Biotechnology Commons^™

Targeted Deletion Of The Mouse Mitoferrin1 Gene: From Anemia To Protoporphyria, Marie-Berengere Troadec, David Warner, Jared Wallace, Kirk Thomas, Gerald J. Spangrude, John Phillips, Oleh Khalimonchuk, Barry H. Paw, Diane Mcvey Ward, Jerry Kaplan

Department of Biochemistry: Faculty Publications

Mitoferrin1 is 1 of 2 homologous mitochondrial iron transporters and is required for mitochondrial iron delivery in developing erythroid cells. We show that total deletion of Mfrn1 in embryos leads to embryonic lethality. Selective deletion of Mfrn1 in adult hematopoietic tissues leads to severe anemia because of a deficit in erythroblast formation. Deletion of Mfrn1 in hepatocytes has no phenotype or biochemical effect under normal conditions. In the presence of increased porphyrin synthesis, however, deletion of Mfrn1 in hepatocytes results in a decreased ability to convert protoporphyrin IX into heme, leading to protoporphyria, cholestasis, and bridging cirrhosis. Our results show …

Reduced Utilization Of Selenium By Naked Mole Rats Due To A Specific Defect In Gpx1 Expression, Marina V. Kasaikina, Alexei V. Lobanov, Mikalai I. Malinouski, Byung Cheon Lee, Javier Seravalli, Dmitri E. Fomenko, Anton A. Turanov, Lydia Finney, Stefan Vogt, Thomas J. Park, Richard A. Miller, Dolph L. Hatfield, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Naked mole rat (MR) Heterocephalus glaber is a rodent model of delayed aging because of its unusually long life span (>28 years). It is also not known to develop cancer. In the current work, tissue imaging by x-ray fluorescence microscopy and direct analyses of trace elements revealed low levels of selenium in the MR liver and kidney, whereas MR and mouse brains had similar selenium levels. This effect was not explained by uniform selenium deficiency because methionine sulfoxide reductase activities were similar in mice and MR. However, glutathione peroxidase activity was an order of magnitude lower inMRliver and kidney …

A 4-Selenocysteine, 2-Selenocysteine Insertion Sequence (Secis) Element Methionine Sulfoxide Reductase From Metridium Senile Reveals A Non-Catalytic Function Of Selenocysteines, Byung Cheon Lee, Alexey V. Lobanov, Stefano M. Marino, Alaattin Kaya, Javier Seravalli, Dolph L. Hatfield, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Selenocysteine (Sec) residues occur in thiol oxidoreductase families, and functionally characterized selenoenzymes typically have a single Sec residue used directly for redox catalysis. However, how new Sec residues evolve and whether non-catalytic Sec residues exist in proteins is not known. Here, we computationally identified several genes with multiple Sec insertion sequence (SECIS) elements, one of which was a methionine-Rsulfoxide reductase (MsrB) homolog from Metridium senile that has four in-frame UGA codons and two nearly identical SECIS elements. One of the UGA codons corresponded to the conserved catalytic Sec or Cys in MsrBs, whereas the three other UGA codons evolved recently …

Escherichia Coli Thioredoxin-Like Protein Ybbn Contains An Atypical Tetratricopeptide Repeat Motif And Is A Negative Regulator Of Groel, Jiusheng Lin, Mark A. Wilson

Department of Biochemistry: Faculty Publications

Many proteins contain a thioredoxin (Trx)-like domain fused with one or more partner domains that diversify protein function by the modular construction of new molecules. The Escherichia coli protein YbbN is a Trx-like protein that contains a C-terminal domain with low homology to tetratricopeptide repeat motifs. YbbN has been proposed to act as a chaperone or co-chaperone that aids in heat stress response andDNAsynthesis. We report the crystal structure of YbbN, which is an elongated molecule with a mobile Trx domain and four atypical tetratricopeptide repeat motifs. The Trx domain lacks a canonical CXXC active site architecture and is not …

