DOCSLIB.ORG

Are CLCA Proteins Metalloproteases? Comparison of the Cleavage Processes of the Secreted Mclca3 and the Transmembrane Mclca6

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Are CLCA Proteins Metalloproteases? Comparison of the Cleavage Processes of the Secreted Mclca3 and the Transmembrane Mclca6 Institute of Veterinary Pathology, Department of Veterinary Medicine, Freie Universität Berlin Are CLCA Proteins Metalloproteases? Comparison of the Cleavage Processes of the Secreted mCLCA3 and the Transmembrane mCLCA6 Proteins Thesis submitted for the fulfillment of a Doctor of Philosophy (Ph.D.) degree in Veterinary Medicine at the Freie Universität Berlin submitted by Melanie Kathrin Bothe Veterinarian from Braunschweig Berlin 2011 Journal-No.: 3553 Printed with permission of the Department of Veterinary Medicine of the Freie Universität Berlin Dean: Univ.-Prof. Dr. Leo Brunnberg Supervision: Univ.-Prof. Dr. Achim D. Gruber, Ph.D.(Cornell Univ.) First Reviewer: Univ.-Prof. Dr. Achim D. Gruber, Ph.D.(Cornell Univ.) Second Reviewer: Univ.-Prof. Dr. Dr. Ralf Einspanier Third Reviewer: PD Dr. Michael Veit Descriptors (according to CAB-Thesaurus): cystic fibrosis; asthma; chronic obstructive pulmonary disease; mice; metalloproteinases; zinc; protein transport Day of Doctorate: 23.11.2012 This document is protected by copyright law. No part of this document may be reproduced in any form by any means without prior written authorization of the publisher. All rights reserved © Mensch und Buch Verlag 2012 Choriner Str. 85 - 10119 Berlin [email protected] – www.menschundbuch.de Aus dem Institut für Tierpathologie des Fachbereichs Veterinärmedizin der Freien Universität Berlin Sind CLCA-Proteine Metalloproteasen? Vergleich der Spaltungsprozesse des sezernierten mCLCA3- und des transmembranären mCLCA6-Proteins Inaugural-Dissertation zur Erlangung des Grades eines Doctor of Philosophy (Ph.D.) in Veterinärmedizin an der Freie Universität Berlin vorgelegt von Melanie Kathrin Bothe Tierärztin aus Braunschweig Berlin 2011 Journal-Nr.: 3553 Gedruckt mit Genehmigung des Fachbereichs Veterinärmedizin der Freie Universität Berlin Dekan: Univ.-Prof. Dr. Leo Brunnberg Betreuung: Univ.-Prof. Dr. Achim D. Gruber, Ph.D.(Cornell Univ.) Erster Gutachter: Univ.-Prof. Dr. Achim D. Gruber, Ph.D.(Cornell Univ.) Zweiter Gutachter: Univ.-Prof. Dr. Dr. Ralf Einspanier Dritter Gutachter: PD Dr. Michael Veit Deskriptoren (nach CAB-Thesaurus): cystic fibrosis; asthma; chronic obstructive pulmonary disease; mice; metalloproteinases; zinc; protein transport Tag der Promotion: 23.11.2012 Bibliografische Information der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über <http://dnb.ddb.de> abrufbar. ISBN: 978-3-86387-252-6 Zugl.: Berlin, Freie Univ., Diss., 2011 Dissertation, Freie Universität Berlin D 188 Dieses Werk ist urheberrechtlich geschützt. Alle Rechte, auch die der Übersetzung, des Nachdruckes und der Vervielfältigung des Buches, oder Teilen daraus, vorbehalten. Kein Teil des Werkes darf ohne schriftliche Genehmigung des Verlages in irgendeiner Form reproduziert oder unter Verwendung elektronischer Systeme verarbeitet, vervielfältigt oder verbreitet werden. Die Wiedergabe von Gebrauchsnamen, Warenbezeichnungen, usw. in diesem Werk berechtigt auch ohne besondere Kennzeichnung nicht zu der Annahme, dass solche Namen im Sinne der Warenzeichen- und Markenschutz-Gesetzgebung als frei zu betrachten wären und daher von jedermann benutzt werden dürfen. Alle Rechte vorbehalten © Mensch und Buch Verlag 2012 Choriner Str. 85 - 10119 Berlin [email protected] – www.menschundbuch.de Für Verena Contents Contents Contents ................................................................................................................................ VII List of abbreviations ................................................................................................................ X 1 INTRODUCTION ...................................................................................................... 11 2 LITERATURE ........................................................................................................... 13 2.1 Tissue expression patterns of CLCA proteins .......................................................... 13 2.1.1 CLCA cluster 1 ......................................................................................................... 