Characterization and interaction studies of Bcl-2-associated athanogene 3 (Bag3) and its P209L mutant Jeffrey Li Department of Biochemistry McGill University, Montreal August 2018 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science © Jeffrey Li 2018 1 Abstract: A genetic form of myofibrillar myopathy (MFM) has been linked to a P209L mutation in Bag3, with patients exhibiting childhood-onset progressive skeletal muscle weakness and associated cardiomyopathy. Microscopy studies on MFM patients show myofibril disintegration beginning at the Z-disk and formation of protein aggregates. Bag3 is a member of the BAG family of Hsp70 nucleotide exchange factors, and is associated with a number of pathways, such as selective autophagy, aggresome formation and Hippo pathway regulation. Previous work in zebrafish suggests Bag3-P209L is aggregation-prone and can sequester wildtype Bag3 into aggregates, leading to a loss of Bag3 function. How a loss in Bag3 function leads to the disease is unknown. We identified changes in Bag3 phosphorylation at residues linked to regulation of the heat shock response. BioID comparison of wildtype Bag3 and Bag3-P209L interactors in the soluble and insoluble fraction show potential changes in pathways such as autophagy, Hippo and Wnt pathway regulation, signal transduction, and actin cytoskeleton maintenance. Interestingly, a Bag3 function in Wnt signaling has never been reported. With these findings, we propose several hypotheses describing how the observed gain and loss of interaction caused by the P209L mutation may affect Bag3-associated pathways, and how these aberrancies could contribute to the MFM phenotype. 2 Résumé: Une forme génétique de la myopathie myofibrillaire (MMF) a été liée à une mutation P209L dans la protéine Bag3. Les patients avec cette version particulière de la myopathie ont démontré une faiblesse progressive débutant à l’enfance des muscles squelettiques avec une cardiomyopathie associée. Les études microscopiques sur les patients atteints du MMF démontrent une désintégration myofibrille commençante a la ligne Z et des formations d’agrégats de protéines. Bag3 est un membre de la famille BAG des facteurs d’échanges de nucléotides de Hsp70, et est associée avec un nombre de processus cellulaires tels que l’autophagie sélective, la formation des agrésomes (« aggresomes », en anglais) et la voie de signalisation Hippo. Les travaux antérieurs sur le poisson-zèbre suggèrent que Bag3-P209L est propice à former des agrégats et peut aussi pousser la version naturelle de Bag3 à former des agrégats. Cela conduit la perte de fonction de Bag3. Nous ne savons pas encore pourquoi la perte de fonction de Bag3 cause cette myopathie. Nous avons identifié des variations de phosphorylation de Bag3 sur des acides aminés liées au contrôle de la réponse au choc thermique. BioID de la version naturelle Bag3 et de Bag3-P209L a demontré des différences entre leurs interactions de protéines dans les fractions solubles et insolubles. Ces différences démontrent des changements potentiels dans les processus cellulaires tels que l’autophagie, le contrôle de Hippo et Wnt, transduction de signal, et l’entretien du cytosquelette d'actine. Bag3 n’a jamais été démontré auparavant à avoir une fonction dans la voie de signalisation Wnt. Avec ces résultats, nous proposons plusieurs hypothèses décrivant comment les gains ou pertes d’interactions observées causés par la mutation P209L peuvent affecter les processus biochimiques associés à Bag3, et comment ces changements en interactions peuvent contribuer au phénotype MMF. 3 Acknowledgements: First and foremost, I would like to thank my supervisor Dr. Jason C. Young. His mentorship, insight, and patience in guiding me through this project has left me a more analytical and organized scientist and individual, and for that, I am deeply indebted. I also thank Jason for his time in editing this thesis. I would also like to thank all members of the Young Lab, past and present, for technical assistance and friendship over the years: Michael Wong, Dr. Imad Baaklini, Dr. Patrick Kim- Chiaw, Dr. Conrado Gonçalves, Sam Lee, Brittany Williamson, Kevin Guo, Eva Wang, and Yogita Patel. Special thanks to Yogita Patel, for the close friendship and support, as well as being an excellent laboratory mentor during my early years as a fledging scientist. Also, thank you to Kevin Simpson-Poirier for translating my abstract. My thanks to Dr. Kurt Dejgaard for providing help with my proteomic studies, as well as Dr. Simon Wing and Dr. Imed Gallouzi for their mentorship in my supervisory committee. Thank you to our collaborator Dr. Josée Lavoie for generously providing me with Bag3 plasmids and antibodies, as well as Dr. Gregor Jansen for BioID plasmids. Thank you to the Bellini Foundation for project funding. Special thanks to Derek Hall, Amr Omer, Dr. Sergio DiMarco and the rest of the Gallouzi lab for advice in microscopy and working with the C2C12 cell line. To my friends in the Schmeing lab, especially Frederik, thanks for the board games, roasting, and blessed beer hour, as well as the occasional ÄKTA assistance. To my skookum homies- Cynthia, Brittany, Colten, Shane, and Camille, thank you for the fantastic memories, support, and copious amounts of food and drink. I do apologize to and thank all friends and mentors that I have not mentioned. Most importantly, I thank my family- Mom, Dad, and Mark, for their undying love and believing in me throughout this project. I could have not made it through graduate school, or life for that matter, without their support. 4 Table of Contents: Abstract: ........................................................................................................................................ 2 Résumé: .......................................................................................................................................... 3 Acknowledgements: ...................................................................................................................... 4 Preface and Contribution of Authors: ........................................................................................ 7 List of Figures and Tables:........................................................................................................... 8 List of Abbreviations: ................................................................................................................... 9 Chapter 1 : Introduction ............................................................................................................ 12 Myofibrillar myopathy: ............................................................................................................. 12 Skeletal muscle structure and mechanism: ............................................................................... 13 Skeletal muscle differentiation: ................................................................................................ 15 Signaling pathways in myogenesis: .......................................................................................... 16 Wnt signaling: ....................................................................................................................... 16 Hippo signaling: .................................................................................................................... 18 Protein homeostasis: ................................................................................................................. 21 Chaperones: .............................................................................................................................. 21 Hsp70 family: ........................................................................................................................... 23 Hsp70 co-chaperones: ............................................................................................................... 25 Small heat shock proteins (sHsps): ........................................................................................... 26 Protein degradation: .................................................................................................................. 28 Ubiquitin proteasome system (UPS): .................................................................................... 28 Autophagy: ............................................................................................................................ 29 Bag3: ......................................................................................................................................... 31 Domains of Bag3: ..................................................................................................................... 33 IPV motifs: ............................................................................................................................ 33 WW domain: ......................................................................................................................... 33 PXXP domains: ..................................................................................................................... 33 Bag3 functions: ......................................................................................................................... 34 Chaperone assisted selective autophagy (CASA): ................................................................ 34 Hippo pathway regulation: ...................................................................................................