Defining A Relationship Between Dietary Fatty Acids And The Cytochrome P450 System In A Mouse Model Of Fatty Liver Disease, Monika Gonzalez, Whitney Sealls, Elliot D. Jesch, M. Julia Brosnan, Istvan Ladunga, Xinxin Ding, Paul N. Black, Concetta C. Dirusso

Department of Biochemistry: Faculty Publications

Liver-specific ablation of cytochrome P450 reductase in mice (LCN) results in hepatic steatosis that can progress to steatohepatitis characterized by inflammation and fibrosis. The specific cause of the fatty liver phenotype is poorly understood but is hypothesized to result from elevated expression of genes encoding fatty acid synthetic genes. Since expression of these genes is known to be suppressed by polyunsaturated fatty acids, we performed physiological and genomics studies to evaluate the effects of dietary linoleic and linolenic fatty acids (PUFA) or arachidonic and decosahexaenoic acids (HUFA) on the hepatic phenotypes of control and LCN mice by comparison with a …

Functional Diversification Of Thylakoidal Processing Peptidases In Arabidopsis Thaliana, Shih-Chi Hsu, Joshua K. Endow, Nicholas J. Ruppel, Rebecca Roston, Amy J. Baldwin, Kentaro Inoue

Department of Biochemistry: Faculty Publications

Thylakoidal processing peptidase (TPP) is responsible for removing amino-terminal thylakoid-transfer signals from several proteins in the thylakoid lumen. Three TPP isoforms are encoded by the nuclear genome of Arabidopsis thaliana. Previous studies showed that one of them termed plastidic type I signal peptidase 1 (Plsp1) was necessary for processing three thylakoidal proteins and one protein in the chloroplast envelope in vivo. The lack of Plsp1 resulted in seedling lethality, apparently due to disruption of proper thylakoid development. The physiological roles of the other two TPP homologs remain unknown. Here we show that the three A. thaliana TPP isoforms …

Enzymatic Defects Underlying Hereditary Glutamate Cysteine Ligase Deficiency Are Mitigated By Association Of The Catalytic And Regulatory Subunits, Melanie Neely Willis, Yilin Liu, Ekaterina I. Biterova, Melanie A. Simpson, Heejeong Kim, Jaekwon Lee, Joseph J. Barycki

Department of Biochemistry: Faculty Publications

Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises

production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly

reduced levels of erythrocyte glutathione, leading to hemolytic anemia and in some cases,

impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a

catalytic (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate limiting step

in glutathione production. Four clinical missense mutations have been identified within GCLC:

Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Here, we have evaluated the impacts of

these mutations on enzymatic function in vivo and in vitro …

The Lyr Protein Mzm1 Functions In The Insertion Of The Rieske Fe/S Protein In Yeast Mitochondria, Aaron Atkinson, Pamela Smith, Jennifer L. Fox, Tie-Shong Cui, Oleh Khalimonchuk, Dennis R. Winge

Department of Biochemistry: Faculty Publications

The assembly of the cytochrome bc1 complex in Saccharomyces cerevisiae is shown to be conditionally dependent on a novel factor, Mzm1. Cells lacking Mzm1 exhibit a modest bc₁ defect at 30°C, but the defect is exacerbated at elevated temperatures. Formation of bc₁ is stalled in mzm1 Δ cells at a late assembly intermediate lacking the Rieske iron-sulfur protein Rip1. Rip1 levels are markedly attenuated in mzm1 Δ cells at elevated temperatures. Respiratory growth can be restored in the mutant cells by the overexpression of the Rip1 subunit. Elevated levels of Mzm1 enhance the stabilization of Rip1 through …

Rpir Homologues May Link Staphylococcus Aureus Rnaiii Synthesis And Pentose Phosphate Pathway Regulation, Yefei Zhu, Nandakumar Madayiputhiya, Marat R. Sadykov, Nandakumar Madayiputhiya, Thanh T. Luong, Rosmarie Gaupp, Chia Y. Lee, Greg Somerville

