Structural modeling and functional characterization of mammalian cytosolic carboxypeptidases 2 and 3 Doctoral thesis presented by Olívia Tort Regàs for the degree of PhD in Biochemistry, Molecular Biology and Biomedicine from the Universitat Autònoma de Barcelona Institut de Biotecnologia i Biomedicina Unitat d’Enginyeria de Proteïnes i Proteòmica Thesis supervised by Prof. Francesc Xavier Avilés Puigvert, Dra. Julia Lorenzo Rivera and Dr. Sebastian Tanco Olívia Tort Regàs Prof. Francesc Xavier Dra. Julia Lorenzo Rivera Dr. Sebastian Tanco Avilés Puigvert Barcelona, September 2014 Table of contents TABLE OF CONTENTS LIST OF FIGURES ...................................................................................... 1 LIST OF TABLES ........................................................................................ 3 LIST OF ABBREVIATIONS .......................................................................... 6 PREFACE .................................................................................................. 9 CHAPTER I: INTRODUCTION ................................................................... 12 1 PEPTIDASES ...................................................................................................... 12 1.1 Nomenclature of peptidases ..................................................................................... 12 1.2 Functions of proteolytic enzymes .............................................................................. 13 1.2.1 Proteases in life processes .............................................................................................. 13 1.2.2 Peptidases as drug targets .............................................................................................. 14 1.3 Specificity of proteolytic enzymes ............................................................................. 14 1.3.1 Diversity of proteolytic processes .................................................................................. 14 1.3.2 Peptide bound hydrolysis ............................................................................................... 15 1.3.3 Peptidases substrate specificity ...................................................................................... 15 1.4 Classification of peptidases ....................................................................................... 16 1.4.1 Point of cleavage ............................................................................................................. 17 1.4.2 Catalytic mechanism ....................................................................................................... 17 1.4.3 Structural and evolutionary relationships ...................................................................... 19 1.5 Carboxypeptidases.................................................................................................... 20 1.5.1 Structure and mechanism ............................................................................................... 22 1.5.2 Specificity and the active site ......................................................................................... 23 1.5.3 Functions of carboxypeptidases ..................................................................................... 24 2 MICROTUBULES ................................................................................................ 26 2.1 Types of microtubules ............................................................................................... 26 2.2 Microtubules: structure and dynamics ....................................................................... 28 2.2.1 The tubulin superfamily .................................................................................................. 28 2.2.2 Tubulin dynamics ............................................................................................................ 29 2.2.3 Microtubules in organization and transport ................................................................... 31 2.3 Cilia and flagella........................................................................................................ 33 2.3.1 Basal body and the axoneme .......................................................................................... 33 2.3.2 Motile cilia/flagella and ciliopathies ............................................................................... 35 2.3.3 Primary cilia .................................................................................................................... 36 3 THE TUBULIN CODE ........................................................................................... 39 3.1 Tubulin isoforms: a first layer of diversity .................................................................. 39 3.1.1 α-Tubulin isotypes .......................................................................................................... 40 3.1.2 β-Tubulin isotypes........................................................................................................... 42 3.2 Tubulin posttranslational modifications increase microtubule diversity ...................... 43 3.2.1 Acetylation ...................................................................................................................... 45 3.2.2 Tyrosination/detyrosination ........................................................................................... 46 3.2.3 Δ2- and Δ3-tubulin .......................................................................................................... 48 3.2.4 Polyglutamylation ........................................................................................................... 49 3.2.5 Polyglycylation ................................................................................................................ 52 4 CYTOSOLIC CARBOXYPEPTIDASES ...................................................................... 53 4.1 Structural domains of CCPs ....................................................................................... 54 4.1.1 Crystal structure of CCPs ................................................................................................ 56 4.2 CCP1 and the pcd mouse model ................................................................................ 57 4.2.1 Hyperglutamylation ........................................................................................................ 57 4.2.2 Neuronal death of the Purkinje cells .............................................................................. 58 4.3 Deglutamylases in the CCP family .............................................................................. 59 4.3.1 CCPs in pathologies ......................................................................................................... 59 4.3.2 CCPs regulation and interaction partners ....................................................................... 60 4.4 CCPs and axoneme of cilia and flagella ...................................................................... 61 4.4.1 Male infertility and CCP disfunctions .............................................................................. 62 OBJECTIVES ............................................................................................................ 63 CHAPTER II: EXPERIMENTAL PROCEDURES ............................................ 67 1 CLONING AND DNA METHODS .......................................................................... 67 1.1 Polymerase chain reaction (PCR) ............................................................................... 67 1.2 Digestion with restriction enzymes ............................................................................ 68 1.3 Ligation and transformation ...................................................................................... 69 1.4 Site-directed mutagenesis ......................................................................................... 69 1.5 DNA purification and sequencing .............................................................................. 70 2 CELL CULTURE ................................................................................................... 70 2.1 Cell lines and maintenance ........................................................................................ 70 2.2 Transfection ............................................................................................................. 71 2.3 shRNA analysis ......................................................................................................... 71 2.4 Cell lysis .................................................................................................................... 71 3 PROTEIN PURIFICATION .................................................................................... 72 3.1 Affinity purification with Strep-tag ............................................................................ 72 3.2 Size exclusion chromatography ................................................................................. 72 4 PROTEIN ANALYSIS ........................................................................................... 73 4.1 1D SDS-PAGE ............................................................................................................ 73 4.2 2D SDS-PAGE ............................................................................................................ 74 4.3 Western-blot ............................................................................................................ 74 5 CONFOCAL