UNIVERSITY OF CALIFORNIA SAN DIEGO Regulation of Ubiquitylation and Ubiquitin-like Post-Translational Modifications A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry by Ryan James Lumpkin Committee in charge: Professor Elizabeth A. Komives, Chair Professor Rommie Amaro Professor Gouri Ghosh Professor Randy Hampton Professor Susan Taylor 2019 Copyright Ryan James Lumpkin, 2019 All rights reserved The dissertation of Ryan James Lumpkin is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California San Diego 2019 iii DEDICATION To my parents who have helped me get this far, my friends who supported me along the way, my PI who believed in me, and my cat who was always waiting for me. iv TABLE OF CONTENTS Signature page.....................................................................................................................iii Dedication............................................................................................................................iv Table of Contents..................................................................................................................v List of Abbreviations............................................................................................................xi Lists of Figures...................................................................................................................xiii Lists of Tables....................................................................................................................xvi Acknowledgements...........................................................................................................xvii Vita...................................................................................................................................xviii Abstract of the Dissertation................................................................................................xix Chapter I Introduction..................................................................................................1 A. Proteome Complexity and Post-Translational Modifications........................2 B. Ubiquitylation................................................................................................2 C. Ubiquitin-like proteins...................................................................................3 D. E3 Ubiquitin Ligases......................................................................................3 E. The ASB family of Protein Receptors...........................................................5 F. References.....................................................................................................6 Chapter II Site-specific identification and quantitation of endogenous SUMO modifications under native conditions.......................................................8 A. Introduction....................................................................................................9 B. Materials and methods.................................................................................12 v 1. WaLP.....................................................................................................12 2. SENP1 and SENP2 deSUMOylation....................................................13 3. Enrichment of KGG-containing peptides..............................................14 4. MS analysis of in vitro deSUMOylation experiment............................15 5. SILAC quantitation of Ub and SUMO..................................................17 6. Preparation of tissue samples................................................................18 7. nLC-MS/MS for SILAC experiments...................................................19 8. Data Availability...................................................................................21 C. Results.........................................................................................................21 1. SUMOylation site profiling by WaLP digestion...................................21 2. Identification of novel SUMOylation sites using WaLP.......................22 3. Validation of SUMOylation sites using in vitro deSUMOylation.........24 4. Parallel Ub and SUMO site identification.............................................29 5. Parallel quantification of Ub and SUMO sites......................................29 6. Identification of SUMOylation in tissue samples..................................34 D. Discussion....................................................................................................35 E. Acknowledgements......................................................................................36 F. References...................................................................................................36 Chapter III DECA, a comprehensive, automatic post-processing program for HDX-MS data.............................................................................................41 A. Introduction..................................................................................................42 B. Methods........................................................................................................44 vi 1. Software Design.....................................................................................44 2. Data Format............................................................................................45 3. Data Analysis.........................................................................................45 4. Sample Dataset.......................................................................................46 5. Fitting of deuterium uptake plots...........................................................46 C. Results..........................................................................................................47 1. Graphical User Interface........................................................................47 2. File Merging...........................................................................................47 3. Back Exchange Correction..............................................................................................48 4. Generation of coverage maps from back-exchange corrected data........51 5. Generation of publication-quality deuterium uptake plots from back-exchange corrected data................................................................51 6. Comparison of overlapping peptides for increased resolution of deuterium uptake data............................................................................52 7. Visualizing Highest Resolution Deuterium Exchange Data..................52 8. Statistical Significance...........................................................................54 9. Data Export............................................................................................56 D. Discussion....................................................................................................57 E. Data Accessibility........................................................................................58 F. Acknowledgements......................................................................................58 G. References....................................................................................................59 vii Chapter IV Structure and dynamics of the ASB9 CUL-RING E3 Ligase................62 A. Introduction..................................................................................................63 B. Materials and methods.................................................................................65 1. Expression Vectors................................................................................65 2. Protein Expression..................................................................................65 3. Protein Purification.................................................................................66 4. Hydrogen Deuterium Exchange.............................................................67 5. Cryo-EM sample preparation and data collection..................................69 6. Homology Modeling..............................................................................74 C. Results..........................................................................................................74 1. The Structure of the CKB-ASB9-ELOB-ELOC Complex by Cryo-EM................................................................................................74 2. Structure of full-length CUL5-RBX2....................................................75 3. Full Model of the ASB9-CRL...............................................................78 4. HDX-MS mapping of the protein-protein interfaces in the CKB-bound ASB9-CRL........................................................................78 5. HDX-MS reveals crosstalk between non-adjacent subunits of the E3 ligase.................................................................................................82 D. Discussion....................................................................................................86 1. Structure of the substrate-bound ASB9-CRL based on Cryo-EM data.........................................................................................................86 2. Allosteric Changes in the ASB9-CRL...................................................89 E. Acknowledgements......................................................................................91