analyse structurale et fonctionnelle de la … · secrétase étroitement liée à la formation des...
TRANSCRIPT
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
Analyse de l’organisation structurale de la PS1 ..................................................23
Revue de littérature ..........................................................................................23
Hypothèse et objectif .......................................................................................24
Développement d’un essai fonctionnel enzymatique pour la γ-secrétase............24
vii
Revue de la littérature ......................................................................................24
Hypothèses et objectifs ....................................................................................25
Méthodologie (résumé)........................................................................................25
DÉVELOPPEMENT ...................................................................................................27
CHAPITRE III .........................................................................................................27
Résumé.................................................................................................................28
Dimerization of presenilin-1 in vivo: suggestion of novel regulatory mechanisms
leading to higher order complexes.......................................................................29
CHAPITRE IV.........................................................................................................52
Résumé.................................................................................................................53
Oligomerization of human presenilin-1 fragments. .............................................54
CHAPITRE V ..........................................................................................................71
Résumé.................................................................................................................72
Presenilin-1 interacts directly with the beta-site APP cleaving enzyme (BACE1).
..............................................................................................................................73
CHAPITRE VI.........................................................................................................92
Résumé.................................................................................................................93
BACE1 immature species regulate PS1 holoprotein levels.................................94
CONCLUSIONS ET PERSPECTIVES ....................................................................124
CHAPITRE VII .....................................................................................................124
RÉFÉRENCES BIBLIOGRAPHIQUES...................................................................128
ANNEXE 1 ................................................................................................................145
RÉSULTATS COMPLÉMENTAIRES.................................................................145
Résumé...............................................................................................................145
Introduction........................................................................................................145
Matériels et méthodes ........................................................................................146
Résultats.............................................................................................................149
Interprétations, conclusion et perspectives ........................................................150
ANNEXE 2 ................................................................................................................152
ANNEXE 3 ................................................................................................................153
viii
LISTE DES ABRÉVIATIONS
Unité de mesure :
M : molaire
mM : millimolaire
µg : microgramme
mg : milligramme
g : gramme
µl : microlitre
ml : millilitre
kDa : kilodalton
PM : poids moléculaire
Divers :
α alpha
β bêta
∆ delta
γ gamma
Aβ amyloïde bêta
AβPP « amyloid bêta precursor protein »
AD « Alzheimer’s disease »
ADN acide désoxyribonucléique
ADNc ADN complémentaire
Ade adenine
AICD « AβPP intracellular domain »
Aph-1 « anterior-pharynx defective-1 »
BACE « beta site APP cleaving enzyme »
β-gal β-galactosidase
C83 fragment C83 (possédant 83 acides aminés) de l’AβPP
C99 fragment C99 (possédant 99 acides aminés) de l’AβPP
ix
C° degrée Celcius
C.elegans Caenorhabditis elegans
CO2 « carbon dioxide »
CTF « C-terminal fragment »
cDNA « complementory DNA »
D. melanogaster Drosophila melanogaster
DAPI « 4', 6-diamidino-2-phenylindole »
DAPT « N-S-phenyl-glycine-t-butyl ester »
DMEM «Dulbecco’s Modified Eagle Medium»
DNA « deoxyribonucleotide acid »
EDTA « ethylenediaminetetraacetic acid »
EOFAD « early onset familial Alzheimer’s disease »
ER « endoplasmic reticulum »
FBS «foetal bovine serum»
FFMA forme familiale de la maladie d’Alzheimer
FL « full-length »
GSK-3 « glycogen synthase kinase-3 »
GST « glutathione S-transferase »
His histidine
HMW « high molecular weight »
Leu leucine
LMW « low molecular weight »
NTF « N-terminal fragment »
PMSF « phenyl-methyl sulfonyl fluoride »
PS1 préséniline-1
PS2 préséniline-2
PVDF « polyvinyldifluoride »
RE réticulum endoplasmique
RNA « ribonucleic acid »
RNAi « RNA interference »
SDS « sodium deodecyl sulfate »
Trp tryptophane
TM transmembranaire
x
LISTE DES FIGURES
Figure 1 Les lésions neuropathologiques de la maladie d’Alzheimer.. .........................2
Figure 2 La cascade amyloïde........................................................................................5
Figure 3 Métabolisme de l’AβPP et la génération des peptides Aβ.. ............................8
Figure 4 Structure putative de la PS1. .........................................................................11
Figure 5 Les mutations associées aux formes familiales de la maladie d’Alzheimer et le clivage γ-secrétase.....................................................................................17
Figure 6 Les différents constituants du complexe γ-secrétase.. ...................................18
Figure 7 Mutations de la préséniline-1 et l’AβPP (C99).. ...........................................19
Figure 8 La β-secrétase (BACE)..................................................................................22
Figure 9 Les vecteurs « BD-matchmakers™ » utilisés dans le système « Two- Hybrid system 3 » de Clontech……………………………………….......147
Figure 10 Stratégie utilisée pour étudier le métabolisme de l’AβPP dans la levure..148
Figure 11 Métabolisme de la protéine AβPP dans la levure………………………..149
xi
LISTE DES TABLEAUX
Tableau 1 Indices des cas familiaux de la maladie d’Alzheimer…………………..... 6
Tableau 2 Ressemblances biologiques et biochimiques entre la « présénilinase » et la BACE (β-secrétase)……………………………………………………………127
xii
LISTE DES ANNEXES
Annexe 1 Résultats complémentaires……………………………………………….145 Annexe 2 Mutations de la préséniline-2………………….........................................152
Annexe 3 Les acides aminés et leurs symboles………………………….………….153
INTRODUCTION
CHAPITRE I
La maladie d’Alzheimer
Origine et statistiques
À la fin du 19ème siècle, l'état de démence d’une personne âgée était considéré par la
grande majorité des gens comme habituel et lié à l'usure normale du temps (Berchtold
and Cotman 1998). Cependant, de plus en plus de médecins croyaient qu’il était
nécessaire d’étudier de façon plus approfondie l’histologie du cerveau dans les
maladies mentales, afin de mieux comprendre ces maladies.
Ce fut en 1906, lors d'une réunion de psychiatres allemands, que le docteur Alois
Alzheimer (1864 – 1915) décrit pour la première fois les symptômes d’une patiente
âgée de 51 ans, atteinte de démence et présentant des hallucinations et des troubles
d'orientation (Gottfries 1988; Boller and Forbes 1998). Puisque cette maladie était
inconnue jusque là, le docteur Alzheimer l’appela « maladie particulière du cortex
cérébral ». À l’aide du microscope, l’analyse histologique du cerveau de la patiente
2
avait révélé des dépôts protéiques (plaques), des enchevêtrements neurofibrillaires et
une perte (mort) neuronale, en particulier dans le cortex cérébral et l’hippocampe
(Figure 1). Peu de temps après ces observations, les gens de la communauté médicale
de l’époque donna à cette nouvelle maladie le nom d'Alois Alzheimer (la maladie
d'Alzheimer). Cette description histologique initiale de la maladie d'Alzheimer n’est
guère différente de la description actuelle. Et, comme nous allons le voir, il a été
possible d’avancer plusieurs hypothèses sur les causes de la maladie en s’appuyant sur
ces observations suivantes.
A B
C D
Figure 1 Les lésions neuropathologiques de la maladie d’Alzheimer. A) Schémas originaux des études histologiques du docteur Alois Alzheimer démontrant la présence de lésions (obscurcissement) intracellulaires (enchevêtrements). B) Enchevêtrements neurofibrillaires visualisés avec des nouvelles techniques de coloration et d’observation. C) Schémas originaux des études histologiques du docteur Alois Alzheimer démontrant la présence de corps circulaires à allure fibrilleux avec un noyau dense (plaques). D) Visualisation contemporaine, par technique de coloration au rouge de Congo (« Congo Red »), d’une plaque amyloïde (sénile). La région dense centrale (et sa périphérie) est principalement constituée de peptides Aβ42 insolubles. (Images tirées d’Internet et pouvant être protégées par « copywrite » ©.)
3
Aujourd’hui, des études statistiques démontrent que les hommes autant que les
femmes, toutes ethnies confondues, sont à risque de développer la maladie. Dans les
pays occidentaux, la maladie d’Alzheimer est la première cause de démence et la
quatrième cause de décès, précédée par les maladies cardiaques, le cancer et les
accidents vasculaires cérébraux (AVC). Au Canada, cette affection touche près de 30
% des canadiens âgés de plus de 85 ans1. Actuellement, on estime à au-delà de 300
000 Canadiens atteints de la maladie d’Alzheimer. Chose certaine, en raison du
vieillissement rapide de la population, le nombre de cas et les coûts liés aux soins des
gens atteints de la maladie prendront des proportions alarmantes dans les années
futures.
Causes et facteurs de risque
À ce jour, les causes exactes de la maladie d’Alzheimer demeurent inconnues. En
quête de réponses, les chercheurs étudient les facteurs qui semblent avoir une
influence quelconque sur la progression de la maladie. Ce sont les «facteurs de
risque».
Le plus important facteur de risque de la maladie d’Alzheimer est l’hérédité. En effet,
il a été possible de démontrer que certains facteurs génétiques (gènes) influencent le
développement de la maladie. Dans certains cas, ces facteurs sont directement
responsables d'une forme familiale de la maladie. C'est le cas des mutations
autosomiques dominantes des gènes AβPP, préséniline-1 (PS1) et préséniline-2 (PS2)
situés respectivement sur les chromosomes 21, 14 et 1 (Goate 1997; Selkoe 2001).
Dans d'autres cas, les facteurs génétiques influencent le déroulement de la maladie, ce
sont des facteurs de susceptibilité génétique. C'est le cas du gène codant pour
l'apolipoprotéine E sur le chromosome 19 (Higgins, Large et al. 1997).
1 Source tirée de : www.alzheimer.ca
4
Cependant, ajoutons que les chercheurs actuels s’accordent pour dire que, pour la
majorité des cas, la maladie d'Alzheimer est de causes multifactorielles, c’est à dire
occasionnée par plusieurs facteurs à la fois héréditaires et non héréditaires (Goate
1997; Ling, Morgan et al. 2003). En effet, des études ont permis de démontrer que
seulement 5-10% des cas de la maladie d’Alzheimer représentent des cas familiaux.
