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
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
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
