Earlier Articles

Gladyshev VN, Kryukov GV, Fomenko DE, Hatfield DL. (2004) Identification of trace element-containing proteins in genomic databases. Annu. Rev. Nutr. 24, 579-596.

AbstractDevelopment of bioinformatics tools provided researchers with the ability to identify full sets of trace element-containing proteins in organisms for which complete genomic sequences are available. Recently, independent bioinformatics methods were used to identify all, or almost all, genes encoding selenocysteine-containing proteins in human, mouse, and Drosophila genomes, characterizing entire selenoproteomes in these organisms. It also should be possible to search for entire sets of other trace element-associated proteins, such as metal-containing proteins, although methods for their identification are still in development. More Information

Wang C, Scott S, Tao Q, Fomenko DE, Gladyshev VN. (2004) New Techniques for Generation and Analysis of Evolutionary Trees. International Conference on Mathematics and Engineering Techniques in Medicine and Biological Sciences 283-289.

Su D, Gladyshev VN. (2004) Alternative splicing involving the thioredoxin reductase module in mammals: a glutaredoxin-containing thioredoxin reductase 1. Biochemistry 43, 12177-12188.

AbstractThioredoxin reductase 1 (TR1) is a key component in the thioredoxin system, one of major redox systems in mammals that links NADPH and thiol-dependent processes. Mammalian TR1 genes are known to be regulated by alternative splicing. In this report, comparative genomic analyses were used to identify and characterize species-specific and common alternative forms of mammalian TR1 genes. Six human TR1 isoforms were identified that were derived from a large number of transcripts and differed in their N-terminal sequences. One isoform resulted from exons located 30-70 kb upstream of the previously identified core TR1 promoter and was composed of a basic TR1 module fused to a glutaredoxin (Grx) domain that contained an unusual active site CTRC sequence. This TR1 form occurred in humans, dogs, and chimpanzees but was inactivated in mice and rats. The CTRC motif in the human enzyme made the N-terminal domain inactive in the Grx assays tested. However, when mutated to CPYC, an active site present in most Grxs, the Grx domain was active. In addition, the presence of the Grx domain interfered with the TR1 activity, distinguishing this enzyme from other proteins with Grx and TR fusions. The data suggest that the fusion of the basic TR1 module and variable N-terminal sequences links the pyridine nucleotide thiol/disulfide oxidoreductase pathway to specific cellular redox functions and may control spatial and temporal expression of TR1 transcripts. Our data also suggest that various N-terminal extensions in mammalian TRs are often expressed in testes. More Information

Carlson BA, Xu XM, Kryukov GV, Rao M, Berry MJ, Gladyshev VN, Hatfield,DL. (2004) Identification and characterization of phosphoseryl-tRNA[Ser]Sec kinase. Proc. Natl. Acad. Sci. USA 101, 12848-12853.

AbstractIn 1970, a kinase activity that phosphorylated a minor species of seryl-tRNA to form phosphoseryl-tRNA was found in rooster liver [Maenpaa, P. H. & Bernfield, M. R. (1970) Proc. Natl. Acad. Sci. USA 67, 688-695], and a minor seryl-tRNA that decoded the nonsense UGA was detected in bovine liver. The phosphoseryl-tRNA and the minor UGA-decoding seryl-tRNA were subsequently identified as selenocysteine (Sec) tRNA[Ser]Sec, but the kinase activity remained elusive. Herein, by using a comparative genomics approach that searched completely sequenced archaeal genomes for a kinase-like protein with a pattern of occurrence similar to that of components of Sec insertion machinery, we detected a candidate gene for mammalian phosphoseryl-tRNA[Ser]Sec kinase (pstk). Mouse pstk was cloned, and the gene product (PSTK) was expressed and characterized. PSTK specifically phosphorylated the seryl moiety on seryl-tRNA[Ser]Sec and, in addition, had a requirement for ATP and Mg2+. Proteins with homology to mammalian PSTK occur in Drosophila, Caenorhabditis elegans, Methanopyrus kandleri, and Methanococcus jannaschii, suggesting a conservation of its function across archaea and eukaryotes that synthesize selenoproteins and the absence of this function in bacteria, plants, and yeast. The fact that PSTK has been highly conserved in evolution suggests that it plays an important role in selenoprotein biosynthesis and/or regulation. Copyright 2004 The National Academy of Sciencs of the USA More Information

Kim HY, Gladyshev VN. (2004) Characterization of mouse endoplasmic reticulum methionine-R-sulfoxide reductase. Biochem. Biophys. Res. Commun. 320, 1277-1283.

AbstractMethionine-R-sulfoxide reductases (MsrBs) catalyze a stereospecific reduction of methionine-R-sulfoxides to methionines in proteins. Mammals possess three MsrB genes. MsrB1 (SelR) is a selenoprotein located in the cytosol and nucleus, MsrB2 (CBS-1) is a mitochondrial protein, and MsrB3 is a recently identified protein with an unusual localization pattern. Human MsrB3 occurs in two protein forms, MsrB3A and MsrB3B, which can be targeted to the endoplasmic reticulum (ER) and mitochondria, respectively. These forms are generated by alternative first exon splicing that introduces contrasting N-terminal signal peptides. Herein, we characterized mouse MsrB3 and found no evidence of alternative splicing of its gene. The ER signal was located upstream of the predicted mitochondrial signal sequence in a single coding region, whose product was targeted to the ER. Although the mitochondrial signal could function if placed at the N-terminus, it did not target MsrB3 to mitochondria as part of the entire coding region. In addition, immunoblot assays detected no mitochondrial MsrB3 in examined mouse tissues. The data suggest that, in mice, MsrB3 is largely or exclusively an ER-resident protein, and that the reduction of methionine-R-sulfoxides in different cellular compartments is provided by individual MsrB isozymes. More Information

Koc A, Gasch AP, Rutherford JC, Kim HY, Gladyshev VN. (2004) Methionine sulfoxide reductase regulation of yeast lifespan reveals reactive oxygen species-dependent and -independent components of aging. Proc. Natl. Acad. Sci. USA 101, 7999-8004.

AbstractAging is thought to be caused by the accumulation of damage, primarily from oxidative modifications of cellular components by reactive oxygen species (ROS). Here we used yeast methionine sulfoxide reductases MsrA and MsrB to address this hypothesis. In the presence of oxygen, these antioxidants could increase yeast lifespan and did so independent of the lifespan extension offered by caloric restriction. However, under ROS-deficient, strictly anaerobic conditions, yeast lifespan was shorter, not affected by MsrA or MsrB, and further reduced by caloric restriction. In addition, we identified changes in the global gene expression associated with aging in yeast, and they did not include oxidative stress genes. Our findings suggest how the interplay between ROS, antioxidants, and efficiency of energy production regulates the lifespan. The data also suggest a model wherein factors implicated in aging (for example, ROS) may influence the lifespan yet not be the cause of aging. More Information

Kryukov GV, Gladyshev VN. (2004) The prokaryotic selenoproteome. EMBO Rep. 5, 538-543.

AbstractIn the genetic code, the UGA codon has a dual function as it encodes selenocysteine (Sec) and serves as a stop signal. However, only the translation terminator function is used in gene annotation programs, resulting in misannotation of selenoprotein genes. Here, we applied two independent bioinformatics approaches to characterize a selenoprotein set in prokaryotic genomes. One method searched for selenoprotein genes by identifying RNA stem-loop structures, selenocysteine insertion sequence elements; the second approach identified Sec/Cys pairs in homologous sequences. These analyses identified all or almost all selenoproteins in completely sequenced bacterial and archaeal genomes and provided a view on the distribution and composition of prokaryotic selenoproteomes. In addition, lineage-specific and core selenoproteins were detected, which provided insights into the mechanisms of selenoprotein evolution. Characterization of selenoproteomes allows interpretation of other UGA codons in completed genomes of prokaryotes as terminators, addressing the UGA dual-function problem. More Information

Castellano S, Novoselov SV, Kryukov GV, Lescure A, Blanco E, Krol A, Gladyshev VN, Guigo R. (2004) Reconsidering the evolution of eukaryotic selenoproteins: a novel nonmammalian family with scattered phylogenetic distribution. EMBO Rep. 7, 71-77.

