UNIVERSITY OF CALIFORNIA, SAN DIEGO Characterizing Postranslational Regulatory Mechanisms of the Ubiquitin Proteasome System A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry by Frankie Robert Gonzales Committee in charge: Professor Gentry Patrick, Chair Professor Elizabeth Komives, Co-Chair Professor Gourisankar Ghosh Professor Judy Kim Professor Dong-Er Zhang Professor Brian Zid 2017 Copyright Frankie Robert Gonzales, 2017 All rights reserved The dissertation of Frankie Robert Gonzales is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Co-Chair Chair University of California, San Diego 2017 iii DEDICATION I dedicate this work to my mom who has supported me in every aspect of my life. And to the countless educators who have taken special interest in my and helped advance me and my career far further than I had ever expected. Mitch Markowitz, Dr. Jennifer Whiles- Lillig, and Dr. Betsy Komives have been among these mentors who have propped me up and I owe my entire educational path and career to them. iv TABLE OF CONTENTS Signature Page ................................................................................................................... iii Dedication .......................................................................................................................... iv Table of Contents .................................................................................................................v List of Abbreviations ......................................................................................................... ix List of Figures ......................................................................................................................x List of Tables .................................................................................................................... xii Acknowledgements .......................................................................................................... xiii Vita ................................................................................................................................... xiv Abstract of the Dissertation ............................................................................................. xvi CHAPTER I: INTRODUCTION .....................................................................................1 A. Protein degradation in eukaryotic cells .....................................................................2 B. Structure of the 26S proteasome. ................................................................................3 C. Functions of the ubiquitin proteasome system. .........................................................4 D. Regulation of the UPS. ..................................................................................................6 E. Protein homeostasis at synapses...................................................................................6 F. References ......................................................................................................................9 CHAPTER II: INVESTIGATING PROTEASOME SUBUNIT RPT6 PHOSPHORYLATION DYNAMICS USING NOVEL MOUSE LINES THAT ABROGATE OR MIMIC PHOSPHORYLATION .....................................................12 A. Abstract .......................................................................................................................14 B. Introduction ................................................................................................................15 C. Materials and Methods ..............................................................................................17 Generation of S120A and S120D knock-in mice ......................................................... 17 v Antibodies and reagents ....................................................................................................... 17 Proteasome purification ....................................................................................................... 18 Peptidase and ATPase assays ............................................................................................. 18 Western blot and native gel assays ................................................................................... 19 Slice Electrophysiology ......................................................................................................... 19 Whole-cell Electrophysiology ............................................................................................. 21 Behavior ...................................................................................................................................... 21 Spine density, and immunohistochemistry ................................................................... 22 Preparation and transfection of organotypic slice cultures ................................... 22 Time-Lapse Imaging and Image Analysis for spine dynamics ............................. 22 D. Results .........................................................................................................................23 1. Generation, purification, and analysis of Rpt6 S120A and S120D mutant proteasome composition and activity .............................................................................. 23 2. Abrogation of phosphorylation dynamics do not effect basal synaptic transmission nor LTP paradigms of synaptic plasticity. ......................................... 25 3. Small changes in proteasome ATPase and peptidase kinetic activity have no effect on fear conditioning, nor context discrimination paradigms of learning and memory ............................................................................................................ 27 4. Elimination of phosphorylation dynamics do not alter basal spine density 28 5. Elimination of phosphorylation dynamics does not have a significant effect on spine outgrowth ................................................................................................................. 29 E. Discussion ....................................................................................................................30 E. References ...................................................................................................................32 vi F. Figures and Figure Legends .......................................................................................35 CHAPTER III: PROTEASOME PHOSPHORYLATION REGULATES COCAINE-INDUCED SENSITIZATION ....................................................................44 A. Abstract ........................................................................................................................46 B. Introduction .................................................................................................................47 C. Materials and methods ...............................................................................................48 Generation of S120D Knockin mice ................................................................................ 48 Antibodies and Reagents ...................................................................................................... 49 Neuronal cultures .................................................................................................................... 49 Proteasome activity assays .................................................................................................. 49 Western blot analysis ............................................................................................................. 50 qPCR ............................................................................................................................................ 50 Behavioral Sensitization ....................................................................................................... 51 Histology ..................................................................................................................................... 51 Data Analysis ............................................................................................................................ 52 D. Results ..........................................................................................................................52 1. Cocaine increases Rpt6 S120 phosphorylation and proteasome activity ..... 52 2. Generation of Rpt6 S120D Knockin (KI) mice ....................................................... 53 3. Rpt6 S120 phosphorylation and peptidase activity is increased in NAc and PFC in cocaine treated wild type mouse brains, but not in S120D mutant mice ............................................................................................................................................... 54 4. Behavioral sensitization is completely absent in Rpt6 S120D KI mutant mice ............................................................................................................................................... 55 E. Discussion .....................................................................................................................56 vii F. Conclusions ..................................................................................................................59 G. References ....................................................................................................................59 CHAPTER IV: PHOSPHORYLATION OF THE 19S