Life Sciences Commons^™

Evidence For Direct Roles Of Two Additional Factors, Secp43 And Soluble Liver Antigen, In The Selenoprotein Synthesis Machinery, Xue-Ming Xu, Heiko Mix, Bradley A. Carlson, Paula J. Grabowski, Vadim N. Gladyshev, Marla J. Berry, Dolph L. Hatfield

Vadim Gladyshev Publications

Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA ^[Ser]Sec, but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2’-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA ^[Ser]Sec, and consequently reduced glutathione peroxidase …

Computer-Assisted Docking Of Flavodoxin With The Atp:Co(I)Rrinoid Adenosyltransferase (Coba) Enzyme Reveals Residues Critical For Protein-Protein Interactions But Not For Catalysis*, Nicole R. Buan, Jorge C. Escalante-Semerena

Department of Biochemistry: Faculty Publications

The activity of the housekeeping ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica sv. Typhimurium is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium. In vitro, reduced flavodoxin (FldA) provides an electron to generate the co(I)rrinoid substrate in the CobA active site. To understand how CobAand FldA interact, a computer model of aCobA∙FldA complex was generated. This model was used to guide the introduction of mutations into CobA using site-directed mutagenesis and the synthesis of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of CobA …

Different Catalytic Mechanisms In Mammalian Selenocysteine- And Cysteine-Containing Methionine-R-Sulfoxide Reductases, Hwa-Young Kim, Vadim Gladyshev

Vadim Gladyshev Publications

Selenocysteine (Sec) is found in active sites of several oxidoreductases in which this residue is essential for catalytic activity. However, many selenoproteins have fully functional orthologs, wherein cysteine (Cys) occupies the position of Sec. The reason why some enzymes evolve into selenoproteins if the Cys versions may be sufficient is not understood. Among three mammalian methionine-R-sulfoxide reductases (MsrBs), MsrB1 is a Sec-containing protein, whereas MsrB2 and MsrB3 contain Cys in the active site, making these enzymes an excellent system for addressing the question of why Sec is used in biological systems. In this study, we found that residues, which are …

A Novel Cysteine-Rich Domain Of Sep15 Mediates The Interaction With Udp-Glucose:Glycoprotein Glucosyltransferase, Vyacheslav M. Labunskyy, Andrew D. Ferguson, Dmitri E. Fomenko, Yogarany Chelliah, Dolph L. Hatfield, Vadim N. Gladyshev

Vadim Gladyshev Publications

Selenium is an essential trace element with potent cancer prevention activity in mammals. The 15-kDa selenoprotein (Sep15) has been implicated in the chemopreventive effect of dietary selenium. Although the precise function of Sep15 remains elusive, Sep15 copurifies with UDP-glucose:glycoprotein glucosyltransferase (GT), an essential regulator of quality control mechanisms within the endoplasmic reticulum. Recent studies identified two GT and two Sep15 homologues in mammals. We characterize interactions between these protein families in this report. Sep15 and GT form a tight 1:1 complex, and these interactions are conserved between mammals and fruit flies. In mammalian cells, Sep15 co-immunoprecipitates with both GT isozymes. …

Crystal Structures Of Oxidized And Reduced Mitochondrial Thioredoxin Reductase Provide Molecular Details Of The Reaction Mechanism, Ekaterina I. Biterova, Anton A. Turanov, Vadim N. Gladyshev, Joseph J. Barycki

Vadim Gladyshev Publications

Thioredoxin reductase (TrxR) is an essential enzyme required for the efficient maintenance of the cellular redox homeostasis, particularly in cancer cells that are sensitive to reactive oxygen species. In mammals, distinct isozymes function in the cytosol and mitochondria. Through an intricate mechanism, these enzymes transfer reducing equivalents from NADPH to bound FAD and subsequently to an active-site disulfide. In mammalian TrxRs, the dithiol then reduces a mobile C-terminal selenocysteine- containing tetrapeptide of the opposing subunit of the dimer. Once activated, the C-terminal redox center reduces a disulfide bond within thioredoxin. In this report, we present the structural data on a …

