2007 Articles

Yoo MH, Xu XM, Carlson BA, Patterson AD, Gladyshev VN, Hatfield DL. (2007) Targeting thioredoxin reductase 1 reduction in cancer cells inhibits self-sufficient growth and DNA replication. PLoS ONE 2, e1112, 1-7.

AbstractThioredoxin reductase 1 (TR1) is a major redox regulator in mammalian cells. As an important antioxidant selenoprotein, TR1 is thought to participate in cancer prevention, but is also known to be over-expressed in many cancer cells. Numerous cancer drugs inhibit TR1, and this protein has been proposed as a target for cancer therapy. We previously reported that reduction of TR1 levels in cancer cells reversed many malignant characteristics suggesting that deficiency in TR1 function is antitumorigenic. The molecular basis for TR1’s role in cancer development, however, is not understood. Herein, we found that, among selenoproteins, TR1 is uniquely overexpressed in cancer cells and its knockdown in a mouse cancer cell line driven by oncogenic k-ras resulted in morphological changes characteristic of parental (normal) cells, without significant effect on cell growth under normal growth conditions. When grown in serum-deficient medium, TR1 deficient cancer cells lose self-sufficiency of growth, manifest a defective progression in their S phase and a decreased expression of DNA polymerase alpha, an enzyme important in DNA replication. These observations provide evidence that TR1 is critical for self-sufficiency in growth signals of malignant cells, that TR1 acts largely as a pro-cancer protein and it is indeed a primary target in cancer therapy. More Information

Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Marechal-Drouard L, Marshall WF, Qu LH, Nelson DR, Sanderfoot AA, Spalding MH, Kapitonov VV, Ren Q, Ferris P, Lindquist E, Shapiro H, Lucas SM, Grimwood J, Schmutz J, Cardol P, Cerutti H, Chanfreau G, Chen CL, Cognat V, Croft MT, Dent R, Dutcher S, Fernandez E, Fukuzawa H, Gonzalez-Ballester D, Gonzalez-Halphen D, Hallmann A, Hanikenne M, Hippler M, Inwood W, Jabbari K, Kalanon M, Kuras R, Lefebvre PA, Lemaire SD, Lobanov AV., Lohr M, Manuell A, Meier I, Mets L, Mittag M, Mittelmeier T, Moroney JV, Moseley J, Napoli C, Nedelcu AM, Niyogi K, Novoselov SV., Paulsen IT, Pazour G, Purton S, Ral JP, Riano-Pachon DM, Riekhof W, Rymarquis L, Schroda M, Stern D, Umen J, Willows R, Wilson N, Zimmer SL, Allmer J, Balk J, Bisova K, Chen CJ, Elias M, Gendler K, Hauser C, Lamb MR, Ledford H, Long JC, Minagawa J, Page MD, Pan J, Pootakham W, Roje S, Rose A, Stahlberg E, Terauchi AM, Yang P, Ball S, Bowler C, Dieckmann CL, Gladyshev VN., Green P, Jorgensen R, Mayfield S, Mueller-Roeber B, Rajamani S, Sayre RT, Brokstein P, Dubchak I, Goodstein D, Hornick L, Huang YW, Jhaveri J, Luo Y, Martinez D, Ngau WC, Otillar B, Poliakov A, Porter A, Szajkowski L, Werner G, Zhou K, Grigoriev IV, Rokhsar DS, Grossman AR. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318, 245-250.

AbstractChlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella. More Information

Gladyshev VN. (2007) Methionine Sulfoxide Reductases. In Redox Biochemistry. Ed., Banerjee R. (ed., Gladyshev VN, Ragsdale SW, Becker DF, Dickman MB), John Wiley & Sons, pp. 84-87.
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Gladyshev VN. (2007) Selenoproteins. In Redox Biochemistry. Ed., Banerjee R. (ed., Gladyshev VN, Ragsdale SW, Becker DF, Dickman MB), John Wiley & Sons, pp. 127-131.
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Fomenko DE, Gladyshev VN. (2007) Bioinformatics Methods to Study Thiol-Based Oxidoreductases. In Redox Biochemistry. Ed., Banerjee R. (ed., Gladyshev VN, Ragsdale SW, Becker DF, Dickman MB), John Wiley & Sons, pp. 251-256.
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Aachmann FL, Fomenko DE, Soragni A, Gladyshev VN, Dikiy A. (2007) Structural analysis of selenoprotein W and NMR analysis of its interaction with 14-3-3 proteins. J. Biol. Chem. 282, 37036-37044.

