Life Sciences Commons^™

Concurrent Expression Of Hyaluronan Biosynthetic And Processing Enzymes Promotes Growth And Vascularization Of Prostate Tumors In Mice, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

Aggressive cells in prostate cancer secrete extracellular hyaluronan (HA) as a result of up-regulated HA synthase enzymes HAS2 and HAS3. Combined detection of HA and the HA processing hyaluronidase enzyme Hyal1 in prostate tumors correlates with poor outcome. HA oligomers produced by hyaluronidases are potent angiogenic stimuli. We investigated the respective roles of HAS2 and Hyal1 using 22Rv1 human prostate tumor cells that lack both enzyme activities. Stable transfectants were selected for overexpression of Hyal1 or HAS2 and for coexpression of Hyal1 and HAS2. HAS2 overexpression elevated HA production and excess pericellular HA retention. However, HAS2-transfected tumor cell growth in …

Visualization Of Intracellular Transport Of Vesicular Stomatitis Virus Nucleocapsids In Living Cells, Subash C. Das, Debasis Nayak, You Zhou, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the viral RNA polymerase. In previous studies, we demonstrated that insertion of 19 amino acids in the hinge region of the protein had no significant effect on P protein function. In the present study, we inserted full-length enhanced green fluorescent protein (eGFP) in frame into the hinge region of P and show that the fusion protein (PeGFP) is functional in viral genome transcription and replication, albeit with reduced activity. A recombinant vesicular stomatitis virus encoding PeGFP in place of the P protein (VSV-PeGFP), which possessed reduced growth kinetics …

Is There A Twenty Third Amino Acid In The Genetic Code?, Alexey V. Lobanov, Gregory V. Kryukov, Dolph L. Hatfield, Vadim Gladyshev

Vadim Gladyshev Publications

The universal genetic code includes 20 common amino acids. In addition, selenocysteine (Sec) and pyrrolysine (Pyl), known as the twenty first and twenty second amino acids, are encoded by UGA and UAG, respectively, which are the codons that usually function as stop signals. The discovery of Sec and Pyl suggested that the genetic code could be further expanded by reprogramming stop codons. To search for the putative twenty third amino acid, we employed various tRNA identification programs that scanned 16 archaeal and 130 bacterial genomes for tRNAs with anticodons corresponding to the three stop signals. Our data suggest that the …

Selenoproteins And Selenoproteomes, Vadim N. Gladyshev

Vadim Gladyshev Publications

In the past several years, progress in genome sequencing and development of specialized bioinformatics tools allowed efficient identification of selenocysteine-containing proteins encoded in completely sequenced genomes. Information is currently available on selenoproteomes from a variety of organisms, including humans, which contain 25 known selenoprotein genes. This review provides basic information about mammalian selenoproteins and other known selenoprotein families. Analysis of full sets of selenoproteins in organisms provides exciting avenues for examining selenoprotein evolution and dependence of organisms on the trace element selenium and allows linking selenoproteins with specific biological and biomedical effects of dietary selenium.

Mammalian And Other Eukaryotic Selenocysteine Trnas, Bradley A. Carlson, Xue-Ming Xu, Rajeev Shrimali, Aniruddha Sengupta, Min-Hyuk Yoo, Robert Irons, Nianxin Zhong, Dolph L. Hatfield, Byeong Jae Lee, Alexei V. Lobanov, Vadim N. Gladyshev

Vadim Gladyshev Publications

Selenocysteine (Sec) tRNA occupies a prominent position in the expression of selenoproteins as it is essential for their synthesis and it provides the means by which selenium is co-translationally inserted into protein as the amino acid, Sec. Thus, Sec tRNA is regarded as the principle constituent in selenoprotein synthesis. Many features unique to this tRNA have been characterized over the years in mammals and other eukaryotes. In the last five years, the major advances have been in an elucidation of the different roles that the two major Sec tRNA isoforms play in selenoprotein biosynthesis and in Sec biosynthesis. One isoform …

