Biochemistry, Biophysics, and Structural Biology Commons^™

Enhancement Of Efficacy Of Prrsv Vaccines By Altering The Glycosylation Pattern Of Viral Glycoproteins, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

PRRSV, the causative agent of PRRS is of major economic significance to the pork industry in the USA and around the world. Current commercial vaccine does not provide adequate protection against PRRSV outbreaks. Therefore, there is an urgent need for development of more efficacious vaccine to combat PRRS. Our previous studies have suggested that (i) induction of neutralizing antibody response is an important correlate of evaluating the efficacy of a vaccine; (ii) neutralizing antibodies can be enhanced by hypoglycosylation of the major surface glycoprotein (GP5). While it is known that GP5 plays a prominent role in neutralizing antibody induction, it …

High-Throughput Screening For Fatty Acid Uptake Inhibitors In Humanized Yeast Identifies Atypical Antipsychotic Drugs That Cause Dyslipidemias, Hong Li, Paul N. Black, Aalap Chokshi, Angel Sandoval-Alvarez, Ravi Vatsyayan, Whitney Sealls, Concetta Dirusso

Department of Biochemistry: Faculty Publications

Fatty acids are implicated in the development of dyslipidemias, leading to type 2 diabetes and cardiovascular disease. We used a standardized small compound library to screen humanized yeast to identify compounds that inhibit fatty acid transport protein (FATP)-mediated fatty acid uptake into cells. This screening procedure used live yeast cells expressing human FATP2 to identify small compounds that reduced the import of a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12). The library used consisted of 2,080 compounds with known biological activities. Of these, ~1.8% reduced cell-associated C1-BODIPY-C12 fluorescence and were selected as potential inhibitors of human FATP2- mediated fatty acid …

New Developments In Selenium Biochemistry: Selenocysteine Biosynthesis In Eukaryotes And Archaea, Xue-Ming Xu, Bradley A. Carlson, Yan Zhang, Heiko Mix, Gregory V. Kryukov, Richard S. Glass, Marla J. Berry, Vadim N. Gladyshev, Dolph L. Hatfield

Vadim Gladyshev Publications

We 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, Ophosphoseryl- tRNA ^[Ser]Sec, and selenophosphate as substrates to generate selenocysteyltRNA ^[Ser]Sec. Sec could be synthesized on tRNA ^[Ser]Sec from selenide, adenosine triphosphate (ATP), and serine using tRNA ^[Ser]Sec, seryl-tRNA synthetase, …

Distinct Mechanisms For Ctr1-Mediated Copper And Cisplatin Transport, Devis Sinani, David J. Adle, Heejeong Kim, Jaekwon Lee

Department of Biochemistry: Faculty Publications

The Ctr1 family of integral membrane proteins is necessary for high affinity copper uptake in eukaryotes. Ctr1 is also involved in cellular accumulation of cisplatin, a platinum-based anticancer drug. Although the physiological role of Ctr1 has been revealed, the mechanism of action of Ctr1 remains to be elucidated. To gain a better understanding of Ctr1-mediated copper and cisplatin transport, we have monitored molecular dynamics and transport activities of yeast Saccharomyces cerevisiae Ctr1 and its mutant alleles. Co-expression of functional Ctr1 monomers fused with either cyan or yellow fluorescent protein resulted in fluorescence resonance energy transfer (FRET), which is consistent with …

Selective Restoration Of The Selenoprotein Population In A Mouse Hepatocyte Selenoproteinless Background With Different Mutant Selenocysteine Trnas Lacking, Bradley A. Carlson, Mohamed E. Moustafa, Aniruddha Sengupta, Ulrich Schweizer, Rajeev Shrimali, Mahadev Rao, Nianxin Zhong, Shulin Wang, Lionel Feigenbaum, Byeong Jae Lee, Vadim N. Gladyshev, Dolph L. Hatfield

Vadim Gladyshev Publications

Novel 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 then selenoprotein expression was 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 (T34->A34) or 37 (A37->G37) 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 N⁶-isopentenyladenosine. Both mutant tRNAs …

The Effect Of Residue C:N Ratio On The Turnover Of N And C In Various Soil Organic Matter Fractions, Ana B. Wingeyer

Department of Agronomy and Horticulture: Dissertations, Theses, and Student Research

Identifying soil organic matter (SOM) fractions that contribute to soil indigenous nitrogen (N) supply and understanding their turnover under different management constitute necessary tools toward an efficient N use. The objectives of this study were: i) trace the endpoint of carbon (C) flux from residue inputs into SOM; and ii) asses the role of the light fraction (LF), mobile humic acid (MHA) fraction and calcium humate (CaHA) fraction as N sources for heterotrophic decomposition of fresh plant residues with contrasting C:N ratio. A long-term aerobic soil incubation was carried out on ¹⁵N-labeled soil samples from Lincoln and Mead, NE. …