L’autre 90-95% des cas de la maladie sont sporadiques2, sans antécédents familiaux et
à étiologie inconnue (Rocchi, Pellegrini et al. 2003). Par exemple, l’âge est un autre
important facteur de risque pour la maladie d’Alzheimer (Ling, Morgan et al. 2003).
En effet, la prédominance de la pathologie double à chaque 5 ans après l’âge de 65
ans. Toutefois, je crois important de mentionner que, contrairement à la croyance
populaire, la démence de type Alzheimer n’est pas un processus normal de
vieillissement.
D’autres facteurs de risque tels l’éducation, l’activité physique, les blessures à la tête,
les métaux lourds, et même la prise de certains médicaments ont été soupçonnés
d’induire et/ou d’accélérer le développement de la maladie (Jorm 1997; Salib 2000).
Cependant, la majorité de ces hypothèses sont basées sur des études statistiques qui,
malheureusement, manquent souvent de preuves convaincantes et reproductibles.
Comme il a été mentionné précédemment, la grande majorité des connaissances sur
les causes probables de la maladie d’Alzheimer proviennent d’études histologiques du
cerveau et de ses pathologies. Donc, il est possible d’imaginer que se sont les facteurs
– génétiques ou non – qui favorisent l’accumulation des soi-disant plaques séniles et
enchevêtrements neurofibrillaires qui sont les vraies causes de la maladie
d’Alzheimer. Effectivement, de fortes évidences in vivo et in vitro (présenté dans
cette thèse) tendent à démontrer que se sont les plaques séniles, et plus
particulièrement les constituants des plaques, nommés peptides amyloïdes Aβ42, qui
sont responsables d’une cascade d’évènements menant à la maladie, la «cascade
amyloïde » (Figure 2) (Hardy and Selkoe 2002). Un bon exemple qui appuie cette
2 En fait, les formes familiales et sporadiques présentent les mêmes empreintes anatomiques (signes neuropathologiques) mais se distinguent par la cinétique de leur apparition. Les formes familiales se caractérisent par un début beaucoup plus précoce et par une évolution plus rapide.
5
hypothèse est que les trois gènes présentement associés à une forme familiale de la
maladie d’Alzheimer lorsque mutés, soit l’AβPP et les présénilines, (Tableau 1) ont
tous un rôle propre à jouer dans la production des peptides amyloïdes neurotoxiques.
Figure 2 La cascade amyloïde3. Une accumulation graduelle et l’oligomérisation des peptides Aβ42 (voir origine ci-dessous) entraînent une chaîne d’événements complexes qui ont comme effet final l’apparition graduelle et non réversible des signes neuropathologiques (en caractères gras) et par conséquent des symptômes cliniques de la maladie d’Alzheimer (ex : démence).
3 Source tirée de Hardy, J. and D. J. Selkoe (2002) et adaptée par Sébastien Hébert (2003)
6
Tableau 1. Indices des cas familiaux de la maladie d’Alzheimer. Pour chaque gène donné, le nombre de mutations associées au développement d’une forme familiale de la maladie d’Alzheimer ainsi que le nombre de familles porteures du dit gène est indiqué (ainsi que le pourcentage respectif). Note : les mutations non pathogéniques ne sont pas présentées. (Donnés tirés de http://molgen-www.uia.ac.be/ADMutations)
Protéines associées à la forme familiale de la maladie d’Alzheimer
Étant donné que les séquelles neuropathologiques de la forme familiale de la maladie
d’Alzheimer sont les mêmes que celles observées dans la forme sporadique (Sommer
2002), il est possible d’imaginer que l’étude des voies biologiques impliquant les
protéines codées par les gènes associés à une forme familiale de la maladie pourra
nous permettre de mieux comprendre les mécanismes responsables du développement
de celle-ci.
Gène
Nombre de mutations et pourcentages respectifs (%)
Nombre de familles et pourcentages respectifs (%)
AβPP
PS1
PS1
TOTAL
16
133 9
158
10.13
84.18
5.70
43
268
15
326
13.19
82.21
4.60
7
L’AβPP, son métabolisme et la génération des peptides amyloïdes
L’AβPP (« Amyloid β Precursor Protein ») fut le premier gène isolé (Kang, Lemaire
et al. 1987; Robakis, Ramakrishna et al. 1987) et associé à une forme familiale de la
maladie d’Alzheimer (Goate, Chartier-Harlin et al. 1991). Aujourd’hui, on a
répertorié plus de 15 mutations différentes dans ce gène, ce qui représentent environ
5-10% de tous les cas familiaux connus (Tableau 1) (Kowalska 2003). L’AβPP est
une glycoprotéine de type 1 transmembranaire à fonction peu connue, bien que de
plus en plus de travaux lui associe un rôle dans le transport protéique kinésine-
dépendante (Annaert and De Strooper 2002; Wolfe 2003). Elle est exprimée de
façon ubiquitaire et trois (3) isoformes principales peuvent être générées par épissage
alternatif. L’isoforme neuronale de l’AβPP a 695 acides aminés (Kitaguchi,
Takahashi et al. 1988).
La protéine AβPP est métabolisée de façon constitutive via deux voies biologiques :
la voie non-amyloïdogénique et la voie amyloïdogénique (Figure 2) (Nunan and
Small 2002; Ling, Morgan et al. 2003) (Evin and Weidemann 2002). Ces deux voies
se distinguent principalement par les enzymes qui clivent l’AβPP et par les
métabolites (fragments) produits. En bref, la voie plus commune dite non-
amyloïdogénigue (c’est à dire qui ne produit pas de peptides amyloïdes) est
caractérisée par deux clivages endoprotéolytiques orchestrés par l’α-secrétase, puis la
γ-secrétase, ayant comme résultat final la production de trois fragments distincts : la
partie AβPP soluble α (AβPPs-α), le domaine AICD («AβPP intracellular domain»)
et le peptide P3. Alternativement, l’AβPP peut être clivée par la β-secrétase (suivi
pareillement par la γ-secrétase) pour produire deux autres fragments dont les peptides
amyloïdes (la voie amyloïdogénique): la partie AβPP soluble β (AβPPs-β) et les
peptides Aβ (en rouge, voir figure 3). Outre un rôle suggéré pour le fragment AICD
dans la transmission des signaux de la membrane vers le noyau (signalisation
cellulaire), l’ensemble de ces fragments n’ont toujours pas de fonction physiologique
précise. Plusieurs mutations retrouvées dans AβPP affecte le clivage γ-secrétase
8
ayant pour effet de produire des peptides légèrement plus long (Aβ42) que les
peptides Aβ404 générés par la AβPP sauvage (non mutée) (Figure 4). L’Aβ42 est
considéré neurotoxique par sa propriété hautement insoluble et oligomérisante
(Jarrett, Berger et al. 1993; Jarrett and Lansbury 1993).
Figure 3 Métabolisme de l’AβPP et la génération des peptides Aβ. La voie non-amyloidogénique est initiée par le clivage d’une α-secrétase dans le domaine cytosolique de l’AβPP. Ensuite, la γ-secrétase clive dans le domaine transmembranaire du fragment résiduel (C83) pour générer les peptides P3 et AICD. La voie amyloidogénique, quant à elle, est initiée par la β-secrétase qui clive elle aussi (mais à un endroit différent) la partie cytosolique de l’AβPP. Ensuite, l’action de la γ-secrétase libère les peptides Aβ (et l’AICD) du fragment C99.
4 Suite à l’isolement et la caractérisation des peptides amyloïdes (provenant des plaques séniles), on a pu mettre en évidence que les peptides existent sous deux principales formes, soit de 40 ou 42 acides aminés en longueur (dès lors appelé AβX, « X » correspondant à la longueur en acides aminés).
9
De nombreuses études ont montré que la β-secrétase était l’aspartyl protéase
membranaire BACE (voir ci-dessous). Un groupe de métalloprotéases incluant TACE
(« Tumor necrosis factor α-Converting Enzyme ») serait quant à lui responsable
d’une fraction significative de l’activité α-secrétase (Evin and Weidemann 2002).
Comme nous allons le voir, la γ-secrétase est dépendante des présénilines, plus
particulièrement de la préséniline-1.
Les présénilines
En 1995, le gène préséniline-1 (PS1) fut identifié et associé au développement d’une
forme familiale de la maladie d’Alzheimer lorsque muté (Sherrington, Rogaev et al.
1995). À ce jour, plus de 100 mutations différentes sont identifiés dans le gène PS1
(Tableau 1 et www.alzforum.org). Elles sont dominantes, fréquentes (environ 80 %
de tous les cas familiaux connus) et conduisent à une forme très agressive de la
maladie, affectant les individus entre 24-50 ans. PS1 est transcrit dans plusieurs types
de cellules du système nerveux central ainsi qu’au niveau des tissus périphériques
(Sherrington, Rogaev et al. 1995). L’identification d’orthologues dans C. elegans
(Levitan and Greenwald 1995) et D. melanogaster (Boulianne, Livne-Bar et al. 1997)
indique que le gène est très conservé dans l’évolution des organismes pluricellulaires.
Peu de temps après la découverte de PS1, le gène préséniline-2 (PS2) fut isolé et
caractérisé (Rogaev, Sherrington et al. 1995; Sherrington, Froelich et al. 1996). En
fait, l’identification de PS2 reposa sur sa forte homologie avec PS1 (67% chez
l’humain). Contrairement à PS1, les mutations dans le gène PS2 sont rares (9
mutations identifiées) (Tableau 1) et associées à une forme familiale moins agressive
(entre 40-70 ans).
10
Propriétés biologiques et biochimiques de la préséniline-1 (PS1)
Distribution cellulaire de la PS1
Dans la cellule, la protéine PS1 est principalement localisée dans les membranes
intracellulaires de l’enveloppe périnucléaire, du réticulum endoplasmique (RE), et de
l’appareil de Golgi (Walter, Capell et al. 1996; De Strooper, Beullens et al. 1997).
Toutefois, avec l’aide de techniques de visualisation et d’imagerie plus modernes,
comme la microscopie électronique de type « immunogold » et la microscopie à
immunofluorescence FLIM («Fluorescence Lifetime Imaging Microscopy »), des
études récentes ont permis de démontrer qu’une petite quantité de la PS1 peut
également se situer dans certaines vésicules intracytoplasmiques (ex : lysosomes)
(Pasternak, Bagshaw et al. 2003) et dans la membrane cytoplasmique (surface
membranaire) (Kaether, Lammich et al. 2002). Des études immuno-histologiques sur
des cerveaux ont démontré que la protéine PS1 est présente de façon prédominante
dans les neurones (Kovacs, Fausett et al. 1996) et les cellules gliales (Lah, Heilman et
al. 1997).