"Preface" To Selenium: Its Molecular Biology And Role In Human Health, Second Edition, Dolph L. Hatfield, Marla J. Berry, Vadim N. Gladyshev

Vadim Gladyshev Publications

The purpose of the new edition book is to inform the reader of many new discoveries and to examine our present knowledge of the molecular biology of selenium, its incorporation into proteins as selenocysteine and the role that this element and selenium-containing proteins (selenoproteins) play in health. The book's emphasis is on our understanding of selenium metabolism in mammals and the role of this element in human health. The book begins with a brief history of selenium and how its face has changed through the years fiom one of a toxin and possible carcinogen to one of an essential micronutrient …

Mouse Models For Assessing The Role Of Selenoproteins In Health And Development, Bradley A. Carlson, Xue-Ming Xu, Rajeev Shrimali, Aniruddha Sengupta, Min-Hyuk Yoo, Nianxin Zhong, Dolph L. Hatfield, Robert Irons, Cindy D. Davis, Byeong Jae Lee, Sergey V. Novoselov, Vadim N. Gladyshev

Vadim Gladyshev Publications

Mouse models have been generated to assess the roles of selenoproteins involved with housekeeping tasks and/or stress-related phenomena in development and health. Each mouse model has taken advantage of the fact that the synthesis of all selenoproteins is dependent on the expression of two selenocysteine ^{(Sec) tRNA[Ser]Sec} isoforms that differ fiom each other by a single methyl group on the ribosyl moiety at position 34. The endogenous ^{(Sec) tRNA[Ser]Sec} population was selectively altered by generating mouse models involving 1) transgenic animals carrying mutant or wild type ^{(Sec) tRNA[Ser]Sec} transgenes, 2) conditional knockout animals carrying a floxed ^{(Sec) tRNA[Ser]Sec} gene that …

Selenium And Methionine Sulfoxide Reduction, Hwa-Young Kim, Vadim N. Gladyshev

Vadim Gladyshev Publications

Methionine residues in proteins can be readily oxidized to a diastereomeric mixture of methionine sulfoxides by reactive oxygen species. In most organisms, methionine sulfoxides are reversibly and stereospecifically reduced back to methionine by two distinct classes of repair enzymes, methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). Methionine sulfoxide reduction is thought to be an essential pathway that protects cells from oxidative stress and regulates protein function. This pathway is also implicated in delaying the aging process in organisms from yeast to mammals. The first selenoprotein identified using bioinformatics methods, SelR (also known as SelX or MsrB1), was recently found to …

Evolution Of Selenocysteine Decoding And The Key Role Of Selenophosphate Synthetase In The Pathway Of Selenium Utilization, Gustavo Salinas, Héctor Romero, Xue-Ming Xu, Bradley A. Carlson, Dolph L. Hatfield, Vadim N. Gladyshev

Vadim Gladyshev Publications

The complete sequencing of genomes and the development of in silico methods for identification of genes encoding selenocysteine (Sec)-containing proteins have greatly contributed to shape our view on the evolution of selenium utilization in nature. Current evidence is consistent with the idea that Sec decoding is a late addition to the genetic code and it evolved once, before the separation of archaeal, bacterial and eukaryal domains. Many organisms have lost the Sec decoding trait, but recent evidence has shown that the loss is not irreversible. The distribution of organisms that use UGA as a Sec codon suggests that Sec decoding …

Selenoproteins In Parasites, Gustavo Salinas, Alexei V. Lobanov, Vadim N. Gladyshev

Vadim Gladyshev Publications

Parasites, which cause an enormous burden in the population of the third world, are a diverse group of organisms, many of which are sensitive to oxidative stress imposed by their hosts. In recent years, several selenoprotein families, some with antioxidant properties, have been described and characterized in metazoan parasites. Glutathione peroxidase and thioredoxin glutathione reductase (TGR) appear to be essential selenoproteins in flatworms (phylum Platyhelminthes). TGR is the single enzyme that provides reducing equivalents to both thioredoxin and glutathione pathways, in contrast to hosts, which evolve parallel pathways. In roundworms (phylum Nematoda), selenoproteins have recently been described, revealing …