Charge-Switch Nucleotides, John G. K. Williams, Gregory R. Bashford, Jiyan Chen, Dan Draney, Nara Narayanan, Bambi L. Reynolds, Pamela Sheaff

Biomedical Imaging and Biosignal Analysis Laboratory

The present invention provides compounds, methods and systems for sequencing nucleic acid using single molecule detection. Using labeled NPs that exhibit charge-switching behavior, single-molecule DNA sequencing in a microchannel sorting system is realized. In operation, sequencing products are detected enabling real-time sequencing as successive detectable moieties flow through a detection channel. By electrically sorting charged molecules, the cleaved product molecules are detected in isolation Without interference from unincorporated NPs and Without illuminating the polymerase-DNA complex.

Evolution Of Selenium Utilization Traits, Hector Romero, Yan Zhang, Vadim Gladyshev, Gustavo Salinas

Vadim Gladyshev Publications

Background: The essential trace element selenium is used in a wide variety of biological processes. Selenocysteine (Sec), the 21st amino acid, is co-translationally incorporated into a restricted set of proteins. It is encoded by an UGA codon with the help of tRNA^Sec (SelC), Sec-specific elongation factor (SelB) and a cis-acting mRNA structure (SECIS element). In addition, Sec synthase (SelA) and selenophosphate synthetase (SelD) are involved in the biosynthesis of Sec on the tRNA^Sec. Selenium is also found in the form of 2-selenouridine, a modified base present in the wobble position of certain tRNAs, whose synthesis is …

Mammalian Selenoprotein Thioredoxin-Glutathione Reductase, Dan Su, Sergey V. Novoselov, Qi-An Sun, Mohamed E. Moustafa, You Zhou, Richard Oko, Dolph L. Hatfield, Vadim N. Gladyshev

Vadim Gladyshev Publications

Thioredoxin reductases (TRs) are important redox regulatory enzymes, which control the redox state of thioredoxins. Mammals have cytosolic and mitochondrial TRs, which contain an essential selenocysteine residue and reduce cytosolic and mitochondrial thioredoxins. In addition, thioredoxin/glutathione reductase (TGR) was identified, which is a fusion of an N-terminal glutaredoxin domain and the TR module. Here we show that TGR is expressed at low levels in various tissues but accumulates in testes after puberty. The protein is particularly abundant in elongating spermatids at the site of mitochondrial sheath formation but is absent in mature sperm. We found that TGR can catalyze isomerization …

Glycoprotein Gp130 Of Dictyostelium Discoideum Influences Macropinocytosis And Adhesion, Catherine P. Chia, Sujatha Gomathinayagam, Robert J. Schmaltz, Laura K. Smoyer

Department of Biochemistry: Faculty Publications

Glycoprotein gp130, found on the plasma membrane of Dictyostelium discoideum amoebae, was postulated previously to play a role in phagocytosis. The gene for gp130 was cloned and when translated, yielded a 768 amino acid preproprotein of 85.3 kDa. It had nearly 40% similarity to the 138 kDa family of glycoproteins implicated in sexual cell fusion during macrocyst formation in D. discoideum. The difference between the calculated size and observed Mr of 130 kDa on protein gels likely was due to N-glycosylation that was confirmed by lectin blots. Consistent with its surface-exposure, an antibody raised against recombinant protein stained …

Pyrrolysine And Selenocysteine Use Dissimilar Decoding Strategies, Yan Zhang, Pavel V. Baranov, John F. Atkins, Vadim N. Gladyshev

Vadim Gladyshev Publications

Selenocysteine (Sec) and pyrrolysine (Pyl) are known as the 21st and 22nd amino acids in protein. Both are encoded by codons that normally function as stop signals. Sec specification by UGA codons requires the presence of a cis-acting selenocysteine insertion sequence (SECIS) element. Similarly, it is thought that Pyl is inserted by UAG codons with the help of a putative pyrrolysine insertion sequence (PYLIS) element. Herein, we analyzed the occurrence of Pyl-utilizing organisms, Pyl-associated genes, and Pyl-containing proteins. The Pyl trait is restricted to several microbes, and only one organism has both Pyl and Sec. We found that methanogenic …