AbstractSelenium is a trace element with significant biomedical potential. It is essential in mammals due to its occurrence in several proteins in the form of selenocysteine (Sec). One of the most abundant mammalian Sec-containing proteins is selenoprotein W (SelW). This protein of unknown function has a broad expression pattern and contains a candidate CXXU (where U represents Sec) redox motif. Here, we report the solution structure of the Sec13–>Cys variant of mouse SelW determined through high resolution NMR spectroscopy. The protein has a thioredoxin-like fold with the CXXU motif located in an exposed loop similarly to the redox-active site in thioredoxin. Protein dynamics studies revealed the rigidity of the protein backbone and mobility of two external loops and suggested a role of these loops in interaction with SelW partners. Molecular modeling of structures of other members of the Rdx family based on the SelW structure identified new conserved features in these proteins, including an aromatic cluster and interacting loops. Our previous study suggested an interaction between SelW and 14-3-3 proteins. In the present work, with the aid of NMR spectroscopy, we demonstrated specificity of this interaction and identified mobile loops in SelW as interacting surfaces. This finding suggests that 14-3-3 are redox-regulated proteins. More Information

Kim HY, Gladyshev VN. (2007) Methionine sulfoxide reductases: selenoprotein forms and roles in antioxidant protein repair in mammals. Biochem. J. 407, 321-329.

AbstractMsrs (methionine sulfoxide reductases), MsrA and MsrB, are repair enzymes that reduce methionine sulfoxide residues in oxidatively damaged proteins to methionine residues in a stereospecific manner. These enzymes protect cells from oxidative stress and have been implicated in delaying the aging process and progression of neurodegenerative diseases. In recent years, significant efforts have been made to explore the catalytic properties and physiological functions of these enzymes. In the current review, we present recent progress in this area, with the focus on mammalian MsrA and MsrBs including their roles in disease, evolution and function of selenoprotein forms of MsrA and MsrB, and the biochemistry of these enzymes. More Information

Lobanov AV, Fomenko DE, Zhang Y, Sengupta A, Hatfield DL, Gladyshev VN. (2007) Evolutionary dynamics of eukaryotic selenoproteomes: large selenoproteomes may associate with aquatic and small with terrestrial life. Genome Biol. 8, R198.

AbstractBACKGROUND: Selenocysteine (Sec) is a selenium-containing amino acid that is co-translationally inserted into nascent polypeptides by recoding UGA codons. Selenoproteins occur in both eukaryotes and prokaryotes, but the selenoprotein content of organisms (selenoproteome) is highly variable and some organisms do not utilize Sec at all. RESULTS: We analyzed the selenoproteomes of several model eukaryotes and detected 26 and 29 selenoprotein genes in the green algae Ostreococcus tauri and Ostreococcus lucimarinus, respectively, five in the social amoebae Dictyostelium discoideum, three in the fly Drosophila pseudoobscura, and 16 in the diatom Thalassiosira pseudonana, including several new selenoproteins. Distinct selenoprotein patterns were verified by metabolic labeling of O. tauri and D. discoideum with 75Se. More than half of the selenoprotein families were shared by unicellular eukaryotes and mammals, consistent with their ancient origin. Further analyses identified massive, independent selenoprotein losses in land plants, fungi, nematodes, insects and some protists. Comparative analyses of selenoprotein-rich and -deficient organisms revealed that aquatic organisms generally have large selenoproteomes, whereas several groups of terrestrial organisms reduced their selenoproteomes through loss of selenoprotein genes and replacement of Sec with cysteine. CONCLUSION: Our data suggest many selenoproteins originated at the base of the eukaryotic domain and show that the environment plays an important role in selenoproteome evolution. In particular, aquatic organisms apparently retained and sometimes expanded their selenoproteomes, whereas the selenoproteomes of some terrestrial organisms were reduced or completely lost. These findings suggest a hypothesis that, with the exception of vertebrates, aquatic life supports selenium utilization, whereas terrestrial habitats lead to reduced use of this trace element due to an unknown environmental factor. More Information

Carlson BA, Moustafa ME, Sengupta A, Schweizer U, Shrimali R, Rao M, Zhong N, Wang S, Feigenbaum L, Lee BJ, Gladyshev VN, Hatfield DL. (2007) Selective restoration of the selenoprotein population in a mouse hepatocyte selenoproteinless background with different mutant selenocysteine tRNAs lacking Um34. J. Biol. Chem. 282, 32591-32602.