AbstractWhile the genome sequence and gene content are available for an increasing number of organisms, eukaryotic selenoproteins remain poorly characterized. The dual role of the UGA codon confounds the identification of novel selenoprotein genes. Here, we describe a comparative genomics approach that relies on the genome-wide prediction of genes with in-frame TGA codons, and the subsequent comparison of predictions from different genomes, wherein conservation in regions flanking the TGA codon suggests selenocysteine coding function. Application of this method to human and fugu genomes identified a novel selenoprotein family, named SelU, in the puffer fish. The selenocysteine-containing form also occurred in other fish, chicken, sea urchin, green algae and diatoms. In contrast, mammals, worms and land plants contained cysteine homologues. We demonstrated selenium incorporation into chicken SelU and characterized the SelU expression pattern in zebrafish embryos. Our data indicate a scattered evolutionary distribution of selenoproteins in eukaryotes, and suggest that, contrary to the picture emerging from data available so far, other taxa-specific selenoproteins probably exist. More Information

Kim HY, Gladyshev VN. (2004) Methionine sulfoxide reduction in mammals: characterization of methionine-R-sulfoxide reductases. Mol. Biol. Cell 15, 1055-1064.

AbstractMethionine residues in proteins are susceptible to oxidation by reactive oxygen species, but can be repaired via reduction of the resulting methionine sulfoxides by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). However, the identity of all methionine sulfoxide reductases involved, their cellular locations and relative contributions to the overall pathway are poorly understood. Here, we describe a methionine-R-sulfoxide reduction system in mammals, in which two MsrB homologues were previously described. We found that human and mouse genomes possess three MsrB genes and characterized their protein products, designated MsrB1, MsrB2, and MsrB3. MsrB1 (Selenoprotein R) was present in the cytosol and nucleus and exhibited the highest methionine-R-sulfoxide reductase activity because of the presence of selenocysteine (Sec) in its active site. Other mammalian MsrBs contained cysteine in place of Sec and were less catalytically efficient. MsrB2 (CBS-1) resided in mitochondria. It had high affinity for methionine-R-sulfoxide, but was inhibited by higher concentrations of the substrate. The human MsrB3 gene gave rise to two protein forms, MsrB3A and MsrB3B. These were generated by alternative splicing that introduced contrasting N-terminal and C-terminal signals, such that MsrB3A was targeted to the endoplasmic reticulum and MsrB3B to mitochondria. We found that only mitochondrial forms of mammalian MsrBs (MsrB2 and MsrB3B) could compensate for MsrA and MsrB deficiency in yeast. All mammalian MsrBs belonged to a group of zinc-containing proteins. The multiplicity of MsrBs contrasted with the presence of a single mammalian MsrA gene as well as with the occurrence of single MsrA and MsrB genes in yeast, fruit flies, and nematodes. The data suggested that different cellular compartments in mammals maintain a system for repair of oxidized methionine residues and that this function is tuned in enzyme- and stereo-specific manner. More Information

Carlson BA, Novoselov SV, Kumaraswamy E, Lee BJ, Anver MR, Gladyshev VN, Hatfield DL. (2004) Specific excision of the selenocysteine tRNA[Ser]Sec (Trsp) gene in mouse liver demonstrates an essential role of selenoproteins in liver function. J. Biol. Chem. 279, 8011-8017.

AbstractSelenium is essential in mammalian embryonic development. However, in adults, selenoprotein levels in several organs including liver can be substantially reduced by selenium deficiency without any apparent change in phenotype. To address the role of selenoproteins in liver function, mice homozygous for a floxed allele encoding the selenocysteine (Sec) tRNA([Ser]Sec) gene were crossed with transgenic mice carrying the Cre recombinase under the control of the albumin promoter that expresses the recombinase specifically in liver. Recombination was nearly complete in mice 3 weeks of age, whereas liver selenoprotein synthesis was virtually absent, which correlated with the loss of Sec tRNA([Ser]Sec) and activities of major selenoproteins. Total liver selenium was dramatically decreased, whereas levels of low molecular weight selenocompounds were little affected. Plasma selenoprotein P levels were reduced by about 75%, suggesting that selenoprotein P is primarily exported from the liver. Glutathione S-transferase levels were elevated in the selenoprotein-deficient liver, suggesting a compensatory activation of this detoxification program. Mice appeared normal until about 24 h before death. Most animals died between 1 and 3 months of age. Death appeared to be due to severe hepatocellular degeneration and necrosis with concomitant necrosis of peritoneal and retroperitoneal fat. These studies revealed an essential role of selenoproteins in liver function. More Information

Fomenko DE, Gladyshev VN. (2003) Genomics perspective on disulfide bond formation. Antioxid. Redox Signal. 5, 397-402.

AbstractDisulfide bond formation, reduction, and isomerization in substrate proteins are catalyzed by designated pathways composed of thiol-dependent enzymes. Disulfides are generated in oxidizing environments, such as bacterial periplasm and eukaryotic endoplasmic reticulum (ER), but could also be formed in the cytosol. Major contributors to the formation of intramolecular disulfides in proteins are thiol/disulfide oxidoreductases containing a conserved CxxC motif (two cysteines separated by two other residues), which in turn transfer reducing equivalents to adapter or membrane-bound oxidoreductases. Disulfide bond formation is accompanied by disulfide bond reduction and isomerization processes, allowing disulfide repair and quality control. Higher eukaryotes evolved a complex network of thiol/disulfide oxidoreductases that are involved in disulfide bond formation and isomerization and thiol-dependent protein retention. Emerging evidence suggests that these ER functions might be assisted by mammalian selenocysteine-containing oxidoreductases Sep15 and SelM. More Information

Kwon SY, Badenhorst P, Martin-Romero FJ, Carlson BA, Paterson BM, Gladyshev VN, Lee BJ, Hatfield DL. (2003) The Drosophila selenoprotein BthD is required for survival and has a role in salivary gland development. Mol. Cell. Biol. 23, 8495-8504.

AbstractSelenium is implicated in many diseases, including cancer, but its function at the molecular level is poorly understood. BthD is one of three selenoproteins recently identified in Drosophila. To elucidate the function of BthD and the role of selenoproteins in cellular metabolism and health, we analyzed the developmental expression profile of this protein and used inducible RNA interference (RNAi) to ablate function. We find that BthD is dynamically expressed during Drosophila development. bthD mRNA and protein are abundant in the ovaries of female flies and are deposited into the developing oocyte. Maternally contributed protein and RNA persist during early embryonic development but decay by the onset of gastrulation. At later stages of embryogenesis, BthD is expressed highly in the developing salivary gland. We generated transgenic fly lines carrying an inducible gene-silencing construct, in which an inverted bthD genomic-cDNA hybrid is under the control of the Drosophila Gal4 upstream activation sequence system. Duplex RNAi induced from this construct targeted BthD mRNA for destruction and reduced BthD protein levels. We found that loss of BthD compromised salivary gland morphogenesis and reduced animal viability. More Information

Fomenko DE, Gladyshev VN. (2003) Identity and functions of CxxC-derived motifs. Biochemistry 42, 11214-11225.

AbstractTwo cysteines separated by two other residues (the CxxC motif) are employed by many redox proteins for formation, isomerization, and reduction of disulfide bonds and for other redox functions. The place of the C-terminal cysteine in this motif may be occupied by serine (the CxxS motif), modifying the functional repertoire of redox proteins. Here we found that the CxxC motif may also give rise to a motif, in which the C-terminal cysteine is replaced with threonine (the CxxT motif). Moreover, in contrast to a view that the N-terminal cysteine in the CxxC motif always serves as a nucleophilic attacking group, this residue could also be replaced with threonine (the TxxC motif), serine (the SxxC motif), or other residues. In each of these CxxC-derived motifs, the presence of a downstream alpha-helix was strongly favored. A search for conserved CxxC-derived motif/helix patterns in four complete genomes representing bacteria, archaea, and eukaryotes identified known redox proteins and suggested possible redox functions for several additional proteins. Catalytic sites in peroxiredoxins were major representatives of the TxxC motif, whereas those in glutathione peroxidases represented the CxxT motif. Structural assessments indicated that threonines in these enzymes could stabilize catalytic thiolates, suggesting revisions to previously proposed catalytic triads. Each of the CxxC-derived motifs was also observed in natural selenium-containing proteins, in which selenocysteine was present in place of a catalytic cysteine. More Information

Thisse C, Degrave A, Kryukov GV, Gladyshev VN, Obrecht-Pflumio S, Krol A, Thisse B, Lescure A. (2003) Spatial and temporal expression patterns of selenoprotein genes during embryogenesis in zebrafish. Gene Expr. Patterns 3, 525-532.