Department of Biochemistry: Faculty Publications

Staphylococcus aureus is a medically important pathogen that synthesizes a wide range of virulence determinants. The synthesis of many staphylococcal virulence determinants is regulated in part by stress-induced changes in the activity of the tricarboxylic acid (TCA) cycle. One metabolic change associated with TCA cycle stress is an increased concentration of ribose, leading us to hypothesize that a pentose phosphate pathway (PPP)-responsive regulator mediates some of the TCA cycle-dependent regulatory effects. Using bioinformatics, we identified three potential ribose-responsive regulators that belong to the RpiR family of transcriptional regulators. To determine whether these RpiR homologues affect PPP activity and virulence determinant …

In Vivo Liver Endocytosis Followed By Purification Of Liver Cells By Liver Perfusion, Sandhya Gopalakrishnan, Edward N. Harris

Department of Biochemistry: Faculty Publications

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure ¹ in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (˜100 μm) within the cells so that the …

Synthesis, Kinetic And Photocatalytic Studies Of Porphyrin-Ruthenium-Oxo Complexes, Yan Huang

Masters Theses & Specialist Projects

Macrocyclic ligand-complexed transition metal-oxo intermediates are the active oxidizing species in a variety of important biological and catalytic oxidation reactions. Many transition metal catalysts have been designed to mimic the predominant oxidation catalysts in Nature, namely the cytochrome P450 enzymes. Ruthenium porphyrin complexes have been the center of the research and have successfully been utilized, as catalysts, in major oxidation reactions such as the hydroxylation of alkanes. This study focuses on kinetic and photocatalytic studies of oxidation reactions with wellcharacterized high-valent ruthenium-oxo porphyrin complexes.
The trans-dioxoruthenium(VI) porphyrins have been among the best characterized metal-oxo intermediates and their involvement as …

The Genomics Education Partnership: Successful Integration Of Research Into Laboratory Classes At A Diverse Group Of Undergraduate Institutions, Christopher D. Shaffer, Consuelo Alvarez, Cheryl Bailey, Daron Barnard, Satish Bhalla, Chitra Chandrasekaran, Vidya Chandrasekaran, Hui-Min Chung, Douglas R. Dorer, Chunguang Du, Todd T. Eckdahl, Jeff L. Poet, Donald Frohlich, Anya L. Goodman, Yuying Gossner, Charles Hauser, Laura L.M. Hoopes, Diana Johnson, Christopher J. Jones, Marian Kaehler, Nighat Kokan, Olga R. Kopp, Gary A. Kuleck, Gerard Mcneil, Robert Moss, Jennifer L. Myka, Alexis Nagengast, Robert Morris, Paul J. Overvoorde, Elizabeth Shoop, Susan Parrish, Kelynne Reed, E. Gloria Regisford, Dennis Revie, Anne G. Rosenwald, Ken Saville, Stephanie Schroeder, Mary Shaw, Gary Skuse, Christopher Smith, Mary Smith, Eric P. Spana, Mary Spratt, Joyce Stamm, Jeff S. Thompson, Matthew Wawersik, Barbara A. Wilson, Jim Youngblom, Wilson Leung, Jeremy Buhler, Elaine R. Mardis, David Lopatto, Sarah C.R. Elgin

Department of Biochemistry: Faculty Publications

Genomics is not only essential for students to understand biology but also provides unprecedented opportunities for undergraduate research. The goal of the Genomics Education Partnership (GEP), a collaboration between a growing number of colleges and universities around the country and the Department of Biology and Genome Center of Washington University in St. Louis, is to provide such research opportunities. Using a versatile curriculum that has been adapted to many different class settings, GEP undergraduates undertake projects to bring draft-quality genomic sequence up to high quality and/or participate in the annotation of these sequences. GEP undergraduates have improved more than 2 …