AbstractNovel mouse models were developed in which the hepatic selenoprotein population was targeted for removal by disrupting the selenocysteine (Sec) tRNA([Ser]Sec) gene (trsp), and selenoprotein expression was then restored by introducing wild type or mutant trsp transgenes. The selenoprotein population was partially replaced in liver with mutant transgenes encoding mutations at either position 34 (34T–>A) or 37 (37A–>G) in tRNA([Ser]Sec). The A34 transgene product lacked the highly modified 5-methoxycarbonylmethyl-2′-O-methyluridine, and its mutant base A was converted to I34. The G37 transgene product lacked the highly modified N(6)-isopentenyladenosine. Both mutant tRNAs lacked the 2′-methylribose at position 34 (Um34), and both supported expression of housekeeping selenoproteins (e.g. thioredoxin reductase 1) in liver but not stress-related proteins (e.g. glutathione peroxidase 1). Thus, Um34 is responsible for synthesis of a select group of selenoproteins rather than the entire selenoprotein population. The ICA anticodon in the A34 mutant tRNA decoded Cys codons, UGU and UGC, as well as the Sec codon, UGA. However, metabolic labeling of A34 transgenic mice with (75)Se revealed that selenoproteins incorporated the label from the A34 mutant tRNA, whereas other proteins did not. These results suggest that the A34 mutant tRNA did not randomly insert Sec in place of Cys, but specifically targeted selected selenoproteins. High copy numbers of A34 transgene, but not G37 transgene, were not tolerated in the absence of wild type trsp, further suggesting insertion of Sec in place of Cys in selenoproteins. More Information

Xu XM, Carlson BA, Zhang Y, Mix H, Kryukov GV, Glass RS, Berry MJ, Gladyshev VN, Hatfield DL. (2007) New developments in selenium biochemistry: selenocysteine biosynthesis in eukaryotes and archaea. Biol. Trace Elem. Res. 119, 234-241.

AbstractWe used comparative genomics and experimental analyses to show that (1) eukaryotes and archaea, which possess the selenocysteine (Sec) protein insertion machinery contain an enzyme, O-phosphoseryl-transfer RNA (tRNA) [Ser]Sec kinase (designated PSTK), which phosphorylates seryl-tRNA [Ser]Sec to form O-phosphoseryl-tRNA [Ser]Sec and (2) the Sec synthase (SecS) in mammals is a pyridoxal phosphate-containing protein previously described as the soluble liver antigen (SLA). SecS uses the product of PSTK, O-phosphoseryl-tRNA[Ser]Sec, and selenophosphate as substrates to generate selenocysteyl-tRNA [Ser]Sec. Sec could be synthesized on tRNA [Ser]Sec from selenide, adenosine triphosphate (ATP), and serine using tRNA[Ser]Sec, seryl-tRNA synthetase, PSTK, selenophosphate synthetase, and SecS. The enzyme that synthesizes monoselenophosphate is a previously identified selenoprotein, selenophosphate synthetase 2 (SPS2), whereas the previously identified mammalian selenophosphate synthetase 1 did not serve this function. Monoselenophosphate also served directly in the reaction replacing ATP, selenide, and SPS2, demonstrating that this compound was the active selenium donor. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that contain selenoproteins. More Information

Shchedrina VA, Novoselov SV, Malinouski MY, Gladyshev VN. (2007) Identification and characterization of a selenoprotein family containing a diselenide bond in a redox motif. Proc. Natl. Acad. Sci. USA 104, 13919-13924.