AbstractSelenium is important for embryogenesis in vertebrates but little is known about the expression patterns and biological functions of most selenoprotein genes. Taking advantage of the zebrafish model, systematic analysis of selenoprotein gene expression was performed by in situ hybridization on whole-mount embryos at different developmental stages. Twenty-one selenoprotein mRNAs were analyzed and all of them exhibited expression patterns restricted to specific tissues. Moreover, we demonstrated that highly similar selenoprotein paralogs were expressed within distinct territories. Therefore, tissue- and development-specific expression patterns provided new information for selenoproteins of unknown function. More Information

Rao M, Carlson BA, Novoselov SV, Weeks DP, Gladyshev VN, Hatfield DL. (2003) Chlamydomonas reinhardtii selenocysteine tRNA[Ser]Sec. RNA 9, 923-930.

AbstractEukaryotic selenocysteine (Sec) protein insertion machinery was thought to be restricted to animals, but the occurrence of both Sec-containing proteins and the Sec insertion system was recently found in Chlamydomonas reinhardtii, a member of the plant kingdom. Herein, we used RT-PCR to determine the sequence of C. reinhardtii Sec tRNA[Ser]Sec, the first non-animal eukaryotic Sec tRNA[Ser]Sec sequence. Like its animal counterpart, it is 90 nucleotides in length, is aminoacylated with serine by seryl-tRNA synthetase, and decodes specifically UGA. Evolutionary analyses of known Sec tRNAs identify the C. reinhardtii form as the most diverged eukaryotic Sec tRNA[Ser]Sec and reveal a common origin for this tRNA in bacteria, archaea, and eukaryotes. More Information

Kryukov GV, Castellano S, Novoselov SV, Lobanov AV, Zehtab O, Guigo R, Gladyshev VN. (2003) Characterization of mammalian selenoproteomes. Science 300, 1439-1443.

AbstractIn the genetic code, UGA serves as a stop signal and a selenocysteine codon, but no computational methods for identifying its coding function are available. Consequently, most selenoprotein genes are misannotated. We identified selenoprotein genes in sequenced mammalian genomes by methods that rely on identification of selenocysteine insertion RNA structures, the coding potential of UGA codons, and the presence of cysteine-containing homologs. The human selenoproteome consists of 25 selenoproteins. More Information

Kumaraswamy E, Carlson BA, Morgan F, Miyoshi K, Robinson GW, Su D, Wan, S, Southo, E, Tessaroll, L, Le, B J, Gladyshe, VN, Hennighausen L, Hatfield DL. (2003) Selective removal of the selenocysteine tRNA [Ser]Sec gene (Trsp) in mouse mammary epithelium. Mol. Cell. Biol. 23, 1477-1488.

AbstractMice homozygous for an allele encoding the selenocysteine (Sec) tRNA [Ser]Sec gene (Trsp) flanked by loxP sites were generated. Cre recombinase-dependent removal of Trsp in these mice was lethal to embryos. To investigate the role of Trsp in mouse mammary epithelium, we deleted this gene by using transgenic mice carrying the Cre recombinase gene under control of the mouse mammary tumor virus (MMTV) long terminal repeat or the whey acidic protein promoter. While both promoters target Cre gene expression to mammary epithelium, MMTV-Cre is also expressed in spleen and skin. Sec tRNA [Ser]Sec amounts were reduced by more than 70% in mammary tissue with either transgene, while in skin and spleen, levels were reduced only with MMTV-Cre. The selenoprotein population was selectively affected with MMTV-Cre in breast and skin but not in the control tissue, kidney. Moreover, within affected tissues, expression of specific selenoproteins was regulated differently and often in a contrasting manner, with levels of Sep15 and the glutathione peroxidases GPx1 and GPx4 being substantially reduced. Expression of the tumor suppressor genes BRCA1 and p53 was also altered in a contrasting manner in MMTV-Cre mice, suggesting greater susceptibility to cancer and/or increased cell apoptosis. Thus, the conditional Trsp knockout mouse allows tissue-specific manipulation of Sec tRNA and selenoprotein expression, suggesting that this approach will provide a useful tool for studying the role of selenoproteins in health. More Information

Novoselov SV, Gladyshev VN. (2003) Non-animal origin of animal thioredoxin reductases: Implications for selenocysteine evolution and evolution of protein function through carboxy-terminal extensions. Protein Sci. 12, 372-378.

AbstractThioredoxin reductase (TR) and thioredoxin constitute a major cellular redox system present in all organisms. In contrast to a single form of thioredoxin, there are two TR types: One (bacterial type or small TR) is present in bacteria, archaea, plants, and most unicellular eukaryotes, whereas the second (animal or large TR) is only found in animals and typically contains a carboxy-terminal penultimate selenocysteine encoded by TGA. Surprisingly, we detected sequences of large TRs in various unicellular eukaryotes. Moreover, green algae Chlamydomonas reinhardtii had both small and large TRs, with the latter being a selenoprotein, but no examples of horizontal gene transfer from animals to the green algae could be detected. In addition, phylogenetic analyses revealed that large TRs formed a subgroup of lower eukaryotic glutathione reductases (GRs). The data suggest that the large TR evolved in a lower eukaryote capable of selenocysteine insertion rather than in an animal. The enzyme appeared to evolve by a carboxy-terminal extension of GR such that the resulting carboxy-terminal glutathionelike peptide became an intramolecular substrate for GR and a reductant for thioredoxin. Subsequently, small TRs were lost in an organism that gave rise to animals, large TRs were lost in plants and fungi, and selenocysteine/cysteine replacements took place in some large TRs. Our data implicate carboxy-terminal extension of proteins as a general mechanism of evolution of new protein function. More Information

Xu XM, Carlson BA, Grimm TA, Kutza J, Berry MJ, Arreola R, Fields, KH, Shanmugam I, Jeang KT, Oroszlan S, Combs GF, Marx PA, Gladyshev VN, Clouse KA, Hatfield DL. (2002) Rhesus monkey simian immunodeficiency virus infection as a model for assessing the role of selenium in AIDS. J. Acquir. Immune. Defic. Syndr. 31, 453-463.

AbstractThe objective of this study was to determine whether simian immunodeficiency virus (SIV) infection of macaques could be used as a model system to assess the role of selenium in AIDS. Plasma and serum selenium levels were determined by standard assays in monkeys before and after inoculation of SIV. SIV-infected cells or cells expressing the HIV Tat protein were labeled with 75Se, and protein extracts were prepared and electrophoresed to analyze selenoprotein expression. Total tRNA was isolated from CEMx174 cells infected with SIV or from KK1 cells infected with HIV, and selenocysteine tRNA isoforms were characterized by reverse phase chromatography. SIV-infected monkeys show a decrease in blood selenium levels similar to that observed in AIDS with development of SAIDS. Cells infected with SIV in vitro exhibit reduced selenoprotein levels and an accumulation of small molecular weight selenium compounds relative to uninfected cells. Examination of the selenocysteine tRNA isoforms in HIV-infected KK1 cells or SIV-infected CEMx174 cells reveals an isoform distribution characteristic of selenium-deficient cells. Furthermore, transfection of Jurkat E6 cells with the Tat gene selectively altered selenoprotein synthesis, with GPX4 and Sep15 being the most inhibited and TR1 the most enhanced. Taken together, the data show that monkeys infected with SIV in vivo and cells infected with SIV in vitro will provide appropriate models for investigating the mechanism(s) responsible for reduced selenium levels that accompany the progression of AIDS in HIV disease. More Information

Fomenko DE, Gladyshev VN. (2002) CxxS: fold-independent redox motif revealed by genome-wide searches for thiol/disulfide oxidoreductase function. Protein Sci. 11, 2285-2296.