Mzm1 Influences A Labile Pool Of Mitochondrial Zinc Important For Respiratory Function, Aaron Atkinson, Oleh Khalimonchuk, Pamela Smith, Hana Sabic, David Eide, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Zinc is essential for function of mitochondria as a cofactor for

several matrix zinc metalloproteins. We demonstrate that a

labile cationic zinc component of low molecular mass exists in

the yeast mitochondrial matrix. This zinc pool is homeostatically

regulated in response to the cellular zinc status. This pool

of zinc is functionally important because matrix targeting of a

cytosolic zinc-binding protein reduces the level of labile zinc

and interferes with mitochondrial respiratory function. We

identified a series of proteins that modulate the matrix zinc

pool, one of which is a novel conserved mitochondrial protein

designated Mzm1. Mutant mzm1∆ cells …

Identification And Characterization Of Small Compound Inhibitors Of Human Fatp2, Angel Sandoval, Aalap Chokshi, Elliot D. Jesch, Paul N. Black, Concetta C. Dirusso

Department of Biochemistry: Faculty Publications

Fatty acid transport proteins (FATPs) are bifunctional proteins, which transport long chain fatty acids into cells and activate very long chain fatty acids by esterification with coenzyme A. In an effort to understand the linkage between cellular fatty acid transport and the pathology associated with excessive accumulation of exogenous fatty acids, we targeted FATP-mediated fatty acid transport in a high throughput screen of more than 100,000 small diverse chemical compounds in yeast expressing human FATP2 (hsFATP2). Compounds were selected for their ability to depress the transport of the fluorescent long chain fatty acid analogue, C₁-BODIPY-C₁₂. Among …

Evolution Of New Enzymatic Function By Structural Modulation Of Cysteine Reactivity In Pseudomonas Fluorescens Isocyanide Hydratase, Mahadevan Lakshminarasimhan, Peter Madzelan, Ruth Nan, Nicole Marie Milkovic, Mark A. Wilson

Department of Biochemistry: Faculty Publications

Isocyanide (formerly isonitrile) hydratase (EC 4.2.1.103) is an enzyme of the DJ-1 superfamily that hydrates isocyanides to yield the corresponding N-formamide. In order to understand the structural basis for isocyanide hydratase (ICH) catalysis, we determined the crystal structures of wild-type and several site-directed mutants of Pseudomonas fluorescens ICH at resolutions ranging from 1.0 to 1.9 Å. We also developed a simple UV-visible spectrophotometric assay for ICH activity using 2-naphthyl isocyanide as a substrate. ICH contains a highly conserved cysteine residue (Cys101) that is required for catalysis and interacts with Asp17, Thr102, and an ordered water molecule in the active …

Identification And Characterization Of Oxalate Oxidoreductase, A Novel Thiamine Pyrophosphate-Dependent 2-Oxoacid Oxidoreductase That Enables Anaerobic Growth On Oxalate, Elizabeth Pierce, Donald F. Becker, Stephen W. Ragsdale

Department of Biochemistry: Faculty Publications

Moorella thermoacetica is an anaerobic acetogen, a class of bacteria that is found in the soil, the animal gastrointestinal tract, and the rumen. This organism engages the Wood-Ljungdahl pathway of anaerobic CO₂ fixation for heterotrophic or autotrophic growth. This paper describes a novel enzyme, oxalate oxidoreductase (OOR), that enables M. thermoacetica to grow on oxalate, which is produced in soil and is a common component of kidney stones. Exposure to oxalate leads to the induction of three proteins that are subunits of OOR, which oxidizes oxalate coupled to the production of two electrons and CO₂ or bicarbonate. Like …

Functional Hybrid Rubisco Enzymes With Plant Small Subunits And Algal Large Subunits Engineered Rbcs Cdna For Expression In Chlamydomonas, Todor Genkov, Moritz Meyer, Howard Griffiths, Robert J. Spreitzer

Department of Biochemistry: Faculty Publications

There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO₂ fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO₂/O₂ specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, …

Diversity Of Protein And Mrna Forms Of Mammalian Methionine Sulfoxide Reductase B1 Due To Intronization And Protein Processing, Xinwen Liang, Dmitri E. Fomenko, Deame Hua, Alaattin Kaya, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Background: Methionine sulfoxide reductases (Msrs) are repair enzymes that protect proteins from oxidative stress by catalyzing stereospecific reduction of oxidized methionine residues. MsrB1 is a selenocysteine-containing cytosolic/nuclear Msr with high expression in liver and kidney.

