Structure-Function Studies of the Proprotein Convertases: the Pro- and P-Domains BY
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Structure-Function Studies of the Proprotein Convertases: The Pro- and P-domains BY Mei Zhong A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirernents of the degree of Doctoor of Philosopby O Mei Zhong, November 1999 Department of Medicine Division of Experimental Medicine McGill University Mootreal, Québec NationaJ Library Bibliothèque nationale 1*1 ofCanâda du Canada Acquisitions and Acquisitions et Bibliographie Seivices services bibliographiques 385 Wdington Street 395,nie Wellington OmwaON KlAOW OUawaON K1AûN4 canada Canada The author has granted a non- L'auteur a accordé une Licence non exclusive licence aiiowing the exclusive permettant à la National Libraty of Canada to Bibliothèque nationale du Canada de reproduce, loaa, distribute or seli reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electroaic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Acknowledgments I am greatly indebted to my supervisor, Dr. Nobil 6. Seidah for giving me the chance to join his lob, for his guidance and support throughout the years, for his invaluable advice, encouragement and criticism. His enthusiasm, tirelessness will be a positive influence throughout rny career. It was an honor to work with him in a highly motivated, diverse environment. 1 thank al1 the members, former and present, in Chétien-Lazure- Seidah laboratories for their assistance, support and friendship throughout the years. Especially Dr. Majambu Mbikay, Dr. Claude Lazure, Dr. Michel Chrétien, Dr. Ajoy Basak, Dr. Scott Munzer, Suzanne Benjannet, Seyed J. Mowla, Maya Mamarbassi, Josée Hamelin, Diane Savaria, Annie Lemieux, Dany Gauthier, Eric Bergeron and Andrew Chen. 1 am also grateful to Ms. Sylvie Ernond for her cheerful and patient assistance in al1 secretarial matters. Special thanks to Jim Rochernont to whom 1 will always be indebted and with whom I always laughed, because his trip to China bound me to such a great lab with the most great people. Without him, it would have been impossible to have such a great memory. 1 thank the Protein Engineering Network Centres of Excellence of Canada for its f inancial support. I thank my thesis committee members, Dr. James Chromlish, Dr. Hugh Bennett, Dr. Yogesh Patel for their advice and directions. Last but not Icast, 1 thank my parents for raising and supporting me and giving me the f reedom to pursue my dream. My beloved boyf riend Dr. Hongyan An for his caring love, patient, and constant support. Abstract The proprotein convertases (PCs) are involved in the activation of a wide variety of precursors via limited proteolysis at either single or paired basic residues. a crucial regdatory process in both normal and disease States. Seven members of this farnily were identified including £brin, PC1, PC2, PC4, PACE4, PC5 and PC7. Al1 of which exhibit a signal peptide, a prosegment, a catalytic domain, a P-domain and a specific C-terminal segment. The present work concentrated on the characterization of two structural elernents, namely the prosegment and the P-domain, which are cntical for enzymatic function and cellular trafficking. We examined the biosynthesis, functional activity and cellular localization of two PACE4 isoforms generated by differential splicing, the Full length PACE4-A and the C-terminally truncated PACECCS that lacks 1 1 arnino acids at the end of its chaperone-like P-domain. Cellular expression demonstrated that PACE4-A codes for a functional secretable enzyme capable of cleaving pro7B2 into 7B2. However. PACE4-CS is not secreted and remains in the endoplasmic reticulurn as an inactive zymogen form, therefore emphasizing the importance of the integrity of the P-domain. The prosegment is presumed to act both as an intramolecular chaperone and an inhibitor of its parent enzyme. We purified recombinant prosegments of funn @Furin) and PC7 (pPC7) fiom bacteria. The inhibitory potencies and selectivities of the prosegments were tested in vitro and ex vivo. The pFurin and pPC7 are potent inhibitors (ICso low nM) of their parent enzymes. Whereas pPC7 is more selective than pFurin. Most of the inhibitory potency seems to reside at the C-terminal region immediately preceding the pcimary cleavage site of the prosegment with the Pl Arg playing a critical role. Furthemore, overexpression of prosegments in ce11 lines efficiently blocked precursor processing of the neurotrophins NGF and BDNF. For the first tirne our results showed that PC prosegments expressed