Structure-Function Analysis of the Prosegment and the Cysteine-Rich Domain of the Proprotein Convertase PC5A: Its

Structure-function analysis of the prosegment and the cysteine-rich domain of the proprotein convertase PC5A: its interaction with TIMP-2 By Nadia Nour Department of Medicine Division of Experimental Medicine McGill University Montreal, Quebec August 2004 A thesis submitted to McGill University in fulfilment of the requirements for the degree of Doctor of Philosophy © Nadia Nour, 2004 ACKNOWLEDGMENTS This work was carried out at the Clinical Research Institute of Montreal, Mc Gill University, during the years 1998-2004. I am sincerely grateful to have worked under the supervision of Dr. Nabil G. Seidah. I am very indebted to Nabil G. Seidah for his valuable contributions and his encouragement throughout the years. It was a pleasure to work with someone that has a vast knowledge of the field of proprotein processing and several other fields. Moreover, his enthusiastic dedication to the scientific world has been of great importance to me in completing this thesis. I also want to express my appreciation for my thesis committee members: Dr. Hugues Bennett, Dr. Mirek Cygler and Dr. David Lohnes for providing their time, scientific insight and guidance throughout my research. Their optimism and encouragements helped me to improve and surpass myself. I wish to thank all my former colleagues in the Chretien-Seidah laboratories, especially Jwadiga Marcienkiewicz, Dr. Michel Chrétien, Dr. Ajoy Basak, Suzanne Benjannet, Josée Hamelin, Marie-Claude Asselin, Dr. Claude Lazure, Dr. Majambu Mbikay, Philomena Pullikotil and Andrew Chen. I am also grateful to Brigitte Mary for her assistance in all secretarial matters and to Christian Charbonneau and Dr. Gaétan Thibault for sharing their invaluable knowledge in confocal analysis. This study was rendered possible by the generous contributions of the Canadian Institutes of Health Research (CIHR) for a five year studentship and the Protein Engineering Network Centers of Excellence of Canada (PENCE). Je voudrais remercier mes parents ainsi que mes deux soeurs Sandra et Sonia pour m’ avoir encouragée et soutenue à travers cette période de ma vie. I am most grateful to my future husband Yves for his unfailing love and support throughout these years. His patience and understanding attitude regarding my work as well as his faith in me have been essential for the completion of this ii thesis and to become Dr. Nad. I also thank you for careful readings of my articles and of my thesis. All my friends deserve my special thanks for bringing joy into my life. I wish to thank Dominique Nolet, Sonia Brault, Melisa Pham, Marie-Elizabeth Desourdy and Veronique Sanscoucy for sharing such enjoyable moments with me outside the laboratory. I would like to express my deep gratitude to Eric Bergeron, Ahmed Zaid, Daniel Gauthier and Nadia Rabah for their friendship and support. Enlightening scientific and non-scientific discussions with them have been helpful and a joy during my graduate years. A special thanks to Ann Chamberland and Dani Gauthier for their friendship, their sense of humour and their great technical assistance. Their positive attitudes are inspiring and were very supportive throughout the years. iii Abstract The mammalian proprotein convertases (PCs) of the secretory pathway are calcium-dependent serine proteinases related to bacterial subtilisin and yeast Kexin. As Kexin and subtilisin, all basic aa-specific convertases contain a comparable N-terminal structure starting with a signal peptide followed by a prosegment and a conserved catalytic domain. The convertases possess additional domains at their C-terminus following the catalytic domain. All PCs exhibit a P-domain prior to the C-terminal architecture, which is specific to each convertase. Three of the basic-aa-specific convertases, furin, PACE4 and PC5 display the presence of a Cysteine-rich domain (CRD). The function of the CRD has not been studied extensively and remains unclear whereas the inhibitory function of the prosegments has been investigated previously. In this manuscript, both the function of the prosegment and the CRD of PC5A were investigated as very little was known about the zymogen activation of PC5A, the inhibitory role of its prosegment and the characterisation of the function of its CRD. Previous subtilisin- and furin-based studies demonstrated that the prosegment acts as an intramolecular chaperone and as a potent inhibitor of its cognate enzyme. From these results, the inhibitory function of the prosegment of PC5A was investigated. The data revealed that the prosegment of PC5 is a potent inhibitor in vitro and ex vivo of its parent enzyme, but is not iv selective. Introduction of single point mutations within the prosegment did not increase the potency or the selectivity of the inhibitor. We next characterised the relevance of the CRD of PC5A as it implication in the cell surface localisation of the enzyme. The plasma membrane retention was demonstrated to be via the interaction of the CRD of PC5A with