AbstractRedox reactions involving thiol groups in proteins are major participants in cellular redox regulation and antioxidant defense. Although mechanistically similar, thiol-dependent redox processes are catalyzed by structurally distinct families of enzymes, which are difficult to identify by available protein function prediction programs. Herein, we identified a functional motif, CxxS (cysteine separated from serine by two other residues), that was often conserved in redox enzymes, but rarely in other proteins. Analyses of complete Escherichia coli, Campylobacter jejuni, Methanococcus jannaschii, and Saccharomyces cerevisiae genomes revealed a high proportion of proteins known to use the CxxS motif for redox function. This allowed us to make predictions in regard to redox function and identity of redox groups for several proteins whose function previously was not known. Many proteins containing the CxxS motif had a thioredoxin fold, but other structural folds were also present, and CxxS was often located in these proteins upstream of an alpha-helix. Thus, a conserved CxxS sequence followed by an alpha-helix is typically indicative of a redox function and corresponds to thiol-dependent redox sites in proteins. The data also indicate a general approach of genome-wide identification of redox proteins by searching for simple conserved motifs within secondary structure patterns. More Information

Kumar RA, Koc A, Cerny RL, Gladyshev VN. (2002) Reaction mechanism, evolutionary analysis, and role of zinc in Drosophila methionine-R-sulfoxide reductase. J. Biol. Chem. 277, 37527-37535.

AbstractMethionine residues in proteins are susceptible to oxidation, and the resulting methionine sulfoxides can be reduced back to methionines by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). Herein, we have identified two MsrB families that differ by the presence of zinc. Evolutionary analyses suggested that the zinc-containing MsrB proteins are prototype enzymes and that the metal was lost in certain MsrB proteins later in evolution. Zinc-containing Drosophila MsrB was further characterized. The enzyme was found to employ a catalytic Cys(124) thiolate, which directly interacted with methionine sulfoxide, resulting in methionine and a Cys(124) sulfenic acid intermediate. A subsequent reaction of this intermediate with Cys(69) generated an intramolecular disulfide. Dithiothreitol could reduce either the sulfenic acid or the disulfide, but the disulfide was a preferred substrate for thioredoxin, a natural electron donor. Interestingly, the C69S mutant could complement MsrA/MsrB deficiency in yeast, and the corresponding natural form of mouse MsrB was active with thioredoxin. These data indicate that MsrB proteins employ alternative mechanisms for sulfenic acid reduction. Four other conserved cysteines in Drosophila MsrB (Cys(51), Cys(54), Cys(101), and Cys(104)) were found to coordinate structural zinc. Mutation of any one or a combination of these residues resulted in complete loss of metal and catalytic activity, demonstrating an essential role of zinc in Drosophila MsrB. In contrast, two conserved histidines were important for thioredoxin-dependent activity, but were not involved in zinc binding. A Drosophila MsrA gene was also cloned, and the recombinant enzyme was found to be metal-free and specific for methionine S-sulfoxide and to employ a similar sulfenic acid/disulfide mechanism. More Information

Novoselov SV, Rao M, Onoshko NV, Zhi H, Kryukov GV, Xiang Y, Weeks DP, Hatfield DL, Gladyshev VN. (2002) Selenoproteins and selenocysteine insertion system in the model plant cell system. Chlamydomonas reinhardtii. EMBO J. 21, 3681-3693.

AbstractKnown eukaryotic selenocysteine (Sec)-containing proteins are animal proteins, whereas selenoproteins have not been found in yeast and plants. Surprisingly, we detected selenoproteins in a member of the plant kingdom, Chlamydomonas reinhardtii, and directly identified two of them as phospholipid hydroperoxide glutathione peroxidase and selenoprotein W homologs. Moreover, a selenocysteyl-tRNA was isolated that recognized specifically the Sec codon UGA. Subsequent gene cloning and bioinformatics analyses identified eight additional selenoproteins, including methionine-S-sulfoxide reductase, a selenoprotein specific to Chlamydomonas: Chlamydomonas selenoprotein genes contained selenocysteine insertion sequence (SECIS) elements that were similar, but not identical, to those of animals. These SECIS elements could direct selenoprotein synthesis in mammalian cells, indicating a common origin of plant and animal Sec insertion systems. We found that selenium is required for optimal growth of Chlamydomonas: Finally, evolutionary analyses suggested that selenoproteins present in Chlamydomonas and animals evolved early, and were independently lost in land plants, yeast and some animals. More Information

Kryukov GV, Kumar RA, Koc A, Sun Z, Gladyshev VN. (2002) Selenoprotein R is a zinc-containing stereo-specific methionine sulfoxide reductase. Proc. Natl. Acad. Sci. USA 99, 4245-4250.

AbstractSelenoprotein R (SelR) is a mammalian selenocysteine-containing protein with no known function. Here we report that cysteine homologs of SelR are present in all organisms except certain parasites and hyperthermophiles, and this pattern of occurrence closely matches that of only one protein, peptide methionine sulfoxide reductase (MsrA). Moreover, in several genomes, SelR and MsrA genes are fused or clustered, and their expression patterns suggest a role of both proteins in protection against oxidative stress. Consistent with these computational screens, growth of Saccharomyces cerevisiae SelR and MsrA mutant strains was inhibited, and the strain lacking both genes could not grow, in the presence of H2O2 and methionine sulfoxide. We found that the cysteine mutant of mouse SelR, as well as the Drosophila SelR homolog, contained zinc and reduced methionine-R-sulfoxide, but not methionine-S-sulfoxide, in in vitro assays, a function that is both distinct and complementary to the stereo-specific activity of MsrA. These findings identify a function of the conserved SelR enzyme family, define a pathway of methionine sulfoxide reduction, reveal a case of convergent evolution of similar function in structurally distinct enzymes, and suggest a previously uncharacterized redox regulatory role of selenium in mammals. More Information

Korotkov KV, Novoselov SV, Hatfield DL, Gladyshev VN. (2002) Mammalian selenoprotein in which selenocysteine (Sec) incorporation is supported by a new form of Sec insertion sequence element. Mol. Cell. Biol. 22, 1402-1411.

AbstractSelenocysteine (Sec), the 21st amino acid in protein, is encoded by UGA. The Sec insertion sequence (SECIS) element, which is the stem-loop structure present in 3′ untranslated regions (UTRs) of eukaryotic selenoprotein-encoding genes, is essential for recognition of UGA as a codon for Sec rather than as a stop signal. We now report the identification of a new eukaryotic selenoprotein, designated selenoprotein M (SelM). The 3-kb human SelM-encoding gene has five exons and is located on chromosome 22 but has not been correctly identified by either Celera or the public Human Genome Project. We characterized human and mouse SelM cDNA sequences and expressed the selenoprotein in various mammalian cell lines. The 3′ UTR of the human, mouse, and rat SelM-encoding genes lacks a canonical SECIS element. Instead, Sec is incorporated in response to a conserved mRNA structure, in which cytidines are present in place of the adenosines previously considered invariant. Substitution of adenosines for cytidines did not alter Sec incorporation; however, other mutant structures did not support selenoprotein synthesis, demonstrating that this new form of SECIS element is functional. SelM is expressed in a variety of tissues, with increased levels in the brain. It is localized to the perinuclear structures, and its N-terminal signal peptide is necessary for protein translocation. More Information

Gladyshev VN. (2002) Thioredoxin and peptide methionine sulfoxide reductase: convergence of similar structure and function in distinct structural folds. Proteins 146, 149-152.

AbstractThioredoxin (Trx) and peptide methionine sulfoxide reductase (PMSR) are small thiol oxidoreductases implicated in antioxidant defense and redox regulation of cellular processes. Here we show that the structures of Trx and PMSR exhibit resemblance in their alphabeta core regions and that the active site cysteines in two proteins occupy equivalent positions downstream of a central beta-strand and at the N-terminus of an alpha-helix. Moreover, we identified a PMSR subfamily that contains an active site CxxC motif (two cysteines separated by two other amino acids) positioned similarly to the catalytic redox active CxxC motif in Trx. However, Trx and PMSR are characterized by distinct ancient folds that differ in both orientation of secondary structures and their patterns. Trx is a member of the Trx-fold superfamily, whereas PMSR has a unique fold not found in other proteins. The data suggest that similar structures and functions of Trx and PMSR were acquired independently during evolution and point to a general strategy of identifying new redox regulatory proteins. Copyright 2001 Wiley-Liss, Inc. More Information
Kumaraswamy E, Korotkov KV, Diamond AM, Gladyshev VN, Hatfield DL. (2002) Genetic and functional analysis of mammalian Sep15 selenoprotein. Methods Enzymol. 347, 187-197.
Kryukov GV, Gladyshev VN. (2002) Mammalian Selenoprotein Gene Signature: identification and functional analysis of selenoprotein genes using bioinformatics methods. Methods Enzymol. 347, 84-100.