The 15-Kda Selenoprotein (Sep15): Functional Analysis And Role In Cancer, Vyacheslav M. Labunskyy, Vadim N. Gladyshev, Dolph L. Hatfield

Vadim Gladyshev Publications

The 15-kDa selenoprotein (Sep15) was identified several years ago as a protein of unknown function. In recent years, several lines of evidence implicated Sepl5 in the effect of dietary selenium in cancer prevention. These lines of evidence include: 1) protein expression patterns in normal and malignant cells; 2) identification of polymorphic sites that regulate Sep15 levels and differentially respond to selenium supplementation; 3) location of the Sep15 gene in the human genome; and 4) correlation between Sep15 haplotype and susceptibility to cancer. Functional analyses revealed a specific interaction between Sep15 and a protein folding sensor in the endoplasmic reticulum of …

Thioredoxin Reductase 1 Deficiency Reverses Tumor Phenotype And Tumorigenicity Of Lung Carcinoma Cells, Min-Hyuk Yoo, Xue-Ming Xu, Bradley A. Carlson, Vadim N. Gladyshev, Dolph L. Hatfield

Vadim Gladyshev Publications

Dietary selenium has potent cancer prevention activity. Both low molecular weight selenocompounds and selenoproteins are implicated in this effect. Thioredoxin reductase 1 (TR1) is one of the major antioxidant and redox regulators in mammals that supports p53 function and other tumor suppressor activities. However, this selenium-containing oxidoreductase is also overexpressed in many malignant cells and has been proposed as a target for cancer therapy. To further assess the role of TR1 in the malignancy process, we used RNA interference technology to decrease its expression in mouse lung carcinoma (LLC1) cells. Stable transfection of LLC1 cells with a small interfering RNA …

Alternative First Exon Splicing Regulates Subcellular Distribution Of Methionine Sulfoxide Reductases, Hwa-Young Kim, Vadim Gladyshev

Vadim Gladyshev Publications

Background: Methionine sulfoxide reduction is an important protein repair pathway that protects against oxidative stress, controls protein function and has a role in regulation of aging. There are two enzymes that reduce stereospecifically oxidized methionine residues: MsrA (methionine-Ssulfoxide reductase) and MsrB (methionine-R-sulfoxide reductase). In many organisms, these enzymes are targeted to various cellular compartments. In mammals, a single MsrA gene is known, however, its product is present in cytosol, nucleus, and mitochondria. In contrast, three mammalian MsrB genes have been identified whose products are located in different cellular compartments.
Results: In the present study, we identified and characterized alternatively spliced …

Identification And Characterization Of Fep15, A New Selenocysteine-Containing Member Of The Sep15 Protein Family, Sergey V. Novoselov, Deame Hua, A. V. Lobanov, Vadim N. Gladyshev

Vadim Gladyshev Publications

Sec (selenocysteine) is a rare amino acid in proteins. It is co-translationally inserted into proteins at UGA codons with the help of SECIS (Sec insertion sequence) elements. A full set of selenoproteins within a genome, known as the selenoproteome, is highly variable in different organisms. However, most of the known eukaryotic selenoproteins are represented in the mammalian selenoproteome. In addition, many of these selenoproteins have cysteine orthologues. Here, we describe a new selenoprotein, designated Fep15, which is distantly related to members of the 15 kDa selenoprotein (Sep15) family. Fep15 is absent in mammals, can be detected only in fi sh …

Nmr Structures Of The Selenoproteins Sep15 And Selm Reveal Redox Activity Of A New Thioredoxin-Like Family, Andrew D. Ferguson, Vyacheslav Labunskyy, Dmitri E. Fomenko, Demet Arac, Yogarany Chelliah, Carlos A. Amexcua, Josep Rizo, Vadim N. Gladyshev, Johann Deisenhofer

