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A Putative Cystathionine Beta-Synthase Homolog of Mycolicibacterium Smegmatis Is Involved in De Novo Cysteine Biosynthesis
University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 5-2020 A Putative Cystathionine Beta-Synthase Homolog of Mycolicibacterium smegmatis is Involved in de novo Cysteine Biosynthesis Saroj Kumar Mahato University of Arkansas, Fayetteville Follow this and additional works at: https://scholarworks.uark.edu/etd Part of the Cell Biology Commons, Molecular Biology Commons, and the Pathogenic Microbiology Commons Citation Mahato, S. K. (2020). A Putative Cystathionine Beta-Synthase Homolog of Mycolicibacterium smegmatis is Involved in de novo Cysteine Biosynthesis. Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/3639 This Thesis is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected]. A Putative Cystathionine Beta-Synthase Homolog of Mycolicibacterium smegmatis is Involved in de novo Cysteine Biosynthesis A thesis submitted in partial fulfillment of the requirement for the degree of Master of Science in Cell and Molecular Biology by Saroj Kumar Mahato Purbanchal University Bachelor of Science in Biotechnology, 2016 May 2020 University of Arkansas This thesis is approved for recommendation to the Graduate Council. ___________________________________ Young Min Kwon, Ph.D. Thesis Director ___________________________________ ___________________________________ Suresh Thallapuranam, Ph.D. Inés Pinto, Ph.D. Committee Member Committee Member ABSTRACT Mycobacteria include serious pathogens of humans and animals. Mycolicibacterium smegmatis is a non-pathogenic model that is widely used to study core mycobacterial metabolism. This thesis explores mycobacterial pathways of cysteine biosynthesis by generating and study of genetic mutants of M. smegmatis. Published in vitro biochemical studies had revealed three independent routes to cysteine synthesis in mycobacteria involving separate homologs of cysteine synthase, namely CysK1, CysK2, and CysM. -
Comparative Analysis of Mutants in the Mycothiol Biosynthesis Pathway in Mycobacterium Smegmatis
Biochemical and Biophysical Research Communications 363 (2007) 71–76 www.elsevier.com/locate/ybbrc Comparative analysis of mutants in the mycothiol biosynthesis pathway in Mycobacterium smegmatis Mamta Rawat a, Chantale Johnson a, Vanessa Cadiz a, Yossef Av-Gay b,* a Department of Biology, California State University-Fresno, Fresno, CA 937401, USA b Department of Medicine, Division of Infectious Diseases, University of British Columbia, Vancouver, BC, Canada V5Z 3J5 Received 17 August 2007 Available online 31 August 2007 Abstract The role of mycothiol in mycobacteria was examined by comparative analysis of mutants disrupted in the four known genes encoding the protein machinery needed for mycothiol biosynthesis. These mutants were sensitive to acid stress, antibiotic stress, alkylating stress, and oxidative stress indicating that mycothiol and mycothiol-dependent enzymes protect the mycobacterial cell against attack from various different types of stresses and toxic agents. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Mycothiol; Mycothiol deacetylase; Mycothiol ligase; Mycothiol synthase; Oxidative stress; Toxins; Xenobiotics Mycobacteria, like most other Gram-positive bacteria do We have previously reported that Mycobacterium not make glutathione but produce another low molecular smegmatis mutants disrupted in the four known genes weight thiol, mycothiol (MSH) (Fig. 1), 1-D-myoinosityl- [3,9–11] involved in mycothiol biosynthesis are resistant to 2-(n-acetyl-L-cysteinyl)-amido-2-deoxy-a-D-glucopyranoside. isoniazid, a front-line drug used in the treatment of tubercu- Since MSH is unique to actinomycetales [1], enzymes losis. We have also reported that M. smegmatis mutants involved in MSH biosynthesis and metabolism are poten- lacking mycothiol ligase activity and thus mycothiol are tial targets for drugs directed against pathogenic mycobac- sensitive to a wide range of antibiotics, alkylating agents, teria like Mycobacterium tuberculosis. -
A New Michaelis Complex Leads to Efficient Transition State Charge Offset
Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset Charlotte M. Mitona,1, Stefanie Jonasa,2, Gerhard Fischera,3, Fernanda Duarteb,3,4, Mark F. Mohameda, Bert van Looa,5, Bálint Kintsesa,6, Shina C. L. Kamerlinb, Nobuhiko Tokurikia,c, Marko Hyvönena, and Florian Hollfeldera,7 aDepartment of Biochemistry, University of Cambridge, CB2 1GA Cambridge, United Kingdom; bDepartment of Chemistry, Biomedicinskt Centrum (BMC), Uppsala University, 751 23 Uppsala, Sweden; and cMichael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada Edited by Daniel Herschlag, Stanford University, Stanford, CA, and accepted by Editorial Board Member Michael A. Marletta May 31, 2018 (received for review January 31, 2018) The recruitment and evolutionary optimization of promiscuous catalytic residues but also occurs at the periphery of the catalytic enzymes is key to the rapid adaptation of organisms to changing machinery, even in positions as remote as the second and third environments. Our understanding of the precise mechanisms shells of the active site (23). In studies examining detailed evo- underlying enzyme repurposing is, however, limited: What are lutionary transitions, function-altering mutations led to the dis- the active-site features that enable the molecular recognition of placement of a catalytic metal ion or the repositioning of a multiple substrates with contrasting catalytic requirements? To nucleophile (24–26). In further cases, the position of the catalytic gain insights into the molecular determinants of adaptation in residues remained unaltered, but other structural features, for promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate Significance hydrolase activity. -
1 Metabolic Dysfunction Is Restricted to the Sciatic Nerve in Experimental
Page 1 of 255 Diabetes Metabolic dysfunction is restricted to the sciatic nerve in experimental diabetic neuropathy Oliver J. Freeman1,2, Richard D. Unwin2,3, Andrew W. Dowsey2,3, Paul Begley2,3, Sumia Ali1, Katherine A. Hollywood2,3, Nitin Rustogi2,3, Rasmus S. Petersen1, Warwick B. Dunn2,3†, Garth J.S. Cooper2,3,4,5* & Natalie J. Gardiner1* 1 Faculty of Life Sciences, University of Manchester, UK 2 Centre for Advanced Discovery and Experimental Therapeutics (CADET), Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Sciences Centre, Manchester, UK 3 Centre for Endocrinology and Diabetes, Institute of Human Development, Faculty of Medical and Human Sciences, University of Manchester, UK 4 School of Biological Sciences, University of Auckland, New Zealand 5 Department of Pharmacology, Medical Sciences Division, University of Oxford, UK † Present address: School of Biosciences, University of Birmingham, UK *Joint corresponding authors: Natalie J. Gardiner and Garth J.S. Cooper Email: [email protected]; [email protected] Address: University of Manchester, AV Hill Building, Oxford Road, Manchester, M13 9PT, United Kingdom Telephone: +44 161 275 5768; +44 161 701 0240 Word count: 4,490 Number of tables: 1, Number of figures: 6 Running title: Metabolic dysfunction in diabetic neuropathy 1 Diabetes Publish Ahead of Print, published online October 15, 2015 Diabetes Page 2 of 255 Abstract High glucose levels in the peripheral nervous system (PNS) have been implicated in the pathogenesis of diabetic neuropathy (DN). However our understanding of the molecular mechanisms which cause the marked distal pathology is incomplete. Here we performed a comprehensive, system-wide analysis of the PNS of a rodent model of DN. -
Investigations Into Intracellular Thiols of Biological Importance
Investigations into Intracellular Thiols of Biological Importance by Christine Elizabeth Hand A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Doctor of Philosophy in Chemistry Waterloo, Ontario, Canada, 2007 © Christine Elizabeth Hand 2007 AUTHOR'S DECLARATION I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract The presence of thiols in living systems is critical for the maintenance of cellular redox homeostasis, the maintenance of protein thiol-disulfide ratios and the protection of cells from reactive oxygen species. In addition to the well studied tripeptide glutathione (γ-Glu-Cys-Gly), a number of compounds have been identified that contribute to these essential cellular roles. Many of these molecules are of great clinical interest due to their essential role in the biochemistry of a number of deadly pathogens, as well as their possible role as therapeutic agents in the treatment of a number of diseases. A series of studies were undertaken using theoretical, chemical and biochemical approaches on a selection of thiols, ergothioneine, the ovothiols and mycothiol, to further our understanding of these necessary biological components. Ergothioneine is present at significant physiological levels in humans and other mammals; however, a definitive role for this thiol has yet to be determined. It has been implicated in radical scavenging in vivo and shows promise as a therapeutic agent against disease states caused by oxidative damage. -