Hatfield DL, Gladyshev VN. (2002) How selenium has altered our understanding of the genetic code. Mol. Cell. Biol. 22, 3565-3576.

AbstractSelenium is an essential micronutrient in the diet of many life forms, including humans and other mammals. Significant health benefits have been attributed to this element. It is rapidly becoming recognized as one of the more promising cancer chemopreventive agents (19), and there are strong indications that it has a role in reducing viral expression (4), in preventing heart disease and other cardiovascular and muscle disorders (23), and in delaying the progression of AIDS in human immunodeficiency virus-infected patients (3). Additional evidence suggests that selenium may have a role in mammalian development (51), in immune function (70), in male reproduction (30), and in slowing the aging process (70). More Information

Sun Q, Gladyshev VN. (2002) Redox regulation of cell signaling by thioredoxin reductase. Methods Enzymol. 347, 451-461.

Gladyshev VN, Liu A, Novoselov SV, Krysan K, Sun QA, Kryukov VM, Kryukov GV, Lou MF (2001) Identification and characterization of a new mammalian glutaredoxin (thioltransferase) Grx2. J. Biol. Chem. 276, 30374-30380.

AbstractA thiol/disulfide oxidoreductase component of the GSH system, glutaredoxin (Grx), is involved in the reduction of GSH-based mixed disulfides and participates in a variety of cellular redox pathways. A single cytosolic Grx (Grx1) was previously described in mammals. We now report identification and characterization of a second mammalian Grx, designated Grx2. Grx2 exhibited 36% identity with Grx1 and had a disulfide active center containing the Cys-Ser-Tyr-Cys motif. Grx2 was encoded in the genomes of mammals and birds and expressed in a variety of cell types. The gene for human Grx2 consisted of four exons and three introns, spanned 10 kilobase pairs, and localized to chromosome 1q31.2-31.3. The coding sequence was present in all exons, with the first exon encoding a mitochondrial signal peptide. The mitochondrial leader sequence was also present in mouse and rat Grx2 sequences and was shown to direct either Grx2 or green fluorescent protein to mitochondria. Alternative splicing forms of mammalian Grx2 mRNAs were identified that differed in sequences upstream of exon 2. To functionally characterize the new protein, human and mouse Grx2 proteins were expressed in Escherichia coli, and the purified proteins were shown to reduce mixed disulfides formed between GSH and S-sulfocysteine, hydroxyethyldisulfide, or cystine. Grx1 and Grx2 were sensitive to inactivation by iodoacetamide and H(2)O(2) and exhibited similar pH dependence of catalytic activity. However, H(2)O(2)-inactivated Grx2 could only be reactivated with 5 mm GSH, whereas Grx1 could also be reactivated with dithiothreitol or thioredoxin/thioredoxin reductase. The Grx2 structural model suggested a common reaction mechanism for this class of proteins. The data provide the first example of a mitochondrial Grx and also indicate the occurrence of a second functional Grx in mammals. More Information

Martin-Romero FJ, Kryukov GK, Lobanov AV, Carlson BA, Lee BJ, Gladyshev VN, Hatfield DL. (2001) Selenium metabolism in Drosophila: selenoproteins, selenoprotein mRNA expression, fertility and mortality. J. Biol. Chem. 276, 29798-29804.

AbstractSelenocysteine is a rare amino acid in protein that is encoded by UGA with the requirement of a downstream mRNA stem-loop structure, the selenocysteine insertion sequence element. To detect selenoproteins in Drosophila, the entire genome was analyzed with a novel program that searches for selenocysteine insertion sequence elements, followed by selenoprotein gene signature analyses. This computational screen and subsequent metabolic labeling with (75)Se and characterization of selenoprotein mRNA expression resulted in identification of three selenoproteins: selenophosphate synthetase 2 and novel G-rich and BthD selenoproteins that had no homology to known proteins. To assess a biological role for these proteins, a simple chemically defined medium that supports growth of adult Drosophila and requires selenium supplementation for optimal survival was devised. Flies survived on this medium supplemented with 10(-8) to 10(-6) m selenium or on the commonly used yeast-based complete medium at about twice the rate as those on a medium without selenium or with >10(-6) m selenium. This effect correlated with changes in selenoprotein mRNA expression. The number of eggs laid by Drosophila was reduced approximately in half in the chemically defined medium compared with the same medium supplemented with selenium. The data provide evidence that dietary selenium deficiency shortens, while supplementation of the diet with selenium normalizes the Drosophila life span by a process that may involve the newly identified selenoproteins. More Information

Moustafa ME, Carlson BA, El-Saadani MA, Kryukov GV, Sun Q.-A, Harney JW, Hill KE, Combs GF, Feigenbaum L, Mansur DB, Burk RF, Berry MJ, Diamond AM, Lee BJ, Gladyshev VN, Hatfield DL. (2001) Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine mutant selenocysteine tRNA transgenes. Mol. Cell. Biol. 21, 3840-3852.

AbstractSelenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins. More Information

Sun Q-A, Kirnarsky L, Sherman S, Gladyshev VN. (2001) Selenoprotein oxidoreductase with specificity for thioredoxin and glutathione systems. Proc. Natl. Acad. Sci. USA 98, 3673-3678.

AbstractThioredoxin (Trx) and glutathione (GSH) systems are considered to be two major redox systems in animal cells. They are reduced by NADPH via Trx reductase (TR) or oxidized GSH (GSSG) reductase and further supply electrons for deoxyribonucleotide synthesis, antioxidant defense, and redox regulation of signal transduction, transcription, cell growth, and apoptosis. We cloned and characterized a pyridine nucleotide disulfide oxidoreductase, Trx and GSSG reductase (TGR), that exhibits specificity for both redox systems. This enzyme contains a selenocysteine residue encoded by the TGA codon. TGR can reduce Trx, GSSG, and a GSH-linked disulfide in in vitro assays. This unusual substrate specificity is achieved by an evolutionary conserved fusion of the TR and glutaredoxin domains. These observations, together with the biochemical probing and molecular modeling of the TGR structure, suggest a mechanism whereby the C-terminal selenotetrapeptide serves a role of a protein-linked GSSG and shuttles electrons from the disulfide center within the TR domain to either the glutaredoxin domain or Trx. More Information

Korotkov KV, Kumaraswamy E, Zhou Y, Hatfield DL, Gladyshev VN. (2001) Association between the 15 kDa selenoprotein and UDP-glucose:glycoprotein glucosyltransferase in the endoplasmic reticulum of mammalian cells. J. Biol. Chem. 276, 15330-15336.

AbstractMammalian selenocysteine-containing proteins characterized with respect to function are involved in redox processes and exhibit distinct expression patterns and cellular locations. A recently identified 15-kDa selenoprotein (Sep15) has no homology to previously characterized proteins, and its function is not known. Here we report the intracellular localization and identification of a binding partner for this selenoprotein which implicate Sep15 in the regulation of protein folding. The native Sep15 isolated from rat prostate and mouse liver occurred in a complex with a 150-kDa protein. The latter protein was identified as UDP-glucose:glycoprotein glucosyltransferase (UGTR), the endoplasmic reticulum (ER)-resident protein, which was previously shown to be involved in the quality control of protein folding. UGTR functions by glucosylating misfolded proteins, retaining them in the ER until they are correctly folded or transferring them to degradation pathways. To determine the intracellular localization of Sep15, we expressed a green fluorescent protein-Sep15 fusion protein in CV-1 cells, and this protein was localized to the ER and possibly other perinuclear compartments. We determined that Sep15 contained the N-terminal signal peptide that was essential for translocation and that it was cleaved in the mature protein. However, C-terminal sequences of Sep15 were not involved in trafficking and retention of Sep15. The data suggest that the association between Sep15 and UGTR is responsible for maintaining the selenoprotein in the ER. This report provides the first example of the ER-resident selenoprotein and suggests a possible role of the trace element selenium in the quality control of protein folding. More Information

Hu YJ, Korotkov KV, Mehta R, Hatfield DL, Rotimi C, Luke A, Prewitt TE, Cooper RS, Stock W, Vokes EE, Dolan ME, Gladyshev VN, Diamond AM. (2001) Distribution and functional consequences of nucleotide polymorphisms in the 3′-untranslated region of the human Sep15 gene. Cancer Res. 61, 2307-2310.

