Other Biochemistry, Biophysics, and Structural Biology Commons^™

Mechanistic Details Of Glutathione Biosynthesis Revealed By Crystal Structures Of Saccharomyces Cerevisiae Glutamate Cysteine Ligase, Ekaterina I. Biterova, Joseph J. Barycki

Department of Biochemistry: Faculty Publications

Glutathione is a thiol-disulfide exchange peptide critical for

buffering oxidative or chemical stress, and an essential cofactor

in several biosynthesis and detoxification pathways. The ratelimiting

step in its de novo biosynthesis is catalyzed by glutamate

cysteine ligase, a broadly expressed enzyme for which limited

structural information is available in higher eukaryotic species.

Structural data are critical to the understanding of clinical glutathione

deficiency, as well as rational design of enzyme modulators

that could impact human disease progression. Here, we

have determined the structures of Saccharomyces cerevisiae glutamate

cysteine ligase (ScGCL) in the presence of glutamate and

MgCl2 (2.1 Å; R …

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 …

A Role For The Atp7a Copper-Transporting Atpase In Macrophage Bactericidal Activity, Carine White, Jaekwon Lee, Taiho Kambe, Kevin Fritsche, Michael J. Petris

Department of Biochemistry: Faculty Publications

Copper is an essential micronutrient that is necessary for healthy immune function. This requirement is underscored by an increased susceptibility to bacterial infection in copper-deficient animals; however, a molecular understanding of its importance in immune defense is unknown. In this study, we investigated the effect of proinflammatory agents on copper homeostasis in RAW264.7 macrophages. Interferon-γ was found to increase expression of the high affinity copper importer, CTR1, and stimulate copper uptake. This was accompanied by copper-stimulated trafficking of the ATP7A copper exporter from the Golgi to vesicles that partially overlapped with phagosomal compartments. Silencing of ATP7A expression attenuated bacterial killing, …

Editorial: Dietary Lipid Absorption, Concetta Dirusso, Paul N. Black

Department of Biochemistry: Faculty Publications

The composition of dietary fat influences tissue fatty acid composition, which in turn impacts cellular function through a number of different processes. This includes changes in signaling, lipid metabolism, and transcriptional activities that normally function to maintain intracellular fatty acid homeostasis. The consumption of high levels of dietary fat in excess of caloric expenditure is linked with obesity and the disruption of normal homeostatic mechanisms governing lipid metabolism. Data from the Centers for Disease Control and Prevention show that obesity (defined as a BMI≥30) represents a considerable health concern in the United States. Of particular note is that for adult …

Chemchains: A Platform For Simulation And Analysis Of Biochemical Networks Aimed To Laboratory Scientists, Tomáš Helikar, Jim A. Rogers

Department of Biochemistry: Faculty Publications

Background: New mathematical models of complex biological structures and computer simulation software allow modelers to simulate and analyze biochemical systems in silico and form mathematical predictions. Due to this potential predictive ability, the use of these models and software has the possibility to compliment laboratory investigations and help refine, or even develop, new hypotheses. However, the existing mathematical modeling techniques and simulation tools are often difficult to use by laboratory biologists without training in high-level mathematics, limiting their use to trained modelers.

Results: We have developed a Boolean network-based simulation and analysis software tool, ChemChains, which combines the advantages of …

Ulk-Atg13-Fip200 Complexes Mediate Mtor Signaling To The Autophagy Machinery, Chang Hwa Jung, Chang Bong Jun, Seung-Hyun Ro, Young-Mi Kim, Neil Michael Otto, Jing Cao, Mondira Kundu, Do-Hyung Kim

Department of Biochemistry: Faculty Publications

Autophagy, the starvation-induced degradation of bulky cytosolic components, is up-regulated in mammalian cells when nutrient supplies are limited. Although mammalian target of rapamycin (mTOR) is known as the key regulator of autophagy induction, the mechanism by which mTOR regulates autophagy has remained elusive. Here, we identify that mTOR phosphorylates a mammalian homologue of Atg13 and the mammalian Atg1 homologues ULK1 and ULK2. The mammalian Atg13 binds both ULK1 and ULK2 and mediates the interaction of the ULK proteins with FIP200. The binding of Atg13 stabilizes and activates ULK and facilitates the phosphorylation of FIP200 by ULK, whereas knockdown of Atg13 …

