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Activation Mechanisms for Zymogens Belonging to the Papain Family of Cysteine Proteases Were Investigated

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Activation Mechanisms for Zymogens Belonging to the Papain Family of Cysteine Proteases Were Investigated ACTIVATION MECHANISMS FOR ZYMOGENS BELONGING TO THE PAPAIN FAMILY OF CYSTEINE PROTEASES OMAR QURAISHI Biochemistry Department McGill University, Montreal September 1999 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements of the degree of Doctor of Philosophy O Omar Quraishi, 1999 National Library Bibliothèque nationale I*I of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON KIA ON4 Ottawa ON KIA ON4 Canada Canada Yow file Votre rélérence Our fi& Notre rdférence The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distibuer ou copies of this thesis in microfom, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/fXm, 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. PAGE Acknowiedgments Abstract Résumé Introduction and Literature Review - Serine Proteases - Activation of Trypsinogen and Chymotrypsinogen - Prothrombin Activation - Proprotein Convertases - Aspartic Proteases - Zinc Metailoproteases - Procarboxypeptidase A and B - Prostromelysin-1 - Cysteine Proteases - Caspase Family - Pap ain Family Chapter 1: The Occluding Loop in Cathepsin B Defines the pH Dependence of Inhibition by Its Propeptide - Connecting Text for Chapters 1 and 2 Cha~ter2: Identification of Interna1 Autoproteolytic Cleavage Sites Within the Prosegments of Recombinant Procathepsin B and Procathepsin S - Connecting Text for Chapters 2 and 3 Chapter 3: Functional Expression of Human Procathepsin H in Pichiapastoris and Attempts at its Correct Processing Summary References LIST OF FIGURES AND TABLES FOLLOWING PAGE WTRODUCTION AND LITERATURE REVIEW FIGURE 1: Activation of Chyrnotrypsinogen IFIGURIF 2: Prothrombinase Cornplex FIGURE 3 :Progastrïcsin Activation FIGURE 4: Sequence Alignment of Cathepsin L-like Prodomains FIGURE 5: Prosegments of Cathepsin L and Cathepsin B FIGURE 6: Structures of Procathepsin L and Procathepsin B FIGURE 7: Structure of Procathepsin B FIGURE 8: Model of Cystatins in Cornplex with Papain ILLUSTRATIONS FOR CHAPTER 1 FIGURE 1: Prosequences of Rat and Human Cathepsin B FIGURE 2: Conformations Adopted by the Occluding Loop FIGURE 3: Autocatalytic Processing of Procathepsin B TABLE 1: Propeptide Inhibition of Cathepsin B Mutants TABLE 2: Role of Aspartic Acid Residues in Propeptide TABLE 3: Activity of Cathepsin B Mutants Towards 2-Phe-Arg-MCA ILLUSTRATIONS FOR CHAPTER 2 FIGURE 1: Am3-stained SDS-PAGE of Procathepsin B Processing FIGURE 2: PVDF-membranes of Procathepsins B and S FIGURE 3: Autoproteolytic Cleavage Sites in Procathepsins B and S FIGURE 4: Active Site Cleft in Rat Cys29Ser Procathepsin B FIGURE 5: Plots of k,b, versus Proenzyme Concentration FXGURE 6: Western Blots of Wild-Type and Arg8Ala Propapain ILLUSTRATIONS FOR CHAPTER 3 FIGURE 1: Homology Mode1 of Procathepsin H 111 FLGURE 2 (A,B):Non-Reducing SDS-PAGE of Glycosylated Procathepsin H 111 FIGURE 3 : Structural Alignment of Prosegments Composed of Cys82p 111 FIGURE 4: Progress Curves of Aminopeptidase Activity for Cathepsin H Isoforms 111 The research was fûnded in part by the Govemment of Canada's Network of Centres of Excellence Program supported by the Medical Research Council of Canada and the Natural Sciences and Engineering Research Council of Canada through Pence Inc. (the Protein Engineering Network of Centres of Excellence). The author gratefully acknowledges the mentorship and encouragement provided by Dr. Andrew C- Storer who made sure that he left the laboratory with the ability to 'think independently' in order to be prepared to take on firture challenges. The author also wishes to thank the encouragement and advice provided by several members of the laboratory and other employees of the Biotechnology Research Institute, National Research Council Canada @ast and present) : Drs. Marko Pregel, Dorit K. Nagler, Robert Ménard, Edmund Ziomek, Richard Hrabal, J. Sivaraman, Shahul NiIar, and Dr- John S. Mort (Joint Diseases Laboratory, Shnners Hospital for Children). The author wishes to thank Mr. Robert Dupras for introducing him to the hockey and softball games which enabled him to interact with the fiendly employees of the BRI. The author would also like to reserve special mention for his wife, Dr. Katharine A. Cacpenter, who demonstrated tremendous patience during the course of these studies. To his beloved parents, (late) Abdul