AbstractSelenium has been shown to prevent cancer in a variety of animal model systems. Both epidemiological studies and supplementation trials have supported its efficacy in humans. However, the mechanism by which selenium suppresses tumor development remains unknown. Selenium is present in known human selenoproteins as the amino acid selenocysteine (Sec). Sec is inserted cotranslationally in response to UGA codons within selenoprotein mRNAs in a process requiring a sequence within the 3′-untranslated region (UTR), referred to as a Sec insertion sequence (SECIS) element. Recently, a human Mr 15,000 selenoprotein (Sep15) was identified that contains an in-frame UGA codon and a SECIS element in the 3′-UTR. Examination of the available cDNA sequences for this protein revealed two polymorphisms located at position 811 (C/T) and at position 1125 (G/A) located within the 3′-UTR. Here, we demonstrate significant differences in Sep15 allele frequencies by ethnicity and that the identity of the nucleotides at the polymorphic sites influences SECIS function in a selenium-dependent manner. This, together with genetic data indicating loss of heterozygosity at the Sep15 locus in certain human tumor types, suggests that Sep15 may be involved in cancer development, risk, or both. More Information

Sun Q-A, Zappacosta F, Factor VM, Wirth P, Hatfield DL, Gladyshev VN. (2001) Heterogeneity within animal thioredoxin reductases: evidence for alternative first exon splicing. J. Biol. Chem. 276, 3106-3114.

AbstractAnimal thioredoxin reductases (TRs) are selenocysteine-containing flavoenzymes that utilize NADPH for reduction of thioredoxins and other protein and nonprotein substrates. Three types of mammalian TRs are known, with TR1 being a cytosolic enzyme, and TR3, a mitochondrial enzyme. Previously characterized TR1 and TR3 occurred as homodimers of 55-57-kDa subunits. We report here that TR1 isolated from mouse liver, mouse liver tumor, and a human T-cell line exhibited extensive heterogeneity as detected by electrophoretic, immunoblot, and mass spectrometry analyses. In particular, a 67-kDa band of TR1 was detected. Furthermore, a novel form of mouse TR1 cDNA encoding a 67-kDa selenoprotein subunit with an additional N-terminal sequence was identified. Subsequent homology analyses revealed three distinct isoforms of mouse and rat TR1 mRNA. These forms differed in 5′ sequences that resulted from the alternative use of the first three exons but had common downstream sequences. Similarly, expression of multiple mRNA forms was observed for human TR3 and Drosophila TR. In these genes, alternative first exon splicing resulted in the formation of predicted mitochondrial and cytosolic proteins. In addition, a human TR3 gene overlapped with the gene for catechol-O-methyltransferase (COMT) on a complementary DNA strand, such that mitochondrial TR3 and membrane-bound COMT mRNAs had common first exon sequences; however, transcription start sites for predicted cytosolic TR3 and soluble COMT forms were separated by approximately 30 kilobases. Thus, this study demonstrates a remarkable heterogeneity within TRs, which, at least in part, results from evolutionary conserved genetic mechanisms employing alternative first exon splicing. Multiple transcription start sites within TR genes may be relevant to complex regulation of expression and/or organelle- and cell type-specific location of animal thioredoxin reductases. More Information
Gladyshev VN. (2001) Comparison of selenium-containing molybdoenzymes. In Molybdenum and Tungsten. Their roles in biological systems”. “ Metal Ions in Biological Systems 39, 655-672.

Gladyshev VN, Kryukov GV. (2001) Evolution of selenocysteine-containing proteins: significance of identification and functional characterization of selenoproteins. BioFactors 14, 87-92.

AbstractIn the genetic code, UGA serves as either a signal for termination or a codon for selenocysteine (Sec). Sec rarely occurs in protein and is different from other amino acids in that much of the biosynthetic machinery governing its incorporation into protein is unique to this amino acid. Sec-containing proteins have diverse functions and lack a common amino acid motif or consensus sequence. Sec has previously been considered to be a relic of the primordial genetic code that was counter-selected by the presence of oxygen in the atmosphere. In the present report, it is proposed that Sec was added to the already existing genetic code and its use has accumulated during evolution of eukaryotes culminating in vertebrates. The more recently evolved selenoproteins appear to take advantage of unique redox properties of Sec that are superior to those of Cys for specific biological functions. Further understanding of the evolution of selenoproteins as well as biological properties and biomedical applications of the trace element selenium requires identification and functional characterization of all mammalian selenoproteins. More Information
Gladyshev VN. (2001) Identity, evolution and functions of selenoproteins and selenoprotein genes. In Selenium: its molecular biology and role in human health (ed., Hatfield DL), Kluwer Academic Publishers, pp. 99-113.

Gladyshev VN, Diamond DL, Hatfield DL. (2001) The 15 kDa selenoprotein (Sep15): functional studies and a role in cancer etiology. In Selenium: its molecular biology and role in human health (ed., Hatfield DL), Kluwer Academic Publishers, pp. 147-155.

AbstractThe 15 kDa selenoprotein (Sepl5) is one of several recently identified selenoproteins. It contains a single selenocysteine residue in the middle of a 162-amino acid open reading frame and has no detectable homology to known proteins. The human Sepl5 gene spans 5 1 kb, has 5 exons and is located on chromosome 1 at position p3 1. The gene contains two single nucleotide polymorphisms in the 3\’-untranslated region (3\’-UTR) including one in the SECIS element, that are distributed differently between Caucasians and African Americans. Sep15 localizes to the endoplasmic reticulum where it is tightly bound to UDP-glucose:glycoprotein glucosyltransferase, a protein involved in the quality control of protein folding. Sepl5 may be involved in the chemopreventive effect of dietary selenium. This hypothesis is based on its differential expression in normal and malignant tissues, the distribution and functional consequences of natural polymorphisms within its gene, and the location of the Sepl5 gene in a region that is often altered in a variety of cancers. More Information

Gladyshev VN. (2001) Selenium in biology and human health: controversies and perspectives. In Selenium: its molecular biology and role in human health (ed., Hatfield DL), Kluwer Academic Publishers, pp. 313-317.

AbstractImportant unresolved questions raised by the contributors of this book and addressing roles of selenium in biology and human health are discussed. Resolving major scientific controversies in the field should further highlight a bright future for selenium in fundamental science, biotechnology and medicine. More Information

Gladyshev VN, Hatfield DL. (2001) Analysis of selenocysteine-containing proteins. Curr. Protoc. Protein Sci. 3, 3.8.

AbstractRepresentatives of three primary life domains–bacteria, archaea, and eukaryotes–possess specific selenium-containing proteins. The majority of naturally occurring selenoproteins contain an amino acid, selenocysteine, that is incorporated into protein in response to the code word UGA. The presence of selenium in natural selenoproteins and in proteins in which this element is introduced by chemical or biological manipulations provides additional opportunities for characterizing structure, function, and mechanism of action. This unit provides an overview of known selenocysteine-containing proteins, examples of targeted incorporation of selenium into proteins, and methods specific for selenoprotein identification and characterization. More Information

Kumaraswamy E, Malykh A, Korotkov KV, Kozyavkin S, Hu Y, Moustafa ME, Carlson B, Berry MJ, Lee BJ, Hatfield DL, Diamond AM, Gladyshev VN. (2000) Structure-expression relationships of the 15 kDa selenoprotein gene: possible role of the protein in cancer etiology. J. Biol. Chem. 275, 35540-35547.

AbstractSelenium has been implicated in cancer prevention, but the mechanism and possible involvement of selenoproteins in this process are not understood. To elucidate whether the 15-kDa selenoprotein may play a role in cancer etiology, the complete sequence of the human 15-kDa protein gene was determined, and various characteristics associated with expression of the protein were examined in normal and malignant cells and tissues. The 51-kilobase pair gene for the 15-kDa selenoprotein consisted of five exons and four introns and was localized on chromosome 1p31, a genetic locus commonly mutated or deleted in human cancers. Two stem-loop structures resembling selenocysteine insertion sequence elements were identified in the 3′-untranslated region of the gene, and only one of these was functional. Two alleles in the human 15-kDa protein gene were identified that differed by two single nucleotide polymorphic sites that occurred within the selenocysteine insertion sequence-like structures. These 3′-untranslated region polymorphisms resulted in changes in selenocysteine incorporation into protein and responded differently to selenium supplementation. Human and mouse 15-kDa selenoprotein genes manifested the highest level of expression in prostate, liver, kidney, testis, and brain, and the level of the selenoprotein was reduced substantially in a malignant prostate cell line and in hepatocarcinoma. The expression pattern of the 15-kDa protein in normal and malignant tissues, the occurrence of polymorphisms associated with protein expression, the role of selenium in differential regulation of polymorphisms, and the chromosomal location of the gene may be relevant to a role of this protein in cancer. More Information

Kryukov GV, Gladyshev VN. (2000) Selenium metabolism in zebrafish: multiplicity of selenoprotein genes and expression of a protein containing seventeen selenocysteine residues. Genes Cells 5, 1049-1060.

