Regulation and function of the mitochondrial protease HtrA2 1 Omi in the control of cell death Kristina Klupsch A thesis submitted toward the degree of Doctor of Philosophy February 2007 Signal Transduction Laboratory, CANCER RESEARCH UK Cancer Research UK - London Research Institute, 44 Lincoln’s Inn Fields, London. Department of Biochemistry and Molecular Biology, University College London, UCL Gower Street, London. UMI Number: U592206 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U592206 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Declaration I, Kristina Klupsch, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. London, February 2007 2 Abstract The serine protease HtrA2 is released from mitochondria following apoptotic stimuli. Once in the cytosol, HtrA2 has been implicated in promoting cell death by a caspase- dependent and -independent mechanism. However, mice lacking expression of HtrA2 show no evidence of reduced rates of cell death. On the contrary, loss of HtrA2 causes mitochondrial dysfunction leading to a neurodegenerative disorder with parkinsonian features. This suggests that the protease function of HtrA2 in the mitochondria, and not its pro-apoptotic action in the cytosol, is critical. Mammalian HtrA2 is therefore likely to function in vivo in a manner similar to its bacterial homologues, which are involved in protection against cell stress. The bacterial DegS homologue senses unfolded proteins, activating a proteolytic cascade leading to induction of stress response genes. Transcriptional profiling of wild type and HtrA2 knockout (KO) cells identified the stress-inducible transcription factor CHOP being differentially regulated when mitochondrial stress was triggered. CHOP up-regulation was found in HtrA2 KO mouse brains but not in other tissues. Transcriptional profiling of brain tissue revealed a number of putative ATF4 target genes being up-regulated in HtrA2 KO, among these CHOP. Promoter analysis identified a C/EBP-ATF composite site in the majority of the genes within this signature. Therefore, loss of HtrA2 might impact on nuclear gene expression specifically in brain, subverting normal cellular homeostasis leading to disease. In humans, point mutations in HtrA2 are a susceptibility factor for Parkinson’s disease (PD) resulting in partial loss of proteolytic activity. Affinity purification shows that the mitochondrial kinase PINK1 interacts with HtrA2. PINK1 mutations are associated with the PARK6 PD susceptibility locus. HtrA2 is phosphorylated in a PINK1- dependent manner at residues adjacent to positions found mutated in PD patients. Phosphorylation of HtrA2 and thereby modulation of its proteolytic activity seems necessary for the function of HtrA2 in the mitochondria contributing to increased resistance of cells to mitochondrial stress. 3 Acknowledgements Firstly, 1 would like to thank my supervisor, Julian Downward, for giving me the opportunity to work in his laboratory, for his support, advice and patience and also for giving me the freedom to develop and explore my own ideas. In particular, I would like to thank Miguel Martins for his invaluable advice, encouragement and enthusiasm that have helped and shaped the progress of this work. Very special thanks go to Helene Plun-Favreau for support and discussions, shared experiments, and a lot of fascination for the proteins at centre-stage of this thesis. I would also like to thank Dave Hancock for giving feed-back and providing an incomparable source of knowledge. Thanks to all the other past and present members of the Signal Transduction Laboratory and beyond - Thomas, Barbara, Julie, Caro, Pat, Michy, Surbhi, Megan, Justin, Charlie, Michael, Tony, Patrick, Olivier, Sophie, Subham, Boon Tin, Oona and Almut - for scientific and social contributions which have made the past years very enjoyable. I would like to thank the staff of the research services, especially Phil East and Simon Tomlinson in the Bioinformatics Group, Stuart Pepper and Yvonne Hey in the GeneChip Service, Derek Davies and Ayad Eddaoudi in the FACS Laboratory, and David Frith in the Protein Analysis Laboratory. In addition, I would like to thank Cancer Research UK for providing funding and an excellent research environment, and the Boehringer Ingelheim Fonds for building a great network with other students and for providing support as well as funding. Special thanks go to the people who have enriched my time in London and all the friends everywhere else. Especially, 1 would like to thank Johannes for his steadfast support and patience, Sarah for being a great friend, and Angi for providing a warm welcome and a lot of enthusiasm for living in London. Finally, I would like to thank my parents for their constant support and encouragement, helping me through difficult times, and making everything possible. 4 Table of Contents ABSTRACT........................................................................................................................................ 3 ACKNOWLEDGEMENTS............................................................................................................. 4 TABLE OF CONTENTS..................................................................................................................5 LIST OF FIGURES......................................................................................................................... 11 LIST OF TABLES.......................................................................................................................... 13 ABBREVIATIONS......................................................................................................................... 14 1 CHAPTER 1: INTRODUCTION...........................................................................................18 1.1 APOPTOSIS, p r o g r a m m e d c e l l d e a t h ....................................................................................................18 1.1.1 Caspases, central executioners of apoptosis............................................................18 1.2 C e l l u l a r s u b s t r a t e s o f c a s p a s e s .......................................................................................................2 0 1.2.1 Formation of the apoptosome....................................................................................20 1.2.2 Bcl-2 proteins.............................................................................................................. 21 1.2.3 Inhibitor o f Apoptosis proteins..................................................................................22 1.2.4 IAP antagonists ........................................................................................................... 23 1.2.5 Identification o f HtrA2 as a Reaper-related protein............................................... 23 1.3 HTRA2 BELONGS TO THE HTRA FAMILY OF SERINE PROTEASES.............................................. 25 1.3.1 HtrA2 structure and regulation of its protease activity.......................................... 26 1.3.2 E. coli DegP can function as a chaperone or protease........................................... 27 1.3.3 E. coli DegS is involved in the periplasmic stress response................................... 28 1.4 P r o t e i n s b in d i n g t o a n d c l e a v e d b y H t r A 2 .................................................................................30 1.5 C e l l u l a r s t r e s s p a t h w a y s ............................................................................................... 32 1.5.1 The unfolded protein response (UPR)....................................................................... 32 1.5.2 elF2a kinases and translational control ...................................................................33 1.5.3 CHOP and Herp are regulated by the ER stress-specific as well as the shared branch of the UPR ....................................................................................................................34 1.5.4 A TF3 is induced by multiple stresses........................................................................35 1.6 P a r k i n s o n ’s d i s e a s e .......................................................................................................................................... 37 1.6.1 Mitochondrial dysfunction in Parkinson ’s disease ................................................. 37 1.6.2 Activation o f the UPR in Parkinson’s disease models ............................................ 38 5 1.6.3 Genetic forms o f Parkinson’s disease .......................................................................39 1.6.3.1 Parkin, an E3 ligase ............................................................................................... 39 1.6.3.2 DJ-1, involved in oxidative stress protection .......................................................40 1.6.3.3