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Targeting of Heat Shock Protein Hspa6

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Targeting of Heat Shock Protein Hspa6 TARGETING OF HEAT SHOCK PROTEIN HSPA6 (HSP70B') TO THE PERIPHERY OF NUCLEAR SPECKLES IS DISRUPTED BY A TRANSCRIPTION INHIBITOR FOLLOWING THERMAL STRESS IN HUMAN NEURONAL CELLS by Larissa Becirovic A thesis submitted in conformity with the requirements for the degree of Master of Science Cell and Systems Biology Department University of Toronto © Copyright by Larissa Becirovic (2016) ABSTRACT Targeting of Heat Shock Protein HSPA6 (HSP70B') to the Periphery of Nuclear Speckles is Disrupted by a Transcription Inhibitor Following Thermal Stress in Human Neuronal Cells Larissa Becirovic Master of Science Cell and Systems Biology Department University of Toronto 2016 Heat shock proteins (Hsps) are a set of conserved proteins involved in cellular repair and protection. Localization of inducible members of the HSPA (HSP70) family can be used as an index to identify stress-sensitive sites in differentiated human neuronal cells. Following thermal stress, the little studied HSPA6 (HSP70B') associated with the periphery of nuclear speckles (perispeckles) that are sites of transcription factories, however the widely studied HSPA1A (HSP70-1) did not. Triptolide, a fast-acting transcription inhibitor, knocked down levels of the large subunit of RNA polymerase II, RPB1, during the time-frame when HSPA6 localized to perispeckles. Administration of triptolide to heat shocked human neuronal SH-SY5Y cells, disrupted HSPA6 localization to perispeckles, suggesting involvement of HSPA6 in transcriptional recovery after stress. The HSPA6 gene is present in the human genome but not in mouse and rat. Hence, current animal models of neurodegenerative diseases lack a potentially protective member of the HSPA family. ii ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. Ian R. Brown, for providing me the opportunity to study in his laboratory. I will be forever grateful for his guidance and support throughout my MSc. degree. Dr. Brown has taught me how to improve my communication skills, as well as how to think critically. His strong work ethic and thorough pursuit of knowledge are objectives that I will strive to model through the rest of my career. I would also like to thank my supervisory committee members, Dr. Rongmin Zhao and Dr. Maurice Ringuette, for their support throughout the completion of my degree. I would also like to thank the other members of the Brown lab, Catherine Deane and Sadek Shorbagi. Their invaluable encouragement tied together with the scientific discussions and advice we exchanged proved to be indispensible during my time at the University of Toronto. I would also like to thank former members of the Brown lab, Dr. Sam Khalouei, Dr. Ari Chow, and Hashwin Ganesh, for teaching me the scientific techniques that I needed to know. I would also like to thank my fellow graduate students at the University of Toronto for their moral support. A special thanks to my parents, Ennis and Kyana Becirovic, and my brother, Allan Becirovic, for their love and ongoing support. Finally, thanks to all of my close friends for their encouragement. iii TABLE OF CONTENTS ABSTRACT ................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................ iii LIST OF FIGURES ...................................................................................................................... v LIST OF ABBREVIATIONS ..................................................................................................... vi 1 INTRODUCTION .................................................................................................................. 1 1.1 Heat shock response (HSR) ............................................................................................. 1 1.2 Heat shock proteins (Hsps) ............................................................................................. 2 1.3 Hsps and neurodegenerative diseases ............................................................................ 4 1.4 HSPA (HSP70) Family .................................................................................................... 5 1.4.1 HSPA6 (HSP70B') ...................................................................................................... 6 1.5 Selection of triptolide as a fast-acting transcription inhibitor ..................................... 8 2 OBJECTIVES ......................................................................................................................... 9 3 MATERIALS AND METHODS ......................................................................................... 11 3.1 Growth of human neuronal SH-SY5Y cells ................................................................ 