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Regulation of Human 69-Kda Choline Acetyltransferase Protein Stability and Function by Molecular Chaperones and the Ubiquitin- Proteasome System

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Regulation of Human 69-Kda Choline Acetyltransferase Protein Stability and Function by Molecular Chaperones and the Ubiquitin- Proteasome System Western University Scholarship@Western Electronic Thesis and Dissertation Repository 1-25-2017 2:00 PM Regulation of Human 69-kDa Choline Acetyltransferase Protein Stability and Function by Molecular Chaperones and the Ubiquitin- Proteasome System Trevor M. Morey The University of Western Ontario Supervisor Rylett, R. Jane The University of Western Ontario Graduate Program in Physiology A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Trevor M. Morey 2017 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Molecular and Cellular Neuroscience Commons Recommended Citation Morey, Trevor M., "Regulation of Human 69-kDa Choline Acetyltransferase Protein Stability and Function by Molecular Chaperones and the Ubiquitin-Proteasome System" (2017). Electronic Thesis and Dissertation Repository. 5218. https://ir.lib.uwo.ca/etd/5218 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. ABSTRACT: The enzyme choline acetyltransferase (ChAT) mediates synthesis of the neurotransmitter acetylcholine required for cholinergic neurotransmission. ChAT mutations are linked to congenital myasthenic syndrome (CMS), a rare neuromuscular disorder. One CMS-related mutation, V18M, reduces ChAT enzyme activity and cellular protein levels, and is located within a highly-conserved N-terminal proline-rich motif at residues 14PKLPVPP20. It is currently unknown if this motif regulates ChAT function. In this thesis, I demonstrate that disruption of this proline-rich motif in mouse cholinergic SN56 cells reduces both the protein levels and cellular enzymatic activity of mutated P17A/P19A- and V18M-ChAT. The cellular loss of mutant ChAT protein appears to be a result of increased proteasome- dependent degradation due to enhanced ChAT ubiquitination. Using a novel fluorescent- biorthogonal pulse-chase protocol, I determined that the cellular protein half-life of P17A/P19A-ChAT (2.2 h) is substantially reduced compared to wild-type ChAT (19.7 h), and that proteasome inhibition by MG132 treatment increases the half-life and steady-state levels of ChAT protein. By proximity-dependent biotin identification (BioID), co- immunoprecipitation, and in situ proximity-ligation assay (PLA), I identified the heat shock proteins HSC/HSP70 and HSP90 as novel ChAT protein-interactors that are enriched in cells expressing mutant P17A/P19A-ChAT. Pharmacological inhibition of these HSPs by treatment with the HSC/HSP70 inhibitors 2-phenylethynesulfonamide (PES) or VER- 155008, or the HSP90 inhibitor 17-AAG reduced cellular ChAT activity and solubility, and enhanced ubiquitination and proteasomal loss of ChAT protein. The effects of HSP inhibition were greatest for mutant P17A/P19A- and V18M-ChAT. While I observed that ChAT interacts in situ with the HSP-associated E3 ubiquitin ligase CHIP, siRNA-mediated knock-down of CHIP had no effect on ChAT protein levels. Inhibition of HSC/HSP70 by PES treatment sensitized ChAT to H2O2-induced insolubilization, and ChAT ubiquitination was enhanced following H2O2 treatment. Lastly, inhibition of the endoplasmic reticulum (ER)- and HSP-associated co-chaperone Cdc48/p97/Valosin-containing protein (VCP) prevented the degradation of ubiquitinated ChAT. Together my results identify novel mechanisms for the functional regulation of wild-type and CMS-related mutant ChAT by ii multiple molecular chaperones and the ubiquitin-proteasome system that, importantly, may have broader implications for ChAT function during cellular stress and disease. KEYWORDS: Choline acetyltransferase (ChAT), molecular chaperones, protein degradation, protein quality control, proteasome, ubiquitination, click chemistry, BioID, proximity-ligation assay (PLA), HSC/HSP70, HSP90, CHIP/Stub1, Cdc48/p97/VCP, oxidative stress. iii CO-AUTHORSHIP STATEMENT The studies detailed within this thesis were completed by Trevor M. Morey in the laboratory of Dr. R. Jane Rylett with the following co-author assistance: 1. Shawn Albers, M.Sc., assisted with cellular ChAT activity assays. 2. Dr. Brian Shilton, Department of Biochemistry (UWO), provided resources for, assisted with, and provided expertise for studies utilizing bacterially-expressed and purified recombinant ChAT protein. 3. Claudia Seah (Dr. Pasternak, UWO), assisted with in situ PLA experiments by performing confocal microscopy. 