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Regulation of Nrf2 by a Keap1-Dependent E3 Ubiquitin Ligase

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Regulation of Nrf2 by a Keap1-Dependent E3 Ubiquitin Ligase REGULATION OF NRF2 BY A KEAP1-DEPENDENT E3 UBIQUITIN LIGASE A Dissertation presented to The Faculty of the Graduate School at the University of Missouri-Columbia In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy by SHIH-CHING (JOYCE) LO Dr. Mark Hannink, Dissertation Adviser DECEMBER 2007 The undersigned, appointed by the Dean of the Graduate School, have examined the dissertation entitled REGULATION OF NRF2 BY A KEAP1-DEPENDENT E3 UBIQUITIN LIGASE Presented by Shih-Ching (Joyce) Lo A candidate for the degree of Doctor of Philosophy And hereby certify that in their opinion it is worthy of acceptance. Mark Hannink Thomas Guilfoyle David Pintel Grace Sun Richard Tsika DEDICATION Okay. Mom, I got you the Ph.D. you asked, at the place you picked. Can I now please do something else? Dad, this is to you, too… you accomplice! ACKNOWLEDGEMENTS Dr. Mark Hannink. This work would never have been possible without his guidance, criticism and encouragement. His sheer enthusiasm and critical thinking of science are the most valuable lessons that I could ever obtain during my graduate studies. I should also extend my appreciation to my committee members, Drs. Thomas Guilfoyle, David Pintel, Grace Sun and Richard Tsika, as well as Drs. Lesa Beamer, Joan Conaway, Beverly DaGue, Marc Johnson, Alan Diehl and Michael Henzl, for their insightful advice and thoughtful comments on this work. I thank the present and previous members in Dr. Hannink’s laboratory, including Drs. Donna Zhang, Rick Sachdev, Sang-Hyun Lee, Xuchu Li, as well as Brittany Angle, Carolyn Eberle, Zheng Sun, Jordan Wilkins, Marquis Patrick, Xiaofang Jin, Casey Williams, Joel Pinkston, Julie Unverferth, and Benjamin Creech. They provided me with technical help and inspiring discussions on this work. Toward completion of my graduate study, I have also received copious amounts of help and encouragement from friends and colleagues both on campus and elsewhere. While it is not possible to list all the names due to space constraint, I would like to express my appreciation to them all. ii TABLES OF CONTENTS ACKNOWLEDGEMENTS ..…………………………………………………………..…ii LIST OF TABLE ..………………………………………………………………………vii LIST OF FIGURE ……………………………………………………………………...viii ABSTRACT …..………………………………………………………………………...xii CHAPTER I. INTRODUCTION …..……………………………………………………………..…...1 A. A snapshot of the ubiquitin/proteasome system (UPS) B. General features of cullin-RING ligases (CRLs) B.1. The cullin family B.2. The RING finger proteins in cullin-RING ligases B.3. Substrate adaptors of cullin-RING ligases B.4. Recognition of degradation signals in substrates by substrate adaptors B.5. Architecture of cullin-RING ligases and the ‘positioning’ model B.6. Polygamy among substrates and substrate adaptors, and the ‘processivity’ model B.7. Cyclic assembly and disassembly of cullin-RING ligase complex, and the major mediator CAND1 C. Modulation of cullin-RING ligase activity toward substrates C.1. Modulation of substrate binding events C.2. Dimerization of culiin-RING ligases C.3. Dynamic subcellular localization of cullin-RING ligases: circuit riders in CRL systems. iii C.4. Degradation of substrate adaptors C.5. Nedd8, and regulation of cullin-RING ligases via neddylation C.6. COP9 signalosome (CSN) and its role in regulation of CRLs C.7. Multilateral interplay among regulatory factors in CRLs D. Nrf2 and Keap1 D.1. The transcription factor Nrf2 D.2. The modular protein Keap1, and its sensor system E. Regulation of Nrf2 by Keap1 E.1. Ubiquitination and degradation of Nrf2 by Keap1-dependent Cul3-RING ligases E.2. Structure of the Nrf2-Keap1 complex, and its binding stoichiometry E.3. Dynamic subcellular localization of the Nrf2-Keap1 system F. A preview of the following chapters in this dissertation II. KEAP1 IS A REDOX-REGULATED SUBSTRATE ADAPTOR PROTEIN FOR A CUL3-DEPENDENT UBIQUITIN LIGASE COMPLEX …………………..………….90 Abstract Introduction Materials and methods Results Discussion References III. UBIQUITINATION OF KEAP1, A BTB-KELCH SUBSTRATE ADAPTOR PROTEIN FOR CUL3, TARGETS KEAP1 FOR DEGRADATION BY A PROTEASOME-INDEPENDENT PATHWAY ……………………………………....135 iv Abstract Introduction Materials and methods Results Discussion References IV. CAND1-MEDIATED SUBSTRATE ADAPTOR RECYCLING IS REQUIRED FOR EFFICIENT REPRESSION OF NRF2 BY KEAP1 ………………………...…...174 Abstract Introduction Materials and methods Results Discussion References V. STRUCTURE OF THE KEAP1;NRF2 INTERFACE PROVIDES MECHANISTIC INSIGHT INTO NRF2 SIGNALING …………………………………………..