11 3.2 Western blotting ............................................................................................................. 11 3.3 Heat shock treatment ..................................................................................................... 12 3.4 Immunocytochemistry ................................................................................................... 12 4 RESULTS .............................................................................................................................. 14 4.1 The transcriptional inhibitor triptolide is effective at nanomolar concentrations .. 15 4.2 Targeting of YFP-HSPA6 to nuclear structures following thermal stress in differentiated human neuronal cells ...................................................................................... 18 4.3 Localization of YFP-HSPA6 is disrupted following triptolide administration in human neuronal cells .............................................................................................................. 18 5 DISCUSSION ........................................................................................................................ 25 6 REFERENCES ..................................................................................................................... 29 iv LIST OF FIGURES Figure 1. Nanomolar concentrations of triptolide knocked down levels of RPB1, the large subunit of RNA polymerase II, in differentiated human neuronal cells………...………...….… 16 Figure 2. Association of YFP-tagged HSPA6 protein with nuclear structures in differentiated human neuronal cells following thermal stress…………………………………………………. 19 Figure 3. Triptolide disrupted localization of YFP-tagged HSPA6 to perispeckles………….... 22 v LIST OF ABBREVIATIONS °C Degree Celsius ALS Amyotrophic lateral sclerosis APP Amyloid precursor protein ATP Adenosine triphosphate CDK7 Cyclin dependent kinase-7 FBS Fetal bovine serum hr Hour HOP HSP70/HSP90 organizing protein Hsp Heat shock protein HSPA HSP70 family HSPA1A HSP70-1 protein HSPA6 HSP70B' protein HSE Heat shock element HSF Heat shock transcription factor HSR Heat shock response kDa kilodalton mRNA Messenger ribonucleic acid nM Nanomolar concentration vi PBS Phosphate buffered saline RPB1 Large subunit of RNA polymerase II SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SOD-1 Superoxide dismutase-1 YFP Yellow fluorescent protein vii 1 1 INTRODUCTION 1.1 Heat shock response (HSR) After exposure to a range of stressful stimuli, cells exhibit a highly conserved heat shock response during which protein production is inhibited and a set of heat shock proteins (Hsps) is induced (Lindquist and Craig, 1988; Pardue et al., 1992; Welch, 1992; Feder and Hofmann, 1999; Powers and Workman, 2007; Richter et al., 2010; Velichko et al., 2013). Hsp induction is regulated at the level of transcription by heat shock transcription factor-1 (HSF-1) in mammalian cells (Wu, 1995; Morimoto, 1998; Morano and Thiele, 1999). Present as a monomer under normal conditions, HSF-1 becomes trimerized, phosphorylated, and bound to heat shock elements (HSEs) in the promoter region of stress-inducible heat shock genes following exposure to stress, which induces their transcription (Fernandes et al., 1994; Holmberg et al., 2002; Soncin et al., 2003; Guettouche et al., 2005; Kim et al., 2005). The heat shock response has been investigated in relation to human aging and neurodegenerative diseases (Anckar and Sistonen, 2011; Neef et al., 2011; Heimberger et al., 2013). Up-regulation of Hsps has been suggested as a potential therapeutic approach to counter protein misfolding and aggregation that are characteristic of neurodegenerative disorders (Selkoe, 2004b; Brown, 2007b; Haass and Selkoe, 2007; Asea and Brown, 2008; Brown, 2008). 2 1.2 Heat shock proteins (Hsps) Heat shock proteins (Hsps) bind to misfolded proteins, and thus have been associated with protein quality control mechanisms that minimize the cytotoxic effects triggered by the accumulation of misfolded proteins (Zhang and Qian, 2011; Dreiseidler et al., 2012; Kim et al., 2013). Hsps were discovered following the observed change in chromosome puffing patterns in the chromosomes of Drosophila salivary gland cells following an increase in temperature (Ritossa, 1962). Subsequently, studies on the transcription and expression of Hsps emerged (Tissieres et al., 1974; Moran et al., 1978). Despite a variety of stressors having the ability to induce Hsp expression, they historically have been referred to as heat shock proteins (Lindquist, 1986; Morimoto, 1993; Velichko et al., 2013). Hsps are composed of both constitutively expressed and stress-inducible members. Constitutively
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