Selenocysteine Insertion Directed By The 3’-Utr Secis Element In Escherichia Coli, Dan Su, Yehua Li, Vadim Gladyshev

Vadim Gladyshev Publications

Co-translational insertion of selenocysteine (Sec) into proteins in response to UGA codons is directed by selenocysteine insertion sequence (SECIS) elements. In known bacterial selenoprotein genes, SECIS elements are located in the coding regions immediately downstream of UGA codons. Here, we report that a distant SECIS element can also function in Sec insertion in bacteria provided that it is spatially close to the UGA codon. We expressed a mammalian phospholipid hydroperoxide glutathione peroxidase in Escherichia coli from a construct in which a natural E.coli SECIS element was located in the 3’-untranslated region (3’-UTR) and adjacent to a sequence complementary to the …

Nematode Selenoproteome: The Use Of The Selenocysteine Insertion System To Decode One Codon In An Animal Genome?, Kalin Taskov, Charles Chapple, Gregory V. Kryukov, Sergi Castenello, Alexei V. Lobanov, Konstantin V. Korotkov, Roderic Guigo, Vadim Gladyshev

Vadim Gladyshev Publications

Selenocysteine (Sec) is co-translationally inserted into selenoproteins in response to codon UGA with the help of the selenocysteine insertion sequence (SECIS) element.The number of selenoproteinsinanimals varies, with humans having 25 and mice having 24 selenoproteins. To date, however, only one selenoprotein, thioredoxin reductase, has been detected in Caenorhabditis elegans, and this enzyme contains only one Sec. Here, we characterize the selenoproteomes of C.elegans and Caenorhabditis briggsae with three independent algorithms,one searching for pairs of homologous nematode SECIS elements, another searching for Cys- or Sec-containing homologs of potential nematode selenoprotein genes and the third identifying Sec-containing homologs of annotated nematode …

The Microbial Selenoproteome Of The Sargasso Sea, Yan Zhang, Dmitri E. Fomenko, Vadim Gladyshev

Vadim Gladyshev Publications

Background: Selenocysteine (Sec) is a rare amino acid which occurs in proteins in major domains of life. It is encoded by TGA, which also serves as the signal for termination of translation, precluding identification of selenoprotein genes by available annotation tools. Information on full sets of selenoproteins (selenoproteomes) is essential for understanding the biology of selenium. Herein, we characterized the selenoproteome of the largest microbial sequence dataset, the Sargasso Sea environmental genome project.
Results: We identified 310 selenoprotein genes that clustered into 25 families, including 101 new selenoprotein genes that belonged to 15 families. Most of these proteins were predicted …

Nucleic Acid Sequencing Using Charge-Switch Nucleotides, John G. K. Williams, Gregory R. Bashford

Biomedical Imaging and Biosignal Analysis Laboratory

The present invention provides compounds, methods and systems for sequencing nucleic acid using single molecule detection. Using labeled NPs that exhibit charge-switching behavior, single-molecule DNA sequencing in a microchannel sorting system is realized. In operation, sequencing products are detected enabling real-time sequencing as successive detectable moieties flow through a detection channel. By electrically sorting charged molecules, the cleaved product molecules are detected in isolation without interference from unincorporated NPs and without illuminating the polymerase-DNA complex.

