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Kallikrein-Related Peptidase Signalling Via Proteinase-Activated Receptors

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Kallikrein-Related Peptidase Signalling Via Proteinase-Activated Receptors KALLIKREIN-RELATED PEPTIDASE SIGNALLING VIA PROTEINASE-ACTIVATED RECEPTORS by Aikaterini Oikonomopoulou A thesis submitted in conformity with the requirements for the Degree of Doctor of Philosophy Graduate Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Aikaterini Oikonomopoulou 2008 Kallikrein-related peptidase signalling via proteinase-activated receptors Aikaterini Oikonomopoulou Doctor of Philosophy, 2008 Department of Laboratory Medicine and Pathobiology University of Toronto ABSTRACT The family of human kallikrein-related peptidases (KLKs) numbers 15 serine proteinases implicated in tumour progression. Despite the wide tissue distribution of KLKs and the numerous reports of their differential expression in pathological settings, the signalling mechanism(s) whereby these enzymes regulate tissue function are not yet known. Further, knowledge of the levels of their activity, as well as of their potential endogenous targets, has only been extracted from in vitro studies and cell culture systems. We hypothesized that KLKs can trigger tumour signalling via proteinase-activated receptors (PARs), a family of G-protein-coupled receptors. To test our hypothesis, we evaluated the ability of KLKs 5, 6, and 14: to activate or prevent signalling via PARs 1, 2, and 4 in cells and tissues expressing these receptors. Further, we used a novel activity-based probe approach, coupled with conventional immunoassay (ELISA), to determine the abundance of active KLK6 relative to total immunoreactive KLK6 in cancer-related biological fluids. We concluded that KLKs can regulate multiple signalling pathways triggered by PARs 1, 2, and 4, resulting in calcium release, platelet aggregation and vascular relaxation, and they can cause murine inflammation. Further, our activity-based ELISA demonstrated the presence of active KLK6 in ovarian cancer ascites fluids and cancer cell supernatants. We, therefore, suggest that tumours can produce active KLKs, which can potentially control tumour behaviour by regulating PAR activity. ii ACKNOWLEDGEMENTS Completing a PhD thesis is not a matter of receiving another degree to hang on the wall but rather a justification of being able to deal with difficult questions and generate problem- solving ideas. It is a path which requires wise mentorship and guidance, as well as putting into practice your intuition and self-confidence. I, therefore, consider myself extremely lucky that I was able to have the best mentorship from two supervisors, Dr. Morley D. Hollenberg and Dr. Eleftherios P. Diamandis, with different scientific backgrounds, both being experts in their field. It is the successful bonding of these two different fields that ensured my adventurous but fruitful journey during the past 4 years. I would also like to extend my appreciation to Dr. Harry Elsholtz, my graduate coordinator, and my committee advisors Dr. Bharati Bapat, Dr. Joanne McLaurin, and Dr. Alex Romaschin for their useful advices and scientific discussions throughout this project, as well as to Dr. Christopher Power and Dr. Martin McGavin for valuable input on the content of my thesis. I am also very grateful to Antoninus Soosaipillai, Dr. Mahmoud Saiffedine, Dr. Kristina K. Hansen, Dr. Amos Baruch, Dr. Nathalie Vergnolle, Kevin Chapman, Dr. Rithwik Ramachandran, and Eric Huyan for sharing their knowledge and expertise with me. Also, I would like to thank Linda Grass, Tammy Earle, Bernard Renaux, and Dr. John (Zhenguo) Yu for their help and time. Furthermore, I would like to thank the members of both laboratories and my friends for sharing ideas and moments in and outside the lab. Above all, I would like to express my sincere gratitude to my father, mother, sister, and my husband, who all led my way from undergraduate to graduate university, managed to put up with my ambitions and constant lack of time, and supported me all the way to the completion of this thesis. iii TABLE OF CONTENTS ABSTRACT............................................................................................................II ACKNOWLEDGEMENTS ................................................................................ III TABLE OF CONTENTS .................................................................................... IV LIST OF TABLES............................................................................................... IX LIST OF FIGURES............................................................................................... X LIST OF ABBREVIATIONS............................................................................. XI LIST OF ORIGINAL PUBLICATIONS ........................................................XIV INTRODUCTION ..................................................................................................2 1.1 Overview ................................................................................................2 1.2 Serine Proteinases .................................................................................2 1.2.1 General classification of proteinases......................................................................... 2 1.2.2 Catalytic activity of serine proteinases ..................................................................... 5 1.2.3 Regulation of serine proteinase activity.................................................................... 7 1.2.4 Role of serine proteinases in physiology and pathophysiology................................ 8 1.3 Proteinase Signalling via Proteinase-Activated Receptors (PARs) ....................................................................................................9 1.3.1 Non-proteolytic pathways of proteinase signalling................................................. 10 1.3.2 Proteolytic pathways of proteinase signalling......................................................... 10 1.3.3 Proteinase-activated receptors................................................................................. 13 1.3.4 Activation versus inhibition or dis-arming of PARs............................................... 17 1.3.5 Role of PARs in pathophysiology........................................................................... 18 1.3.6 Proteinases and PARs as therapeutic targets........................................................... 22 iv 1.4 Kallikrein-related Peptidases (KLKs) ..............................................24 1.4.1 Historical Overview ................................................................................................ 24 1.4.2 Genes and proteins of kallikrein-related peptidases................................................ 26 1.4.3 Enzymatic activity and KLK substrates.................................................................. 28 1.4.4 Tissue expression.................................................................................................... 31 1.4.5 Role in physiology and pathobiology ..................................................................... 32 1.4.6 KLK expression in cancer....................................................................................... 35 1.4.7 Role of kallikrein-related peptidases in carcinogenesis.......................................... 40 1.5 Rationale, Hypothesis, and Objectives..............................................41 1.5.1 Rationale ................................................................................................................. 41 1.5.2 Hypothesis............................................................................................................... 42 1.5.3 Objectives................................................................................................................ 42 PRODUCTION, PURIFICATION, AND ACTIVATION OF KLK5, KLK6, AND KLK14..........................................................................................................44 2.1 Introduction.........................................................................................44 2.1.1 Kallikrein-related peptidase 5 ................................................................................. 44 2.1.2 Kallikrein-related peptidase 6 ................................................................................. 46 2.1.3 Kallikrein-related peptidase 14 ............................................................................... 48 2.2 Material and Methods ........................................................................51 2.2.1 Reagents .................................................................................................................. 51 2.2.2 Immunofluorometric quantification of KLKs 5, 6, and 14 ..................................... 53 2.2.3 Production, purification, and activation of kallikrein-related peptidase 5 and 14 ..................................................................................................................... 55 2.2.4 Production of active kallikrein-related peptidase 6 in baculovirus......................... 56 2.2.5 Production, purification, and activation of KLK6 in HEK-293 cells ..................... 57 2.2.6 Storage optimization of the newly produced active KLK6..................................... 60 v 2.3 Results ..................................................................................................61 2.3.1 Recombinant active KLK5 and KLK14.................................................................. 61 2.3.2 Production, purification, and activation of KLK6 zymogen in HEK-293 cells...... 62 2.3.3 Production and purification of active
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