Exploring the Microbiome of the Mediterranean Sponge Aplysina Aerophoba by Single-Cell and Metagenomics
Exploring the microbiome of the Mediterranean sponge Aplysina aerophoba by single-cell and metagenomics Untersuchungen am Mikrobiom des Mittelmeerschwamms Aplysina aerophoba mittels Einzelzell- und Metagenomik Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences Julius-Maximilians-Universität Würzburg Section: Integrative Biology Submitted by Beate Magdalena Slaby from München Würzburg, March 2017 Submitted on: ……………………………………………………… Members of the Promotionskomitee Chairperson: Prof. Dr. Thomas Müller Primary Supervisor: Prof. Dr. Ute Hentschel Humeida Supervisor (Second): Prof. Dr. Thomas Dandekar Supervisor (Third): Prof. Dr. Frédéric Partensky Date of public defense: ……………………………………………………… Date of receipt of certificates: ……………………………………………………… ii Affidavit I hereby confirm that my thesis entitled ‘Exploring the microbiome of the Mediterranean sponge Aplysina aerophoba by single-cell and metagenomics’ is the result of my own work. I did not receive any help or support from commercial consultants. All sources and / or materials applied are listed and specified in the thesis. Furthermore, I confirm that this thesis has not yet been submitted as part of another examination process neither in identical nor in similar form. Place, Date Signature iii Acknowledgements I received financial support for this thesis project by a grant of the German Excellence Initiative to the Graduate School of Life Sciences of the University of Würzburg through a PhD fellowship, and from the SponGES project that has received funding from the European Union’s Horizon 2020 research and innovation program. I would like to thank: Dr. Ute Hentschel Humeida for her support and encouragement, and for providing so many extraordinary opportunities. Dr. Thomas Dandekar and Dr. Frédéric Partensky for the supervision and a number of very helpful discussions. -
S. Nagel Et Al., Microarray Analysis of the Global Gene Expression Profile Following Hypothermia and Transient Focal Cerebral Ischemia Neuroscience 2012
S. Nagel et al., Microarray analysis of the global gene expression profile following hypothermia and transient focal cerebral ischemia Neuroscience 2012 Supplementary Table 1 Gene Symbol Entrez Gene Name Affymetrix SNP ID Fold Change PEBP1 phosphatidylethanolamine binding protein 1 E05646cds_s_at 36,571 KIFC1 kinesin family member C1 AF035951_at 29,307 SERP1 stress-associated endoplasmic reticulum protein 1 AF100470_at 23,16 FDXR ferredoxin reductase D63761_g_at 22,504 SYNPO synaptopodin AB013130_at 21,174 ID1 inhibitor of DNA binding 1, dominant negative helix-loop-helix protein L23148_g_at 16,233 GTF2F2 general transcription factor IIF, polypeptide 2, 30kDa L01267_at 15,592 PPP2R2C protein phosphatase 2, regulatory subunit B, gamma D38261_at 15,356 RASGRP1 RAS guanyl releasing protein 1 (calcium and DAG-regulated) AF081196_at 15,142 UBB -- D16554_at 14,996 SLC37A4 solute carrier family 37 (glucose-6-phosphate transporter), member 4 AF080468_g_at 14,757 LIMK2 LIM domain kinase 2 D31874_at 14,254 PPP1R15A protein phosphatase 1, regulatory (inhibitor) subunit 15A AF020618_g_at 13,988 HPCAL4 hippocalcin like 4 D13125_at 13,128 FAIM2 Fas apoptotic inhibitory molecule 2 AF044201_at 12,77 GJA5 gap junction protein, alpha 5, 40kDa AF022136_at 12,751 SLC2A3 solute carrier family 2 (facilitated glucose transporter), member 3 D13962_g_at 12,245 TRIM23 tripartite motif containing 23 L04760_at 12,122 PIP4K2C phosphatidylinositol-5-phosphate 4-kinase, type II, gamma AF030558_at 11,698 SBK1 SH3-binding domain kinase 1 AB010154_at 11,523 PLEKHA1 pleckstrin -
N-Acetylgalactosamine-6-Sulfate Sulfatase in Man. Absence of the Enzyme in Morquio Disease
