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Characterizing the Bcl-2 Associated Athanogene 5 Interactome in the Context of Parkinson’S Disease

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Characterizing the Bcl-2 Associated Athanogene 5 Interactome in the Context of Parkinson’S Disease Characterizing the Bcl-2 Associated Athanogene 5 Interactome in the Context of Parkinson’s Disease by Erik Loewen Friesen A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Laboratory Medicine & Pathobiology University of Toronto © Copyright by Erik Loewen Friesen 2018 Characterizing the Bcl-2 Associated Athanogene 5 Interactome in the Context of Parkinson’s Disease Erik Loewen Friesen Master of Science Department of Laboratory Medicine & Pathobiology University of Toronto 2018 Abstract Aberrant alpha-synuclein aggregation is associated with the onset and progression of Parkinson’s disease (PD). This has made molecular chaperones, a class of proteins responsible for maintaining proteostasis, an enticing therapeutic target. BAG5 is a co-chaperone protein that inhibits the chaperone, Hsp70, and promotes PD-like alpha-synuclein aggregation and neurodegeneration. The mechanisms of how BAG5 impairs proteostasis and promotes apoptosis are unclear. The purpose of this project was to characterize the BAG5 interactome to guide further studies of its role in physiological and disease states. A novel interaction between BAG5 and the autophagy adaptor protein, p62, was discovered and investigated, as it pointed to a potential mechanism by which BAG5 could modify alpha-synuclein aggregation. p62 reduced and BAG5 enhanced alpha-synuclein self-association in vitro. BAG5 also promoted p62 stability, suggesting a function of the interaction on other p62-dependent proteostasis pathways. ii Acknowledgments I would like to thank all of the members of the Kalia and Lozano labs for their continuous support throughout this process. This has been a tremendous learning experience that was made very positive by the team members that have helped me along the way. A special thanks goes to Hien Chau for teaching me virtually every basic science technique needed to complete this thesis, and being a great mentor to me for over three years. Thanks also goes to my fellow graduate students, Mitch, Greg, Krystal, Shirley, Stanley and Kevin, for all the support and good times we have had throughout my time in the lab. I would also like to acknowledge the entire group of graduate students and postdocs on the 8th floor of Krembil, who continuously made this process fun and worthwhile. A special thanks also goes to Mitch De Snoo for his extensive technical assistance and helping me get through the thick and thin of laboratory research. The continuous support from both Suneil and Lorraine Kalia has been instrumental to my success in this program. Thank you for having the trust and patience to allow me to independently explore new ideas and providing guidance when needed. Thanks also to the members of the Schmitt-Ulms lab, namely Gerold, Louisa, and Declan, for the time and effort you put into taking my proteomics data to the next level. Lastly, I would like to thank the people who supported me on the home front throughout this process. Thank you Katrina for putting up with my ramblings about obscure topics in neuroscience research, and understanding when I had to put in long hours at the lab. Thanks to my parents, Brad, Shelagh, Louise and Peter for your continuous support on multiple fronts. iii Table of Contents Acknowledgments .......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Abbreviations .................................................................................................................... vii List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix List of Appendices ......................................................................................................................... xi Chapter 1 Introduction .....................................................................................................................1 1.1 Parkinson’s Disease .............................................................................................................1 1.1.1 Overview ..................................................................................................................1 1.1.2 Familial PD ..............................................................................................................2 1.1.3 Alpha-synuclein and Proteostasis in PD ..................................................................5 1.1.4 Mitochondrial Dysfunction in PD ............................................................................8 1.2 Molecular Chaperones in PD .............................................................................................10 1.2.1 The Nature and Function of Molecular Chaperones ..............................................10 1.2.2 Molecular Chaperones and Alpha-Synuclein Pathology .......................................15 1.2.3 Molecular Chaperones and Mitochondrial Dysfunction ........................................17 1.2.4 BAG Family Co-chaperones ..................................................................................18 1.2.5 BAG5 .....................................................................................................................21 1.3 Summary and Research Objectives ...................................................................................23 Chapter 2 Characterizing the BAG5 Interactome ..........................................................................25 2.1 Introduction ........................................................................................................................25 2.2 Materials & Methods .........................................................................................................31 2.2.1 Antibodies & Reagents ..........................................................................................31 2.2.2 Cell Culture ............................................................................................................32 iv 2.2.3 Western Blotting ....................................................................................................32 2.2.4 Generation of the H4 Stable Cell Lines .................................................................32 2.2.5 Immunoprecipitation and Mass Spectrometry: H4 Cells .......................................33 2.2.6 Generation of the SH-SY5Y Stable Cell Lines ......................................................33 2.2.7 Immunoprecipitation and Mass Spectrometry: SH-SY5Y Cells ...........................34 2.2.8 Bioinformatic Analysis ..........................................................................................35 2.3 Results ................................................................................................................................36 2.3.1 Characterization of the BAG5 Interactome: H4 Cells ...........................................36 2.3.2 Characterization of the BAG5 Interactome: SH-SY5Y Cells ................................41 2.4 Discussion ..........................................................................................................................44 Chapter 3 Validating the Interaction Between BAG5 and p62 ......................................................48 3.1 Introduction ........................................................................................................................48 3.2 Materials & Methods .........................................................................................................52 3.2.1 Antibodies & Reagents ..........................................................................................52 3.2.2 Cell Culture ............................................................................................................53 3.2.3 Western Blotting ....................................................................................................53 3.2.4 GST Pull-down Assay ...........................................................................................53 3.2.5 Immunoprecipitation ..............................................................................................54 3.2.6 Immunohistochemistry ..........................................................................................55 3.3 Results ................................................................................................................................55 3.3.1 Validation and Visualization of the BAG5-p62 Interaction ..................................55 3.3.2 p62 Interacts with BAG5 via its C-terminal Domains ...........................................56 3.4 Discussion ..........................................................................................................................61 Chapter 4 Investigating the BAG5-p62 Interaction in the Context of Alpha-synuclein aggregation ................................................................................................................................63 4.1 Introduction ........................................................................................................................63 v 4.2 Materials & Methods .........................................................................................................66
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