AbstractSelenocysteine (Sec, U) insertion into proteins is directed by translational recoding of specific UGA codons located upstream of a stem-loop structure known as Sec insertion sequence (SECIS) element. Selenoproteins with known functions are oxidoreductases containing a single redox-active Sec in their active sites. In this work, we identified a family of selenoproteins, designated SelL, containing two Sec separated by two other residues to form a UxxU motif. SelL proteins show an unusual occurrence, being present in diverse aquatic organisms, including fish, invertebrates, and marine bacteria. Both eukaryotic and bacterial SelL genes use single SECIS elements for insertion of two Sec. In eukaryotes, the SECIS is located in the 3′ UTR, whereas the bacterial SelL SECIS is within a coding region and positioned at a distance that supports the insertion of either of the two Sec or both of these residues. SelL proteins possess a thioredoxin-like fold wherein the UxxU motif corresponds to the catalytic CxxC motif in thioredoxins, suggesting a redox function of SelL proteins. Distantly related SelL-like proteins were also identified in a variety of organisms that had either one or both Sec replaced with Cys. Danio rerio SelL, transiently expressed in mammalian cells, incorporated two Sec and localized to the cytosol. In these cells, it occurred in an oxidized form and was not reducible by DTT. In a bacterial expression system, we directly demonstrated the formation of a diselenide bond between the two Sec, establishing it as the first diselenide bond found in a natural protein. More Information

See commentary in: Drahl C. (2007) Selenium Doubles Up In Proteins. Chemical & Engineering News 85, 14. Commentary

Zhang Y, Gladyshev VN. (2007) High content of proteins containing 21st and 22nd amino acids, selenocysteine and pyrrolysine, in a symbiotic deltaproteobacterium of gutless worm Olavius algarvensis. Nucleic Acids Res. 35, 4952-4963.

AbstractSelenocysteine (Sec) and pyrrolysine (Pyl) are rare amino acids that are cotranslationally inserted into proteins and known as the 21st and 22nd amino acids in the genetic code. Sec and Pyl are encoded by UGA and UAG codons, respectively, which normally serve as stop signals. Herein, we report on unusually large selenoproteomes and pyrroproteomes in a symbiont metagenomic dataset of a marine gutless worm, Olavius algarvensis. We identified 99 selenoprotein genes that clustered into 30 families, including 17 new selenoprotein genes that belong to six families. In addition, several Pyl-containing proteins were identified in this dataset. Most selenoproteins and Pyl-containing proteins were present in a single deltaproteobacterium, delta1 symbiont, which contained the largest number of both selenoproteins and Pyl-containing proteins of any organism reported to date. Our data contrast with the previous observations that symbionts and host-associated bacteria either lose Sec utilization or possess a limited number of selenoproteins, and suggest that the environment in the gutless worm promotes Sec and Pyl utilization. Anaerobic conditions and consistent selenium supply might be the factors that support the use of amino acids that extend the genetic code. More Information

See commentary in: Atkins JF. Baranov PV. (2007) Duality in the genetic code. Nature 448, 1004-1005. Commentary

Sal LS, Aachmann FL, Kim H, Gladyshev VN, Dikiy A. (2007) NMR assignments of 1H, 13C and 15N spectra of methionine sulfoxide reductase B1 from Mus musculus. Biomol NMR Assign, 1, 131-133.

AbstractIsotopically labeled, 15N and 15N/13C forms of recombinant methionine-r-sulfoxide reductase 1 (MsrB1, SelR) from Mus musculus were produced, in which catalytic selenocysteine was replaced with cysteine. We report here the 1H, 15N and 13C NMR assignment of the reduced form of this mammalian protein. More Information

Su D, Berndt C, Fomenko DE, Holmgren A, Gladyshev VN. (2007) A Conserved cis-Proline Precludes Metal Binding by the Active Site Thiolates in Members of the Thioredoxin Family of Proteins. Biochemistry 46, 6903-6910.