AbstractBACKGROUND: Fish are an important source of selenium in human nutrition and the zebrafish is a potentially useful model organism for the study of selenium metabolism and its role in biology and medicine. Selenium is present in vertebrate proteins in the form of selenocysteine (Sec), the 21st natural amino acid in protein which is encoded by UGA. RESULTS: We report here the detection of 18 zebrafish genes for Sec-containing proteins. We found two zebrafish orthologs of human SelT, glutathione peroxidase 1 and glutathione peroxidase 4, and single orthologs of several other selenoproteins. In addition, new zebrafish selenoproteins were identified that were distant homologues of SelP, SelT and SelW, but their direct orthologs in other species are not known. This multiplicity of selenoprotein genes appeared to result from gene and genome duplications, followed by the retention of new selenoprotein genes. We found a zebrafish selenoprotein P gene (designated zSelPa) that contained two Sec insertion sequence (SECIS) elements and encoded a protein containing 17 Sec residues, the largest number of Sec residues found in any known protein. In contrast, a second SelP gene (designated zSelPb) was also identified that contained one SECIS element and encoded a protein with a single Sec. We found that zSelPa could be expressed and secreted by mammalian cells. CONCLUSIONS: The occurrence of zSelPa and zSelPb suggested that the function of the N-terminal domain of mammalian SelP proteins may be separated from that of the C-terminal Sec-rich sequence: the N-terminal domain containing the UxxC motif is likely involved in oxidoreduction, whereas the C-terminal portion of the protein may function in selenium transport or storage. Our data also suggest that the utilization of Sec is more common in zebrafish than in previously characterized species, including mammals. More Information

Mansur DB, Hao H, Gladyshev VN, Korotkov K, Hu Y, Moustafa ME, El-Saadani MA. Carlson BA, Hatfield DL, Diamond AM. (2000) Multiple levels of regulation of selenoprotein biosynthesis revealed from the analysis of human glioma cell lines. Biochem. Pharm. 60, 489-497.

AbstractTo gain a better understanding of the biological consequences of the exposure of tumor cells to selenium, we evaluated the selenium-dependent responses of two selenoproteins (glutathione peroxidase and the recently characterized 15-kDa selenoprotein) in three human glioma cell lines. Protein levels, mRNA levels, and the relative distribution of the two selenocysteine tRNA isoacceptors (designated mcm(5)U and mcm(5)Um) were determined for standard as well as selenium-supplemented conditions. The human malignant glioma cell lines D54, U251, and U87 were maintained in normal or selenium-supplemented (30 nM sodium selenite) conditions. Northern blot analysis demonstrated only minor increases in steady-state GSHPx-1 mRNA in response to selenium addition. Baseline glutathione peroxidase activity was 10.7 +/- 0.7, 7.6 +/- 0.7, and 4.3 +/- 0.7 nmol NADPH oxidized/min/mg protein for D54, U251, and U87, respectively, as determined by the standard coupled spectrophotometric assay. Glutathione peroxidase activity increased in a cell line-specific manner to 19.7 +/- 1.4, 15.6 +/- 2.1, and 6. 7 +/- 0.5 nmol NADPH oxidized/min/mg protein, respectively, as did a proportional increase in cellular resistance to H(2)O(2), in response to added selenium. The 15-kDa selenoprotein mRNA levels likewise remained constant despite selenium supplementation. The selenium-dependent change in distribution between the two selenocysteine tRNA isoacceptors also occurred in a cell line-specific manner. The percentage of the methylated isoacceptor, mcm(5)Um, changed from 35.5 to 47.2 for D54, from 38.1 to 47.3 for U251, and from 49.0 to 47.6 for U87. These data represent the first time that selenium-dependent changes in selenoprotein mRNA and protein levels, as well as selenocysteine tRNA distribution, were examined in human glioma cell lines. More Information
Kryukov GV, Kryukov VM, Gladyshev VN. (1999) New Mammalian Selenocysteine-containing Proteins Identified with an Algorithm That Searches for Selenocysteine Insertion Sequence Elements. J. Biol. Chem. 274, 33888-33897.
Sun QA, Wu Y, Zappacosta F, Jeang KT, Lee BJ, Hatfield DL, Gladyshev VN. (1999) Redox regulation of cell signaling by selenocysteine in mammalian thioredoxin reductases. J. Biol. Chem. 274, 24522-24530.
Gladyshev VN, Krause M, Xu XM, Korotkov KV, Kryukov GV, Sun QA, Lee BJ, Wootton JC, Hatfield DL. (1999) Selenocysteine-containing thioredoxin reductase in C. elegans. Biochem. Biophys. Res. Commun. 259, 244-249.

Gladyshev VN, Stadtman TC, Hatfield DL, Jeang KT. (1999) Levels of major selenoproteins in T cells decrease during HIV infection and low molecular mass selenium compounds increase. Proc. Natl. Acad. Sci. USA 96, 835-839.

AbstractIt has been observed previously that plasma selenium and glutathione levels are subnormal in HIV-infected individuals, and plasma glutathione peroxidase activity is decreased. Under these conditions the survival rate of AIDS patients is reduced significantly. In the present study, using 75Se-labeled human Jurkat T cells, we show that the levels of four 75Se-containing proteins are lower in HIV-infected cell populations than in uninfected cells. These major selenoproteins migrated as 57-, 26-, 21-, and 15-kDa species on SDS/PAGE gels. In our earlier studies, the 57-kDa protein was purified from T cells and identified as a subunit of thioredoxin reductase. The 26- and 21-kDa proteins were identified in immunoblot assays as the glutathione peroxidase (cGPX or GPX1) subunit and phospholipid hydroperoxide glutathione peroxidase (PHGPX or GPX4), respectively. We recently purified the 15-kDa protein and characterized it as a selenoprotein of unknown function. In contrast to selenoproteins, low molecular mass [75Se]compounds accumulated during HIV infection and migrated as a diffuse band near the front of SDS/PAGE gels. More Information
Gladyshev VN, Martin-Romero FJ, Xu XM, Kumaraswamy E, Carlson BA, Hatfield DL, Lee BJ. (1999) Molecular biology of selenium and its role in cancer, AIDS and other human diseases. Recent Research Developments in Biochemistry 1, 145-167
Gladyshev VN, Hatfield DL. (1999) Selenocysteine-containing proteins in mammals. J. Biomed. Sci. 6, 151-160.
Hatfield DL, Gladyshev VN, Park J, Park SI, Chittum HS, Baek HJ, Carlson BA, Yang ES, Moustafa ME, Lee BJ. (1999) Biosynthesis of selenocysteine and its incorporation into protein as the 21st amino acid. Comprehensive Natural Products Chemistry 4, 353-380.
Gladyshev VN, Factor VM, Housseau F, Hatfield DL. (1998) Contrasting patterns of regulation of the antioxidant selenoproteins, thioredoxin reductase, and glutathione peroxidase, in cancer cells. Biochem. Biophys. Res. Commun. 251, 488-93.

Gladyshev VN, Jeang KT, Wootton JC, Hatfield DL. (1998) A new human selenium-containing protein: purification, characterization and cDNA sequence. J. Biol. Chem. 273, 8910-8915.