Identification And Characterization Of A Selenoprotein Family Containing A Diselenide Bond In A Redox Motif, Valentina A. Shchedrina, Sergey V. Novoselov, Mikalai I. Malinouski, Vadim N. Gladyshev

Vadim Gladyshev Publications

Selenocysteine (Sec, U) insertion into proteins is directed by translational recoding of specific UGA codons located upstream of a stemloop 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 fi sh, invertebrates, and marine bacteria. Both eukaryotic and bacterial SelL genes use single SECIS elements for insertion of two …

Low Molecular Weight Protein Tyrosine Phosphatase (Lmw-Ptp) And Its Possible Physiological Functions Of Redox Signaling In The Eye Lens, Kuiyi Xing, Ashraf Raza, Stefan Lofgren, M. Rohan Fernando, Ye-Shih Ho, Marjorie F. Lou

School of Veterinary and Biomedical Sciences: Faculty Publications

Low molecular weight protein tyrosine phosphatase (LMW-PTP) was cloned from human lens epithelial B3 cells (HLE B3) and the recombinant enzyme was purified to homogeneity. The pure enzyme reacted positively with anti-LMW-PTP antibody, displayed tyrosine-specific phosphatase activity and was extremely sensitive to H2O2. The inactivated LMW-PTP could be regenerated by thioltransferase (TTase)/GSH system as demonstrated by both activity assay and by mass spectrometry (MS). The MS study also showed that an intramolecular disulfide bond was formed between C13 and C18 at the active site, and was reduced by the TTase/GSH system. The putative role of LMW-PTP in regulating platelet derived …

Structural Basis Of Allosteric And Intrasteric Regulation In Human Cystathionine Β-Synthase And Its Regulation By A Cxxc Motif, Suvajit Sen

Department of Biochemistry: Dissertations, Theses, and Student Research

The human cystathionine β-synthase (CBS) is a 5’ pyridoxal phosphate (PLP) dependent protein that catalyzes the condensation reaction between serine and homocysteine to yield cystathionine. This is the first step in the transsulfuration pathway which connects the methionine cycle to cysteine production. Mutations in CBS are the single most common cause of severe hereditary hyperhomocystenemia and over one hundred pathogenic mutations have been described so far. These mutations represent residues which are important for the structural and functional integrity of the enzyme. The biochemical characterization of these mutants thus leads to further insights into structure-function correlations in CBS.

CBS has …

A Systematic Approach To The Development Of Fluorescent Contrast Agents For Optical Imaging Of Mouse Cancer Models, Joy L. Kovar, Melanie A. Simpson, Amy Schutz-Geschwender, D. Michael Olive

Department of Biochemistry: Faculty Publications

In the past decade, our increased elucidation of the molecular basis of cancer has led to the development of novel targeted strategies for specific inhibition of cancer signaling pathways that control growth, proliferation, apoptosis, and angiogenesis. Several monoclonal-antibody-based therapeutics and small-molecule drugs have received clearance for use as human therapeutics [1]. However, among these successes are many candidate drugs that have failed in clinical trials despite promising preclinical results [2]. The development of targeted therapeutics is expensive and time consuming. In their Critical Path Initiative, the United States Food and Drug Administration emphasized the need for more effective tools to …

High Content Of Proteins Containing 21st And 22nd Amino Acids, Selenocysteine And Pyrrolysine, In A Symbiotic Deltaproteobacterium Of Gutless Worm Olavius Algarvensis, Yan Zhang, Vadim N. Gladyshev

Vadim Gladyshev Publications

Selenocysteine (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 …

Nmr Assignments Of 1H, 13C And 15N Spectra Of Methionine Sulfoxide Reductase B1 From Mus Musculus, Lena S. Sal, Finn L. Aachmann, Hwa-Young Kim, Vadim N. Gladyshev, Alexander Dikiy

Vadim Gladyshev Publications

Isotopically 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.

Crystal Structure Of Formate Dehydrogenase H: Catalysis Involving Mo, Molybdopterin, Selenocysteine, And An Fe4S4 Cluster, Jeffrey C. Boyington, Vadim Gladyshev, Sergei V. Khangulov, Thressa C. Stadtman, Peter D. Sun

Vadim Gladyshev Publications

Formate dehydrogenase H from Escherichia coli contains selenocysteine (SeCys), molybdenum, two molybdopterin guanine dinucleotide (MGD) cofactors, and an Fe₄S₄ cluster at the active site and catalyzes the two-electron oxidation of formate to carbon dioxide. The crystal structures of the oxidized [Mo( VI), Fe₄S_4(ox)] form of formate dehydrogenase H (with and without bound inhibitor) and the reduced [Mo(IV ), Fe₄S_4(red)] form have been determined, revealing a four-domain ab structure with the molybdenum directly coordinated to selenium and both MGD cofactors. These structures suggest a reaction mechanism that directly involves SeCys …