N-acetylgalactosamine-6-sulfate sulfatase in man. Absence of the enzyme in Morquio disease. J Singh, … , P Niebes, D Tavella J Clin Invest. 1976;57(4):1036-1040. https://doi.org/10.1172/JCI108345. Research Article Human N-acetylgalactosamine-6-sulfate sulfatase (6-sulfatase) activity is measured by using as a substrate a sulfated tetrasaccharide obtained by digesting purified chondroitin-6-sulfate (C-6-S) with testicular hyaluronidase. The amount of inorganic sulfate released is measured turbidimetrically. The enzyme from human kidney has a pH optimum of 4.8; its activity is augmented by low levels of NaCl and inhibited by phosphate and high levels of NaCl. Free glucuronate, acetylgalactosamine, inorganic sulfate, polymeric C-6-S, or tetrasaccharide obtained from chondroitin-4-sulfate do not affect the enzyme activity. The method may be used for the diagnosis of Morquio disease since extracts of Morquio fibroblasts are devoid of 6-sulfatase activity. Find the latest version: https://jci.me/108345/pdf N-Acetylgalactosamine-6-Sulfate Sulfatase in Man ABSENCE OF THE ENZYME IN MORQUIO DISEASE JAGAT SINGH, NICOLA Di FERRANTE, PAUL NIEBES, and DANIELA TAVELLA From the Departments of Biochemistry and Medicine, and the Division of Orthopedic Surgery of the Department of Surgery, Baylor College of Medicine, Houston, Texas 77025 and Zyma, S.A., Nyon, Switzerland A B S T R A C T Human N-acetylgalactosamine-6-sulfate measurement, and the need to ascertain whether the sulfatase (6-sulfatase) activity is measured by using as sulfate released was in position 4 or 6 of the galactos- a substrate a sulfated tetrasaccharide obtained by di- amine moieties make the method (1) rather laborious gesting purified chondroitin-6-sulfate (C-6-S) with tes- and not ideal for the routine assay of the enzyme ac- ticular hyaluronidase. -
Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT)
International Journal of Molecular Sciences Review Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT) Abu Iftiaf Md Salah Ud-Din 1, Alexandra Tikhomirova 1 and Anna Roujeinikova 1,2,* 1 Infection and Immunity Program, Monash Biomedicine Discovery Institute; Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia; [email protected] (A.I.M.S.U.-D.); [email protected] (A.T.) 2 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia * Correspondence: [email protected]; Tel.: +61-3-9902-9194; Fax: +61-3-9902-9222 Academic Editor: Claudiu T. Supuran Received: 30 May 2016; Accepted: 20 June 2016; Published: 28 June 2016 Abstract: General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) catalyze the transfer of an acyl moiety from acyl coenzyme A (acyl-CoA) to a diverse group of substrates and are widely distributed in all domains of life. This review of the currently available data acquired on GNAT enzymes by a combination of structural, mutagenesis and kinetic methods summarizes the key similarities and differences between several distinctly different families within the GNAT superfamily, with an emphasis on the mechanistic insights obtained from the analysis of the complexes with substrates or inhibitors. It discusses the structural basis for the common acetyltransferase mechanism, outlines the factors important for the substrate recognition, and describes the mechanism of action of inhibitors of these enzymes. It is anticipated that understanding of the structural basis behind the reaction and substrate specificity of the enzymes from this superfamily can be exploited in the development of novel therapeutics to treat human diseases and combat emerging multidrug-resistant microbial infections. -
Suzanne Pfeffer
July 2010 ASBMB PreSidentiAl PriMer: Suzanne Pfeffer American Society for Biochemistry and Molecular Biology AAdjuvdjuvAAntnt IImmunothermmunotherAApypy ususIIngng KKrnrn70007000 KRN7000 (α-Galactosyl Ceramide) Avanti Number 867000 Supplier: Funakoshi Co. Ltd. Hepatic metastasis is a major clinical problem in cancer treatment. We examined antitumor ac- tivity of alpha-galactosylceramide (KRN7000) on mice with spontaneous liver metastases of re- ticulum cell sarcoma M5076 tumor cells (spontaneous metastasis model). In this model, all mice that were s.c. challenged with one million tumor cells developed a solid s.c. mass by day 7 and died of hepatic metastases. In the current study, we administered 100 microg/kg of KRN7000 to the model mice on days 7, 11, and 15. This