14 2.1.2 CLCA cluster 2 ......................................................................................................... 15 2.1.3 CLCA cluster 3 ......................................................................................................... 16 2.1.4 CLCA cluster 4 ......................................................................................................... 18 2.2 Biomedical relevance of CLCA proteins ................................................................... 19 2.2.1 Diseases with secretory dysfunction ........................................................................ 19 2.2.1.1 Modulation of calcium activated chloride channels in cystic fibrosis ........................ 19 2.2.1.2 Mucus hypersecretion in cystic fibrosis, asthma and chronic obstructive pulmonary disease ................................................................................................... 22 2.2.2 Cell adhesion under physiologic conditions and in metastasizing tumor cells ......... 24 2.2.3 Role in apoptosis and tumor growth ......................................................................... 25 2.3 Structure and biochemical characteristics of CLCA proteins .................................... 26 2.3.1 n-CLCA domain ........................................................................................................ 26 2.3.2 Von Willebrand factor A domain ............................................................................... 27 2.3.3 Fibronectin type III domain ....................................................................................... 27 2.4 Cellular processing of CLCA proteins ...................................................................... 28 2.4.1 Signal sequence ....................................................................................................... 28 2.4.2 Glycosylation ............................................................................................................ 28 2.4.3 Cleavage of CLCA proteins ...................................................................................... 29 2.4.4 Secretion of whole protein versus ectodomain shedding of transmembrane CLCA proteins .......................................................................................................... 30 VII Contents 3 AIMS OF THIS PH.D. PROJECT ............................................................................. 32 3.1 Aim no. 1: Characterization of the cleavage process of the secreted mCLCA3 protein ...................................................................................................................... 32 3.2 Aim no. 2: Identification of a murine transmembrane CLCA protein in tissues relevant for cystic fibrosis ......................................................................................... 33 3.2.1 Expression pattern analysis and biochemical characterization of mCLCA5 ............. 33 3.2.2 Expression pattern analysis and biochemical characterization of mCLCA6 ............. 34 3.3 Aim no. 3: Comparison of the cleavage processes of a secreted CLCA protein versus a transmembrane CLCA protein ................................................................... 34 4 RESEARCH PUBLICATIONS IN PEER-REVIEWED JOURNALS ......................... 35 4.1 Murine mCLCA6 is an Integral Apical Membrane Protein of Non-goblet Cell Enterocytes and Co-localizes with the Cystic Fibrosis Transmembrane Conductance Regulator ............................................................................................ 35 4.2 Murine mCLCA5 is Expressed in Granular Layer Keratinocytes of Stratified Epithelia .................................................................................................................... 36 4.3 The Murine mCLCA3 is a Zinc-dependent Metalloprotease with Autoproteolytic Activity ...................................................................................................................... 37 4.4 Impaired Autoproteolytic Cleavage of mCLCA6, a Murine Integral Membrane Protein Expressed in Enterocytes, Leads to Cleavage at the Plasma Membrane Instead of Cleavage in the Endoplasmic Reticulum ................................................. 38 5 CONCLUDING DISCUSSION .................................................................................. 83 5.1 Hypothesis I: CLCA proteins show characteristics of metalloproteases ................... 