Um34 In Selenocysteine Trna Is Required For The Expression Of Stress-Related Selenoproteins In Mammals, Bradley A. Carlson, Xue-Ming Xu, Vadim N. Gladyshev, Dolph L. Hatfield

Vadim Gladyshev Publications

Selenium is an essential micronutrient in the diet of mammals and has many health benefits. Selenium-containing proteins are responsible for most, if not all, of these benefits. This element is incorporated into protein as selenocysteine (Sec), the 21^st amino acid in the genetic code. There are two species of Sec tRNA in mammalian cells that differ by a single 2’-O-hydroxymethyl group on the ribosyl moiety at position 34 (Um34). The relationship between this modification and selenoprotein synthesis was examined in mice in which the wild type Sec tRNA gene was replaced with a mutant Sec tRNA transgene incapable of …

Arabidopsis Atspl14, A Plant-Specific Sbp-Domain Transcription Factor, Participates In Plant Development And Sensitivity To Fumonisin B1, Julie M. Stone, Xinwen Liang, Emily R. Nekl, Justin J. Stiers

Department of Biochemistry: Faculty Publications

The recessive Arabidopsis thaliana fumonisin B1-resistant (fbr6) mutant was identified by its ability to survive in the presence of a programmed cell death (PCD)-inducing fungal toxin FB1. The fbr6 mutant also displays altered plant architecture in the absence of FB1, most notably elongated petioles and enhanced leaf margin serration. These phenotypes are a result of a T-DNA insertion in the SQUAMOSA promoter binding protein (SBP) domain gene, AtSPL14. AtSPL14 encodes a plant-specific protein with features characteristic of a transcriptional regulator, including a nuclear localization signal sequence, a plant-specific DNA binding domain (the SBP box), and a protein …

Selective Rescue Of Selenoprotein Expression In Mice Lacking A Highly Specialized Methyl Group In Selenocysteine Trna, Bradley A. Carlson, Xue-Ming Xu, Vadim N. Gladyshev, Dolph L. Hatfield

Vadim Gladyshev Publications

Selenocysteine (Sec) is the 21st amino acid in the genetic code. Its tRNA is variably methylated on the 2’-Ohydroxyl site of the ribosyl moiety at position 34 (Um34). Herein, we identified a role of Um34 in regulating the expression of some, but not all, selenoproteins. A strain of knock-out transgenic mice was generated, wherein the Sec tRNA gene was replaced with either wild type or mutant Sec tRNA transgenes. The mutant transgene yielded a tRNA that lacked two base modifications, N⁶- isopentenyladenosine at position 37 (i⁶A37) and Um34. Several selenoproteins, including glutathione peroxidases 1 and 3, …

5-Ht1b Receptor-Mediated Presynaptic Inhibition Of Gaba Release In The Suprachiasmatic Nucleus, Jayne R. Bramley, Patricia J. Sollars, Gary E. Pickard, F. Edward Dudek

School of Veterinary and Biomedical Sciences: Faculty Publications

The suprachiasmatic nucleus (SCN) receives a dense serotonergic innervation that modulates photic input to the SCN via serotonin 1B (5-HT_1B) presynaptic receptors on retinal glutamatergic terminals. However, the majority of 5-HT_1B binding sites in the SCN are located on nonretinal terminals and most axonal terminals in the SCN are GABAergic. We therefore tested the hypothesis that 5-HT_1B receptors might also be located on SCN GABAergic terminals by examining the effects of the highly selective 5-HT_1B receptor agonist CP-93,129 on SCN miniature inhibitory postsynaptic currents (mIPSCs). Whole cell patch-clamp recordings of mIPSCs were obtained from rat …

Role Of The Hypervariable Hinge Region Of Phosphoprotein P Of Vesicular Stomatitis Virus In Viral Rna Synthesis And Assembly Of Infectious Virus Particles, Subash C. Das, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

The phosphoprotein (P protein) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNAdependent RNA polymerase and has multiple functions residing in its different domains. In the present study, we examined the role of the hypervariable hinge region of P protein in viral RNA synthesis and recovery of infectious VSV by using transposon-mediated insertion mutagenesis and deletion mutagenesis. We observed that insertions of 19-amino-acid linker sequences at various positions within this region affected replication and transcription functions of the P protein to various degrees. Interestingly, one insertion mutant was completely defective in both transcription and replication. Using …

Ph-Dependent Substrate Preference Of Pig Heart Lipoamide Dehydrogenase Varies With Oligomeric State: Response To Mitochondrial Matrix Acidification, Natalia L. Klyachko, Valentina A. Shchedrina, Alexander V. Efimov, Sergey V. Kazakov, Irina G. Gazaryan, Bruce S. Kristal, Abraham M. Brown