4. Dr. Warren Winick-Ng assisted with in situ PLA experiments by performing confocal microscopy and assisting with PLA optimization. Initial experiments related to the effects of H2O2-induced oxidative stress on ChAT protein function were designed, completed, and analyzed previously by Shawn Albers, M.Sc.1 All other experiments were designed, and data was analyzed and interpreted by Trevor M. Morey and Dr. R. Jane Rylett. Experiments were performed by Trevor M. Morey in collaboration with the above co-authors. All authors contributed to the preparation of manuscripts for publication. This thesis was written by Trevor M. Morey. 1Albers, Shawn. “The role of choline acetyltransferase variants in Alzheimer’s disease models” (2013). Electronic Thesis and Dissertation Repository. 1844. http://ir.lib.uwo.ca/etd/1844. iv PUBLICATION STATEMENT This thesis contains text, data, and figures that have been published in the followed peer- reviewed first-author publications: 1. Morey, T.M., Winick-Ng, W., Seah, C., and Rylett, R.J. (2017). Chaperone-mediated regulation of choline acetyltransferase protein stability and activity by HSC/HSP70, HSP90 and p97/VCP. Front Mol Neurosci. 10:415. doi: 10.3389/fnmol.2017.00415. 2. Morey, T.M., Albers, S., Shilton, B.H., and Rylett, R.J. (2016). Enhanced ubiquitination and proteasomal-degradation of catalytically-deficient human choline acetyltransferase mutants. J. Neurochem. 137, 630-646. v ACKNOWLEDGEMENTS I am extremely grateful for the opportunity to complete this Ph.D thesis under the supervision of Dr. R. Jane Rylett. Her continued support, expertise, insight, and guidance throughout my studies provided me with the opportunity to learn, develop critical research skills, and most importantly to be successful during my time in her laboratory. I wish Jane all the best in her future research and look forward to future collaborations. I would also like to thank my advisory committee members, Drs. Lina Dagnino, John Di Guglielmo, Susan Meakin, and Brian Shilton for providing invaluable critique, direction, and suggestions throughout my degree. Special thanks are extended to Dr. John Di Guglielmo for his generous read-through of my preliminary thesis and for providing his thoughts and expertise. To Drs. Brian Shilton and Martin Duennwald, thank you for providing me with your scientific and technical expertise that ultimately helped me design, implement, and interpret certain experiments completed within this thesis. To Dr. Warren Winick-Ng, my colleague and friend, thank you for the many stimulating conversations, both research and non-research related, as well as for the laughs and good times while at work. I wish you all the best and success on your post-doctoral adventures in Berlin and beyond. In addition, I thank all my peers, friends, and colleagues for fostering a joyful, successful, and memorable learning and working experience. To my parents Debi McKellar and Gary Morey - thank you for always being there to lend an ear, for your love and guidance, and for your belief that anything is possible. Lastly, to my wife Suze, thank you for joining me on this incredible journey and for always motivating me to better myself, to take chances, and to always give my 100% in all things. Without your unwavering love and support, your patience and understanding, and your faith in me, this study would not have been possible. I hope one day, in your future endeavours, to extend the same supports as that have given to me throughout my studies. Funding for this study was provided to R.J.R. by the Canadian Institutes of Health Research. T.M.M is a two-time recipient of the Ontario Graduate Scholarship. vi TABLE OF CONTENTS Abstract…………………………………………………………….……………………...ii Keywords…………………………………………………………………………………iii Co-authorship Statement………………………………………………………………….iv Publication Statement………………………………………………………………….…..v Acknowledgements…………………………………………………………………….....vi Table of Contents…………………………………………………………………………vii List of Figures……………………………………………………………………….……xii List of Abbreviations……………………………………………………………...…….xvii Chapter 1: Introduction……………………………………………………….………...….1 1.1: Proteostasis and protein quality control mechanisms……………….…………..1 1.2: The ubiquitin-proteasome system…………………..……………….…………..3 1.2.1: The proteasome…………………………………………..…….……….3 1.2.2: Ubiquitin…………………………….………………………………….3 1.2.3: Ubiquitination mechanisms……………………………………….……5 1.2.4: The ubiquitin code………………………………………..…………….8 1.2.5: Autophagy-lysosomal degradation………………………...………….10 1.3: Protein folding and molecular chaperones…………………….………….……11 1.3.1: Molecular chaperones overview………………....……………………11 1.3.2: Heat shock proteins and the heat shock response…………..…………..13 vii 1.3.3: The HSP chaperone network……………………………….…...……..14 1.3.4: HSC/HSP70………………………………………….………………..17 1.3.5: HSP90…………………………………………………………………19 1.3.6: Ubiquitin E3 ligase CHIP……………………………….………..……23 1.4: Molecular chaperones in human disease…………………………..….………..25
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