83 5.1.1 Aim no. 1: Characterization of the cleavage process of the secreted mCLCA3 protein ...................................................................................................................... 83 5.1.2 Are CLCA proteins metalloproteases of the zincin family?....................................... 85 5.1.2.1 HEXXH motif of metalloproteases ............................................................................ 85 5.1.2.2 Zinc-dependent cleavage mechanism ...................................................................... 86 5.1.2.3 (Auto-)proteolytic activity .......................................................................................... 87 VIII Contents 5.2 Hypothesis II: The cleavage processes of transmembrane and secreted CLCA proteins differ in consequence of the transmembrane domain................................. 87 5.2.1 Aim no. 2: Identification of a murine transmembrane
Load more

Recommended publications
  • (12) United States Patent (10) Patent No.: US 6,395,889 B1 Robison (45) Date of Patent: May 28, 2002
    USOO6395889B1 (12) United States Patent (10) Patent No.: US 6,395,889 B1 Robison (45) Date of Patent: May 28, 2002 (54) NUCLEIC ACID MOLECULES ENCODING WO WO-98/56804 A1 * 12/1998 ........... CO7H/21/02 HUMAN PROTEASE HOMOLOGS WO WO-99/0785.0 A1 * 2/1999 ... C12N/15/12 WO WO-99/37660 A1 * 7/1999 ........... CO7H/21/04 (75) Inventor: fish E. Robison, Wilmington, MA OTHER PUBLICATIONS Vazquez, F., et al., 1999, “METH-1, a human ortholog of (73) Assignee: Millennium Pharmaceuticals, Inc., ADAMTS-1, and METH-2 are members of a new family of Cambridge, MA (US) proteins with angio-inhibitory activity', The Journal of c: - 0 Biological Chemistry, vol. 274, No. 33, pp. 23349–23357.* (*) Notice: Subject to any disclaimer, the term of this Descriptors of Protease Classes in Prosite and Pfam Data patent is extended or adjusted under 35 bases. U.S.C. 154(b) by 0 days. * cited by examiner (21) Appl. No.: 09/392, 184 Primary Examiner Ponnathapu Achutamurthy (22) Filed: Sep. 9, 1999 ASSistant Examiner William W. Moore (51) Int. Cl." C12N 15/57; C12N 15/12; (74) Attorney, Agent, or Firm-Alston & Bird LLP C12N 9/64; C12N 15/79 (57) ABSTRACT (52) U.S. Cl. .................... 536/23.2; 536/23.5; 435/69.1; 435/252.3; 435/320.1 The invention relates to polynucleotides encoding newly (58) Field of Search ............................... 536,232,235. identified protease homologs. The invention also relates to 435/6, 226, 69.1, 252.3 the proteases. The invention further relates to methods using s s s/ - - -us the protease polypeptides and polynucleotides as a target for (56) References Cited diagnosis and treatment in protease-mediated disorders.
    [Show full text]
  • Serine Proteases with Altered Sensitivity to Activity-Modulating
    (19) & (11) EP 2 045 321 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.04.2009 Bulletin 2009/15 C12N 9/00 (2006.01) C12N 15/00 (2006.01) C12Q 1/37 (2006.01) (21) Application number: 09150549.5 (22) Date of filing: 26.05.2006 (84) Designated Contracting States: • Haupts, Ulrich AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 51519 Odenthal (DE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Coco, Wayne SK TR 50737 Köln (DE) •Tebbe, Jan (30) Priority: 27.05.2005 EP 05104543 50733 Köln (DE) • Votsmeier, Christian (62) Document number(s) of the earlier application(s) in 50259 Pulheim (DE) accordance with Art. 76 EPC: • Scheidig, Andreas 06763303.2 / 1 883 696 50823 Köln (DE) (71) Applicant: Direvo Biotech AG (74) Representative: von Kreisler Selting Werner 50829 Köln (DE) Patentanwälte P.O. Box 10 22 41 (72) Inventors: 50462 Köln (DE) • Koltermann, André 82057 Icking (DE) Remarks: • Kettling, Ulrich This application was filed on 14-01-2009 as a 81477 München (DE) divisional application to the application mentioned under INID code 62. (54) Serine proteases with altered sensitivity to activity-modulating substances (57) The present invention provides variants of ser- screening of the library in the presence of one or several ine proteases of the S1 class with altered sensitivity to activity-modulating substances, selection of variants with one or more activity-modulating substances. A method altered sensitivity to one or several activity-modulating for the generation of such proteases is disclosed, com- substances and isolation of those polynucleotide se- prising the provision of a protease library encoding poly- quences that encode for the selected variants.
