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