……..213 Abstract Introduction Materials and methods Results Discussion References v VI. PGAM5, A BCL-XL-INTERACTING PROTEIN, IS A NOVEL SUBSTRATE FOR THE REDOX-REGULAETD KEAP1-DEPENDENT UBIQUITIN LIGASE COMPLEX …………………………………………………………………………….258 Abstract Introduction Materials and methods Results Discussion References VII. REGULATION OF ANTI-OXIDANT GENE EXPRESSION BY A MITOCHONDRIAL-LOCALIZED COMPLEX CONTAINING KEAP1, NRF2 AND PGAM5 ………………………………………………………………………………...302 Abstract Introduction Materials and methods Results Discussion References VIII. SUMMARY AND FUTURE DIRECTIONS ……………………………………341 VITA …………………………………………………………………………………...373 vi LIST OF TABLES Table Page II-1. Half-lives of the HA-Nrf2 proteins in MDA-MB-231 cells …..………………..115 V-1. Table V-1 ……………………………………………………………………….235 V-2. Table V-2 …………………………………………………………………..…...237 V-3. Table V-3 ……………………………………………………………………….238 VI-1. Table VI-1………………………………………………………………………283 VII-1. Table VII-1 ...……...……………………………………………………………324 vii LIST OF FIGURES Figure Page I-1. Architecture of cullin-RING ligases …………………………………………….63 I-2. The conundrum of a 50-60 Å gap in cullin-RING ligases ………………………65 I-3. A dimeric CRL supercomplex ……………………………………………..…….67 I-4. A tentative model for the cyclic assembly and disassembly of a Cul1-RING ligase by neddylation and deneddylation of Cul1 ……………………………………...69 I-5. Domain structures and motifs of Nrf2 and Keap1 ……………………………….71 I-6. Three-dimensional structures of the Kelch β-propeller bound with the Nrf2- derived peptide …………………………………………………………….…….73 II-1. Domain structures of Nrf2 and Keap1 …...…………..…………………………116 II-2. Association of Keap1 with Cul3 mediates Keap1-dependent ubiquitination of Nrf2 …………………………………………………………………….……….118 II-3. Keap1 functions as a substrate adaptor for a Cul3-Rbx1 E3 ubiquitin ligase complex ………………………………………………………………….……...120 II-4. Mutations within BTB domain of Keap1 decrease ubiquitination of Nrf2 but increase ubiquitination of Keap1 ……………………………………….…...…..122 II-5. Identification of lysine residues in Nrf2 targeted for ubiquitination by Keap1 …124 II-6. Oxidative stress results in reduced ubiquitination of Nrf2 by a Keap1-dependent Cul3-RING ligase ………………………………………………………..……...126 II-7. Oxidative stress does not cause dissociation of Nrf2 from Keap1 ………….......128 III-1. Autoubiquitination is a key feature for BTB-Kelch proteins that assemble into viii Cul3-RING ligase complexes …………….………………………………….....159 III-2. Autoubiquitination of Keap1 by a Cul3-Rbx1 subcomplex ………………...…..161 III-3. Increased ubiquitination results in proteasome-independent degradation of Keap1 …………………………………………………………………………….163 III-4. Quinone-induced oxidative stress increases ubiquitination and turnover of Keap1 …………………………………………………………………………….165 III-5. Quinone-induced degradation of Keap1 is enhanced in cells deficient in glutathione Biosynthesis ……………………………………………………………………...167 III-6. Quinone-induced oxidative stress induces a switch in ubiquitination from substrate to substrate adaptor ………………………………………………………………169 IV-1. CAND1 competes with Keap1 for binding to Cul3 ……………………………..195 IV-2. Increased CAND1 expression reduces Keap1-dependent ubiquitination and degradation of Nrf2 ………………………………………………………………197 IV-3. CAND1 is required for efficient repression of Nrf2 by Keap1 ………………….199 IV-4. CAND1 is required for Nrf2 turnover, and contributes to repression of ARE- dependent gene expression ………………………………………………………201 IV-5. Nedd8 is required for ubiquitination of Nrf2 by a Keap1-Cul3 ligase complex ...203 IV-6. Neddylation of Cul3 on Lys 712 is required for efficient ubiquitination and degradation of Nrf2 by a Keap1-Cul3 ligase complex …………………………..205 IV-7. Catalytic activity of a Keap1-Cul3 ligase complex per se does not require neddylation on the Lys 712 of Cul3 ……………………………………………...207 V-1. Sequence alignments among orthologues of Keap1 or Nrf2 ……………………239 V-2. Specific bindings between Keap1 and the Nrf2-derived peptides ………………241 ix V-3. Three-dimensional structure of the complex consisting of a Kelch β-propeller domain and an Nrf2-derived peptide ……………………………………………243 V-4. Intermolecular and intramolecular contacts within the Kelch domain and the bound Nrf2 peptide ……………………………………………………………………..245 V-5. Identification of amino acids within the Kelch domain of Keap1 that are required for binding to Nrf2 ………………………………………………………………247 V-6. Keap1 forms a homodimer capable of binding two Nrf2 molecules ……………249 V-7. Phosphorylation of the conserved threonine within the DxETGE motif in Nrf2 disrupts binding to Keap1 ……………………………………………………….251 VI-1. Identification of PGAM5 as an interaction partner of Keap1 …………………..284 VI-2. Featured domains and motifs in PGAM5, a member in phosphoglycerate mutase family …………………………………………………………………………....286 VI-3. Subcellular localization of N-terminal FLAG-tagged PGAM5 isoforms ………289 VI-4. Identification of the interaction interface between Keap1 and PGAM5 ………..291 VI-5. PGAM5-L is targeted for ubiquitination by a Keap1-dependent Cul3-RING ligase …………………………………………………………………………….293
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