AbstractMany thioredoxin-fold proteins possess a conserved cis-proline located in their C-terminal portions. This residue, as well as catalytic and resolving cysteines, is a key functional group in the active sites of these thiol-disulfide oxidoreductases. However, the specific function of the proline is poorly understood, and some thioredoxin-fold proteins lack this residue. Herein, we found that mutation of a cis-proline, Pro75, in human thioredoxin to serine, threonine, or alanine leads to the formation of an Fe2-S2 cluster in this protein. Further mutagenesis studies revealed that the first cysteine in the CxxC motif and a cysteine in the C-terminal region of the protein were responsible for metal binding. Replacement of Pro75 with arginine, a residue that occurs in place of Pro in peroxiredoxins, also led to the formation of the cluster in the thioredoxin. In addition, we found that mutation of the TxxC active site in a peroxiredoxin to the CxxC form could lead to coordination of an Fe2-S2 cluster in these proteins in vitro. Sco1, a distantly related thioredoxin-fold protein, has histidine in place of the cis-proline, and this residue binds copper. The Pro75His mutation led to increased copper binding by human thioredoxin when cells were grown in the presence of this trace element. Taken together, our data suggest that an important function of Pro75 in human thioredoxin, and likely other members of this superfamily, is to prevent metal binding by the reactive thiolate-based active site. More Information

Dikiy A, Novoselov SV, Fomenko DE, Sengupta A, Carlson BA, Cerny RL, Ginalski K, Grishin NV, Hatfield DL, Gladyshev VN. (2007) SelT, SelW, SelH, and Rdx12: Genomics and Molecular Insights into the Functions of Selenoproteins of a Novel Thioredoxin-like Family. Biochemistry 46, 6871-6882.

AbstractSelenium is an essential trace element in many life forms due to its occurrence as a selenocysteine (Sec) residue in selenoproteins. The majority of mammalian selenoproteins, however, have no known function. Herein, we performed extensive sequence similarity searches to define and characterize a new protein family, designated Rdx, that includes mammalian selenoproteins SelW, SelV, SelT and SelH, bacterial SelW-like proteins and cysteine-containing proteins of unknown function in all three domains of life. An additional member of this family is a mammalian cysteine-containing protein, designated Rdx12, and its fish selenoprotein orthologue. Rdx proteins are proposed to possess a thioredoxin-like fold and a conserved CxxC or CxxU (U is Sec) motif, suggesting a redox function. We cloned and characterized three mammalian members of this family, which showed distinct expression patterns in mouse tissues and different localization patterns in cells transfected with the corresponding GFP fusion proteins. By analogy to thioredoxin, Rdx proteins can use catalytic cysteine (or Sec) to form transient mixed disulfides with substrate proteins. We employed this property to identify cellular targets of Rdx proteins using affinity columns containing mutant versions of these proteins. Rdx12 was found to interact with glutathione peroxidase 1, whereas 14-3-3 protein was identified as one of the targets of mammalian SelW, suggesting a mechanism for redox regulation of the 14-3-3 family of proteins. More Information

Novoselov SV, Lobanov AV, Hua D, Kasaikina MV, Hatfield DL, Gladyshev VN. (2007) A highly efficient form of the selenocysteine insertion sequence element in protozoan parasites and its use in mammalian cells. Proc. Natl. Acad. Sci. USA 104, 7857-7862.

AbstractSelenoproteins are an elite group of proteins containing a rare amino acid, selenocysteine (Sec), encoded by the codon, UGA. In eukaryotes, incorporation of Sec requires a Sec insertion sequence (SECIS) element, a stem-loop structure located in the 3′-untranslated regions of selenoprotein mRNAs. Here we report identification of a noncanonical form of SECIS element in Toxoplasma gondii and Neospora canine, single-celled apicomplexan parasites of humans and domestic animals. This SECIS has a GGGA sequence in the SBP2-binding site in place of AUGA previously considered invariant. Using a combination of computational and molecular techniques, we show that Toxoplasma and Neospora possess both canonical and noncanonical SECIS elements. The GGGA-type SECIS element supported Sec insertion in mammalian HEK 293 and NIH 3T3 cells and did so more efficiently than the natural mammalian SECIS elements tested. In addition, mammalian type I and type II SECIS elements mutated into the GGGA forms were functional but manifested decreased Sec insertion efficiency. We carried out computational searches for both AUGA and GGGA forms of SECIS elements in Toxoplasma and detected five selenoprotein genes, including one coding for a previously undescribed selenoprotein, designated SelQ, and two containing the GGGA form of the SECIS element. In contrast, the GGGA-type SECIS elements were not detected in mammals and nematodes. As a practical outcome of the study, we developed pSelExpress1, a vector for convenient expression of selenoproteins in mammalian cells. It contains an SBP2 gene and the most efficient tested SECIS element: an AUGA mutant of the GGGA-type Toxoplasma SelT structure. More Information

Yoo MH, Xu XM, Turanov AA, Carlson BA, Gladyshev VN, Hatfield DL. (2007) A new strategy for assessing selenoprotein function: siRNA knockdown/knock-in targeting the 3′-UTR. RNA 13, 921-929.