    [Show full text]
  • Loss of Protease Activity of ADAM15 Abolishes Protective Effects on Plaque Progression in Atherosclerosis
    382 Letters to the Editor Loss of protease activity of ADAM15 abolishes protective effects on plaque progression in atherosclerosis Andreas Bültmann, Zhongmin Li, Silvia Wagner, Meinrad Gawaz, Martin Ungerer, Harald Langer, Andreas E. May ⁎⁎, Götz Münch ⁎ Corimmun, Fraunhofer Str. 17, D-82152 Martinsried, Germany Medizinische Klinik III, Eberhard-Karls Universität Tübingen, D-72076 Tübingen, Germany article info For the induction of atherosclerosis, rabbits were fed with Western Article history: type high cholesterol (0.25%) diet for 8 weeks and vascular gene Received 8 August 2011 transfer to the carotid artery was performed as previously described Accepted 13 August 2011 [7]. Available online 9 September 2011 Animals were sacrificed 4 weeks after the adenovirus delivery. Keywords: The left common carotid arteries, aorta and iliac arteries were Atherosclerosis macroscopically prepared for “en face” evaluation of plaque ADAM15 extension and stained with Sudan III. Serial 6-μm-thick cryosections Sheddase were cut and histological assessment of atherosclerosis after Metalloproteinase hematoxylin eosin (HE) and van Gieson (VG)-elastica staining and GFP expression were performed. Immunohistochemical analysis, with anti rabbit RAM 11 antibody (DAKO, Hamburg, Germany) was The A Disintegrin And Metallporteinases (ADAMs) contain a used for macrophages as previously described [12]. metalloprotease-like and a disintegrin-like domain. Currently 40 After vascular gene transfer into the carotid artery, GFP expression different types of ADAM proteins have been identified. ADAM15 is could be detected with AdGFP and also with Ad-ADAM15 and Ad- found in the myocardium [1,2], endothelial cells and in vascular ADAM15 prot neg, which both co-expressed GFP (Fig. 1). Relative atherosclerotic lesions [3].