TIMP-2, an endogenous MMP inhibitor. This study also showed that PC5A may have an anti-proliferative role as its overexpression diminishes cell proliferation while its inactivation by siRNA causes an increase in the proliferation rate. This study on the structure-function analysis of both the prosegment and the cysteine-rich domain of PC5A will aid us to define the biological function of this enzyme. v Sommaire Les convertases des proprotéines qui résident dans la voie de sécrétion sont des serine protéases de mammifères, apparentées par leur structure aux subtilisines bactériennes et à l’ enzyme de la levure kexine. Comme ceux-ci, les convertases possèdent un peptide signal, un segment pro et une région catalytique conservée. De plus, ces protéases possèdent deux domaines supplémentaires, appelés domaines P et C-terminal qui sont absents chez les subtilisines mais présents chez la kexine. Le domaine C-terminal est unique à chaque convertase. Trois membres de la famille des convertases, furine, PACE4 et PC5 démontrent la présence d’ un domaine riche en cysteine (CRD). La fonction de ce domaine ne fut jamais sérieusement étudiée et demeure inconnue, toutefois, l’ activité inhibitrice du segment pro de la subtilisine ainsi que de certaines convertases furent étudiées extensivement auparavant. Dans ce mémoire, la fonction du segment pro ainsi que du domaine riche en cysteine de PC5A furent étudiées. Des études précédentes sur la subtilisine et la furine ont demontrées que le segment pro agit à la fois de chaperone et d’ inhibiteur de l’ enzyme parental. Inspirée de ces études, la fonction inhibitrice du segment pro de PC5 fut investiguée et révéla que ce domaine est un inhibiteur puissant de PC5 in vitro et ex vivo, cependant l’ inhibition n’ est pas spécifique à une seule convertase. De plus, des mutations ponctuelles vi introduites dans la région pro n’ ont augmenté ni la puissance, ni la spécificité de l’ inhibiteur. Auncune fonction du CRD n’ avait été définie chez les PCs. Ce domaine joue un rôle important dans la localisation de l’ enzyme puisqu’ il permet à PC5A d’ être retenu à la surface cellulaire. Sans ce domain riche en cysteine, PC5A n’ est plus retrouvée à la surface car c’ est au niveau de ce domaine que PC5A interragit avec TIMP-2 qui se trouve à la surface cellulaire. De plus, la surexpression de PC5A dans des cellules tumorales cause une diminution de la proliferation, par contre, les cellules traitées avec un siRNA contre PC5 prolifèrent d’ avantage. Cette recherche sur la structure-fonction du segment pro et du domaine riche en cysteine de PC5A va aider à définir la fonction biologique de cet enzyme en plus de définir le role de ces deux domaines chez les autres convertases. vii Preface This thesis is submitted to the department of Graduate and Postdoctoral studies, McGill University. There are two types of thesis formats accepted for submission. The first option is the traditional thesis format and as an alternative, the second format consists of a dissertation based on a collection of submitted publications. This present dissertation follows the second format consisting of a manuscript- based thesis conforming to the specifications of the faculty of Graduate and Postdoctoral studies. Candidates have the option of including, as part of the thesis, the text of one or more papers submitted, or to be submitted, for publication, or the clearly- duplicated text (not the reprints) of one or more published papers. These texts must conform to the "Guidelines for Thesis Preparation" with respect to font size, line spacing and margin sizes and must be bound together as an integral part of the thesis. The thesis must be more than a collection of manuscripts. All components must be integrated into a cohesive unit with a logical progression from one chapter to the next. In order to ensure that the thesis has continuity, connecting texts that provide logical bridges preceeding and following each manuscript are mandatory. The thesis must conform to all other requirements of the "Guidelines for Thesis Preparation" in addition to the manuscripts: a table of contents; a brief abstract in viii both English and French; an introduction which clearly states the rational and objectives of the research; a comprehensive review of the literature (in addition to that covered in the introduction to each paper); a final conclusion and summary; a thorough bibliography; Appendix containing an ethics certificate in the case of research involving human or animal subjects, microorganisms, living cells, other biohazards and/or radioactive material. As manuscripts for publication are frequently very concise documents, where appropriate, additional material must be provided (e.g., in appendices) in sufficient detail to allow a clear and precise judgement to be made of the importance and originality of the research reported in the thesis. In general, when co-authored papers are included in a thesis the candidate must have made a substantial contribution to all papers included in the thesis.

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