational Redox Biology: Methods and Applications. Springer, 187-211.

Banci B. Editor. (2013) Metallomics and the Cell. Springer.

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Gladyshev VN, Zhang Y. Comparative Genomics Analysis of the Metallomes. Springer, 529-580.

Jager T, Koch O, Flohe L, Selzer PM. Editors. (2013)
Trypanosomatid Diseases: Molecular Routes to Drug Discovery. Wiley-Blackwell.

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Gladyshev VN, Zhang Y. Selenoproteome of Kinetoplastids. Wiley-Blackwell, 237-242.

Hatfield DL, Berry MJ, Gladyshev VN. Editors. (2011) Selenium: Its molecular biology and role in human health, 3rd Edition. Springer.

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Xu XM, Turanov A, Carlson BA, Yoo MH, Gladyshev VN, Hatfield DL. Selenocysteine biosynthesis and the replacement of selenocysteine with cysteine in the pathway. Springer, 23-31.
Gladyshev VN. Selenoproteins and selenoproteomes. Springer, 109-123.
Marino SM, Gladyshev VN, Dikiy A. Structural characterization of mammalian selenoproteins. Springer, 125-136.
Yoo MH, Carlson BA, Tsuji PA, Tobe R, Naranjo-Suarez S, Lee BJ, Davis CD, Gladyshev VN, Hatfield DL. Selenoproteins harboring a split personality in both preventing and promoting cancer. Springer, 325-333.
Turanov AA, Malinouski M, Gladyshev VN. Selenium and male reproduction. Springer, 409-417.
Salinas G, Bonilla M, Otero L, Lobanov AV, Gladyshev VN. Selenoproteins in parasites. Springer, 471-479.
Kim HY, Gladyshev VN. Selenium and methionine sulfoxide reduction. Springer, 481-492.
Carlson BA, Yoo MH, Tsuji PA, Tobe R, Naranjo-Suarez S, Chen F, Feigenbaum L, Tessarollo L, Lee BJ, Gladyshev VN, Hatfield DL. Mouse models that target removal or over-expression of the selenocysteine tRNA ^[Ser]Sec gene to elucidate the role of selenoproteins in health and development. Springer, 561-587.

Atkins JF, Gesteland R. Editors. (2010) Recoding: Expansion of Decoding Rules Enriches Gene Expression. Springer.

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Gladyshev VN, Hatfield DL. Selenocysteine Biosynthesis, Selenoproteins, and Selenoproteomes. Springer, 3-27.

Gladyshev VN, Ragsdale SW, Becker DF, Dickman MB. (2007) Redox Biochemistry. Ed., Banerjee R. John Wiley & Sons.

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Gladyshev VN. Methionine Sulfoxide Reductases. John Wiley & Sons, 84-87.
Gladyshev VN. Selenoproteins. John Wiley & Sons, 127-131.
Fomenko DE, Gladyshev VN. Bioinformatics Methods to Study Thiol-Based Oxidoreductases. John Wiley & Sons, 251-256.

Hatfield DL, Berry MJ, Gladyshev VN. Editors. (2006) Selenium: Its molecular biology and role in human health, 2nd Edition. Springer.

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Carlson BA, Xu, XM, Shrimali R, Sengupta A, Yoo MH, Irons R, Zhong N, Hatfield DL, Lee BJ, Lobanov AV, Gladyshev VN. Mammalian and other eukaryotic selenocysteine tRNAs. Springer, 31-40.
Salinas G, Romero H, Xu XM, Carlson BA, Hatfield DL, Gladyshev VN. Evolution of Sec decoding and the key role of selenophosphate synthetase in the pathway of selenium utilization. Springer, 41-52.
Gladyshev VN. Selenoproteins and selenoproteomes. Springer, 101-112.
Kim HY, Gladyshev VN. Selenium and methionine sulfoxide reduction. Springer, 125-136.
Labunskyy VM, Gladyshev VN, Hatfield DL. The 15-kDa selenoprotein (Sep15): functional analysis and role in cancer. Springer, 143-150.
Carlson BA, Xu XM, Shrimali R, Sengupta A, Yoo MH, Zhong N, Hatfield DL, Irons R, Davis C, Lee BJ, Novoselov SV, Gladyshev VN. Mouse models for assessing the role of selenoproteins in health and development. Springer, 337-346.
Salinas G, Lobanov AV, Gladyshev VN. Selenium in parasites. Springer, 359-370.

Hatfield DL. Editor. (2001) Selenium: Its molecular biology and role in human health. Kluwer Academic Publishers.

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Gladyshev VN. Identity, evolution and functions of selenoproteins and selenoprotein genes. Kluwer Academic Publishers, 31-40.
Gladyshev VN, Diamond DL, Hatfield DL. The 15 kDa selenoprotein (Sep15): functional studies and a role in cancer etiology. Kluwer Academic Publishers, 147-155.
Gladyshev VN. Selenium in biology and human health: controversies and perspectives. Kluwer Academic Publishers, 313-317.