Vadim Gladyshev Publications

Selenium has significant health benefits, including potent cancer prevention activity and roles in immune function and the male reproductive system. Selenium-containing proteins, which incorporate this essential micronutrient as selenocysteine, are proposed to mediate the positive effects of dietary selenium. Presented here are the solution NMR structures of the selenoprotein SelM and an ortholog of the selenoprotein Sep15. These data reveal that Sep15 and SelM are structural homologs that establish a new thioredoxinlike protein family. The location of the active-site redox motifs within the fold together with the observed localized conformational changes after thiol-disulfide exchange and measured redox potential indicate that …

Transcriptome Signature Of Virulent And Attenuated Pseudorabies Virus-Infected Rodent Brain, Christina Paulus, Patricia J. Sollars, Gary E. Pickard, Lynn W. Enquist

School of Veterinary and Biomedical Sciences: Faculty Publications

Mammalian alphaherpesviruses normally establish latent infections in ganglia of the peripheral nervous system in their natural hosts. Occasionally, however, these viruses spread to the central nervous system (CNS), where they cause damaging, often fatal, infections. Attenuated alphaherpesvirus derivatives have been used extensively as neuronal circuit tracers in a variety of animal models. Their circuit-specific spread provides a unique paradigm to study the local and global CNS response to infection. Thus, we systematically analyzed the host gene expression profile after acute pseudorabies virus (PRV) infection of the CNS using Affymetrix GeneChip technology. Rats were injected intraocularly with one of three selected …

The Plasmodium Selenoproteome, Alexey V. Lobanov, Cesar Delgado, Stefan Rahlfs, Sergey V. Novoselov, Gregory V. Kryukov, Stephan Gromer, Dolph L. Hatfield, Katja Becker, Vadim Gladyshev

Vadim Gladyshev Publications

The use of selenocysteine (Sec) as the 21st amino acid in the genetic code has been described in all three major domains of life. However, within eukaryotes, selenoproteins are only known in animals and algae. In this study, we characterized selenoproteomes and Sec insertion systems in protozoan Apicomplexa parasites. We found that among these organisms, Plasmodium and Toxoplasma utilized Sec, whereas Cryptosporidium did not. However, Plasmodium had no homologs of known selenoproteins. By searching computationally for evolutionarily conserved selenocysteine insertion sequence (SECIS) elements, which are RNA structures involved in Sec insertion, we identified four unique Plasmodium falciparum selenoprotein genes. These …

S-Adenosylmethionine Stabilizes Cystathionine Β-Synthase And Modulates Redox Capacity, Anna Prudova, Zachary Bauman, Aaron Braun, Victor Vitvitsky, Shelly C. Lu, Ruma Banerjee

Department of Biochemistry: Faculty Publications

The transsulfuration pathway converts homocysteine to cysteine and represents the metabolic link between antioxidant and methylation metabolism. The first and committing step in this pathway is catalyzed by cystathionine β-synthase (CBS), which is subject to complex regulation, including allosteric activation by the methyl donor, S-adenosylmethionine (AdoMet). In this study, we demonstrate that methionine restriction leads to a >10-fold decrease in CBS protein levels, and pulse proteolysis studies reveal that binding of AdoMet stabilizes the protein against degradation by ≈12 kcal/mol. These observations predict that under pathological conditions where AdoMet levels are diminished, CBS, and therefore glutathione levels, will be reduced. …

Purification And Initial Biochemical Characterization Of Atp:Cob(I)Alamin Adenosyltransferase (Eutt) Enzyme Of Salmonella Enterica, Nicole R. Baun, Jorge C. Escalante-Semerena

Department of Biochemistry: Faculty Publications

ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella

enterica was overproduced and enriched to ~70% homogeneity,

and its basic kinetic parameters were determined. Abundant

amounts of EutT protein were produced, but all of it remained

insoluble. Soluble active EutT protein (~70% homogeneous) was

obtained after treatment with detergent. Under conditions in which

cobalamin (Cbl) was saturating, _Km(ATP) = 10 µM, k_cat = 0.03 s^-1,

and V_max = 54.5 nM min^-1. Similarly, under conditions in which

MgATPwas saturating,K_m(Cbl) = 4.1µM, k_cat = 0.06 s^-1, andV_max= …