Principal Findings: Here, we identified differences in MsrB1 gene structure among mammals. Human MsrB1 gene consists of four, whereas the corresponding mouse gene of five exons, due to occurrence of an additional intron that flanks the stop signal and covers a large part of the 39-UTR. This intron evolved in a subset of rodents through intronization of exonic sequences, whereas the human gene structure represents …

Incorporating Genomics And Bioinformatics Across The Life Sciences Curriculum, Jayna L. Ditty, Christopher A. Kvaal, Brad Goodner, Sharyn K. Freyermuth, Cheryl Bailey, Robert A. Britton, Stuart G. Gordon, Sabine Heinhorst, Kelynne Reed, Zhaohui Xu, Erin R. Sanders-Lorenz, Seth Axen, Edwin Kim, Mitrick Johns, Kathleen Scott, Cheryl A. Kerfeld

Department of Biochemistry: Faculty Publications

Undergraduate life sciences education needs an overhaul, as clearly described in the National Research Council of the National Academies’ publication BIO 2010: Transforming Undergraduate Education for Future Research Biologists. Among BIO 2010’s top recommendations is the need to involve students in working with real data and tools that reflect the nature of life sciences research in the 21st century [1]. Education research studies support the importance of utilizing primary literature, designing and implementing experiments, and analyzing results in the context of a bona fide scientific question [1–12] in cultivating the analytical skills necessary to become a scientist. Incorporating these …

Regulation Of Sealing Ring Formation By L-Plastin And Cortactin In Osteoclasts, Tao Ma, Kavitha Sadashivalah, Nandakumar Madayiputhiya, Meenakshi A. Chellaia

Department of Biochemistry: Faculty Publications

The aim of this study is to identify the exact mechanism(s) by which cytoskeletal structures are modulated during bone resorption. In this study, we have shown the possible role of different actin-binding and signaling proteins in the regulation of sealing ring formation. Our analyses have demonstrated a significant increase in cortactin and a corresponding decrease in L-plastin protein levels in osteoclasts subjected to bone resorption for 18 h in the presence of RANKL, M-CSF, and native bone particles. Time-dependent changes in the localization of L-plastin (in actin aggregates) and cortactin (in the sealing ring) suggest that these proteins may be …

Structural Basis For Feedback And Pharmacological Inhibition Of Saccharomyces Cerevisiae Glutamate Cysteine Ligase, Ekaterina I Biterova, Joseph J. Barycki

Department of Biochemistry: Faculty Publications

Structural characterization of glutamate cysteine ligase (GCL), the enzyme that catalyzes the initial, rate-limiting step in glutathione biosynthesis, has revealed many of the molecular details of substrate recognition. To further delineate the mechanistic details of this critical enzyme, we have determined the structures of two inhibited forms of Saccharomyces cerevisiae GCL (ScGCL), which shares significant sequence identity with the human enzyme. In vivo, GCL activity is feedback regulated by glutathione. Examination of the structure of ScGCL-glutathione complex (2.5 A ; R = 19.9%, R_free = 25.1%) indicates that the inhibitor occupies both the glutamate- and the presumed cysteine- …

Cug Start Codon Generates Thioredoxin/Glutathione Reductase Isoforms In Mouse Testes, Maxim Gerashchenko, Dan Su, Vadim Gladyshev