    [Show full text]
  • Handbook of Proteolytic Enzymes Second Edition Volume 1 Aspartic and Metallo Peptidases
    Handbook of Proteolytic Enzymes Second Edition Volume 1 Aspartic and Metallo Peptidases Alan J. Barrett Neil D. Rawlings J. Fred Woessner Editor biographies xxi Contributors xxiii Preface xxxi Introduction ' Abbreviations xxxvii ASPARTIC PEPTIDASES Introduction 1 Aspartic peptidases and their clans 3 2 Catalytic pathway of aspartic peptidases 12 Clan AA Family Al 3 Pepsin A 19 4 Pepsin B 28 5 Chymosin 29 6 Cathepsin E 33 7 Gastricsin 38 8 Cathepsin D 43 9 Napsin A 52 10 Renin 54 11 Mouse submandibular renin 62 12 Memapsin 1 64 13 Memapsin 2 66 14 Plasmepsins 70 15 Plasmepsin II 73 16 Tick heme-binding aspartic proteinase 76 17 Phytepsin 77 18 Nepenthesin 85 19 Saccharopepsin 87 20 Neurosporapepsin 90 21 Acrocylindropepsin 9 1 22 Aspergillopepsin I 92 23 Penicillopepsin 99 24 Endothiapepsin 104 25 Rhizopuspepsin 108 26 Mucorpepsin 11 1 27 Polyporopepsin 113 28 Candidapepsin 115 29 Candiparapsin 120 30 Canditropsin 123 31 Syncephapepsin 125 32 Barrierpepsin 126 33 Yapsin 1 128 34 Yapsin 2 132 35 Yapsin A 133 36 Pregnancy-associated glycoproteins 135 37 Pepsin F 137 38 Rhodotorulapepsin 139 39 Cladosporopepsin 140 40 Pycnoporopepsin 141 Family A2 and others 41 Human immunodeficiency virus 1 retropepsin 144 42 Human immunodeficiency virus 2 retropepsin 154 43 Simian immunodeficiency virus retropepsin 158 44 Equine infectious anemia virus retropepsin 160 45 Rous sarcoma virus retropepsin and avian myeloblastosis virus retropepsin 163 46 Human T-cell leukemia virus type I (HTLV-I) retropepsin 166 47 Bovine leukemia virus retropepsin 169 48
    [Show full text]
  • Drobiak S ( .Pdf )
    CHARACTERIZATION OF THE ZPS1P CELL WALL PROTEIN FROM Saccharomyces cerevisiae By STEPHANIE L. DROBIAK A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004 Copyright 2004 by Stephanie L. Drobiak This document is dedicated to my fiancé and my family for all their help and support during the last few years. ACKNOWLEDGMENTS I would like to thank my future husband and my family for their moral support, my lab mates for their continuous help and friendship, and my mentor, Dr. Thomas Lyons, for his endless guidance and patience. iv TABLE OF CONTENTS Page ACKNOWLEDGMENTS ................................................................................................. iv LIST OF FIGURES ........................................................................................................... vi ABSTRACT...................................................................................................................... vii 1 INTRODUCTION ...................................................................................................1 Zps1p-like Proteins from Candida albicans and Aspergillus spp. ......................... 2 Zps1p from Saccharomyces cerevisiae................................................................. 10 Zinc-dependent Metalloproteases of the M35 Clan.............................................. 12 Comparison of the Zps1p-like Proteins and the M35 Metalloproteases............... 14 2 RESULTS AND
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2004/0081648A1 Afeyan Et Al
    US 2004.008 1648A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0081648A1 Afeyan et al. (43) Pub. Date: Apr. 29, 2004 (54) ADZYMES AND USES THEREOF Publication Classification (76) Inventors: Noubar B. Afeyan, Lexington, MA (51) Int. Cl." ............................. A61K 38/48; C12N 9/64 (US); Frank D. Lee, Chestnut Hill, MA (52) U.S. Cl. ......................................... 424/94.63; 435/226 (US); Gordon G. Wong, Brookline, MA (US); Ruchira Das Gupta, Auburndale, MA (US); Brian Baynes, (57) ABSTRACT Somerville, MA (US) Disclosed is a family of novel protein constructs, useful as Correspondence Address: drugs and for other purposes, termed “adzymes, comprising ROPES & GRAY LLP an address moiety and a catalytic domain. In Some types of disclosed adzymes, the address binds with a binding site on ONE INTERNATIONAL PLACE or in functional proximity to a targeted biomolecule, e.g., an BOSTON, MA 02110-2624 (US) extracellular targeted biomolecule, and is disposed adjacent (21) Appl. No.: 10/650,592 the catalytic domain So that its affinity Serves to confer a new Specificity to the catalytic domain by increasing the effective (22) Filed: Aug. 27, 2003 local concentration of the target in the vicinity of the catalytic domain. The present invention also provides phar Related U.S. Application Data maceutical compositions comprising these adzymes, meth ods of making adzymes, DNA's encoding adzymes or parts (60) Provisional application No. 60/406,517, filed on Aug. thereof, and methods of using adzymes, Such as for treating 27, 2002. Provisional application No. 60/423,754, human Subjects Suffering from a disease, Such as a disease filed on Nov.