Contribution Of Impaired Myocardial Insulin Signaling To Mitochondrial Dysfunction And Oxidative Stress In The Heart, Sihem Boudina, Heiko Bugger, Sandra Sena, Brian T. O'Neill, Vlad G. Zaha, Olesya Ilkun, Jordan J. Wright, Pradip K. Mazumber, Eric Palfreyman, Timothy J. Tidwell, Heather Theobald, Oleh Khalimonchuk, Benjamin Wayment, Xiaoming Sheng, Kenneth J. Rodnick, Ryan Centini, Dong Chen, Sheldon E. Litwin, Bart E. Weimer, E. Dale Abel

Department of Biochemistry: Faculty Publications

Background—Diabetes-associated cardiac dysfunction is associated with mitochondrial dysfunction and oxidative stress, which may contribute to LV dysfunction. The contribution of altered myocardial insulin action, independently of associated changes in systemic metabolism is incompletely understood. The present study tested the hypothesis that perinatal loss of insulin signaling in the heart impairs mitochondrial function.

Methods and Results—In 8-week-old mice with cardiomyocyte deletion of insulin receptors (CIRKO), inotropic reserves were reduced and mitochondria manifested respiratory defects for pyruvate that was associated with proportionate reductions in catalytic subunits of pyruvate dehydrogenase. Progressive age-dependent defects in oxygen consumption and ATP synthesis with the substrates glutamate …

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 …

Heme Regulation Of Human Cystathionine Β-Synthase Activity: Insights From Fluorescence And Raman Spectroscopy, Colin L. Weeks, Sangita Singh, Peter Madzelan, Ruma Banerjee, Thomas G. Spiro

Department of Biochemistry: Faculty Publications

Cystathionine β-synthase (CBS) plays a central role in cysteine metabolism, and malfunction of the enzyme leads to homocystinuria, a devastating metabolic disease. CBS contains a pyridoxal 5′- phosphate (PLP) cofactor which catalyzes the synthesis of cystathionine from homocysteine and serine. Mammalian forms of the enzyme also contain a heme group, which is not involved in catalysis. It may, however, play a regulatory role, since the enzyme is inhibited when CO or NO are bound to the heme. We have investigated the mechanism of this inhibition using fluorescence and resonance Raman spectroscopies. CO binding is found to induce a tautomeric shift …

Targeting The Fatty Acid Transport Proteins (Fatp) To Understand The Mechanisms Linking Fatty Acid Transport To Metabolism, Paul N. Black, Angel Sandoval, Elsa Arias-Barrau, Concetta C. Dirusso

Department of Biochemistry: Faculty Publications

One principal process driving fatty acid transport is vectorial acylation, where fatty acids traverse the membrane concomitant with activation to CoA thioesters. Current evidence is consistent with the proposal that specific fatty acid transport (FATP) isoforms alone or in concert with specific long chain acyl CoA synthetase (Acsl) isoforms function to drive this energy-dependent process. Understanding the details of vectorial acylation is of particular importance as disturbances in lipid metabolism many times leads to elevated levels of circulating free fatty acids, which in turn increases fatty acid internalization and ectopic accumulation of triglycerides. This is associated with changes in fatty …

Copper Transport Activity Of Yeast Ctr1 Is Down-Regulated Via Its C Terminus In Response To Excess Copper, Xiaobin Wu, Devis Sinani, Heejeong Kim, Jaekwon Lee

Department of Biochemistry: Faculty Publications

Copper is an essential yet toxic trace element. The Ctr1 family of proteins plays a critical role for copper uptake in eukaryotes. However, the mechanisms of action of Ctr1 are largely unknown. Our previous data demonstrated that copper transport induces conformational changes in the cytosolic C terminus of the yeast Saccharomyces cerevisiae Ctr1. To define the physiological significance of this molecular event and gain better insights into the mechanism of Ctr1-mediated copper uptake, we have characterized the functional roles of the Ctr1 C terminus.ACtr1 mutant lacking the entire C-terminal cytosolic tail is functional in high affinity copper uptake; however, yeast …