Topologie et métabolisme de la PS1
La PS1 possède un cadre de lecture de 467 acides aminés et son analyse topologique
prédit une protéine intégralement membranaire. La protéine possède au moins 6
domaines hydrophobiques transmembranaire (TM) et deux domaines acidiques
hydrophiliques; l’un dans la partie N-terminale et l’autre suivant le domaine TM6 (Li
and Greenwald 1996; Lehmann, Chiesa et al. 1997; Li and Greenwald 1998). Ces
deux domaines, ainsi que la partie C-terminale de PS1, sont localisés dans le
cytoplasme (Doan, Thinakaran et al. 1996; De Strooper, Beullens et al. 1997;
11
Lehmann, Chiesa et al. 1997). Certaines données avancent que la PS1 peut posséder
entre 6 et 9 domaines transmembranaires en plus d’un segment hydrophobe
additionnel rattaché à la surface de la membrane (Nakai, Yamasaki et al. 1999). Par
convention et par simplicité, la majorité des gens ont adopté un modèle topologique
de la PS1 incluant 8 domaines transmembranaires et deux domaines hydrophobes
attachés à la membrane (Figure 4) (Thinakaran 2001).
Des analyses in vivo et in vitro du métabolisme de PS1 révèlent que la protéine subit
une coupure endoprotéolytique au niveau de sa partie acidique hydrophilique (entre
les acides aminés 292 et 298) par une enzyme inconnue, la « présénilinase » (Figure
4) (Podlisny, Citron et al. 1997; Selkoe 2001; Thinakaran 2001). Il en résulte une
accumulation de 2 fragments prédominants (stœchiométrie 1 :1) d’un poids
approximatif de 28 kDa (N-terminal, NTF) et 18 kDa (C-terminal, CTF) ainsi qu’une
faible quantité résiduelle (holoprotéine) de PS1 intact d’environ 49 kDa.
Figure 4 Structure putative de la PS1. Schéma (non à l’échelle) de la protéine préséniline-1 avec ses huit (8) domaines transmembranaires et deux domaines hydrophobes rattachés à la membrane. La flèche représente le site de clivage endoprotéolytique (a.a. 292-298) de la PS1 effectué par la « présénilinase ».
Des études plus approfondies de l’état natif de la PS1 ont permis de démontrer que
ces fragments s’assemblent pour former un hétérodimère stable (NTF/CTF) (Capell,
12
Grunberg et al. 1998; Yu, Chen et al. 1998). Ces mêmes études suggèrent que
l’holoprotéine PS1 est un composant d’un complexe d’environ 100-180 kDa, et que
ces fragments endoprotéolytique (28 kDa et 18 kDa) font partie d’un complexe de
plus de 250 kDa. Jusqu’à présent, aucun rôle physiologique précis n’est connu pour
ces complexes.
Fonctions putatives de la PS1
Les premières expériences visant à déterminer le rôle de la protéine PS1 furent basées
sur des observations de comparaison entre les effets d’une PS1 mutante vis-à-vis la
sauvage. Ces expériences, principalement effectuées dans des conditions de
surexpression, ont pu démontrer que la PS1 (du moins certaines mutations associées
au développement d’une forme familiale de la maladie d’Alzheimer) est impliquée,
directement ou non, dans plusieurs voies biologiques. Par exemple, l’équipe de Guo,
Q. et al. (1996) ont démontré qu’une PS1 mutante perturbe l’homéostasie du calcium
des cellules en culture (Guo, Q., K. Furukawa, et al. 1996). En effet, une protéine
PS1 mutante amplifie de façon anormale la réponse d’un agoniste donné (qui induit la
libération du Ca2+ du réticulum endoplasmique) ayant pour effet d’augmenter la
vulnérabilité des cellules aux agents toxiques (comme, par exemple, l’amyloïde
Aβ42). D’autres études suggèrent que la PS1 jouerait en rôle dans la régulation de
l’apoptose. En fait, il a été démontré qu’une protéine PS1 mutante augmente de façon
considérable la vulnérabilité des cellules en culture face aux agents pro-apoptotique
(la staurosporine par exemple) (Kovacs, D. M., R. Mancini, et al. 1999). Il a même
été proposé que la surexpression d’une PS1 sauvage aurait un effet anti-apoptotique
(Kovacs, D. M., R. Mancini, et al. 1999). L’équipe de Janicki, S.M. et al. (2000),
quant à eux, ont montré qu’une préséniline mutée influence le cycle cellulaire. Plus
spécifiquement, ils démontrent que la surexpression d’une PS1 mutante favorise
l’arrêt du cycle cellulaire des cellules Hela (Janicki, S. M., S. M. Stabler, et al. 2000).
Certains résultats suggèrent même que la PS1 mutante affecte le transport (i.e.
13
« trafficking ») protéique. En effet, il a été démontré qu’une PS1 mutante perturbe la
maturation de la protéine AβPP en culture de cellule ayant pour effet d’inhiber et/ou
ralentir le transport de celle-ci, entre autre, à la surface cellulaire (Cai, D., J. Y. Leem,
et al. 2003). Toutefois, un grand nombre de ces études ont été faites dans des
conditions expérimentales particulières (ex : cellules, agents chimiques et approches
expérimentales spécifiques) et, malheureusement, se sont avérées souvent peu
reproductives et non concluantes.
Une des premières évidences associant PS1 à un rôle physiologique précis ont été
obtenues à partir des souris nulles (« knock-out ») pour la PS1 (De Strooper, Saftig et
al. 1998). En bref, leur étude montre que les cellules dérivées de ces souris « knock-
out » ne présentent pas d’activité γ-secrétase et, par conséquent, pas d’accumulation
de peptides Aβ (ni Aβ40 ou Aβ42).
Similairement à ce qui est observé avec le gène AβPP, toutes les mutations retrouvées
dans PS1 (Figure 6) (et PS2 [voir annexe 2]) affectent le clivage γ-secrétase de façon
à favoriser la production du peptide Aβ42 neurotoxique (par rapport à Aβ40) (Figure
5) (Scheuner, Eckman et al. 1996; Selkoe 1999). Ses observations, basées
principalement sur des techniques d’immunobuvardage et d’ELISA (« Enzyme-
Linked Immunosorbent Assay »), ont été reportées dans plusieurs types de cellules
ainsi que dans le cerveau de souris transgéniques surexprimant une PS1 mutée.
À partir des études menées sur le métabolisme de l’AβPP (et Notch (Selkoe 2000)),
les chercheurs ont pu mettre en évidence un nouveau mécanisme biologique répandu
chez les mammifères et qui implique la PS1 (et la PS2) : la RIP (« Regulated
Intramembrane Proteolysis ») (Steiner and Haass 2000; Medina and Dotti 2003; Xia
and Wolfe 2003). En fait, la PS1 joue un rôle évident dans ce processus protéolytique
caractérisé par un clivage (de type γ-secrétase) d’une protéine donnée dans son
domaine transmembranaire (Figure 6). Ce clivage a pour effet de libérer le domaine
intracellulaire de la protéine ainsi clivée (par exemple, dans le cas de l’AβPP, le
fragment AICD est libéré). À part l’AβPP, on évalue aujourd’hui à plus de trente5 le
5 Bart De Strooper, M.D., Ph.D. (2003) Communication personnelle
14
nombre de substrats de type 1 transmembranaire clivés par ce mécanisme
protéolytique (par exemple : les protéines homologues à l’AβPP, les APLPs (Walsh,
Fadeeva et al. 2003), Notch (De Strooper, Annaert et al. 1999), Jagged (LaVoie and
Selkoe 2003), Delta-1 (LaVoie and Selkoe 2003), Erb-B4 (Lee, Jung et al. 2002),
CD44 (Murakami, Okamoto et al. 2003), E-Cadhérine (Marambaud, Shioi et al.
2002), Syndécane 3 (Schulz, Annaert et al. 2003), glycoprotéine LRP (May, Reddy et
al. 2002), etc.).
La γ-secrétase
Les premières études portant sur la caractérisation du métabolisme des présénilines
ont permis de démontrer que l’activité enzymatique de type γ-secrétase était
étroitement liée au complexe de haut poids moléculaire contenant, à posteriori,
l’hérérodimère NTF/CTF (Levitan, Lee et al. 2001). Toutefois, étant donné le haut
poids moléculaire du complexe (dépendamment des auteurs, variant entre 250 et 2000
kDa), l’activité γ-secrétase semblait de toute évidence nécessiter la participation
d’autres protéines (Li, Lai et al. 2000; Steiner, Winkler et al. 2002; De Strooper 2003;
Farmery, Tjernberg et al. 2003; Kimberly, LaVoie et al. 2003). Des recherche
intensives au cours des dernières années ont permis de suggérer que l’activité γ-
secrétase est en fait assurée par un large complexe protéique multimérique, appelé
complexe γ-secrétase. De multiples protéines membranaires participent à sa
formation, parmi lesquelles la Nicastrine, l’Aph-1, la Pen-2 et finalement les
présénilines-1 et -2 (et plus particulièrement les hétérodimères NTFs/CTFs) (Figure 7)
(De Strooper 2003; Kimberly, LaVoie et al. 2003; Wolfe 2003).
Dans un premier temps, la protéine Nicastrine fut isolée parce qu’elle intéragissait
avec PS1 dans le complexe de haut poids moléculaire (par technique de co-
immunoprécipitation et isolement du complexe sur gel d’acrylamide). Le gène
nicastrine code pour une glycoprotéine de type 1 transmembranaire. La Nicastrine
possède un cadre de lecture de 709 acides aminés. Elle est exprimée de façon
15
ubiquitaire mais préférentiellement dans le cerveau. Dans la cellule, la Nicastrine est
principalement localisée dans le réticulum endoplasmique (RE) et l’appareil de Golgi.
Aussi, tel qu’observé avec la PS1, une petite quantité de la Nicastrine peut également
se situer dans certaines vésicules intracytoplasmiques (ex : lysosomes) et à la
membrane cytoplasmique (surface membranaire).