    [Show full text]
  • WO 2012/110564 Al 23 August 2012 (23.08.2012) P O P C T
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2012/110564 Al 23 August 2012 (23.08.2012) P O P C T (51) International Patent Classification: (74) Common Representative: NOVOZYMES A/S; CUD 3/386 (2006.01) C12N 9/58 (2006.01) Krogshoejvej 36, DK-2880 Bagsvaerd (DK). C12N 9/54 (2006.01) (81) Designated States (unless otherwise indicated, for every (21) International Application Number: kind of national protection available): AE, AG, AL, AM, PCT/EP2012/05261 1 AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, (22) International Filing Date: DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, 15 February 2012 (15.02.2012) HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, (25) Filing Language: English KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (26) Publication Language: English OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, SD, (30) Priority Data: SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, 1115471 1.3 16 February 201 1 (16.02.201 1) EP TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicant (for all designated States except US) : NO- (84) Designated States (unless otherwise indicated, for every VOZYMES A S [DK/DK]; Krogshoejvej 36, DK-2880 kind of regional protection available): ARIPO (BW, GH, Bagsvaerd (DK).
    [Show full text]
  • Architecture and Function of Metallopeptidase Catalytic Domains
    Cerdà-Costa et al. 1 !"#$%&'#&("')*+,)-(+#&%.+).-)/'&*00.1'1&%,*2')#*&*03&%#) ,./*%+2) ) 45"%*)6'",786.2&*)9):;)<*=%'")>./%28?@&$)A)) ) ) B".&'.032%2)C*DE)F'1*"&/'+&).-)G&"(#&("*0)H%.0.I3E)J.0'#(0*")H%.0.I3)K+2&%&(&').-) H*"#'0.+*L)6GK6E)H*"#'0.+*)G#%'+#')B*"ME)N'0%O)H(%0,%+IE)#P)H*0,%"%)?'%O*#L)QR8SQE)T8 UVUSV)H*"#'0.+*)WG1*%+X;) ) A)6.""'21.+,'+#'Y)'8/*%0Y)-OI"Z%D/D;#2%#;'2L)1$.+'Y)W[\]X)^\])USU)QV_L)-*OY)W[\]X)^\]) U\])^`^;) ) a$')*(&$."2)2&*&')&$'3)$*=')+.)#./1'&%+I)-%+*+#%*0)%+&'"'2&;) ) b'3c.",2Y)G&"(#&("*0)D%.#$'/%2&"3L)/'&d%+#%+)#0*+L)#*&*03&%#),./*%+2L)*#&%='82%&')#0'-&L) $3,".03&%#)'+d3/'2L)/*&"%O)/'&*00.1".&'*2'2L)*2&*#%+2L)!F!JL)*,*/*032%+2L)2'""*032%+2L) /'&*00.1".&'*2'L)/'&*00.1".&'%+*2';) ! Cerdà-Costa et al. 2 "#$%&"'%! a$')#0'*=*I').-)1'1&%,')D.+,2)D3)/'&*00.1'1&%,*2'2)WJB2X)%2)'22'+&%*0)-.")0%-';)a$'2')(D%e(%&.(2)'+d3/'2) 1*"&%#%1*&')%+)*00)/*f.")1$32%.0.I%#*0)1".#'22'2L)*+,)2.)&$'%"),'"'I(0*&%.+)0'*,2)&.),%2'*2'2)"*+I%+I)-"./)#*+#'") *+,)/'&*2&*2%2L)%+-0*//*&%.+L)*+,)/%#".D%*0)%+-'#&%.+)&.)+'(".0.I%#*0)%+2(0&2)*+,)#*",%.=*2#(0*"),%2.",'"2;)JB2) #0'*=')&$'%")2(D2&"*&'2)c%&$.(&)#.=*0'+&)%+&'"/',%*&')%+)*)2%+I0'82&'1)"'*#&%.+)%+=.0=%+I)*)2.0='+&)/.0'#(0'L)*) I'+'"*0) D*2'P*#%,L) *+,) *) /.+.8) .") ,%+(#0'*") #*&*03&%#) /'&*0) 2%&';) J.2&) /.+./'&*00%#) JB2) #./1"%2') *) 2$."&) /'&*08D%+,%+I)/.&%-)WNT<<NXL)c$%#$)%+#0(,'2)&c.)/'&*08D%+,%+I)$%2&%,%+'2)*+,)*)I'+'"*0)D*2'P*#%,)I0(&*/*&'L) *+,)&$'3)*"')I".(1',)%+&.)&$')d%+#%+)&"%D').-)JB2;)a$')0*&&'"),%=%,'2)/*%+03)%+&.)&$')I0(d%+#%+)*+,)/'&d%+#%+) #0*+2;) J'&d%+#%+2) #.+2%2&)
    [Show full text]
  • Funktionelle Charakterisierung Der Metalloprotease Neprilysin 4 Aus Drosophila Melanogaster