Department of Biochemistry: Faculty Publications

Cycling of intracellular pH has recently been shown to play a critical role in ischemia-reperfusion injury. Ischemia-reperfusion also leads to mitochondrial matrix acidification and dysfunction. However, the mechanism by which matrix acidification contributes to mitochondrial dysfunction, oxidative stress, and the resultant cellular injury has not been elucidated. We observe pH-dependent equilibria between monomeric, dimeric, and a previously undescribed tetrameric form of pig heart lipoamide dehydrogenase (LADH), a mitochondrial matrix enzyme. Dynamic light scattering studies of native LADH in aqueous solution indicate that lowering pH favors a shift in average molecular mass from higher oligomeric states to monomer. Sedimentation velocity of …

An Algorithm For Identification Of Bacterial Selenocysteine Insertion Sequence Elements And Selenoprotein Genes, Yan Zhang, Vadim Gladyshev

Department of Biochemistry: Faculty Publications

Motivation: Incorporation of selenocysteine (Sec) into proteins in response to UGA codons requires a cis-acting RNA structure, Sec insertion sequence (SECIS) element. Whereas SECIS elements in Escherichia coli are well characterized, a bacterial SECIS consensus structure is lacking.

Results: We developed a bacterial SECIS consensus model, the key feature of which is a conserved guanosine in a small apical loop of the properly positioned structure. This consensus was used to build a computational tool, bSECISearch, for detection of bacterial SECIS elements and selenoprotein genes in sequence databases. The program identified 96.5% of known selenoprotein genes in completely sequenced bacterial genomes …

Regulation Of Enteric Endophytic Bacterial Colonization By Plant Defenses, A. Leonardo Iniguez, Yuemei Dong, Heather D. Carter, Brian M. M. Ahmer, Julie M. Stone, Eric W. Triplett

Department of Biochemistry: Faculty Publications

Bacterial endophytes reside within the interior of plants without causing disease or forming symbiotic structures. Some endophytes, such as Klebsiella pneumoniae 342 (Kp342), enhance plant growth and nutrition. Others, such as Salmonella enterica serovar Typhimurium (S. typhimurium), are human pathogens that contaminate raw produce. Several lines of evidence are presented here to support the hypothesis that plant defense response pathways regulate colonization by endophytic bacteria. An ethyleneinsensitive mutant of Medicago truncatula is hypercolonized by Kp342 compared to the parent genotype. Addition of ethylene, a signal molecule for induced systemic resistance in plants, decreased endophytic colonization in Medicago spp. …

Diversity And Functional Plasticity Of Eukaryotic Selenoproteins: Identification And Characterization Of The Selj Family, Sergi Castellano, Alexey V. Lobanov, Charles Chapple, Sergey V. Novoselov, Mario Albrecht, Deame Hua, Alain Lescure, Thomas Lengauer, Alain Krol, Vadim N. Gladyshev, Roderic Guigó

Department of Biochemistry: Faculty Publications

Selenoproteins are a diverse group of proteins that contain selenocysteine (Sec), the 21st amino acid. In the genetic code, UGA serves as a termination signal and a Sec codon. This dual role has precluded the automatic annotation of selenoproteins. Recent advances in the computational identification of selenoprotein genes have provided a first glimpse of the size, functions, and phylogenetic diversity of eukaryotic selenoproteomes. Here, we describe the identification of a selenoprotein family named SelJ. In contrast to known selenoproteins, SelJ appears to be restricted to actinopterygian fishes and sea urchin, with Cys homologues only found in cnidarians. SelJ shows significant …

Phylogenetic Engineering At An Interface Between Large And Small Subunits Imparts Land-Plant Kinetic Properties To Algal Rubisco, Robert J. Spreitzer, Srinivasa R. Peddi, Sriram Satagopan