Peu de temps après la découverte de Nicastrine, les protéines Aph-1 et Pen-2 furent
isolées à partir d’un criblage génétique pour des phénotypes mutants de la voie de
signalisation Notch (« Notch-defective ») chez C.elegans. L’orthologue aph-1
humain code pour une protéine de sept (7) domaines transmembranaires (Figure 6) et
possède un cadre de lecture d’environ 250 acides aminés. Elle est exprimée de façon
ubiquitaire. Trois (3) isoformes peuvent être générées par épissage alternatif : Aph-
1aL (Long), Aph-1aS (Short) et Aph-1b. L’isoforme principale, l’Aph-1aL, a 265
acides aminés et est la principale forme exprimée du cerveau.
L’orthologue Pen-2 (« presenilin-enhancer ») humain code pour une protéine de 2
domaines transmembranaires (Figure 6) et possède un cadre de lecture de 101 acides
aminés. Pen-2 est exprimée de façon ubiquitaire dans plusieurs types de tissus
humains, incluant le cerveau.
Les premières études portant sur la localisation intracellulaire des protéines Aph-1 et
Pen-2 humaines ont permis de démontrer que ces protéines étaient principalement
localisées au niveau du réticulum endoplasmique et de l’appareil de Golgi. Pour
l’instant, il n’est pas connu si ces protéines sont situées à la membrane plasmique.
Même aujourd’hui, le rôle spécifique de ces protéines dans le complexe (et en
général) demeure inconnu. Cependant, l’effet des mutations sur les gènes des
présénilines sur le clivage de l’AβPP en position γ suggère que les présénilines
peuvent elles-mêmes assurer la fonction protéolytique du complexe γ-secrétase
(Wolfe 2003). Toutefois, cette hypothèse est loin de faire l’unanimité, même si de
plus en plus d’évidences biochimiques tendent à prouver ce dogme. Par exemple,
l’équipe de Wolfe et al. (1998) démontra que la PS1 porte des résidus aspartiques
conservés au niveau des domaines transmembranaires 6 et 7, positions qui
s’aligneraient avec le site de clivage γ de l’AβPP (Figure 7) . La mutagenèse dirigée
16
de ces deux résidus aspartates de la PS1 empêche le clivage de l’AβPP (Wolfe, Xia et
al. 1999; Kimberly, Xia et al. 2000). De plus, des inhibiteurs de transition de la γ-
secrétase qui mimiquent le site de clivage γ de l’AβPP se lient spécifiquement à la
PS1, plus particulièrement l’hétérodimère NTF/CTF actif (Esler, Kimberly et al.
2000).
Quoique cette hypothèse reste à confirmer, du moins in vitro, la PS1 assure
incontestablement la RIP de l’AβPP (et des autres substrats mentionnés ci-haut)
tandis que les autres membres du complexe γ-secrétase auraient un rôle régulateur de
cette activité de protéolyse (résumé dans :(De Strooper 2003)).
17
Figure 5 Les mutations associées aux formes familiales de la maladie d’Alzheimer et le clivage γ-secrétase. A) Suite à un premier clivage par la β-secrétase, l’AβPP génère le fragment C99 (qui possède 99 acides aminés) toujours associé à la membrane et substrat directe de la γ-secrétase. Par un mécanisme encore mal compris, presque la totalité des mutations associées aux formes familiales de la maladie d’Alzheimer altère l’activité et/ou le spécificité de la γ-secrétase de façon à favoriser la production de l’amyloïde Aβ42 (versus Aβ40). B) Dans des conditions dites normales (sauvages) la production des peptides Aβ40 est favorisée à Aβ42 par un facteur d’environ 9 fois à 1. Les mutations associées aux formes familiales de la maladie d’Alzheimer ont pour effet de modifier le rapport (d’environ 2 à 5 fois) et non la quantité des peptides Aβ42 produits.
18
Figure 6 Les différents constituants du complexe γ-secrétase. Les homologues présénilines (principalement les hétérodimères), combinés aux protéines Aph-1, Nicastrine et Pen-2 constituent l’ensemble minimal du complexe γ-secrétase actif. Toutes ces protéines sont transmembranaires. La manière dont les protéines s’associent pour cliver la protéine AβPP dans son domaine transmembranaire reste un phénomène toujours inexpliqué. De plus, on ignore si ce complexe est stable et dans quel(s) compartiment(s) souscellulaire(s) il réside. Finalement, la manière dont les mutations associées aux formes familiales de la maladie d’Alzheimer affectent l’ensemble du complexe pour favoriser la production des peptides Aβ42 demeure du domaine de l’inconnu.
19
Figure 7 Mutations de la préséniline-1 et l’AβPP (C99). Schéma (non à l’échelle) des mutations (acides aminés en rouge) associées au développement d’une forme familiale de la maladie d’Alzheimer dans la protéine PS1 ainsi que dans l’AβPP (une portion du fragment C99 est montré ici). La mutagenèse dirigée des résidus aspartiques conservés que porte la PS1 au niveau des domaines transmembranaires 6 et 7 (acides aminés en bleu) empêche le clivage de l’AβPP, et par conséquent, la génération de peptides Aβ. (Image adaptée de John Hardy, National Institute on Aging, Bethesda, MD et mise à jour par Sébastien Hébert 2003).
20
La β-secrétase
En 1999, quatre groupes de chercheurs indépendants ont cloné et caractérisé BACE
(« β-site APP Cleaving Enzyme »), la β-secrétase impliquée dans le premier clivage
de la génération de l’amyloïde-β 42 neurotoxique (Hussain, Powell et al. 1999;
Sinha, Anderson et al. 1999; Vassar, Bennett et al. 1999; Yan, Bienkowski et al.
1999). BACE est une enzyme de la famille des aspartyl protéases. Le gène bace code
pour une protéine de type 1 transmembranaire de 501 acides aminés. BACE est
exprimée dans plusieurs types cellulaires et organes mais plus particulièrement chez
le pancréas et le cerveau. Tel qu’observé avec la PS1, la protéine BACE est localisée
principalement dans la région du réticulum endoplasmique et de l’appareil de Golgi
(Haass, Lemere et al. 1995; Kitazume, Tachida et al. 2001; Huse, Liu et al. 2002).
Toutefois, de petite quantité de BACE semble présente à la surface membranaire ainsi
que dans certaines vésicules cytoplasmiques (endosomes) (Kinoshita, Fukumoto et al.
2003).
La protéine BACE subit une maturation post-traductionnelle de sorte que son pro-
domaine est libéré par l’action d’une furine dans l’appareil du Golgi (Bennett, Denis
et al. 2000). Par son homologie avec d’autres aspartyl protéases (Richter, Tanaka et
al. 1998), les gens croient que l’holoprotéine (protéine avec le pro-domaine) est en
fait un zymogène6. Toutefois, des expériences in vitro avec la BACE purifiée
suggèrent que la présence du pro-domaine n’influence pas ou peu l’activité de BACE
sur l’AβPP (Benjannet, Elagoz et al. 2001; Shi, Chen et al. 2001; Lullau, Kanttinen et
al. 2003). Cette hypothèse reste à être confirmée in vivo.
6 Définition : Un zymogène est une enzyme non active. Les résidus du centre actif se trouvent éloignés dans le zymogène. La scission d'une liaison permet un réarrangement spatial, puis un changement de conformation rend la molécule active. Les zymogènes permettent aux cellules de produire des enzymes et de les activer uniquement quand c'est nécessaire. Les zymogènes font partie des moyens de contrôle de la protéolyse dans les cellules.
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Des analyses de l’expression de BACE dans la cellule confirment que les α- et β-
secrétases sont en compétition pour agir sur leur substrat AβPP (Tyler, Dawbarn et al.
2002). Par ailleurs, la surexpression de cette protéase augmente les clivages aux
niveaux de l'aspartate 1 et du glutamate 11 (sites endoprotéolytiquse précédemment
reconnus comme substrats à la β-secrétase) mais pas à d’autres endroits sur l’AβPP
(Howlett, Simmons et al. 2000). À l’inverse, si on inhibe l’expression de BACE
endogène (par exemple, avec des oligonucleotides anti-senses ou par RNAi), il y a
une baisse très importante de l’activité β-secrétase endogène (Vassar, Bennett et al.
1999; Luo, Bolon et al. 2001; Basi, Frigon et al. 2003). Des expériences in vitro sur
la BACE purifiée montrent que ce n’est pas un simple co-facteur, ni un activateur
pour une β-secrétase inconnue. Son activité directe sur le substrat AβPP démontre
réellement son rôle d’enzyme β-secrétase.
Curieusement, des études récentes sur la souris nulle pour la BACE ne révèlent
aucune aberration phénotypique apparente (Luo, Bolon et al. 2001; Roberds,
Anderson et al. 2001). Outre le manque évident d’activité β-secrétase endogène et de
production de peptides amyloïdes (Aβ40 et Aβ42), il semblerait que l’ablation du
gène bace n’affecte pas, à priori, la santé de la souris. Toutefois, d’autres études
viennent de démontrer que l’AβPP n’est pas le seul substrat de BACE (Kitazume,
Saido et al. 2002; Kametaka, Shibata et al. 2003; Lichtenthaler, Dominguez et al.
2003; Scholefield, Yates et al. 2003) et qu’il soit possible que l’ablation de ce gène
pourrait avoir un effet à long terme sur la santé des souris (et chez d’autres
mammifères) .
À ce jour, aucune mutation ni polymorphisme spécifique de bace n’ont pu être
associée au développement de la maladie d’Alzheimer, soit la forme familiale ou
sporadique (Cruts, Dermaut et al. 2001; Liu, Leu et al. 2003). Toutefois, trois études
très récentes démontrent une augmentation significative de la quantité de la protéine
et de l’activité de la BACE dans les cerveaux des patients atteints de la forme
sporadique de la maladie d’Alzheimer (Fukumoto, Cheung et al. 2002; Holsinger,
McLean et al. 2002; Tyler, Dawbarn et al. 2002). Cette élévation n’a toutefois pas
22
encore été mesurée chez la forme familiale. Le rôle physiologique de cette
augmentation n’est pas connu.