Department of Biochemistry: Faculty Publications

Mammalian cytosolic and mitochondrial thioredoxin reductases are essential selenocysteine-containing enzymes that control thioredoxin functions. Thioredoxin/glutathione reductase (TGR) is a third member of this enzyme family. It has an additional glutaredoxin domain and shows highest expression in testes. Herein, we found that human and several other mammalian TGR genes lack any AUG codons that could function in translation initiation. Although mouse and rat TGRs have such codons, we detected protein sequences upstream of them by immunoblot assays and direct proteomic analyses. Further gene engineering and expression analyses demonstrated that a CUG codon, located upstream of the sequences previously thought to initiate …

Structure Of The Proline Utilization A Proline Dehydrogenase Domain Inactivated By N-Propargylglycine Provides Insight Into Conformational Changes Induced By Substrate Binding And Flavin Reduction, Dhiraj Srivastava, Weidong Zhu, William H. Johnson Jr., Christian P. Whitman, Donald F. Becker, John J. Tanner

Department of Biochemistry: Faculty Publications

Proline utilization A (PutA) from Escherichia coli is a flavoprotein that has mutually exclusive roles as a transcriptional repressor of the put regulon and a membrane-associated enzyme that catalyzes the oxidation of proline to glutamate. Previous studies have shown that the binding of proline in the proline dehydrogenase (PRODH) active site and subsequent reduction of the FAD trigger global conformational changes that enhance PutA-membrane affinity. These events cause PutA to switch from its repressor to enzymatic role, but the mechanism by which this signal is propagated from the active site to the distal membrane-binding domain is largely unknown. Here, it …

Analysis Of Leigh Syndrome Mutations In The Yeast Surf1 Homolog Reveals A New Member Of The Cytochrome Oxidase Assembly Factor Family, Megan Bestwick, Mi-Young Jeong, Oleh Khalimonchuk, Hyung Kim, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Three missense SURF1 mutations identified in patients with Leigh syndrome (LS) were evaluated in the yeast homolog Shy1 protein. Introduction of two of the Leigh mutations, F²⁴⁹T and Y³⁴⁴D, in Shy1 failed to significantly attenuate the function of Shy1 in cytochrome c oxidase (CcO) biogenesis as seen with the human mutations. In contrast, a G¹³⁷E substitution in Shy1 results in a nonfunctional protein conferring a CcO deficiency. The G¹³⁷E Shy1 mutant phenocopied shy1 Δ cells in impaired Cox1 hemylation and low mitochondrial copper. A genetic screen for allele-specific suppressors of the G137E …

Formation Of The Redox Cofactor Centers During Cox1 Maturation In Yeast Cytochrome Oxidase, Oleh Khalimonchuk, Megan Bestwick, Brigitte Meunier, Talina C. Watts, Dennis R. Winge

Department of Biochemistry: Faculty Publications

The biogenesis of cytochrome c oxidase initiates with synthesis and maturation of the mitochondrionencoded Cox1 subunit prior to the addition of other subunits. Cox1 contains redox cofactors, including the low-spin heme a center and the heterobimetallic heme a₃:Cu_B center. We sought to identify the step in the maturation of Cox1 in which the redox cofactor centers are assembled. Newly synthesized Cox1 is incorporated within one early assembly intermediate containing Mss51 in Saccharomyces cerevisiae. Subsequent Cox1 maturation involves the progression to downstream assembly intermediates involving Coa1 and Shy1. We show that the two heme a cofactor sites …

The Role Of Coa2 In Hemylation Of Yeast Cox1 Revealed By Its Genetic Interaction With Cox10, Megan Bestwick, Oleh Khalimonchuk, Fabien Pierrel, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Saccharomyces cerevisiae cells lacking the cytochrome c oxidase (CcO) assembly factor Coa2 are impaired in Cox1 maturation and exhibit a rapid degradation of newly synthesized Cox1. The respiratory deficiency of coa2 Δ cells is suppressed either by the presence of a mutant allele of the Cox10 farnesyl transferase involved in heme a biosynthesis or through impaired proteolysis by the disruption of the mitochondrial Oma1 protease. Cox10 with an N196K substitution functions as a robust gain-of-function suppressor of the respiratory deficiency of coa2 Δ cells but lacks suppressor activity for two other CcO assembly mutant strains, the coa1 Δ and shy1 …