AbstractSelenocysteine insertion into protein in mammalian cells requires RNA elements in the 3′-untranslated regions (3′-UTRs) of selenoprotein genes. The occurrence of these conserved sequences should make selenoproteins particularly amenable for knockdown/knock-in strategies to examine selenoprotein functions. Herein, we utilized the 3′-UTR of various selenoproteins to knock down their expression using siRNAs and then knock in expression using constructs containing mutations within the target region. Thioredoxin reductase 1 (TR1) knockdown in a mouse kidney cell line resulted in the cells growing about 10% more slowly, being more sensitive to UV radiation, and having increased apoptosis in response to UV than control cells. The knockdown cells transfected with a construct encoding the wild-type TR1 gene and having mutations in the sequences targeted by siRNA restored TR1 expression and catalytic activity, rendered the knockdown cells less sensitive to UV, and protected the cells against apoptosis. We also applied this technique to other selenoproteins, selenophosphate synthetase 2 and glutathione peroxidase 1, and found that mRNA and protein levels were restored following transfection of knockdown cells with the corresponding knock-in constructs. In addition to important new insights into the functions of key mammalian selenoproteins, the data suggest that the RNAi-based knock-in technology could distinguish phenotypes due to off-targeting and provide a new method for examining many of the subtleties of selenoprotein function not available using RNAi technology alone. More Information

Koc A, Gladyshev VN. (2007) Methionine sulfoxide reduction and the aging process. Ann. NY Acad. Sci. 1100, 383-386.

AbstractAging has been described for multicellular and asymmetrically dividing organisms, but the mechanisms are poorly understood. Oxidation of proteins is considered to be one of the major factors that leads to aging. Oxidative damage to proteins results in the oxidation of certain amino acid residues, among which oxidation of sulfur-containing amino acids, methionine and cysteine, is notable because of the susceptibility of these residues to damage, and occurrence of repair mechanisms. Methionine sulfoxide reductases, MsrA and MsrB, are thioredoxin-dependent oxidoreductases that reduce oxidized forms of methionine, methionine sulfoxides, in a stereospecific manner. These enzymes are present in all cell types and have shown to be regulating life spans in mammals, insects, and yeast. Here, their roles in modulating yeast life span are discussed. More Information

Labunskyy VM, Hatfield DL, Gladyshev VN. (2007) The Sep15 protein family: Roles in disulfide bond formation and quality control in the endoplasmic reticulum. IUBMB Life 59, 1-5.

AbstractDisulfide bonds play an important role in the structure and function of membrane and secretory proteins. The formation of disulfide bonds in the endoplasmic reticulum (ER) of eukaryotic cells is catalyzed by a complex network of thiol-disulfide oxidoreductases. Whereas a number of ER-resident oxidoreductases have been identified, the function of only a few of them is firmly established. Recently, a selenocysteine-containing oxidoreductase, Sep15, has been implicated in disulfide bond assisted protein folding, and a role in quality control for this selenoprotein has been proposed. This review summarizes up-to-date information on the Sep15 family proteins and highlights new insights into their physiological function. More Information

Xu XM, Carlson BA, Irons R, Mix H, Zhong N, Gladyshev VN, Hatfield DL. (2007) Selenophosphate synthetase 2 is essential for selenoprotein biosynthesis. Biochem. J. 404, 115-20.