ex vivo as independent domains could act in nuns to inhibit precursor maturation by intracellular PCs. in conclusion, the prosegrnent and the P- domain of PCs are cntical for PC zyrnogen activation, enzymatic activity and intracellular traffrcking. The prosegment study also presents a novel enzyme silencing strategy, which provides a novel approach to the treatment of a variety of pathologies. Les proprotéines convertases (PCs) sont impliquées dans l'activation d'une panoplie de précurseurs via la protéolyse limitée à des résidus basiques ou dibasiques, un processus essentiel à l'homéostasie ainsi que dans le développement de maladies. Sept membres de cette famille ont été identifiés dont furine, PCI, PC2, PC4, PACE4, PCS et PC7. Tous possèdent un peptide signal, un prosegment, un domaine catalytique. un P- domaine et un segment C-terminal spécifique. Le présent travail se concentre sur la caractérisation du prosegment et du P-domaine des PCs qui sont critiques pour leur activité enzymatique et leur trafic cellulaire. Nous avons examiné la biosynthèse, I'activité fonctionnelle et localisation cellulaire de deux isoformes de PACE4 générés par épissage alternatif, le PACE4 pleine longueur et la forme tronquée en C-terminal PACE4CS, qui manque 11 acides aminés à la fin de son P-domaine. La surexpression cellulaire de PACE4-A produit un enzyme fonctionnelle, sécrétée et capable de cliver le pro782 en 782. Cependant, PACE4-CS n'est pas sécrétée et reste sous forme de zymogène inactif démontrant l'importance de l'intégrité du P-domaine. Le prosegment agit possiblement comme chaperon intramoléculaire et inhibiteur de l'enzyme parentale. Nous avons purifié les prosegments recombinants de funne @Fuine) et PC7 @PC7) de bactéries. Le potentiel d'inhibition et la sélectivité des prosegrnents ont été testés in vitro et ex vivo. Le pFurine et le pPC7 sont des inhibiteurs efficaces (IClo de l'ordre du nM) de leur enzyme parentale. Le pPC7 montre une plus grande sélectivité que le pFurine. La majorité du potentiel inhibiteur semble résider dans la région C-terminale précédant immédiatement le site de coupure primaire du prosegment avec l'kg en Pl jouant un rôle critique. De plus, la surexpression de prosegments dans des lignées cellulaires bloquent efficacement la maturation des neurothrophines NGF et BDNF. Pour la première fois, nos résultats démontrent que les prosegments de PCs exprimés comme domaine indépendant, ex vivo, peuvent agir en pans pour inhiber la maturation effectuée par des PCs intracellulairement. En conclusion, le prosegrnent et le P-domaine des PCs sont critiques dans l'activation des PCs sous forme de zymogènes, pour leur activité enymatique et leur trafic intracellulaire. L'expression cellulaire du prosegment représente une nouvelle approche afin d'inhiber les convertases et pour le traitement d'une variété de pathologie. Preface This thesis is submitted to the McGill University Faculty of Graduate studies and Research. This thesis is presented according to manuscript-based thesis format: 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 integrai part of the thesis. The thesis must be more than a collection of manuscripis. A11 cornponents must be integrated into a cohesive unit with a logical progression fiom one chapter to the next. In order to ensure that the thesis has continuity. connecting texts that provide logical bridges between the different papers are mandatory . The thesis mut conform to al1 other requirements of the "Guidelines for Thesis Preparation" in addition to the manuscripts The thesis must include the following: a table of contents; an abstract in 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 fdconclusion and summary. As manuscripts for publication are freqwntly very concise documents. where appropriate, additional material mut be provided (e.g.. in appendices) in suficient 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-authoredpapers are included in a thesis the candidate must have made a substantial contribution to al1 papers included in the thesis. In addition, the candidate is required to make an explicit statement in the thesis as to who contributed to such work and to what extent. This statement should appear in a single section entitled "Contributions of Authon" as a preface to the thesis. The supervisor must attest to the accuracy of this staternent at the doctoral oral defence. Since the task of the examiners is made more dificult in these cases, it is in the candidate's interest to clearly specify the responsibilities of al1 the authoa of the CO-authoredPapen.