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Role of RGS2 in Cellular Stress

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Role of RGS2 in Cellular Stress Western University Scholarship@Western Electronic Thesis and Dissertation Repository 8-18-2015 12:00 AM Role of RGS2 in Cellular Stress Chang-Hui Wang The University of Western Ontario Supervisor Dr. Peter Chidiac The University of Western Ontario Graduate Program in Physiology and Pharmacology A thesis submitted in partial fulfillment of the equirr ements for the degree in Master of Science © Chang-Hui Wang 2015 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Cellular and Molecular Physiology Commons Recommended Citation Wang, Chang-Hui, "Role of RGS2 in Cellular Stress" (2015). Electronic Thesis and Dissertation Repository. 3018. https://ir.lib.uwo.ca/etd/3018 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]. ROLE OF RGS2 IN CELLULAR STRESS (Thesis format: Monograph) by Chang-Hui Wang Graduate Program in Physiology and Pharmacology A thesis submitted in partial fulfillment of the requirements for the degree of Masters of Science The School of Graduate and Postdoctoral Studies The University of Western Ontario London, Ontario, Canada © Chang-Hui Wang 2015 Abstract Stresses from the external environment can disrupt cellular processes and result in damaging effects, such as the misfolding of proteins, which have been linked to several diseases. Regulator of G protein signalling 2 (RGS2) is upregulated by several forms of stress and can inhibit protein synthesis, an established response to stress typically achieved via the phosphorylation of the initiation factor, eIF2, to conserve energy and resources. Under reduced translation, some factors are selectively expressed via alternative translation mechanisms and these factors consequently may promote apoptosis. The molecular mechanisms mediating such opposing responses to stress are not well understood. Here, we suggested that RGS2 may be an important regulatory component in the cellular stress response and we hypothesized that RGS2 contributes to the response of cells to stress through its translational control abilities. Previously, we have shown that RGS2 can interact with the translation initiation factor, eIF2B, and inhibit de novo protein synthesis. Here, we demonstrated that the expression of RGS2 decreased total protein levels and significantly increased levels of factors linked to stress-induced apoptosis such as ATF4 and CHOP. Interestingly, expression of the eIF2Bε-interacting domain of RGS2 (RGS2eb) alone resulted in a 20-fold increase in caspase 3 activation which was not seen with full-length RGS2. Furthermore, we showed that these effects are translationally regulated and independent of eIF2 phosphorylation. Thus, we present a novel mechanism in the regulation of stress response by RGS2. These results also suggest that RGS2 may be pro-apoptotic and may potentially be an important target in stress-related pathologies. Keywords Regulator of G protein signalling 2 (RGS2), cell stress response, translation, eukaryotic initiation factor 2 (eIF2), ATF4, CHOP, apoptosis ii Acknowledgments I would like to thank my supervisor, Dr. Peter Chidiac, for all the support and guidance he has provided me throughout my Masters studies and made my research endeavours a very enriching experience full of learning and neat opportunities. I would also like to thank the members of my advisory committee: Dr. Sean Cregan, Dr. Patrick Lajoie, and Dr. Nica Borradaile, for their continuous support and mentorship at every meeting and interaction, each time providing me with great suggestions and insights toward the formation of this current body of work that I am truly proud of. Additionally, this work would not have been possible without the generosity of many labs in providing the materials and tools that I needed to carry out my research, so I would like to give out my sincere thanks and gratitude to them as well. To my fellow lab mates Katherine Lee, Patrick Stockwell, and Drew Wallace, thank you for all your help and friendship, you were there to provide encouragement and support when I needed it the most. I will always remember the fun times we had in and outside of the lab, bouncing off ideas to successfully troubleshoot the crux of the problem in a technique or experiment, the chats in our student office and at the Grad Club. There are also many other people I would like to thank for their help, guidance, and personal support along my scientific journey, without whom I would not be where I am at today. A very special thank you to Dr. Patrick Swan and Dr. Adrian Gunaratne who have been great mentors, providing me with the needed antibodies and primers, and offering scientific insight on my project to fulfill my research goals. Thank you to all the friends and colleagues, especially Dr. Dan Secor, who consistently cheers me up and points out the positives and strengths of my work. To my loving parents and family, thank you for your endless support and being there every step of the way. I have been very fortunate to have had the financial support from CIHR, and I am grateful to the organizations and donors who generously contributed towards the scholarship that funded my research these past two years. Lastly, to all who may be reading this in the future, keep an open mind and be curious, reach for your goals, don’t give up! iii Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iii Table of Contents ............................................................................................................... iv List of Tables .................................................................................................................... vii List of Figures .................................................................................................................. viii List of Abbreviations .......................................................................................................... x List of Appendices ........................................................................................................... xiii Chapter 1 ............................................................................................................................. 1 1 Introduction .................................................................................................................... 1 1.1 Cellular responses to stress: Cell survival and cell death ....................................... 1 1.2 Cellular stress pathways: Endoplasmic reticulum (ER) stress ................................ 2 1.2.1 Proteotoxic stress and associated diseases .................................................. 2 1.2.2 The unfolded protein response (UPR) ........................................................ 3 1.2.3 ER stress induced apoptosis and diseases ................................................... 7 1.3 Other cellular stress pathways and in vitro models of stress ................................ 12 1.4 Regulation of protein synthesis: Translational control at initiation ...................... 14 1.4.1 Translational control by eukaryotic initiation factor 2 (eIF2) .................. 14 1.4.2 Alternative translational mechanisms ....................................................... 18 1.5 G protein coupled receptor (GPCR) mediated signalling and function in cellular stress ...................................................................................................................... 19 1.6 Regulator of G protein signalling (RGS) proteins ................................................ 20 1.7 Regulator of G protein signalling 2 (RGS2) ......................................................... 22 1.7.1 Cellular functions of RGS2 and modulation of GPCR signalling ............ 22 1.7.2 Distribution and regulation of RGS2 expression ...................................... 25 1.7.3 Physiological roles of RGS2 ..................................................................... 25 iv 1.7.4 Translational control by RGS2: Role of RGS2 in cellular stress response26 1.8 Rationale ............................................................................................................... 29 1.8.1 Hypothesis and Predictions ....................................................................... 29 1.8.2 Objectives ................................................................................................. 29 Chapter 2 ........................................................................................................................... 31 2 Materials and Methods ................................................................................................. 31 2.1 Cell culture ............................................................................................................ 31 2.2 Adenoviruses......................................................................................................... 31 2.3 Reagents and drugs ............................................................................................... 32 2.4 Adenoviral infection and protein isolation ........................................................... 33 2.5 Immunoblotting....................................................................................................
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