AbstractSelenophosphate synthetase (SelD) generates the selenium donor for selenocysteine biosynthesis in eubacteria. One homologue of SelD in eukaryotes is SPS1 (selenophosphate synthetase 1) and a second one, SPS2, was identified as a selenoprotein in mammals. Earlier in vitro studies showed SPS2, but not SPS1, synthesized selenophosphate from selenide, whereas SPS1 may utilize a different substrate. The roles of these enzymes in selenoprotein synthesis in vivo remain unknown. To address their function in vivo, we knocked down SPS2 in NIH3T3 cells using small interfering RNA and found that selenoprotein biosynthesis was severely impaired, whereas knockdown of SPS1 had no effect. Transfection of SPS2 into SPS2 knockdown cells restored selenoprotein biosynthesis, but SPS1 did not, indicating that SPS1 cannot complement SPS2 function. These in vivo studies indicate that SPS2 is essential for generating the selenium donor for selenocysteine biosynthesis in mammals, whereas SPS1 probably has a more specialized, non-essential role in selenoprotein metabolism. More Information

Novoselov SV, Kryukov GV, Xu XM, Carlson BA, Hatfield DL, Gladyshev VN. (2007) Selenoprotein H is a nucleolar thioredoxin-like protein with a unique expression pattern. J. Biol. Chem. 282,11960-11968.

AbstractThe human selenoproteome consists of 25 known selenoproteins, but functions of many of these proteins are not known. Selenoprotein H (SelH) is a recently discovered 14-kDa mammalian protein with no sequence homology to functionally characterized proteins. By sensitive sequence and structure analyses, we identified SelH as a thioredoxin fold-like protein in which a conserved CXXU motif (cysteine separated by two other residues from selenocysteine) corresponds to the CXXC motif in thioredoxins. These data suggest a redox function of SelH. Indeed, a recombinant SelH shows significant glutathione peroxidase activity. In addition, SelH has a conserved RKRK motif in the N-terminal sequence. We cloned wild-type and cysteine mutant forms of SelH either upstream or downstream of green fluorescent protein (GFP) and localized this fusion protein to the nucleus in transfected mammalian cells, whereas mutations in the RKRK motif resulted in the cytosolic protein. Interestingly, the full-length SelH-GFP fusion protein localized specifically to nucleoli, whereas the N-terminal sequence of SelH fused to GFP had a diffuse nucleoplasm location. Northern blot analyses revealed low expression levels of SelH mRNA in various mouse tissues, but it was elevated in the early stages of embryonic development. In addition, SelH mRNA was overexpressed in human prostate cancer LNCaP and mouse lung cancer LCC1 cells. Down-regulation of SelH by RNA interference made LCC1 cells more sensitive to hydrogen peroxide but not to other peroxides tested. Overall, these data establish SelH as a novel nucleolar oxidoreductase and suggest that some functions in this compartment are regulated by redox and dependent on the trace element selenium. More Information

Grossman AR, Croft M, Gladyshev VN, Merchant SS, Posewitz MC, Prochnik S, Spalding MH. (2007) Novel metabolism in Chlamydomonas through the lens of genomics. Curr. Opin. Plant Biol. 10, 190-198.

AbstractChlamydomonas has traditionally been exploited as an organism that is associated with sophisticated physiological, genetic and molecular analyses, all of which have been used to elucidate several biological processes, especially photosynthesis and flagella function and assembly. Recently, the genomics of Chlamydomonas has been combined with other technologies to unveil new aspects of metabolism, including inorganic carbon utilization, anaerobic fermentation, the suite and functions of selenoproteins, and the regulation of vitamin biosynthesis. These initial findings represent the first glimpse through a genomic window onto the highly complex metabolisms that characterize a unicellular, photosynthetic eukaryote that has maintained both plant-like and animal-like characteristics over evolutionary time. More Information

Fomenko DE, Xing W, Adair BM, Thomas DJ, Gladyshev VN. (2007) High-Throughput Identification of Catalytic Redox-Active Cysteine Residues. Science 315, 387-389.

AbstractCysteine (Cys) residues often play critical roles in proteins; however, identification of their specific functions has been limited to case-by-case experimental approaches. We developed a procedure for high-throughput identification of catalytic redox-active Cys in proteins by searching for sporadic selenocysteine-Cys pairs in sequence databases. This method is independent of protein family, structure, and taxon. We used it to selectively detect the majority of known proteins with redox-active Cys and to make additional predictions, one of which was verified. Rapid accumulation of sequence information from genomic and metagenomic projects should allow detection of many additional oxidoreductase families as well as identification of redox-active Cys in these proteins. More Information

Mix H, Lobanov AV, Gladyshev VN. (2007) SECIS elements in the coding regions of selenoprotein transcripts are functional in higher eukaryotes. Nucleic Acids Res. 35, 414-423.