AbstractSelenium which occurs in proteins as the amino acid, selenocysteine, is essential for numerous biological processes and for human health. A prominent 75Se-labeled protein detected in human T-cells migrated as a 15-kDa band by SDS-polyacrylamide gel electrophoresis. This protein subunit was purified and subjected to tryptic digestion and peptide sequence analyses. Sequences of tryptic peptides derived from the protein corresponded to a human placental gene sequence containing an open reading frame of 162 residues and a readthrough in-frame TGA codon. Three different peptide sequences of the 15-kDa protein corresponded to a nucleotide sequence located downstream of this codon, suggesting that the T-cell 15-kDa selenoprotein contains a selenocysteine residue encoded by TGA. Post-translational processing of the N-terminal portion of the predicted gene product to give the 15-kDa protein was suggested on the basis of molecular mass, amino acid analysis, and immunoblot assays of the purified protein. The 3-untranslated region (UTR) of the gene encoding the 15-kDa protein contained a sequence that is very similar to the canonical selenocysteine-inserting sequence element. Computer analysis of transcript map data bases indicated that this gene was located on human chromosome 1. Its coding sequence showed no homology to known protein-encoding genes. The 15-kDa protein gene was expressed as mRNA in a wide range of tissues, with increased levels in the thyroid, parathyroid, and prostate-derived cells as evidenced by searches of partial cDNA sequences in public data bases. Genes corresponding to the 15-kDa selenocysteinecontaining protein were found in mice and rats, while the corresponding genes in Caenorhabditis elegans and Brugia malayi contained a cysteine codon in place of TGA. The discovery of a new human selenoprotein provides an additional example of the role of selenium in mammalian systems. More Information
Khangulov SV, Gladyshev VN, Dismukes C, Stadtman TC. (1998) Selenium-containing formate dehydrogenase from E. coli: molybdopterin enzyme that catalyes formate oxidation without oxygen transfer. Biochemistry 37, 3518-3528.
George GN, Colangelo CM, Dong J, Scott RA, Khangulov SV, Gladyshev VN, Stadtman TC. (1998) X-ray absorbtion spectroscopy of the molybdenum site of Escherichia coli formate dehydrogenase. J. Am. Chem. Soc. 120, 1267-1273.

Boyington JC, Gladyshev VN, Khangulov SV, Stadtman TC, Sun PD. (1997) Crystal structure of formate dehydrogenase H: catalysis involving molybdenum, molybdopterin, selenocysteine and an Fe4S4 cluster. Science 275, 1305-1308.

AbstractFormate dehydrogenase H from Escherichia coli contains selenocysteine (SeCys), molybdenum, two molybdopterin guanine dinucleotide (MGD) cofactors, and an Fe4S4 cluster at the active site and catalyzes the two-electron oxidation of formate to carbon dioxide. The crystal structures of the oxidized [Mo( VI), Fe4S4(ox)] form of formate dehydrogenase H (with and without bound inhibitor) and the reduced [Mo(IV ), Fe4S4(red)] form have been determined, revealing a four-domain ab structure with the molybdenum directly coordinated to selenium and both MGD cofactors. These structures suggest a reaction mechanism that directly involves SeCys140 and His141 in proton abstraction and the molybdenum, molybdopterin, Lys44, and the Fe4S4 cluster in electron transfer. More Information
Baek HJ, Chittum HS, Yang ES, Park JM, Gladyshev VN, Moustafa ME, Carlson BA, Diamond AM, Lee BJ, Hatfield DL. (1997) Response of the selenocysteine tRNA population to selenium in mammals and Xenopus oocytes. Nucl. Acids Res. Symp. Ser. 36, 157-158
Gladyshev VN, Lecchi P. (1996) Identification of molybdopterins in molybdenum- and selenium-containing enzymes. BioFactors 5, 94-97.

Gladyshev VN, Jeang KT, Stadtman TC. (1996) Selenocysteine, identified as the penultimate C- terminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene. Proc. Natl. Acad. Sci. USA 93, 6146-6151.

AbstractThe possible relationship of selenium to immunological function which has been suggested for decades was investigated in studies on selenuim metabolism in human T cells. One of the major 75Se-labeled selenoproteins detected was purified to homogeneity and shown to be a homodimer of 55-kDa subunits. Each subunit contained about 1 FAD and at least 0.74 Se. This protein proved to be thioredoxin reductase (TR) on the basis of its catalytic activities, cross-reactivity with anti-rat liver TR antibodies, and sequence identities of several tryptic peptides with the published deduced sequence of human placental TR. Physicochemical characteristics of T-cell TR were similar to those of a selenocysteine (Secys)- containing TR recently isolated from human lung adenocarcinoma cells. The sequence of a 12-residue 75Se-labeled tryptic peptide from T-cell TR was identical with a C-terminaldeduced sequence of human placental TR except that Secys was present in the position corresponding to TGA, previously thought to be the termination codon, and this was followed by Gly-499, the actual C-terminal amino acid. The presence of the unusual conserved Cys-Secys-Gly sequence at the C terminus of TR in addition to the redox active cysteines of the Cys-Val- Asn-Val-Gly-Cys motif in the FAD-binding region may account for the peroxidase activity and the relatively low substrate specificity of mammalian TRs. The finding that T-cell TR is a selenoenzyme that contains Se in a conserved Cterminal region provides another example of the role of selenium in a major antioxidant enzyme system (i.e., thioredoxin- thioredoxin reductase), in addition to the well-known glutathione peroxidase enzyme system. More Information
Gladyshev VN, Khangulov SV, Stadtman TC. (1996) Properties of selenium- and molybdenum-containing nicotinic acid hydroxylase from Clostridium barkeri. Biochemistry 35, 212-223.

Gladyshev VN, Boyington JC, Khangulov SV, Grahame DA, Stadtman TC, Sun PD. (1996) Characterization of crystalline formate dehydrogenase H from Escherichia Coli: stabilization, EPR spectroscopy and preliminary crystallographic analysis. J. Biol. Chem. 271, 8095-8100.

AbstractThe selenocysteine-containing formate dehydrogenase H (FDH) is an 80-kDa component of the Escherichia coli formate-hydrogen lyase complex. The molybdenumcoordinated selenocysteine is essential for catalytic activity of the native enzyme. FDH in dilute solutions (30 µg/ml) was rapidly inactivated at basic pH or in the presence of formate under anaerobic conditions, but at higher enzyme concentrations ( 3 mg/ml) the enzyme was relatively stable. The formate-reduced enzyme was extremely sensitive to air inactivation under all conditions examined. Active formate-reduced FDH was crystallized under anaerobic conditions in the presence of ammonium sulfate and PEG 400. The crystals diffract to 2.6 � resolution and belong to a space group of P41212 or P43212 with unit cell dimensions a = b = 146.1 � and c = 82.7 �. There is one monomer of FDH per crystallographic asymmetric unit. Similar diffraction quality crystals of oxidized FDH could be obtained by oxidation of crystals of formate-reduced enzyme with benzyl viologen. By EPR spectroscopy, a signal of a single reduced FeS cluster was found in a crystal of reduced FDH, but not in a crystal of oxidized enzyme, whereas Mo(V) signal was not detected in either form of crystalline FDH. This suggests that Mo(IV)- and the reduced FeS cluster-containing form of the enzyme was crystallized and this could be converted into Mo(VI)- and oxidized FeS cluster form upon oxidation. A procedure that combines anaerobic and cryocrystallography has been developed that is generally applicable to crystallographic studies of oxygen-sensitive enzymes. These data provide the first example of crystallization of a substrate-reduced form of a Se- and Mo-containing enzyme. More Information
Tamura T, Gladyshev VN, Liu SY, Stadtman TC. (1996) The mutual sparring effects of selenium and vitamin E in animal nutrition may be further explained by the discovery that mammalian thioredoxin reductase is a selenoenzyme. BioFactors 5, 99-102.
Gladyshev VN, Khangulov SV, Stadtman TC. (1994) Nicotinc acid hydroxylase from Clostridium barkeri: Electron paramagnetic resonance studies show that selenium is coordinated with molybdenum in the catalytically active selenium-dependent enzyme. Proc. Natl. Acad. Sci. USA 91, 232-236.
Gladyshev VN, Khangulov SV, Axley MJ, Stadtman TC. (1994) Coordination of selenium to molybdenum in formate dehydrogenase H from Escherichia coli.. Proc. Natl. Acad. Sci. USA 91, 7708-7711.
Tishkov VI, Galkin AG, Gladyshev VN, Karzanov VV, Egorov AM. (1992) Analysis of gene structure, optimization of expression in E. coli, and properties of recombinant formate dehydrogenase from Pseudomonas sp. 101. Biotechnology 5, 52-59.