Department of Biochemistry: Faculty Publications

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO₂ fixation and, thus, limits agricultural productivity. However, Rubisco enzymes from different species have different catalytic constants. If the structural basis for such differences were known, a rationale could be developed for genetically engineering an improved enzyme. Residues at the bottom of the large-subunit α/β-barrel active site of Rubisco from the green alga Chlamydomonas reinhardtii (methyl-Cys-256, Lys-258, and Ile-265) were previously changed through directed mutagenesis and chloroplast transformation to residues characteristic of land-plant Rubisco (Phe-256, Arg-258, and Val-265). The resultant enzyme has decreases in carboxylation efficiency and CO₂/O …

Computer-Assisted Docking Of Flavodoxin With The Atp:Co(I)Rrinoid Adenosyltransferase (Coba) Enzyme Reveals Residues Critical For Protein-Protein Interactions But Not For Catalysis, Nicole R. Baun, Jorge C. Escalante-Semerena

Department of Biochemistry: Faculty Publications

The activity of the housekeeping ATP:co(I)rrinoid adenosyltransferase

(CobA) enzyme of Salmonella enterica sv. Typhimurium

is required to adenosylate de novo biosynthetic intermediates

of adenosylcobalamin and to salvage incomplete and complete

corrinoids from the environment of this bacterium. In vitro,

reduced flavodoxin (FldA) provides an electron to generate the

co(I)rrinoid substrate in the CobA active site. To understand how

CobAand FldA interact, a computer model of aCobA-FldA complex

was generated. This model was used to guide the introduction of

mutations into CobA using site-directed mutagenesis and the synthesis

of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of

CobA …

Imposed Constraints On The Smith-Waterman Alignment Algorithm For Enhanced Modeling Of A Single-Molecule Dna Sequencer, Patrick G. Humphrey, Gregory R. Bashford

Biomedical Imaging and Biosignal Analysis Laboratory

An effort has been underway to develop a system for de novo sequencing of single DNA molecules with very long reads. The system operates by optically detecting the passage of fluorescently tagged DNA bases through a detection zone. A successful system would be revolutionary with respect to speed, read length, cost and minimized laboratory infrastructure. An important part of system development is modeling of the detection process. In particular, predicting the expected error from a set of sequencing parameters is helpful in system design. This paper describes variations on the Smith-Waterman algorithm for subsequence alignment used in a single-molecule detection …

Crystallization And Preliminary X-Ray Analysis Of Na-Asp-1, A Multi-Domain Pathogenesis-Related-1 Protein From The Human Hookworm Parasite Necator Americanus, Oluwatoyin A. Asojo, Alex Loukas, Mehmet Inan, Rick Barent, Jicai Huang, Brad Plantz, Amber Swanson, Mark Gouthro, Michael M. Meagher, Peter J. Hotez

Department of Chemical and Biomolecular Engineering: Faculty Publications

Human hookworm infection is a major cause of anemia and malnutrition in the developing world. In an effort to control hookworm infection, the Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective larval stage (L3) of the parasite, including a family of pathogenesis-related-1 (PR-1) proteins known as the ancylostoma-secreted proteins (ASPs). The functions of the ASPs are unknown. In addition, it is unclear why some ASPs have one while others have multiple PR-1 domains. There are no known structures of a multi-domain ASP and in an effort to remedy this situation, recombinant Na-ASP-1 has been expressed, …

Roles For Nutrients In Epigenetic Events, Anna M. Oommen, Jacob B. Griffin, Gautam Sarath, Janos Zempleni

Gautam Sarath Publications

The field of epigenetics is the study of modifications of DNA and DNA-binding proteins that alter the structure of chromatin without altering the nucleotide sequence of DNA; some of these modifications may be associated with heritable changes in gene function. Nutrients play essential roles in the following epigenetic events. First, folate participates in the generation of S-adenosylmethionine, which acts as a methyl donor in the methylation of cytosines in DNA; methylation of cytosines is associated with gene silencing. Second, covalent attachment of biotin to histones (DNA-binding proteins) plays a role in gene silencing and in the cellular response to DNA …