treatment suppressed the growth of established liver metastases and resulted in the prolongation of survival time. Fluorescence-activated cell sorter analysis of phenotypes of spleen cells, hepatic lymphocytes, and regional lymph node cells around the s.c. tumor revealed that CD3+NK1.1+ (NKT) cells increased in hepatic lym- phocytes of the KRN7000-treated mice. Cytotoxic activity and IFN-gamma production of hepatic lymphocytes were augmented in comparison with those of spleen cells and regional LN cells. At the same time, interleukin (IL)-12 production of hepatic lymphocytes was markedly enhanced. Neutralization of IL-12 using a blocking monoclonal antibody diminished the prolonged survival time. These results showed that the in vivo antitumor effects of KRN7000 on spontaneous liver metastases were dependent on the endogenous IL-12 production, where NKT cells in the liver are suggested to be involved. Adjuvant immunotherapy using KRN7000 could be a promising modality for the prevention of postoperative liver metastases. -
NIH Conflict of Interest Regs Revised
OCTOBEROCTOBER 2005 www.asbmb.org Constituent Society of FASEB AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY NIH Conflict of Interest Regs Revised SEE PAGE 30 FOR NEW CLARA BENSON TRAVEL FELLOWSHIP AWARD Held in conjunction with EB2006 Custom Antibodies Your Way! Choose the protocol that is right for you! QwikScreen ™: 65 day, 2 rabbit protocol - 4 immunizations, 3 bleeds/rabbit (~100ml serum), customer supplied peptide/protein - Options: Peptide synthesis, immunograde Conjugation to carrier u ELISA u u Animal extensionsMS analysis $685 Standard: 80 day, 2 rabbit protocol - 5 immunizations, 5 bleeds/rabbit (~ 200ml ser Options: um), ELISA, customer supplied peptide/pr Peptide synthesis MS Check™ peptide sequence confirmation u HPLC purified peptide Affinity purification otein - Pinnacle: $975 u HPLC and MS analysis u Complete Affinity Purified Protocol- Animal extensions 2 rabbit pr 5 bleeds/rabbitotocol, (~ 200mlepitope serum), design, peptide PhD technical synthesis support, (up to 20mer),5 immunizations, HPLC purified to ~85%, 5+mg peptide to customer, ELISA, evaluation period, affinity purification, and morMS Check™ peptide sequence confirmationNo Hidden Charges! e… - Discounts for Multiple Protocols$1795 , Includes peptide sequencing by CID MS/MS– u Guaranteed Peptide Let our enthusiasm for scienceExpert workTechnical for SupportFidelity! P: 508.303.8222 www.21stcenturybio.com Toll-free: 877.217.8238 F: 508.303.8333 you! E: [email protected] www.asbmb.org AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY OCTOBER -
Development and Validation of a Protein-Based Risk Score for Cardiovascular Outcomes Among Patients with Stable Coronary Heart Disease
Supplementary Online Content Ganz P, Heidecker B, Hveem K, et al. Development and validation of a protein-based risk score for cardiovascular outcomes among patients with stable coronary heart disease. JAMA. doi: 10.1001/jama.2016.5951 eTable 1. List of 1130 Proteins Measured by Somalogic’s Modified Aptamer-Based Proteomic Assay eTable 2. Coefficients for Weibull Recalibration Model Applied to 9-Protein Model eFigure 1. Median Protein Levels in Derivation and Validation Cohort eTable 3. Coefficients for the Recalibration Model Applied to Refit Framingham eFigure 2. Calibration Plots for the Refit Framingham Model eTable 4. List of 200 Proteins Associated With the Risk of MI, Stroke, Heart Failure, and Death eFigure 3. Hazard Ratios of Lasso Selected Proteins for Primary End Point of MI, Stroke, Heart Failure, and Death eFigure 4. 9-Protein Prognostic Model Hazard Ratios Adjusted for Framingham Variables eFigure 5. 9-Protein Risk Scores by Event Type This supplementary material has been provided by the authors to give readers additional information about their work. Downloaded From: https://jamanetwork.com/ on 10/02/2021 Supplemental Material Table of Contents 1 Study Design and Data Processing ......................................................................................................... 3 2 Table of 1130 Proteins Measured .......................................................................................................... 4 3 Variable Selection and Statistical Modeling ........................................................................................