Sdh5, A Gene Required For Flavination Of Succinate Dehydrogenase, Is Mutated In Paraganglioma, Huai-Xiang Hao, Oleh Khalimonchuk, Margit Schraders, Noah Dephoure, Jean-Pierre Bayley, Henricus Kunst, Peter Devilee, Cor W.R.J. Cremers, Joshua D. Schiffman, Brandon G. Bentz, Steven P. Gygi, Dennis R. Winge, Hannie Kremer, Jared Rutter

Department of Biochemistry: Faculty Publications

Mammalian mitochondria contain about 1100 proteins, nearly 300 of which are uncharacterized. Given the well-established role of mitochondrial defects in human disease, functional characterization of these proteins may shed new light on disease mechanisms. Starting with yeast as a model system, we investigated an uncharacterized but highly conserved mitochondrial protein (named here Sdh5). Both yeast and human Sdh5 interact with the catalytic subunit of the succinate dehydrogenase (SDH) complex, a component of both the electron transport chain and the tricarboxylic acid cycle. Sdh5 is required for SDH-dependent respiration and for Sdh1 flavination (incorporation of the flavin adenine dinucleotide cofactor). Germline …

Crystal Structure Of Acivicin-Inhibited Γ-Glutamyltranspeptidase Reveals Critical Roles For Its C-Terminus In Autoprocessing And Catalysis, Kristin Williams, Sierra Cullati, Aaron Sand, Ekaterina I. Biterova, Joseph J. Barycki

Department of Biochemistry: Faculty Publications

Helicobacter pylori γ-glutamyltranspeptidase (HpGT) is a general γ-glutamyl hydrolase and a

demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme. Acivicin is a widely used γ-glutamyltranspeptidase inhibitor that covalently modifies the enzyme, but its precise mechanism of action remains unclear. The time-dependent inactivation of HpGT exhibits a hyperbolic dependence on acivicin concentration with kmax = 0.033 ± 0.006 sec^{−1 …}

Glucose Promotes Stress Resistance In The Fungal Pathogen Candida Albicans, Alexandra Rodaki, Iryna M. Bohovych, Brice Enjalbert, Tim Young, Frank C. Odds, Neil A.R. Gow, Alistair J.P. Brown

Department of Biochemistry: Faculty Publications

Metabolic adaptation, and in particular the modulation of carbon assimilatory pathways during disease progression, is thought to contribute to the pathogenicity of Candida albicans. Therefore, we have examined the global impact of glucose upon the C. albicans transcriptome, testing the sensitivity of this pathogen to wide-ranging glucose levels (0.01, 0.1, and 1.0%). We show that, like Saccharomyces cerevisiae, C. albicans is exquisitely sensitive to glucose, regulating central metabolic genes even in response to 0.01% glucose. This indicates that glucose concentrations in the bloodstream (approximate range 0.05–0.1%) have a significant impact upon C. albicans gene regulation. However, in contrast …

Modulation Of The Heme Electronic Structure And Cystathionine Β-Synthase Activity By Second Coordination Sphere Ligands: The Role Of Heme Ligand Switching In Redox Regulation, Sangita Singh, Peter Madzelan, Jay Stasser, Colin L. Weeks, Donald F. Becker, Thomas G. Spiro, James Penner-Hahn, Ruma Banerjee

Department of Biochemistry: Faculty Publications

In humans, cystathionine β-synthase (CBS) is a hemeprotein, which catalyzes a pyridoxal phosphate (PLP)-dependent condensation reaction. Changes in the heme environment are communicated to the active site, which is ~20 Å away. In this study, we have examined the role of H67 and R266, which are in the second coordination sphere of the heme ligands, H65 and C52 respectively, in modulating the heme's electronic properties and in transmitting information between the heme and active sites. While the H67A mutation is comparable to wild-type CBS, interesting differences are revealed by mutations at the R266 site. The pathogenic mutant, R266K, is moderately …