    Funktionelle Charakterisierung der Metalloprotease Neprilysin 4 aus Drosophila melanogaster Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) vorgelegt am Fachbereich Biologie/Chemie der Universität Osnabrück von Mareike Panz Osnabrück, 2011 Inhaltsverzeichnis Inhaltsverzeichnis 1. Zusammenfassung .............................................................................................................. 2 2. Einleitung ........................................................................................................................... 4 2.1. Die Klassifizierung der Metalloproteasen .................................................................. 4 2.2. Vertreter der ADAM Metalloproteasen in Drosophila .............................................. 4 2.3. Struktureller Aufbau und katalytische Aktivität der Neprilysine ............................... 8 2.4. Struktur und Funktion der intrazellulären Domäne der Neprilysine .......................... 9 2.5. Regulation der physiologischen Peptidhomöostase durch Neprilysine ................... 13 2.6. Muskelentwicklung und Physiologie in Drosophila melanogaster ......................... 18 2.7. Drosophila als Modell für Muskeldegeneration ...................................................... 23 3. Ergebnisse-Publikationen ................................................................................................. 27 3.1. The disintegrin and metalloprotease Meltrin from Drosophila forms oligomers via its protein binding domain and is regulated
    [Show full text]
  • Tesis Doctoral Alejandro Piñeiro Ugalde Oviedo 2011
    Universidad de Oviedo Departamento de Bioquímica y Biología Molecular Identificación de nuevos componentes del degradoma y estudio de la regulación biológica por miRNAs en un modelo animal deficiente en la metaloproteasa Face-1 Tesis Doctoral Alejandro Piñeiro Ugalde Oviedo 2011 Director de la Tesis Carlos López-Otín ABREVIATURAS, SIGLAS Y SÍMBOLOS ADAM metaloproteasa y desintegrina ADAMTS ADAM con dominios trombospondina cDNA DNA copia Ci curio dCTP desoxicitidina trifosfato DNA ácido desoxirribonucleico dNTPs desoxinucleósidos trifosfato GH hormona de crecimiento HGPS síndrome de Hutchinson-Gilford IGF-1 factor de crecimiento similar a insulina 1 kb kilobase MBS sitio de unión de microRNAs MMP metaloproteasa de matriz miRNA microRNA mRNA RNA mensajero pb pares de bases PCR reacción en cadena de la polimerasa RNA ácido ribonucleico ROS especie reactiva del oxígeno RISC complejo de silenciamiento inducido por RNA SSC tampón citrato sódico TAE tampón Tris/acetato con EDTA TBE tampón Tris/borato con EDTA TE tampón Tris con EDTA TIMP inhibidor tisular de metaloproteasa Tris tris(hidroximetil)-aminometano ÍNDICE Introducción ................................................................................................. 1 Los sistemas proteolíticos .................................................................................................. 4 Metaloproteasas ................................................................................................. 6 Aminopeptidasas ...............................................................................................
