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Canada Du Canada Acquisitions and Direction Des Acquisitions Et Bibliographie Services Branch Des Services Bibliographiques 395 Wellington Street 395 National Ubrary Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Direction des acquisitions et Bibliographie Services Branch des services bibliographiques 395 Wellington Street 395. rue Wellington Ottawa, Ontario Ottawa (Ontario) K1A QN4 K1A QN4 Ouf ',Ic Nolrt' réfc'/t'nct' NOTICE AVIS The quality of this microform is La qualité de cette microforme heavily dependent upon the dépend grandement de la qualité quality of the original thesis de la thèse soumise au submitted for microfilming. microfilmage. Nous avons tout Every effort has been made to fait pour assurer une qualité ensure the highest quality of supérieure de reproduction. reproduction possible. If pages are missing, contact the S'il manque des pages, veuillez university which granted the communiquer avec l'université degree. qui a conféré le grade. Some pages may have indistinct La qualité d'impression de print especially if the original certaines pages peut laisser à pages were typed with a poor désirer, surtout si les pages typewriter ribbon or if the originales ont été university sent us an inferior dactylographiées à l'aide d'un photocopy. ruban usé ou si l'université nous a fait parvenir une photocopie de qualité inférieure. Reproduction in full or in part of La reproduction, même partielle, this microform is governed by de cette microforme est soumise the Canadian Copyright Act, à la Loi canadienne sur le droit R.S.C. 1970, c. C-30, and d'auteur, SRC 1970, c. C-30, et subsequent amendments. ses amendements subséquents. Canada • Cysteine proteases: Interaction with cystatin C and sequence to structure-function relationships Paul J. Berti Department of Biochemistry McGill University, Montréal October 1993 • A Thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements of the degree of Ph.D. • ©Paul J. Berti, 1993 National Library Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Direction des acquisitions et Bibliographie Services Branch des services bibliographiques 395 Wellington Street 395, rue Wellington Ottawa, Ontario Ottawa (Ontario) K1A ON4 K1A ON4 Ovt '.le NO/IV t~l~enco The author has granted an L'auteur a accordé une licence irrevocable non-exclusive licence irrévocable et non exclusive allowing the National Library of permettant à la Bibliothèque Canada to reproduce, loan, nationale du Canada de distribute. or sell copies of reproduire, prêter, distribuer ou his/her thesis by any means and vendre des copies de sa thèse in any form or format, making de quelque manière et sous this thesis available to interested quelque forme que ce soit pour persons. mettre des exemplaires de cette thèse à la disposition des personnes intéressées. The author retains ownership of L'auteur conserve la propriété du the copyright in his/her thesis. droit d'auteur qui protège sa Neither the thesis nor substantial thèse. Ni la thèse ni des extraits extracts fram it may be printed or substantiels de celle-ci ne otherwise reproduced without doivent être imprimés ou his/her permission. autrement reproduits sans son autorisation. ISBN 0-315-94581-8 Canada Cysteine proteases: Interaction with cystatii'"1 C and sequence analysis Abstract An alignment/phylogeny of the papain superfamily of eysteine proteases was created • from which the existence of at least 11 families of mammalian eysteine proteases was inferred, including Iwo as yet uncharacterized ones. Character wcighting increased the average speed of maximum parsimony phylogenetic searches Iwo- to three-fold. To study a sequence motif in pro-cysteine proteases and mammalian cystatins, eystatin C, a eysteine protease inhibitor, was expressed and purified using a new affinity method giving > 99% purity. Methionine oxidation to the suIfoxide occurring during protein production was eliminated. Reduced stability caused by Met14<lxidation was demonstrated. No function for the sequence motif was found, however cystatin C underwent a coniàrmational char>ge at acidic pH that rendered the His86-Asp87 pepti~e bond labile to proteolysis. This could constitute amechanism for clearing inappropriately localized cystatins from the acid environment of lysosomé~ andexplain differences between the X-ray and NMR structures of the homologous chicken cystatin. • • Résumé Un alignement/phylogénie de la superfanùlle papaïne des protéases cysteines a été créé, • duquel l'existence d'au moins onze fanùlle" de protéases cysteines mammifères a été inférée, dont deux ne sont pas encore charactérisées. La variation de la pondération des caractèr.,s a augmenté la vitesse des recherches phylogénétiques de deux à trois fois. Pour étudier un motif de séquence présent dans les pro-protéases cysteines et les cystatines mamnùfère, la