Figure 8 La β-secrétase (BACE). Schéma (pas à l’échelle) de la protéine BACE. BACE est une protéine de type 1 transmembranaire. BACE est synthétisée comme protéine précurseur (zymogène) de 501 acides aminés. Au niveau de l’appareil de Golgi, BACE est clivée par la furine ce qui mène à une perte de son pro-domaine. Toutefois, il n’est présentement pas clair si cette action a un rôle à jouer dans la régulation de l’activité de la BACE, et par conséquent, la production des peptides amyloïdes.
HYPOTHÈSES POSÉES ET OBJECTIFS
CHAPITRE II
Les projets de recherche réalisés dans mon laboratoire d’accueil ont pour objectifs
principaux d’apporter des éclaircissements sur la fonction de la préséniline-1 (PS1).
Cette étude avait pour but d’étudier la relation qui existe entre PS1 et l’activité γ-
secrétase. Plus spécifiquement, nous voulions approfondir le rôle qu’exerce PS1 dans
la régulation de l’amyloïde-bêta 42 neurotoxique. Pour atteindre ce but, j’ai
développé les deux objectifs spécifiques ci-dessous.
1) Analyse de l’organisation structurale de la PS1
Revue de littérature
Bien qu’il soit convenu que diverses interactions intra- et intermoléculaires modulent
l’organisation structurale de la PS1 (Ratovitski, Slunt et al. 1997; Saura, Tomita et al.
1999; Thinakaran 2001), aucune littérature ne tente de démontrer les régions de
contact. D’autres études portant sur des protéines multi-transmembranaires, telles les
« G-protein coupled receptors » (GPCRs) et la « cystic fibrosis transmembrane
conductance regulator » (CFTR), ont permis de démontrer que plusieurs régions de
24
contacts intra- et intermoléculaires, et ce, au sein de la même protéine, peuvent
réguler de façon différée le comportement et/ou l’activité de celles-ci (Lomize,
Pogozheva et al. 1999; King and Sorscher 2000; Kirk 2000; Lee, Xie et al. 2000).
Hypothèse et objectif
Étant donné la forte homologie structurale entre la PS1 et les GPCRs/CFTR, nous
pouvons imaginer que différentes régions intramoléculaires peuvent réguler
indépendamment ou collectivement la localisation, la maturation, l’hétérodimérisation
et, par conséquent, l’activité enzymatique étroitement associée à PS1. De plus, étant
donné la disparité et le nombre surprenant des mutations dans le gène de PS1 (figure
6), il semble évident que l’analyse structurale de la PS1 normale et mutée soit une
étape primordiale dans l’étude structuro-comportementale de celle-ci. En
conséquence, un des objectifs premiers de ma recherche est d’étudier l’organisation
structurale de la PS1.
2) Développement d’un essai fonctionnel enzymatique pour la γ-secrétase
Revue de la littérature
Diverses fonctions telles la transmission de signaux intracellulaires,
l’induction/suppression de l’apoptose, et le trafic de protéines ont été suggérées pour
la PS1 (Wolozin, Alexander et al. 1998; Fraser, Yang et al. 2000; Selkoe and Wolfe
2000). De plus, il a été rapporté que la PS1 est impliquée dans la régulation du
25
clivage protéolytique du précurseur de la protéine amyloïde réalisé par une secrétase
peu connue (γ-secrétase) (De Strooper, Saftig et al. 1998). Toutefois, cette dernière
proposition est loin de faire l’unanimité (Small 2001). Comment les fonctions
physiologiques normales de PS1 sont médiées et comment les mutations dans la PS1
altèrent ce clivage de façon telle que cela conduit à une augmentation du niveau de
l'amyloïde-β neurotoxique (la forme Aβ42) demeurent du domaine de l’inconnu.
Hypothèses et objectifs
Contrairement à BACE, il n’a pas été possible de démontrer clairement une activité
enzymatique intrinsèque pour la PS1 in vivo ni in vitro; faute d’essai enzymatique
convenable. Il semblerait que, pour exercer une influence enzymatique de type γ-
secrétase, la PS1 doit se retrouver dans un contexte environnemental précis (pH
acidique, ancrée à la membrane, etc.). De plus, il est possible que la PS1 doit se
retrouver liée à d’autres protéines comme, par exemple, la Nicastrine, la Pen-2 ou
l’Aph-1 qui, elles aussi, influenceraient l’activité γ-secrétase (Esler, Kimberly et al.
2002; De Strooper 2003). Donc, à partir des connaissances structurales de la PS1, je
propose de 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 mutés dans
le clivage de la protéine amyloïde AβPP.
Méthodologie (résumé)
De plus en plus de publications tentent de démontrer que la préséniline-1 et l’AβPP
peuvent s’exprimer et se comporter de façon physiologiquement similaire à une
cellule dans la levure (Zhang, Komano et al. 1994; Evin, Le Brocque et al. 2000;
Song, Ohba et al. 2000; Cervantes, Gonzalez-Duarte et al. 2001; Edbauer, Winkler et
26
al. 2003). Puisqu’il n’existe aucune activité endogène de type β- ni γ-secrétase dans
la levure (ni d’homologue de l’AβPP) (www.ncbi.nlm.nih.gov), il est possible
d’imaginer que la levure puisse être un modèle biologique de choix afin de
développer un essai enzymatique in vivo pour la γ-secretase. De plus, il est possible
de prendre avantage des souches de levure Y187 et AH109 et des vecteurs pGBKT7
et pGADT7 (Clontech) afin de vérifier une interaction entre les protéines impliquées
dans le métabolisme de l’amyloïde-β par le système de double-hybride (décrit par le
manufacturier et brièvement dans les chapitres 3, 4 et 5). Bien que le système de
double hybride en levure présente certaines limites techniques pouvant mener à des
résultats de type « faux-positifs » (par exemple, avec l’étude de protéines
transmembranaires, il est possible d’observer une interaction non spécifique entre
deux régions hautement hydrophobes –i.e. deux domaines transmembranaires –), cette
approche s’est avérée fonctionnelle avec d’autres protéines transmembranaires
incluant la PS1 (Lévesque, G., G. Yu, et al. 1999). Finalement, l’absence de PS1
endogène dans la levure nous permettra de tester la (les) propriété(s)
oligomérisante(s) (analyse structuro-comportementale) de l’holoprotéine et/ou des
fragments PS1 NTFs et CTFs normaux et mutés.
En résumé, la réalisation des différents objectifs du projet fait appel à l’utilisation de
nombreuses techniques de biologie cellulaire et moléculaire, dont les plus importantes
sont la culture de levure, la culture cellulaire, la transfection, la transformation de
levure, l’immunobuvardage, l’immunoprécipitation et l’immunohistochimie.
DÉVELOPPEMENT
CHAPITRE III
Dimérisation de la préséniline-1 in vivo : suggestion de nouveaux
mécanismes de maturation pouvant mener à des complexes de
haut poids moléculaire
Note : Ce chapitre, présenté sous la forme d’un manuscrit publié dans la revue Biochemical and Biophysical Research Communications (Biochem. Biophys. Res. Commun. 2003, Jan 31;301(1):119-26.), résume une partie des travaux effectués dans le cadre de mon projet de doctorat. Plus spécifiquement, ce travail contient des expériences visant à atteindre mon premier objectif de travail (décrit dans le chapitre 2 à la page 23). Comme ma contribution dans la conception (approches expérimentales), la réalisation (constructions plasmidiques et manipulations) et la rédaction (incluant le montage des figures) de ce travail est majoritaire, mon nom apparaît comme premier auteur sur le manuscrit. Toutefois, il est important de souligner l’apport très apprécié de ma consoeur de laboratoire, Mme Chantal Godin, dans la réalisation technique de ce travail. En gros, Mme Godin a amplifié par PCR puis cloné les fragments PS1 NTF et CTF dans les vecteurs d’expression de levures pGBKT7 utilisés dans cet ouvrage. De plus, nous tenons à remercier les Drs. Takami Tomiyama et Hiroshi Mori, de l’Université Médicale Osaka, pour l’anticorps monoclonal PSN2 utilisé dans ce travail. Cet article est disponible sur Internet en version originale anglaise à l’adresse électronique suivante : http://dx.doi.org/10.1016/S0006-291X(02)02984-4
28
Résumé
Traditionnellement, les présénilines sont représentés comme des protéines
monomériques composés de huit domaines transmembranaires. Au cours des
dernières années, plusieurs données biochimiques et fonctionnelles démontrent que
ces protéines peuvent exister sous forme de complexes oligomériques de poids
moléculaire variable. Cette étude avait pour but d’étudier les propriétés
oligomérisantes des présénilines, plus particulièrement la préséniline-1 (PS1). Par
technique de double-hybride en levure, nous démontrons que l’holoprotéine PS1 peut
s’oligomériser pour former des homodimères. Lorsque surexprimée en cellules N2A
et HEK293T, l’holoprotéine PS1, avec ou sans tag, forme des homodimères
spécifiques (correspondant au double de leur poids moléculaire respectifs) tels que
visualisés par immunobuvardage. Les mutations associées à une forme familiale de la
maladie d’Alzheimer Y115H, M146L, L392V et ∆exon 10, ainsi que la mutation
dominante négative pour l’activité γ-secrétase D257A, n’affectent pas les propriétés
homo-oligomérisantes de la PS1. Dans des conditions d’extraction protéique non-
dénaturantes, la PS1 endogène peut former des homodimères spécifiques tels
qu’observées par immunobuvardage dans différentes lignées cellulaires humaines.
L’ensemble des résultats suggèrent que la PS1 peut s’oligomériser pour former des
homodimères. Donc, la PS1 possède des domaines de liaisons intra-moléculaires
essentiels à la formation des complexes oligomériques étroitement associés au clivage
endoprotéolytique de celle-ci et à l’activité γ-secrétase.
29
Dimerization of presenilin-1 in vivo: suggestion of novel regulatory
mechanisms leading to higher order complexes.
Sébastien S. Hébert1, Chantal Godin1, Takami Tomiyama3, Hiroshi Mori3 and
Georges Lévesque1, 2
1Molecular and Human Genetics Unit, CHUQ-Pavillon St-François d'Assise, 10 rue
de l'Espinay, Québec, Canada G1L 3L5, 2 Medical Biology Department, Laval
University, Quebec, Canada, G1K 7P4 and 3 Department of Neuroscience, Osaka City
University Medical School 1-4-3 Asahimachi, Abenoku, Osaka 545-8585, Japan
Address correspondence to: Dr. Georges Lévesque, Molecular and Human Genetics
Unit, CHUQ-Pavillon St-François d'Assise, 10 rue de l' Espinay, Québec, Canada
G1L 3L5.