AbstractExpression of selenocysteine (Sec)-containing proteins requires the presence of a cis-acting mRNA structure, called selenocysteine insertion sequence (SECIS) element. In bacteria, this structure is located in the coding region immediately downstream of the Sec-encoding UGA codon, whereas in eukaryotes a completely different SECIS element has evolved in the 3′-untranslated region. Here, we report that SECIS elements in the coding regions of selenoprotein mRNAs support Sec insertion in higher eukaryotes. Comprehensive computational analysis of all available viral genomes revealed a SECIS element within the ORF of a naturally occurring selenoprotein homolog of glutathione peroxidase 4 in fowlpox virus. The fowlpox SECIS element supported Sec insertion when expressed in mammalian cells as part of the coding region of viral or mammalian selenoproteins. In addition, readthrough at UGA was observed when the viral SECIS element was located upstream of the Sec codon. We also demonstrate successful de novo design of a functional SECIS element in the coding region of a mammalian selenoprotein. Our data provide evidence that the location of the SECIS element in the untranslated region is not a functional necessity but rather is an evolutionary adaptation to enable a more efficient synthesis of selenoproteins. More Information

Shrimali RK, Weaver JA, Miller GF, Starost MF, Carlson BA, Novoselov SV, Kumaraswamy E, Gladyshev VN, Hatfield DL. (2007) Neuromuscul. Disord. 7, 135-142.

AbstractLoxP-Cre technology was used to remove the selenocysteine tRNA gene, trsp, in either endothelial cells or myocytes of skeletal and heart muscle to elucidate the role of selenoproteins in cardiovascular disease. Loss of selenoprotein expression in endothelial cells was embryonic lethal. A 14.5-day-old embryo had numerous abnormalities including necrosis of the central nervous system, subcutaneous hemorrhage and erythrocyte immaturity. Loss of selenoprotein expression in myocytes manifested no apparent phenotype until about day 12 after birth. Affected mice had decreased mobility and an increased respiratory rate, which proceeded rapidly to death. Pathological analysis revealed that mice lacking trsp had moderate to severe myocarditis with inflammation extending into the mediastinitis. Thus, ablation of selenoprotein expression demonstrated an essential role of selenoproteins in endothelial cell development and in proper cardiac muscle function. The data suggest a direct connection between the loss of selenoprotein expression in these cell types and cardiovascular disease. More Information

Xu XM, Carlson BA, Mix H, Zhang Y, Saira K, Glass RS, Berry MJ, Gladyshev VN, Hatfield DL. (2007) Biosynthesis of Selenocysteine on Its tRNA in Eukaryotes. PLoS Biol. 5, e4, 1-9.

AbstractSelenocysteine (Sec) is cotranslationally inserted into protein in response to UGA codons and is the 21st amino acid in the genetic code. However, the means by which Sec is synthesized in eukaryotes is not known. Herein, comparative genomics and experimental analyses revealed that the mammalian Sec synthase (SecS) is the previously identified pyridoxal phosphate-containing protein known as the soluble liver antigen. SecS required selenophosphate and O-phosphoseryl-tRNA([Ser]Sec) as substrates to generate selenocysteyl-tRNA([Ser]Sec). Moreover, it was found that Sec was synthesized on the tRNA scaffold from selenide, ATP, and serine using tRNA([Ser]Sec), seryl-tRNA synthetase, O-phosphoseryl-tRNA([Ser]Sec) kinase, selenophosphate synthetase, and SecS. By identifying the pathway of Sec biosynthesis in mammals, this study not only functionally characterized SecS but also assigned the function of the O-phosphoseryl-tRNA([Ser]Sec) kinase. In addition, we found that selenophosphate synthetase 2 could synthesize monoselenophosphate in vitro but selenophosphate synthetase 1 could not. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that synthesize selenoproteins. More Information