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Identification of Novel Substrates of the Ubiquitin E3 Ligase RNF126 and Characterization of Its Role in Lipid Droplet Homeostasis

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Identification of Novel Substrates of the Ubiquitin E3 Ligase RNF126 and Characterization of Its Role in Lipid Droplet Homeostasis Identification of novel substrates of the ubiquitin E3 ligase RNF126 and characterization of its role in lipid droplet homeostasis by Zhongda Pan A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Medical Biophysics University of Toronto © Copyright by Zhongda Pan 2016 Identification of novel substrates of the ubiquitin E3 ligase RNF126 and characterization of its role in lipid droplet homeostasis Zhongda Pan Master of Science Department of Medical Biophysics University of Toronto 2016 Abstract Ubiquitin E3 ligases confer specificity by recognizing target substrates and mediating the final step of the ubiquitination process. Despite this critical role, our understanding of their physiological partners and biological functions remains limited. Here we use a proximity-based protein screen called BioID to explore novel interacting proteins and substrates of the ubiquitin E3 ligase RNF126. We demonstrate that RNF126 can associate with p97 as well as its co-factors UBXD1 and UBXD8 and show that UBXD1 and UBXD8 are bonafide substrates of RNF126. To determine a functional role, we explored the role of RNF126 in UBXD8 mediated functions. We found that stable knockdown of RNF126 in HeLa cells results in smaller LDs following oleate stimulation and that this is due to defective expansion. Together, this work demonstrates the successful use of BioID to identify substrates of ubiquitin E3 ligases and furthers our understanding of the role of ubiquitin in LD biology. ii Acknowledgments I would like to thank my supervisor, Dr. Jane McGlade, for her outstanding support and mentorship throughout the years. Her patience and guidance came second only to her willingness to place my success as her number one priority. I would also like to thank my committee members, Dr. Brian Raught and Dr. Peter Kim, for their time, guidance, and support. I would like to thank everyone in the McGlade lab for making this time an enjoyable journey through all the ups and downs of graduate school. I have not only learned a lot from everyone, but also truly appreciate the support when things turned sour. I wish you all the best. Finally, I would like to thank my parents for sticking through this with me and listening to my over-detailed explanations about my project. I would also like to thank my best friend Annabelle Sumenap for keeping me afloat during difficult times. iii Table of Contents Acknowledgments.......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................ vii List of Figures .............................................................................................................................. viii List of Appendices ......................................................................................................................... ix Chapter 1 Introduction .....................................................................................................................1 1.1 The ubiquitin regulatory system ..........................................................................................1 1.1.1 RING type ubiquitin E3 ligases ...............................................................................3 1.1.2 The ubiquitin code ...................................................................................................4 1.1.3 RNF126 and RNF115 are two related ubiquitin E3 ligases .....................................5 1.2 The AAA ATPase p97 .........................................................................................................7 1.2.1 Biochemical properties of p97 .................................................................................7 1.2.2 Biological functions of p97 ......................................................................................8 1.2.3 Control of p97 function by co-factors ......................................................................8 1.2.4 The UBX family is the largest family of p97 co-factors .......................................10 1.2.5 UBXD8 is an integral membrane protein ..............................................................10 1.3 Lipid droplets are dynamic cellular organelles ..................................................................13 1.3.1 Lipid droplet life cycle ...........................................................................................15 1.3.2 Regulation of lipid droplets by ubiquitin ...............................................................16 1.3.3 The role of UBXD8 and p97 in the regulation of lipid droplets ............................17 1.4 Thesis rationale and objectives ..........................................................................................20 Chapter 2 Identifying interacting proteins and substrates of RNF126 and RNF115 .....................21 iv 2.1 Introduction ........................................................................................................................21 2.2 Materials and Methods .......................................................................................................21 2.2.1 Cell culture, cDNA constructs, and reagents .........................................................21 2.2.2 Forward and reverse transfection ...........................................................................24 2.2.3 Generation of doxycycline inducible stable cell lines and BioID screen ..............24 2.2.4 Production of GST fusion proteins ........................................................................25 2.2.5 Co-immunoprecipitations, GST pull-downs, in vivo ubiquitination assays, and western blotting ......................................................................................................25 2.3 Results ................................................................................................................................26 2.3.1 Identification of proteins in near proximity to RNF126 and RNF115 by BioID...26 2.3.2 Validation of potential interacting proteins identified by BioID ...........................34 2.3.3 RNF126 associates with p97 and the UBX family of co-factors ...........................36 2.3.4 Over-expression of RNF126 promotes UBXD8 association with p97 ..................40 2.3.5 UBXD8 and UBXD1 are ubiquitinated by RNF126 .............................................42 2.4 Discussion ..........................................................................................................................42 Chapter 3 RNF126 regulates lipid droplet size following fatty acid stimulation ..........................45 3.1 Introduction ........................................................................................................................45 3.2 Materials and Methods .......................................................................................................45 3.2.1 Cell culture, cDNA constructs, and reagents .........................................................45 3.2.2 Preparation of oleate-BSA conjugate.....................................................................46 3.2.3 Generation of doxycycline inducible stable cell lines ...........................................46 3.2.4 Immunocytochemistry ...........................................................................................46 3.2.5 Quantification of lipid droplets ..............................................................................47 3.2.6 Lipid droplet purification and immunoprecipitation ..............................................47 3.2.7 Statistical analysis ..................................................................................................48 3.3 Results ................................................................................................................................48 v 3.3.1 RNF126 regulates lipid droplet size in oleate stimulated cells ..............................48 3.3.2 Re-expression of RNF126 in an inducible cell system can rescue decreased lipid droplet size .....................................................................................................51 3.3.3 RNF126 does not regulate lipid droplet turnover ..................................................55 3.3.4 RNF126 is required for lipid droplet expansion ....................................................58 3.4 Discussion ..........................................................................................................................60 Chapter 4 Conclusions and Future Directions ...............................................................................62 4.1 Summary ............................................................................................................................62 4.2 Regulation of p97 co-factors by RNF126 ..........................................................................62 4.3 The role of RNF126 mediated ubiquitination of KEAP1 and CUL3 ................................67 4.4 UBXD1, p97, and RNF126 in EGFR trafficking ..............................................................71 4.5 The role of RNF126 mediated ubiquitination of UBXD8 in lipid droplet regulation .......72 4.6 RNF126 and other lipid droplet regulatory proteins ..........................................................73
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