Tel: 418-525-4444 ext 3752
Fax: 418-525-4195
Email: [email protected]
Running title: Presenilin-1 self-association.
30
ABSTRACT
A growing body of evidence indicates that presenilins could exist and be active as
oligomeric complexes. Using yeast two-hybrid and cell culture analysis, we provide
evidence that presenilin-1 (PS1) may self-oligomerize giving rise to specific full-
length /full-length homodimers. When expressed in N2A and HEK239T cultured
cells, full-lenght PS1-wt and 5’myc-PS1-wt form specific homodimers corresponding
to twice their molecular weight. The Alzheimer’s disease-associated PS1 mutations
Y115H, M146L, L392V, ∆E10(PS11-289/320-467), the γ-secretase dominant negative
mutant D257A and the PS1 polymorphism mutant E318G do not affect their ability to
self-oligomerize. Under non-denaturing conditions, endogenous PS1 forms specific
homo-oligomers in human cultured cells. The results obtained herein suggest that
PS1 associates intramolecularly to form higher order complexes, which may be
needed for endoproteolytic cleavage and/or γ-secretase-associated activity.
KEYWORDS
Presenilin, γ-secretase, oligomerization, protein dimerization, yeast two-hybrid,
western blotting.
INTRODUCTION
Alzheimer’s Disease (AD) is, among other amyloidosis-related diseases, the most
common cause of presenile death in the elderly. The pathological features of AD are
neuronal loss, neurofibrillary tangles and amyloid (senile) plaques. Most cases of
early onset familial Alzheimer’s Disease (EOFAD) disease are associated with a point
mutation in presenilin 1 (PS1). To date, over one hundred different mutations have
already been found in this gene (for references, see www.alzforum.org). PS1 is a
polytopic transmembrane protein located primarily in the endoplasmic reticulum
(ER), the early Golgi and, to a lesser extent, at the cell surface and nuclear membrane
(1,2-4). The precise role of PS1 is not fully understood as it is associated with
multiple biological functions such as protein folding and trafficking, intracellular
31
adhesion, calcium homeostasis, GSK-3 and Tau metabolism, signal transduction,
neuronal apoptosis and γ-secretase cleavage (4).
The PS1 protein is approximately 45-50 kDa in size and undergoes a series of
proteolytic cleavages by ‘presenilinase’ to generate N-terminal (NTF) and C-terminal
(CTF) fragments of approximately 32-35 and 16-20 kDa, respectively. The
stoichiometry of these fragments is maintained on a 1:1 ratio such as artificial
expression of PS1 results in only a modest increase in these fragments. It has also
been reported that overexpression of PS1 may lead to the formation of aggresomes
(5). Endogenous PS1 forms low (~ 100-180 kDa) and high (> 250 kDa) molecular
weight complexes containing both full-length (fl) and heterodimeric NTF and CTF (6-
9). Recently, studies have identified PS1 key binding partners such as β-catenin and
nicastrin which seem to act on or at least “help” PS1 to maintain these distinct
oligomers (1,8,10). However, even if a growing number of studies is underway to
understand the enzymatic-related properties of PS1, little is known about the kinetics,
the regulation, the contents and the exact function(s) of these complexes, particularly
the low molecular weight (LMW) species. Compelling evidences show that γ-
secretase activity, which seems to be the ‘key’ function of these PS1 complexes, is
associated to the highest (i.e.>250 kDa) molecular weight complex (8,11). However,
certain authors argue this concept (12). Moreover, it has been speculated that a
limited abundance of another unknown component of the PS1 complexes restrains the
amount of PS1 that can be stabilized in complexes (13). Since other numerous
binding partners of PS1 have been elucidated (14) and their relation to presenilin
function have not been fully understood, it is necessary to understand PS1 complex
maturation and its link, if any, in the cellular mechanisms leading to the pathogenesis
of Alzheimer’s disease. To gain new insights on PS1 structure and function in vivo,
we have studied its oligomerization properties. Here, we provide evidence that
human wild-type (wt) or mutant PS1 may self-oligomerize giving rise to full-
length/full-length homodimers. The AD-associated PS1 mutations Y115H, M146L,
L392V, ∆E10(PS11-289/320-467), the γ-secretase dominant negative mutant D257A and
the polymorphism mutant E318G (15-17) do not affect PS1 ability to self-
oligomerize.
32
MATERIALS AND METHODS
1) Yeast two-hybrid assays.
cDNA sequences encoding various parts of human PS1 were amplified by PCR using
the Herculase Taq polymerase (Stratagene). The following PS1 constructs were
prepared: fl-PS1wt (PS11-467), fl-PS1-E318G, fl-PS1-D257A, fl-PS1-L392V, NTF
(PS11-291) and CTF (PS1292-467). Constructs were cloned both into pGBKT7 and
pGADT7 vectors (Clontech) as a fusion to the Gal4-DNA binding domain and Gal4-
DNA activating domain, respectively. Each construct was sequenced and shown to be
free of autonomous Gal-4 activation.
Each of the pGBKT7-construct was co-transformed into Y187 or AH109
Saccharomyces cerevisiae strain (Clontech) together with each pGADT7-construct.
Transformants were selected on yeast drop-out media (Clontech) that lacked both
tryptophan and leucine (-TL) and assayed for β-galactosidase activity (β-gal) in the
case of Y187 or histidine (His) and adenine (Ade) activity in the case of AH109.
Negative control experiments were directed against empty vectors (either pGBKT7 or
pGADT7), or Lamin C (Clontech). Positive controls included pGBKT7-p53/
pGADT7-T antigen and pGBKT7-PS1Loop6-7/pGADT7-hNPRAP as previously
described (1).
2) Preparation of yeast cell lysates and immunoblotting.
Transformed yeasts were harvested by centrifugation and washed twice in sterile
H2O. Yeast pellets were lysed in liquid nitrogen then thawed on ice. Acid washed
glass beads (Sigma) (~250 µl) were added to the cells with 300 µl of yeast extraction
buffer (Clontech), 1 mM PMSF and mini-complete protease inhibitors (Roche-
Diagnostic). The mixture was subjected to four cycles of : vortexing at full-speed 30
sec, then placed on ice for another 30 sec. 5% v/v of β-mercaptoethanol was added to
the samples. Samples were then boiled for 2 min and centrifuged at 14,000 rpm. 10
µl of supernatants were loaded on a 4-20% gel and subjected to SDS-PAGE. Blots
were probed with either the anti-c-myc (Roche Diagnostics), anti-PS1 Ab14 (gift from
Dr P. Fraser), PSN2, N-19 (Santa Cruz) or C-20 (Santa Cruz) antibodies.
33
3) Cell culture and transfection.
Murine neuroblastoma 2A (N2A) and human embryonic kidney 293T (HEK293T)
cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco)
supplemented with 10% (v/v) fetal bovine serum (Biomedia) and Geneticin®
antibiotics (Invitrogen). Stock cultures were maintained at 37 °C in a humidified
atmosphere consisting of 5% CO2, 95% air. For overexpression studies, N2A or
HEK293T cells were transiently transfected with cDNAs corresponding to: human
PS1-wt, human PS1-wt tagged at the N-terminus with c-myc, EQKLISEEDLN,
(PS1wt-5’myc), a FAD-associated splicing mutation, exon 10 deletion, (PS1 ∆E10), a
FAD-associated mutant Y115H (PS1 Y115H) and an aspartate 257 mutant (PS1
D257A) expressed from pcDNA3 (Invitrogen). Constructs (10 µg) were transfected
using LipofectAMINE® according to the manufacturer’s protocol (Life Technologies,
Inc.). Cells were harvested and processed for immunoblot analyses 48 h after
transfection.
4) Preparation of cell lysates and immunoblotting.
For typical (i.e. denaturing and reducing) SDS-PAGE, HEK293T, N2A and PS1 K/O
native cells, as well as PS1-transfected cells, were washed twice with ice-cold
phosphate buffer saline (PBS) then lysed for 30 min on ice in lysis buffer A [25 mM
HEPES, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 1 mM PMSF
and mini-complete protease inhibitors (Roche-Diagnostic)]. Insoluble material was
removed by centrifugation at 13,000 X g for 15 min at 4°C. Equal amounts of
proteins were fractionated on Novex® 4-20% Tris-Glycine gels (Invitrogen) by SDS-
PAGE and transferred onto PVDF membrane (Amersham) using the XCell II™ Mini-
Cell blot module (Invitrogen) according to the manufacturer protocol. For native
PAGE (i.e. non-denaturing), native PS1-K/O mice cells, as well as native and
transfected HEK293T cells, were lysed under non-denaturing conditions using lysis
buffer B [25 mM HEPES, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1mM
PMSF and complete protease inhibitors (Roche-Diagnostic)]. Equal amounts of
proteins were mixed with Novex® 2X native sample buffer (0.5 M Tris-HCl, pH 8.8,
glycerol 20%, bromophenol blue 0.1%), incubated at 37°C for 15 min, then subjected
to native PAGE as described by the manufacturer. For “revisited” native PAGE (i.e.
34
partially denaturing), equal amounts of proteins were mixed with Novex® 2X native
sample buffer then subjected to SDS-PAGE (i.e. with SDS-PAGE running buffer).
Western blots were probed with the appropriate antibodies, c-myc, Ab14, PSN2, N-19
or C-20 and detected using chemiluminescence detection system, ECL plus
(Amersham).
5) Protein Extraction from SDS-PAGE gel.
Migration of protein extracts under partial (i.e. “revisited”) denaturing conditions was
performed as described above. For each sample, approximately 1 mg of total protein
(60 mm confluent dish) was deposited on the gel. After a preliminary electrophoretic
run, the gel was cut at the molecular weight range corresponding to the PS1 dimer
(approximately 3-4 mm wide). Excised polyacrylamide gel bands were crushed,
resuspended in 100 µl of lysis buffer A and incubated at 37°C for 1 h. Following
incubation, 100 µl of Novex® 2X reducing sample buffer was added to each sample.
Samples were boiled for 2 min then 20 µl of the recuperated proteins (surnageant)
were resubjected to SDS-PAGE.