Quantitative Nuclear Proteomics Identifies Mtor Regulation Of Dna Damage Response, Sricharan Bandhakavi, Young-Mi Kim, Seung-Hyun Ro, Hongwei Xie, Getiria Onsongo, Chang-Bong Jun, Do-Hyung Kim, Timothy J. Griffin

Department of Biochemistry: Faculty Publications

Cellular nutritional and energy status regulates a wide range of nuclear processes important for cell growth, survival, and metabolic homeostasis. Mammalian target of rapamycin (mTOR) plays a key role in the cellular responses to nutrients. However, the nuclear processes governed by mTOR have not been clearly defined. Using isobaric peptide tagging coupled with linear ion trap mass spectrometry, we performed quantitative proteomics analysis to identify nuclear processes in human cells under control of mTOR. Within 3 h of inhibiting mTOR with rapamycin in HeLa cells, we observed downregulation of nuclear abundance of many proteins involved in translation and RNA modification. …

Methanogenesis By Methanosarcina Acetivorans Involves Two Structurally And Functionally Distinct Classes Of Heterodisulfide Reductase, Nicole R. Buan, William W. Metcalf

Department of Biochemistry: Faculty Publications

Biochemical studies have revealed two distinct classes of Coenzyme B-Coenzyme M heterodisulfide (CoB-S-S-CoM) reductase (Hdr), a key enzyme required for anaerobic respiration in methaneproducing archaea. A cytoplasmic HdrABC enzyme complex is found in most methanogens, whereas a membrane-bound HdrED complex is found exclusively in members of the order Methanosarcinales. Unexpectedly, genomic data indicate that multiple copies of both Hdr classes are found in all sequenced Methanosarcinales genomes. The Methanosarcina acetivorans hdrED1 operon is constitutively expressed and required for viability under all growth conditions examined, consistent with HdrED being the primary Hdr. HdrABC appears to be specifically involved in methylotrophic …

Proteomic Analysis Of Endodontic Infections By Liquid Chromatography–Tandem Mass Spectrometry, R. Nandakumar, Nandakumar Madayiputhiya, A. F. Fouad

Department of Biochemistry: Faculty Publications

Introduction—Endodontic infections are very prevalent and have a polymicrobial etiology characterized by complex interrelationships between endodontic microorganisms and the host defenses. Proteomic analysis of endodontic infections can provide global insights into the invasion, pathogenicity mechanisms, and multifactorial interactions existing between root canal bacteria and the host in the initiation and progression of apical periodontitis. The purpose of this study was to apply proteomic techniques such as liquid chromatography–tandem mass spectrometry (LC–MS/ MS) for the identification of proteins of bacterial origin present in endodontic infections.

Methods—Endodontic specimens were aseptically obtained from seven patients with root canal infections. Protein mixtures were subjected …

Hyaluronidase Activity Of Human Hyal1 Requires Active Site Acidic And Tyrosine Residues, Ling Zhang, Alamelu G. Bharadwaj, Andrew Casper, Joel Barkley, Joseph J. Barycki, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

Hyaluronidases are a family of endolytic glycoside hydrolases

that cleave the β1–4 linkage between N-acetylglucosamine and

glucuronic acid in hyaluronan polymers via a substrate-assisted

mechanism. In humans, turnover of hyaluronan by this enzyme

family is critical for normal extracellular matrix remodeling.

However, elevated expression of the Hyal1 isozyme accelerates

tumor growth and metastatic progression. In this study, we used

structural information, site-directed mutagenesis, and steady

state enzyme kinetics to probe molecular determinants of

human Hyal1 function. Mutagenesis of active site residues

Glu¹³¹ and Tyr²⁴⁷ to Gln and Phe, respectively, eliminated activity

at all hyaluronan concentrations (to 125 …