A Highly Efficient Form Of The Selenocysteine Insertion Sequence Element In Protozoan Parasites And Its Use In Mammalian Cells, Sergey V. Novoselov, Alexey V. Lobanov, Deame Hua, Marina V. Kasaikina, Dolph L. Hatfield, Vadim Gladyshev

Vadim Gladyshev Publications

Selenoproteins 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 …

Supporting Information For: A Highly Efficient Form Of The Selenocysteine Insertion Sequence Element In Protozoan Parasites And Its Use In Mammalian Cells, Sergey V. Novoselov, Alexey V. Lobanov, Deame Hua, Marina V. Kasaikina, Dolph L. Hatfield, Vadim Gladyshev

Vadim Gladyshev Publications

Selenoproteins 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 …

Selenoprotein H Is A Nucleolar Thioredoxin-Like Protein With A Unique Expression Pattern, Sergey V. Novoselov, Gregory V. Kryukov, Xue-Ming Xu, Bradley A. Carlson, Dolph L. Hatfield, Vadim N. Gladyshev

Vadim Gladyshev Publications

The 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 …

Selenophosphate Synthetase 2 Is Essential For Selenoprotein Biosynthesis, Xue-Ming Xu, Bradley A. Carlson, Robert Irons, Heiko Mix, Nianxin Zhong, Vadim Gladyshev, Dolph L. Hatfield

Vadim Gladyshev Publications

Selenophosphate 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. …

Direct Comparison Of Feature Tracking And Autocorrelation For Velocity Estimation, Gregory R. Bashford, Derek J. Robinson

Biomedical Imaging and Biosignal Analysis Laboratory

Feature tracking is an algorithm for estimating tissue motion and blood flow using pulse-echo ultrasound. It was proposed as a computationally simpler alternative to other techniques such as autocorrelation and time-domain cross correlation. The advantage of feature tracking is that it selectively extracts easily identifiable parts of the speckle signal (e.g., the local maxima), reducing the amount of information being processed. Studies on feature tracking to date have used stationary, specklegenerating targets to simulate blood flow. Also, feature tracking has not been compared with accepted commercial methods. This study directly compares feature tracking performance with the complex autocorrelation method, which …

Novel Metabolism In Chlamydomonas Through The Lens Of Genomics, Arthur R. Grossman, Martin Croft, Vadim N. Gladyshev, Sebeeha S. Merchant, Matthew C. Posewitz, Simon Prochnik, Martin H. Spalding

Vadim Gladyshev Publications

Chlamydomonas 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 fi ndings 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 …

Purification Method Directly Influences Effectiveness Of An Epidermal Growth Factor-Coupled Targeting Agent For Noninvasive Tumor Detection In Mice, Joy L. Kovar, William M. Volcheck, Jiyan Chen, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

Receptor targeting is an effective method of enhancing fluorescence signal in tumors for optical imaging. We previously used epidermal growth factor (EGF) conjugated to IRDye 800CW to detect and track orthotopic prostate tumors in mice. In this study, our goal was to identify a reliable assay for targeting agent integrity in vitro that correlated with signal strength in vivo. Binding of IRDye 800CW EGF to intact A431 human epidermoid carcinoma cells was quantified in a microplate assay. Specificity was confirmed by competition with unlabeled EGF or monoclonal antibody blocking. Biological activity of intact and damaged targeting agents relative to unlabeled …

High-Throughput Identification Of Catalytic Redox-Active Cysteine Residues, Dmitri E. Fomenko, Weibing Xing, Blakely M. Adair, David J. Thomas, Vadim Gladyshev

Vadim Gladyshev Publications

Cysteine (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 redoxactive 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 …

Supporting Online Material For “High-Throughput Identification Of Catalytic Redox-Active Cysteine Residues”, Dmitri E. Fomenko, Weibing Xing, Blakely M. Adair, David J. Thomas, Vadim Gladyshev

Vadim Gladyshev Publications

Identification of redox-active Cys by homology to selenoproteins
Identification of Cys/Sec pairs
Statistical analysis of redox-active Cys neighborhoods
AdoMet-dependent methyltransferase
Activity of AS3MT and its Cys-to-Ser mutants

A Cadmium-Transporting PIb-Type Atpase In Yeast Saccharomyces Cerevisiae, David J. Adle, Devis Sinani, Heejeong Kim, Jaekwon Lee

Department of Biochemistry: Faculty Publications

Detoxification and homeostatic acquisition of metal ions are vital for all living organisms. We have identified PCA1 in yeast Saccharomyces cerevisiae as an overexpression suppressor of copper toxicity. PCA1 possesses signatures of a P_IB-type heavy metal-transporting ATPase that is widely distributed from bacteria to humans. Copper resistance conferred by PCA1 is not dependent on catalytic activity, but it appears that a cysteine-rich region located in the N terminus sequesters copper. Unexpectedly, when compared with two independent natural isolates and an industrial S. cerevisiae strain, the PCA1 allele of the common laboratory strains we have examined possesses a missense …