RESULTS AND DISCUSSION
Human presenilin-1 interacts with itself. Yeast two-hybrid assay, an in vivo system lacking endogenous PS1, was used to
investigate potential homodimerization between PS1 proteins. This initiative was
incited by recent literature showing that overexpression of fl-PS1 leads to low
(approximately 100-180 kDa) molecular weight complexes (6,8,12) suggesting
possible in vivo homodimerization. We assayed numerous wt and mutant PS1
constructs for intramolecular interactions. Interaction between two PS1 holoproteins
is observed following a β-gal assay using the yeast strain Y187, which is very
sensitive for this assay (Fig 1A).
In order to verify if these two-hybrid interactions were specific, and not due to non-
specific β-gal reporter activation from Y187 strain, we tested other reporter genes by
co-transforming the constructs in the AH109 strain. This AH109 strain virtually
35
eliminates false positives by using three reporters, Ade2, His3, and Mel1 (or lacZ),
under the control of distinct Gal4 responsive promotors. These promoters yield
strong and specific responses to Gal4. After plating the co-transformed AH109 strain
on selective medium lacking Ade, His, Trp and Leu (-AHTL), colony growth is
observed (Fig 1B), confirming all β-gal results obtained using the yeast strain Y187.
Taken together, these results suggested possible homo- (fl/fl) dimerization of human
PS1 in vivo. Western blot studies of yeast protein extracts confirmed both PS1
constructs expression and fl-PS1 dimerization in vivo (Fig 1C, lanes 3 and 4,
respectively). To confirm that the dimer observed using anti c-myc probe (Fig 1C,
lane 4) is not an artifact, we reprobed this extract with three diffrent anti-PS1
antibodies. As expected, the dimer is observed using specific antibodies directed
against both N-terminal (Ab14 and N-20) and C-terminal (C-20) regions of PS1 (Fig
1C, lanes 5-7). The apparent increase in molecular weight for both CTF and fl-PS1
reflects their fusion with the Gal-4 DNA binding domain and the c-myc tagged from
the pGBKT7 vector. This binding domain alone is observed in the extract from
empty vector transformed yeast (Fig 1C, lane 2). Interestingly, no endoproteolytic
processing of PS1 could be observed with fusion protein in Saccharomyces
Cerevisiae Y187 and AH109 (personal observation and Fig. 1C, lanes 4-7), implying
that homo-oligomerization was not due to intermolecular associations between
endoproteolyticaly cleaved PS1 fragments in yeast.
Effects of mutations on self-association capacities of PS1. AD-associated PS1 mutations were shown to increased the production of the beta-
amyloid fragments probably by increasing the γ-secretase complex activity. We then
tested the effect of different mutations on binding properties of PS1. We first
investigated if AD-associated PS1 mutants could affect self-association of fl-PS1.
Since AD-associated mutations are dominant (18), we investigated if mutant fl-PS1 is
able to associate with its wild-type counterpart. Our results show that AD-associated
PS1 Y115H, L392V and ∆E10 mutants, as well as the non AD-associated PS1
polymorphism E318G, could dimerize with fl-PS1-wt (Fig 2A). We also verified and
confirmed that these mutants could self-associate with each other (Fig 2B).
Interestingly, the ∆E10 deletion mutant was still able to homodimerize and to
heterodimerize with fl-PS1-wt. This observation suggests that the PS1
36
endoproteolytic cleavage region, which is absent in the ∆E10, is not implicated in PS1
self-association. However, endoproteolytic cleavage of PS1 may play an important
role elsewhere as, for example, in complex stability (9,19,20). Finally, the non AD-
associated PS1 D257A mutant, inhibiting both PS1 endoproteolytic cleavage and γ-
secretase-associated activity (15), was also able to self-oligomerize (Fig 1A and 2B).
These results suggest that all mutants tested here may self-oligomerize in vivo, giving
rise to full-length homodimers. The mutants oligomerization capabilities appeared
similar to their wild-type counterparts.
Formation of PS1 dimers in cultured cells. Expression of fl-PS1 in cultured cells leads mainly to the production of monomeric
species of approximately 45-50 kDa with immuno-specific aggregation at higher
molecular ranges (> 250 kDa) when analysed under normal SDS-PAGE denaturing
and reducing conditions (Fig 3B, lane 2 and [2,21,22]). Since, little is known about
PS1 properties under non-denaturing and non-reducing conditions, we performed
analysis of PS1 dimerization under native electrophoresis conditions (i.e. native-
PAGE). First, we tested and confirmed the specificity of the anti-PS1, Ab14, against
endogenous PS1 in different cell lines (Fig 3A). As expected with polytopic
transmembrane proteins, electrophoresis of endogenous HEK293T protein extracts
under native conditions resulted in specific, high (>250 kDa) PS1 immunoreactive
aggregates when probed with the Ab14 antibody (Fig 3B, lane 4). Overexpression of
PS1 accentuated accumulation of aggregates (Fig 3B, lane 5). Milder extraction
detergents, such as CHAPS (0.5 %-1%) or Digitonin (0.5 %-1%), did not substantially
affect PS1 aggregation under native conditions (data not shown). We next tested if
different non-denaturing and non-reducing variables could “detach” potential PS1
dimers from these high molecular weight complexes. We observed that addition of
0.1% SDS in the electrophoresis running buffer is sufficient to partially denature PS1
electrophoretic aggregates (Fig 3B, lanes 7 and 8). Previous work has shown that
certain LMW PS1-immunoreactive aggregates are SDS resistant (3), implying
possible PS1 homo-oligomers. Moreover, other authors have shown that low amounts
of SDS do not interfere with certain protein’s homodimerization abilities (24,25),
including GPCRs -which are structurally similar to presenilins-. Of particular
37
importance, addition of reducing agents, such as β-mercaptoethanol or DTT (data not
shown), did not significantly interfere with PS1 dimerization as observed by Western
blotting (Fig 3B, lanes 7 and 8), thus suggesting that homo-oligomerization is not
thiol-dependent. Further experiments are underway in our laboratory to address this
issue.
We next tested if PS1-wt-5’myc could homodimerize. Results show that a major
band of approximately 53 kDa corresponding to the expected PS1-wt-5’myc
monomer was detected (Fig 3C, lane 3). In addition, a higher molecular weight band
of approximately 108 kDa was observed. As suspected, this ~ 108 kDa band
corresponded in size to a putative PS1-wt-5’myc dimer. The non-tagged PS1
monomer or dimer migrates slightly lower than its 5’myc-tagged version, hence
demonstrating the immunoreactive specificity of this high molecular weight species
(Fig 3C, lanes 2 and 3, respectively). To rule out the possibility that the ~ 108 kDa
band represented HEK293T cell-specific artifacts, N2A cells over-expressing PS1-wt-
5’myc were subjected to the same treatment. Both the polyclonal anti-PS1 antibody,
Ab14, and the anti-c-myc antibody detected a similar set of PS1 immunoreactive
species (Fig 3D, lanes 2 and 4, respectively). Taken together, these observations
suggests the existence of non-disulfide-linked full-length PS1 dimers or oligomers (in
the case of the > 250 kDa immunoreactive species) that preexist at least in HEK293T
and neuronal type, N2A, cells.
The PS1 Y115H, ∆E10 and D257A mutants dimerize in cultured cells. Using SDS-PAGE under non-reducing and “revisited” non-denaturing conditions, we
investigated the dimerization of different presenilin mutants in N2A cells. The N2A
cells were transfected with different PS1 mutants construct and the analysis showed
that PS1-Y115H, ∆E10 and D257A mutants as well as PS1-wt formed putative dimers
corresponding, in size, to twice their full-length moieties (Fig 4A). In order to
investigate the PS1 immunospecific constituents of this dimer, the band migrating at
approximately 105 kDa was excised from the gel after a first electrophoretic run and
subjected to a second electrophoretic run under reducing and denaturing conditions.
This second run revealed the full-lenght monomer PS1 implying that the original ~
105 kDa band contains full-length presenilins (Fig 4B). Similar results were observed
38
in HEK293T cells (data not shown). Our results demonstrate that PS1 mutants can
form homodimers in vivo.
Evidence for endogenous PS1 self-association. It has previously been shown that, as a consequence of overexpression, the high
molecular weight PS1 complexes are highly unstable and could either be
ubiquitinated then degraded (9,13,25) and/or mature to form aggresomes (5).
Therefore, it cannot be excluded that the dimerization of full-length PS1 is a trigger to
aggregation and breakdown rather than a step prior to complex maturation. Hence, in
order to evaluate if these oligomers are functional derivatives of PS1 or simply
artifacts caused by overexpression, we next tested if endogenous PS1 could
homodimerize. A commercially available anti-PS1 antibody, N-19, directed against
the N-terminal portion of the protein, recognizes specifically full-length PS1 in
endogenous HEK293T cells following western blotting (Fig 4C, lane 1). This
distinctive immunoreactivity for full-length PS1 has been reported elsewhere with
other non-commercial antibodies (26-28). Interestingly, when endogenous HEK293T
non-denaturing and non-reducing protein extracts (i.e. no addition of reducing agents
and no boiling) are subjected to typical SDS-PAGE, an immunoreactive band
corresponding to the putative PS1 dimer is also observed with the N-19 antibody (Fig
4C, lane 2). Similar results were observed with the C-20 antibody and confirmed in
P19 (embryonal carcinoma) cells (data not shown). Taken together, these results are
consistent in showing that endogenous PS1 oligomerizes in vivo to form specific
homodimers.
The oligomerization properties of presenilins observed in vivo (22,29-31) and in vitro
(29) have not been clearly understood. In this report, we clearly show PS1
homodimerization. Our results are consistent at demonstrating that presenilins can
form low molecular weight complexes in vivo. Similar results were observed by
Cervantes S. et al. (2001) using the Drosophilia PS1 when expressed in yeast (32).
We confirmed in vivo PS1 dimerization in HEK293T and N2A cultured cells
overexpressing PS1-wt or mutants. Most importantly, we demonstrated endogenous
PS1 dimerization under non-reducing and non-denaturing conditions in human
cultured cells (HEK293T).
