2009 Articles

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

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

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

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

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

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

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

AbstractNo abstract available. More Information

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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