Mateen Quraishi and Yolande Quraishi, for instilhg the values of obtaining a higher education fiom a very early age. Finally, the author is grateful to the Almighty God :The Creator, Ruler, and Sustainer, Cherisher of Al1 Worlds, The Most Gracious, and The Most Mercifil. ABSTRACT The activation mechanisms for zymogens belonging to the papain family of cysteine proteases were investigated. This was accomplished using site-directed mutagenesis, kinetic measurements, the identification of processing intermediates, and the analysis of the various X-ray crystal structures reported to date. Procathepsin B is a unique precursor of papain-like enzymes in that it is composed of a shorter prodomain ; i-e., 62 residues versus 290 residues for those belonging to the cathepsin L-subfamily, and the mature enzyme is composed of a twenty residue insertion termed the occluding loop. In this study, the pH dependence of cathepsin B inhibition by its propeptide was shown to be eliminated upon the removal of this enzyme's occluding loop. Furthemore, variants of cathepsin B c-g the mutation Asp22Ala or His 11 OAla also displayed a loss of pH dependence for their affinity to the propeptide inhibitor. Sirnilarly, the overall rate of autoprocessing for full-length procathepsin B was shown to be affected by the occluding loop mutations. These results suggest a possible influence of the pH-dependent stability of the occluding loop on the overall rate of processing for this precursor. The addition of the protein-protehase inhibitor, cystatin C, impeded the overall rate of autoactivation for procathepsin B and procathepsin S and caused the accumulation of processing intermediates for these precursors which were subsequently identified by autornated Edrnan degradation. The N-terminal sequences of these processing intermediates correspond to an area of the prodomains which binds through the substrate-binding clefts of these enzymes, thus suggesting a plausible intramolecular step of processing for this family of zymogens. This unimolecular mechanism was found to rely on the confoxmational mobility of prosegment residues (Le., at the C-terminal end). Furthemore, in contrast to what has been observed for zymogens belonging to the aspartic protease family, it was determined that charged residues located at the N-temiuius of the mature segment found in propapain do not contribute to the overall pH-triggering mechanism of activation for this precursor. Procathepsin H was determined to be an unusual mammalian member of the cathepsin L-subfamily due to its inability to autoprocess, and the aminopeptidase activiw of mature cathepsin H was found to be incapable of converting its own precursor. Furthemore, prosegment residues located near the pro/rnature junction of procathepsin H are highly resistant to the proteolytic action of secondary proteases. These findings are consistent with the pre-formation of a disulfide bond within the cathepsin H precursor which links the prodomain to the enzyme using Cys82p and Cys214. Consistent with the fïndings for procathepsins B and S, the unrestncted conformational mobility at the C-terminal end of the prosegment (i-e., near the pro/rnature junction) is an important prerequisite for efficient autoactivation to occur among zymogens of papain-like enzymes. RÉsU1MÉ Les mécanismes d'activation de zymogènes des protéases à cystéine de la famille de la papaine ont été étudiés. Ceci a été accompli en utilisant la mutagénèse dirigée, des mesures de cinétique, l'identification d'intermédiaires de maturation, et l'analyse des diverses structures crystallines connues a ce jour. La procathepsine B est un précurseur unique dans le groupe d'enzymes similaires à la papaine du au fait qu'elle est composée d'une prorégion plus courte, contenant seulement 62 résidus, comparativement aux 90 et plus résidus rencontrés dans les précurseurs appartenant au sous-groupe d'enzymes similaires à la cathepsine L. De plus, la cathepsine B mature contient une insertion de vingt acides amines qui constituent une boucle d'occlusion. Dans cette étude, il a été démontré que l'influence du pH sur l'inhibition de la cathepsine B par son propeptide est éliminée lorsque cette boucle d'occlusion n'est pas présente. De plus, des variantes de la cathepsine B contenant la mutation Asp22Ala ou Hisl lOAla ont aussi perdu cette dépendence du pH pour l'affinité de l'inhibiteur propeptide. Similairement, la vitesse globale d'automaturation de la procathepsine B est affectée par les mutations de la boucle d'insertion.
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