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