Targeting Thioredoxin Reductase 1 Reduction In Cancer Cells Inhibits Self-Sufficient Growth And Dna Replication, Min-Hyuk Yoo, Xue-Ming Xu, Bradley A. Carlson, Andrew D. Patterson, Vadim N. Gladyshev, Dolph L. Hatfield

Vadim Gladyshev Publications

Thioredoxin 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 …

Biosynthesis Of Selenocysteine On Its Trna In Eukaryotes, Xue-Ming Xu, Bradley A. Carlson, Heiko Mix, Yan Zhang, Kazima Saira, Richard S. Glass, Marla J. Berry, Vadim Gladyshev, Dolph L. Hatfield

Vadim Gladyshev Publications

Selenocysteine (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 Ophosphoseryl- 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, Ophosphoseryl- tRNA …

Differential Engagement Of Tim-1 During Activation Can Positively Or Negatively Costimulate T Cell Expansion And Effector Function, Vijay K. Kuchroo, Sheng Xiao, Nader Najafian, Jay Reddy, Monica Albin, Chen Zhu, Eric Jensen, Jaimie Imitola, Thomas Korn, Ana C. Anderson, Zheng Zhang, Cristina Gutierrez, Thomas Moll, Raymond A. Sobel, Dale T. Umetsu, Hideo Yagita, Hisaya Akiba, Terry Strom, Mohamed H. Sayegh, Rosemarie H. Dekruyff, Samia J. Khoury

School of Veterinary and Biomedical Sciences: Faculty Publications

It has been suggested that T cell immunoglobulin mucin (Tim)-1 expressed on T cells serves to positively costimulate T cell responses. However, crosslinking of Tim-1 by its ligand Tim-4 resulted in either activation or inhibition of T cell responses, thus raising the issue of whether Tim-1 can have a dual function as a costimulator. To resolve this issue, we tested a series of monoclonal antibodies specifi c for Tim-1 and identifi ed two antibodies that showed opposite functional effects. One anti–Tim-1 antibody increased the frequency of antigen-specifi c T cells, the production of the proinfl ammatory cytokines IFN-γ and IL-17, …

The Sep15 Protein Family: Roles In Disulfide Bond Formation And Quality Control In The Endoplasmic Reticulum, Vyacheslav M. Labunskyy, Dolph L. Hatfield, Vadim N. Gladyshev

Vadim Gladyshev Publications

Disulfide 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 …

Evolutionary Dynamics Of Eukaryotic Selenoproteomes: Large Selenoproteomes May Associate With Aquatic Life And Small With Terrestrial Life, Alexei Lobanov, Dmitri E. Fomenko, Yan Zhang, Aniruddha Sengupta

Vadim Gladyshev Publications

Background: 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 …

Identification Of Functional Subclasses In The Dj-1 Superfamily Proteins, Ying Wei, Dagmar Ringe, Mark A. Wilson, Mary Jo Ondrechen

Department of Biochemistry: Faculty Publications

Genomics has posed the challenge of determination of protein function from sequence and/or 3-D structure. Functional assignment from sequence relationships can be misleading, and structural similarity does not necessarily imply functional similarity. Proteins in the DJ-1 family, many of which are of unknown function, are examples of proteins with both sequence and fold similarity that span multiple functional classes. THEMATICS (theoretical microscopic titration curves), an electrostatics-based computational approach to functional site prediction, is used to sort proteins in the DJ-1 family into different functional classes. Active site residues are predicted for the eight distinct DJ-1 proteins with available 3-D structures. …

Expression, Processing, And Glycosaminoglycan Binding Activity Of The Recombinant Human 315-Kda Hyaluronic Acid Receptor For Endocytosis (Hare), Ed Harris, Svetlana V. Kyosseva, Janet A. Weigel, Paul H. Weigel

Department of Biochemistry: Faculty Publications

The hyaluronic acid (HA) receptor for endocytosis (HARE; also designated stabilin-2 and FEEL-2) mediates systemic clearance of glycosaminoglycans from the circulatory and lymphatic systems via coated pit-mediated uptake. HARE is primarily found as two isoforms (315- and 190-kDa) in sinusoidal endothelial cells of the liver, lymph node, and spleen. Here we characterize the ligand specificity and function of the large stably expressed 315-HARE isoform in Flp-In 293 cell lines. Like human spleen sinusoidal endothelial cells, Flp-In 293 cell lines transfected with a single cDNA encoding the full-length 315-HARE express both the 315-kDa and the proteolytically truncated 190-kDa isoforms in a …