    [Show full text]
  • Crystal Structure Analysis of a Snake Venom Metalloproteinase In
    Crystal structure analysis of a snake venom metalloproteinase in complex with an inhibitor as basis for considerations on the proteolytic activity and the hemorrhagic mode of action INAUGURALDISSERTATION zur Erlangung der Doktorwürde der Fakultät für Chemie, Pharmazie und Geowissenschaften Albert-Ludwigs Universität Freiburg im Breisgau vorgelegt von Torsten Jens Lingott aus Bayreuth Freiburg im Breisgau November 2010 Tag der Bekanntgabe des Prüfungsergebnisses: 16.12.2010 Dekan: Prof. Dr. H. Hillebrecht Referentin: Prof. Dr. I. Merfort Korreferent: Prof. Dr. J. M. Gutiérrez Drittprüfer: Prof. Dr. A. Bechthold Parts of this thesis have been or are prepared to be published in the following articles: Lingott, T., Schleberger, C., Gutiérrez, J. M., and Merfort, I. (2009). High-resolution crystal structure of the snake venom metalloproteinase BaP1 complexed with a peptidomimetic: insight into inhibitor binding. Biochemistry 48 , 6166-6174. Wallnoefer, H. G., Lingott, T., Gutiérrez, J. M., Merfort, I., and Liedl, K. R. (2010). Backbone flexibility controls the activity and specificity of a protein-protein interface: Specificity in snake venom metalloproteases. J Am Chem Soc 132 , 10330-10337. Lingott, T. and Merfort, I. (xxxx). The catalytic domain of snake venom metalloproteinases - Sequential and structural considerations. in preparation. Wallnoefer, H. G.*, Lingott, T.*, Escalante, T., Ferreira, R. N., Nagem, R. A. P., Gutiérrez, J. M., Merfort, I., and Liedl, K. R. (xxxx). The hemorrhagic activity of P-I snake venom metalloproteinases
    [Show full text]
  • Multiple Architectures and Mechanisms of Latency in Metallopeptidase Zymogens
    Arolas et al. 1 Multiple architectures and mechanisms of latency in metallopeptidase zymogens Joan L. Arolas a,1, Theodoros Goulas 1, Anna Cuppari and F. Xavier Gomis-Rüth * Proteolysis Laboratory; Structural Biology Unit ("María-de-Maeztu" Unit of Excellence); Molecular Biology Institute of Barcelona (CSIC); Barcelona Science Park; c/Baldiri Reixac, 15-21; 08028 Barcelona (Catalonia, Spain). a Present address: Max F. Perutz Laboratories; University of Vienna; Campus Vienna Biocenter 5; 1030 Vienna (Austria). * Corresponding author: Tel.:(+34) 934 020 186; E-mail: [email protected]. 1 These authors contributed equally and share first authorship. ABSTRACT Metallopeptidases cleave polypeptides bound in the active-site cleft of catalytic domains through a general base/acid- mechanism. This involves a solvent molecule bound to a catalytic zinc and general regulation of the mechanism through zymogen-based latency. Sixty reported structures from eleven metallopeptidase families reveal that pro-segments, mostly N-terminally of the catalytic domain, block the cleft regardless of their size. Pro-segments may be peptides (5-14 residues), which are only structured within the zymogens, or large moieties (<227 residues) of one or two folded domains. While some pro-segments globally shield the catalytic domain through a few contacts, others specifically run across the cleft in the same or opposite direction of a substrate, making numerous interactions. Some pro-segments block the zinc by replacing the solvent with particular side chains, others use terminal α-amino or carboxylate groups. Overall, metallopeptidase zymogens employ disparate mechanisms that diverge even within families, which supports that latency is less conserved than catalysis. CONTENTS 1.
    [Show full text]