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Analyse Structurale Et Fonctionnelle De La Préséniline-1 Humaine : Implications Dans La Maladie D'alzheimer

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Analyse Structurale Et Fonctionnelle De La Préséniline-1 Humaine : Implications Dans La Maladie D'alzheimer SÉBASTIEN HÉBERT ANALYSE STRUCTURALE ET FONCTIONNELLE DE LA PRÉSÉNILINE-1 HUMAINE : IMPLICATIONS DANS LA MALADIE D’ALZHEIMER Thèse présentée à la Faculté des études supérieures de l’Université Laval dans le cadre du programme de biologie cellulaire et moléculaire pour l’obtention du grade de Philosophiae Doctor (Ph.D.) FACULTÉ DE MÉDECINE UNIVERSITÉ LAVAL QUÉBEC DÉCEMBRE 2003 © Sébastien S. Hébert, 2003 i RÉSUME COURT La préséniline-1 (PS1) mutée est associée à un développement très précoce de la maladie (chez les individus de 24-55 ans) d’Alzheimer. Dans la cellule, la PS1 existe principalement sous forme de fragments (N-terminaux et C-terminaux, respectivement NTFs et CTFs) qui semblent nécessaires à sa fonction et à l’activité γ- secrétase étroitement liée à la formation des peptides β-amyloïdes neurotoxiques. Nous avons étudié l’organisation structurale et fonctionnelle de la PS1, ses fragments, et leurs relations avec les protéines impliquées dans la production des peptides β- amyloïdes. Dans un premier temps, des expériences en levure, confirmées avec des techniques d’immunobuvardage et d’immunoprécipitation en cellules de mammifères, nous ont permis de mettre en évidence un rôle de l’holoprotéine de la PS1 dans la génération des fragments NTFs et CTFs. De plus, il a été possible de démontrer une interaction directe entre PS1 et BACE (enzyme de type aspartyl protéase aussi appelée β-secrétase), les deux joueurs clés dans la production des peptides β- amyloïdes. Finalement, l’étude du rôle fonctionnel de cette interaction nous a fournis des résultats permettant de suggérer une régulation de la PS1 par BACE. _________________________ _________________________ Dr Georges Lévesque, Ph.D. Sébastien S. Hébert ii RÉSUME LONG Le gène préséniline-1 (PS1) est associé à la forme familiale de la maladie d'Alzheimer lorsqu’il est muté. Les mutations dans le gène PS1 (au-delà de 100) sont dominantes, fréquentes (environ 80% des cas familiaux connus) et conduisent à une forme très agressive de la maladie (chez les individus de 24-50 ans). Malgré des recherches intensives au cours des dernières années, les fonctions précises de la PS1 demeurent inconnues. Des analyses in vivo et in vitro du métabolisme de PS1 révèlent que la protéine subit une coupure endoprotéolytique. Il en résulte une accumulation de 2 fragments prédominants (N-terminal et C-terminal, respectivement NTF et CTF). Il semblerait exister un lien intime entre l’activité enzymatique de type γ-secrétase et l’hérérodimère NTF/CTF de PS1. Dans le cadre de ce projet de recherche, nous avons étudier d’une façon plus approfondie la relation qui existe entre préséniline-1, ses fragments, et l’activité γ-secrétase. Plus spécifiquement, à partir des connaissances structurales acquises de la PS1, nous voulions développer un essai fonctionnel enzymatique in vivo dans le but d’évaluer le rôle de l’holoprotéine et/ou des fragments N- et C-terminal normaux et mutées dans le clivage de la protéine amyloïde. Dans un premier temps, des expériences en levure, confirmées avec des techniques d’immunobuvardage en cellules de mammifères, nous ont permis de mettre en évidence un rôle précurseur qu’exerce l’holoprotéine de la PS1 dans la génération des fragments NTFs et CTFs. De plus, des expériences de type double- hybride en levure, confirmées avec des techniques d'immunoprécipitation in vitro et in vivo, nous ont permis de démontrer une interaction entre PS1 et BACE (enzyme de type aspartyle protéase aussi appelée bêta-secrétase), les deux joueurs clés dans la production du peptide amyloïde bêta-42 toxique, lequel est le constituant majeur des plaques amyloïdes observées chez les patients atteints de la maladie d'Alzheimer. Finalement, l’étude du rôle fonctionnel de cette interaction nous a fourni des résultats permettant de suggérer une régulation de la PS1 par BACE. _________________________ _________________________ Dr Georges Lévesque, Ph.D. Sébastien S. Hébert iii REMERCIEMENTS En premier lieu, je tiens à remercier mon directeur de recherche, le Dr Georges Lévesque, de m'avoir guidé, encouragé, conseillé, tout en me laissant une grande liberté et en me faisant l'honneur de me déléguer plusieurs responsabilités dont j'espère avoir été à la hauteur. De plus, je le remercie pour sa grande disponibilité et son ouverture d’esprit qui m’ont énormément aidé à terminer ce projet. Pour les précieux conseils et l’assistance matérielle, je remercie chaleureusement Madeleine Carreau, Yves Labelle et Edward Khandjian. Je voudrais remercier sincèrement Caroline, Cédric, Chantal, Isabelle, Mélissa et Valérie pour leurs discussions, suggestions, et assistance (c’est à dire « bummage ») matérielle. De plus, je voudrais remercier ma famille, spécialement mes parents, pour leur support unique tout au long de ces études. Finalement, je tiens à remercier tout spécialement mon épouse Maryline. Elle m’a aidé à surmonter les obstacles qui n’ont pas manqué de surgir tout au long de mon parcours. Je n’en serais pas arrivé à ce point sans son soutien (et ses corrections de grammaire !). Je la remercie également pour sa grande patience et ses nombreuses nuits blanches pour « laisser papa dormir afin qu’il soit en forme le lendemain… » Le centre de recherche de l’Hôpital St-François d’Assise, à Québec, et la Société d’Alzheimer du Canada ont généreusement contribué à ce travail par l’entremise de bourses d’études. iv AVANT-PROPOS Cette thèse contient l’ensemble des travaux effectués lors de mon doctorat qui a débuté en janvier 2001. Elle représente l’aboutissement de nombreuses heures de travail réparties entre le laboratoire, la route, l’hôpital et la maison. Il faut dire que ces trois dernières années ont été remplies de toutes sortes d’événements incluant un mariage fortuné et la naissance de ma p’tite choupette. Mon but est d’offrir un ouvrage agréable à lire, aux lecteurs de tout niveau, scientifiques ou non. Pour cela, ce travail a été conçu de façon à offrir un contenu relativement peu pointu. Toutefois, une recherche plus approfondie des références retrouvées dans la bibliographie ainsi que dans les articles publiés, permettra de mieux comprendre, au besoin, les mécanismes biologiques présentés dans ce travail. Dans l’ensemble, ce document contient 7 chapitres répertoriés de façon typique possédant une introduction, un développement et une conclusion. Vous trouverez dans le développement les trois articles scientifiques qui ont été publiés pendant de mon doctorat. Une note précisant mon rôle dans la préparation des articles précède chacun des chapitres 3, 4 et 5. Joints à l’article prêt pour la soumission au chapitre 6 et aux résultats supplémentaires présentés en annexe, ils résument l’ensemble de mes recherches effectuées dans le cadre de ce projet. Ceci étant dit, je vous souhaite bonne lecture v À ma choupette, alias ma p’tite « pitbull », Laeticia vi TABLE DES MATIÈRES RÉSUMÉ COURT..........................................................................................................i RÉSUMÉ LONG ...........................................................................................................ii REMERCIEMENTS.................................................................................................... iii AVANT-PROPOS ........................................................................................................iv TABLE DES MATIÈRES ............................................................................................vi LISTE DES ABRÉVIATIONS.................................................................................. viii LISTE DES FIGURES ..................................................................................................x LISTE DES TABLEAUX.............................................................................................xi LISTE DES ANNEXES …………… …………………………………………...…xii INTRODUCTION .........................................................................................................1 CHAPITRE I..............................................................................................................1 La maladie d’Alzheimer ........................................................................................1 Origine et statistiques.........................................................................................1 Causes et facteurs de risque ...............................................................................3 Protéines associées à la forme familiale de la maladie d’Alzheimer.....................6 L’AβPP, son métabolisme et la génération des peptides amyloïdes .................7 Les présénilines..................................................................................................9 Propriétés biologiques et biochimiques de la préséniline-1 (PS1).......................10 Distribution cellulaire de la PS1 ......................................................................10 Topologie et métabolisme de la PS1................................................................10 Fonctions putatives de la PS1 ..........................................................................12 La γ-secrétase.......................................................................................................14 La β-secrétase ......................................................................................................20 HYPOTHÈSES POSÉES ET OBJECTIFS .................................................................23 CHAPITRE II ..........................................................................................................23
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