cystatine C, un inhibiteur des protéases cysteines, a été produite et purifiée avec une nouvelle méthode d'affinité qui donne une protéine de pureté> 99%. L'oxydation des méthionines aux sulfoxydes pendant la production de la protéine a été élinùnée. L'oxydatian de Metl4 a été demontrée comme diminuant la stabilité. Aucune fonction du motif n'a été decouverte, cependant il y a un changement dans la structure de la cystatine C qui est tributaire du pH et qui la rend sujet à la protéolyse. Ceci peut constituer un méchanisme d'élinùnation des cystatines de l'environnement acide des lysosomes, et peut aussi expliquer les différences entre les structures cristallographique et RMN de l'homologue cystatine de ta poule. • • Table of Contents • List of Tables i List of Figures ii List of Abbreviations iii Preface iv Acknowledgements ; vi Contribution to Original Knowledge viii 1. Introduction 1 Scope of the work 1 Proteolysis 2 Cysteine proteases 4 Mechanism, kinetics description 6 Mechanism, chemical description 7 pH-dependence 9 Thiolate/imidazolium pair 11 Specificity ............................................... 13 Structure 13 • Function "" 14 Cystatins 17 Affinity purification ', 17 Cystatin superfamily 19 Kinetics and mechanism of inhibition 19 Structure 21 Function 23 Sequence analysis 24 Sequence comparison 26 Pairwise alignment ,ft 26 Dayhoff mutation data matrix 28 Current issues 29 Database searching/Homology detection 29 Multiple alignment 30 l'hylogenetic inference 31 • Bootstrapping 34 Examples '' , 35 • 2. Preface to Aligmnent/Plrylogcny of tlrc Papain SlIpcrfalllily of Cystcinc Protcascs 36 3. Alignment/Phylogeny of the Papain Superfamily of Cysteine Protcases 37 Abstract 39 Introduction , 40 Methods " " " 42 Resull''- 45 Discussion 68 4. Preface to Effccts and Elimination of Metlrioninc Oxidation in Rccolllbi'Illllt 1111111all Cystatin C and Local pH-depcndent Conforlllational Clrangcs Lcadillg ta Proteolytic SlIsceptibility of Cystatin C 78 5. Effects and Elimination of Methionine Oxidation in Recombinant Human Cystatin <BI -:- Sti~111ary ..................................................... 83 Introduction 84 Materials and Methods 86 Results 91 Discussion 99 • 6. Local pH-dependent Conformational Changes Leading to Proteolytic Susceptibility of Cystatin C 103 Abstract 104 Introduction ',> .•..••.••.•••• 105 Experimental 107 Results .................................................... .. 108 Discussion ' " ....... 118 7. Summary 124 8. References 128 Appendix A. Alignment of bleomycin hydrolases 142 Appendix B. AÙ~;;ment of calpain-group protease domains. 143 Appendix C. Listing of MLP.BA5 144 Appendix D. Listing of 5TDRA.l\JD.BAS 166 Appendix E. Listing of TIMES.BAS 168 Appendix F. Listing of AREA.BA5 170 • Appendix G. Listing of PAR5E.BA5 ' 176 -i- • List of Tables Table 1. Relative search speeds with weighting data 65 Table Il. Dissociation equilibrium constants of non- and mono-oxidized cC's .. 98 • • -jj- List of Figures • Figure 1. Mechanism of cysteine protease catalyzcd hydrolysis. 8 Figure 2. A simple pH-profile. 10 Figure 3. (k",1KMlob, vs. pH profile for papain. 12 Figure 4. Papain structure :.... 15 Figure 5. E-64 structure and mechanism. 18 Figure 6. Papain-chicken cystatin structure , 22 Figure 7. Example of Needleman-Wunsch dynamic programming algoritlun 27 Figure 8. Hypothetical example of a maximum parsimony phylogcny 33 Figure 9. Structure-based alignment. 46-47 Figure 10. Table of sequence names. 48-50 Figure 11. Alignment of active site regions of bleomycin hydrolase-, papain-, and calpain-groups 51-52 Figure 12. Phylogenetic tree of calpain-group sequences. 54-55 Figure 13. Alignment of the papain-group sequences. 56-60 Figure 14. Phylogenetic tree of papain-group sequences. 61-62 Figure 15. Relative speed of phylogenetic searches with weighted data. 67 • Figure 16. GxNxFxD motif. 79 Figure 17. Analytical reversed phase HPLC of pure cC. 92 Figure 18. IH NMR identification of oxidized forms of cC. 94 Figure 19. Analytical affinity chromatography. .' 96 Figure 20. Sites of c1eavage of cystatin C. 110 Figure 21. Selwyn's test for enzyme inactivation during His86ÎAsp87 hydrolysis. 111 Figure 22. Test of steady state assumption. 112 Figure 23. pH-dependence of His86ÎAsp87 hydrolysis 113 Figure 24. pH-dependence of Gly4ÎLys5 hydrolysis 115 Figure 25. pH-dependence of cC 'papain c.d. spectra. 116 • -' -iii- • List of Abbreviations Standard one- and three-letter aIIÙno acid abbreviations are used throughoutthe thesis. Abbreviated sequence names used in "Alignment/phylogeny of the papain superJamily of cysteine proteases" and Appendices A and B appear in Fig. 10. cC - human cystatin C E-64 .:. l-(L-trans-epoxysuccinylieucylaIIÙno)+guanidinobutane ER - endoplasIIÙc reticulum FPLC - fast protein liguid chromatography Gly4ÎLys5
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