39
The functional role of PS1 dimerization is not clear. However, observations of the
PS1 maturation process may give some clues on this role. It is known that PS1
physiological maturation leads to an endoproteolytic cleavage by a an unknown
‘presenilinase’ resulting in PS1-NTF and CTF that are the major constituents found in
vivo. The mechanisms of this maturation leading to the PS1 complexes are actually
unknown. Different studies have proposed a model in which PS1 molecules are first
stabilized then subsequently cleaved to generate the NTF and CTF (19,33). Based on
this model, it is tempting to speculate that PS1 dimerization may well be a limiting
and/or necessary step in PS1 complex maturation.
We have demonstrated that AD-associated and γ-secretase dominant negative PS1
mutations failed to inhibit self-association of PS1. These observations are in
accordance with in vivo studies suggesting that PS1 mutations lead to a gain of
function (34,35), and not to an abrogated protein function, as seen in PS1 and/or PS2
knock-out mice (36,37). Indeed, our results demonstrate that PS1 mutations do not
prevent complex oligomerization, thus presumably conserving its integrity and basic
function(s). However, AD-associated mutations may lead to distinct and/or perturbed
high molecular weight complexes, hence having new properties. Further studies will
be needed to evaluate this possibility.
All together, our results clearly demonstrate that presenilin 1 may undergo self-
dimerization in vivo. Because fl-PS1 as well as NTF and CTF represent important
AD therapeutic targets (38), their physiological existence as homo- or hetero-
oligomers could have important implications for the development and screening of
new drugs.
ACKNOWLEDGMENTS
We thank Dr Paul Fraser for generously providing the Ab14 antibody, Dr Paul
Mathews for the PS1 K/O cells and Dr Richard Blouin for the N2A cells. This work
40
was supported by grants from Canadian Institutes of Health Research (IRSC) and
Fonds de la Recherche en Santé du Québec (FRSQ).
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45
FIGURE LEGENDS
Figure 1. Two-hybrid assay demonstrating PS1 self-association. (A) Y187 yeast co-transformed colonies were assayed for colony lifts β-galactosidase
activity according to Clontech procedures. Positive interactions were measured by the
blue coloration intensity (++++ represented the highest level and (-) represented
absence of coloration). Positive controls include pGBKT7-p53/pGADT7-T antigen
and pGBKT7-PS1Loop 6-7 loop/pGAD-hNPRAP. (B) AH109 yeast co-transformed
colonies were assayed for His+ and Ade+ activity according to Clontech procedures.
Colony growing on SD/-AHTL indicates positive interactions between two partners.
Growth rate is indicated by positive (+) signs, where +++ represent the fastest (i.e.
early growth observed between 2-4 days). Negative (-) sign indicate absence of
colony growth after 10 days of incubation. Negative controls include pGBKT7-fl-
PS1/pGAD-Lamin and pGBKT7-Lamin/pGAD-fl-PS1. Results shown represent at
least 3 independent experiences. (C) Western blot of yeast protein extract
demonstrating fl-PS1 expression and dimerization in yeast strain AH109. Lane 1
represents an extract from a non-transformed AH109 strain. In lane 2, the AH109
strain was transformed with pGBKT7 empty vector to demonstrate the presence of the
c-myc tagged Gal-4 binding domain expression. Both pGBKT7-PS1-CTF and
pGBKT7-fl-PS1 constructs were transformed in AH109 strain and tested for
expression using anti-c-myc probe (lanes 3 and 4, respectively). To confirm the
specificity of pGBKT7-fl-PS1 expression and dimerization, the same protein extract
used in lane 4 was probed using either anti-PS1 antibody Ab14 (lane 5), N-19 (lane 6)
or C-20 (lane 7).
Figure 2. Dimerization of mutant presenilins in yeast. (A) AH109 strain co-transformed with PS1-wt or mutant constructs were assayed for
His+ and Ade+ activity according to Clontech procedures. Colony growth is observed
with all mutants tested herein. Results shown represent at least 3 independent
experiences.
Figure 3. Dimerization of PS1 in cultured cells. (A) Western blot demonstrating anti-PS1 Ab14 antibody specificity in different cell
types. Equal amounts of proteins extracted from native HEK293T, N2A or PS1 K/O
46
cells were mixed with reducing SDS-PAGE sample buffer, boiled for 2 min, then
fractionated on SDS-PAGE and transferred to PVDF membrane. The membrane was
probed using Ab14 antibody. (B) Western blot following SDS-PAGE of proteins
extracted from non-transfected (lane 1) and PS1-wt transfected (lane 2) HEK293T
cells. Cells were mixed with SDS-PAGE reducing sample buffer and fractionated on
SDS-PAGE. Lanes 3 to 5 represent a western blot following native gel
electrophoresis conditions. Proteins were extracted from both non-transfected PS1
K/O (lane 3) and HEK293T cells (lane 4), and from fl-PS1-wt transfected HEK293T
cells (lane 5). In this case, the cells were lysed in extraction buffer B, mixed with
native sample buffer, incubated at 37°C for 15 min and then subjected to native-
PAGE. Lanes 6 to 8 represent a western blot of protein extracts from both non-
transfected HEK293T cells (lane 6) and from fl-PS1-wt transfected HEK293T cells
(lanes 7 and 8). As above, the cells were lysed using buffer B, mixed with reducing
or non-reducing native sample buffer and incubated at room temperature for 15 min.
Following incubation, SDS-PAGE was carried out using SDS-PAGE running buffer
(containing 0.1 % SDS). The effect of a reducing agent, β-mercaptoethanol, on PS1
dimerization was evaluated (lane 7 vs. 8). (C) Analysis of proteins extracted from
either, empty vector (lane 1), fl-PS1-wt (lane 2) and fl-PS1-5’myc (lane 3) transfected
HEK293T cells and submitted to partial non-denaturing SDS-PAGE. (D) Equal
amounts of proteins extracted from both empty vector (lanes 1 and 3) and fl-PS1-
5’myc (lanes 2 and 4) transfected N2A cells were mixed with native sample buffer
(without reducing agents), incubated at 37°C for 15 min, then fractionated on SDS-
PAGE. Blots were probed with either Ab14 antibody (lanes 1 and 2) or c-myc
antibody (lanes 3 and 4). Sign (#) represents non-specific immunoreactive bands due
to reducing agents. Sign (*) represents non-specific immunoreactive bands due to
native electrophoretic conditions.
Figure 4. Overexpression of wild-type or mutant PS1 as well as endogenous PS1
show dimerization in cultured cells.
(A) Equal amounts of proteins extracted from fl-PS1-wt or mutants transfected N2A
cells were submitted to partial non-denaturing (and non-reducing) SDS-PAGE and
transferred to PVDF membranes. Blot was probed with the Ab14 antibody. (B)
Analysis of the PS1 dimer fragment. The ~ 105 kDa bands were excised from a first
47
electrophoretic run as in (A), resuspended in lysis buffer A and incubated at 37°C for
1h. Following incubation, SDS-PAGE sample buffer was added to the lysates.
Lysates were boiled for 2 min and 20 µl of the suspension were subjected to SDS-
PAGE and transferred to PVDF membranes. To confirm the presence of PS1, blots
were analysed with PSN2 monoclonal antibody. (C) Endogenous PS1 dimerization in
cultured cells. Equal amounts of proteins extracted from HEK293T cells were mixed
with SDS-PAGE sample buffer (without reducing agents), boiled for 2 min (lanes 1)
or incubated at 37°C for 15 min (lanes 2), then fractionated on SDS-PAGE and
transfered to PVDF membranes. Blots were probed with the N-19 antibody.
48
49
50
51
CHAPITRE IV
Oligomérisation des fragments de la préséniline-1 humaine
Note : Ce chapitre, présenté sous la forme d’un manuscrit publié dans la revue FEBS Letters (FEBS Lett. 2003 Aug 28;550(1-3):30-4.), résume une partie des travaux effectués dans le cadre de mon projet de doctorat. Plus spécifiquement, ce travail contient des expériences visant à compléter mon premier objectif de travail (décrit dans le chapitre 2 à la page 23). Comme ma contribution dans la conception (approches expérimentales), la réalisation (constructions plasmidiques et manipulations) et la rédaction (incluant le montage des figures) de ce travail est majoritaire, mon nom apparaît comme premier auteur sur le manuscrit. Toutefois, il est important de souligner l’apport très apprécié de ma consoeur de laboratoire, Mme Chantal Godin, dans la réalisation technique de ce travail. En résumé, Mme Godin a cloné le fragment PS1 CTF dans le vecteur d’expression pGEX-4T1 utilisés dans cet ouvrage. Cet article est disponible sur Internet en version originale anglaise à l’adresse électronique suivante : http://dx.doi.org/10.1016/S0014-5793(03)00813-5
53
Résumé
Dans le but d’acquérir de nouvelles connaissances sur les propriétés structurales et
fonctionnelles de la préséniline-1 (PS1), nous avons étudié les propriétés
oligomérisantes de ces fragments N- et C-terminaux (respectivement, NTF et CTF).
Par technique de double hybride, confirmée avec la technique d’immunoprécipitation
de type « GST pull-down », nous démontrons pour la première fois que les fragments
PS1 peuvent s’associés directement ou entre eux pour former des homodimères
(NTF/NTF ou CTF/CTF). Les mutations associées à une forme familiale de la
maladie d’Alzheimer PS1 Y115H et M146L n’affectent pas les propriétés homo- (ni
hetero-) oligomérisantes des fragments PS1. Ces observations suggèrent que les
mutations associées à la forme familiale de la maladie d’Alzheimer n’altèrent pas (ou
peu) leur(s) fonction(s) physiologique(s) de base. L’ensemble de ces résultats
confirme qu’il existe des liens intramoléculaires complexes qui régulent
l’oligomérisation de la PS1 et possiblement l’activité γ-secrétase.
54
Oligomerization of human presenilin-1 fragments.
Sébastien S. Hébert1, Chantal Godin1 and Georges Lévesque1, 2
1Molecular and Human Genetics Unit, CHUQ-Pavillon St-François d'Assise, 10 rue
de l'Espinay, Québec, Canada G1L 3L5, 2 Medical Biology Department, Laval
University, Quebec, Canada, G1K 7P4 .
ABSTRACT
To gain insight into presenilin-1 (PS1) structural aspects, we explored the structure-
function relationship of its N- and C-terminal (NTF and CTF, respectively)
complexes. We demonstrated that both NTF and CTF act as independent but inter-
changing binding units capable of binding each other (NTF/CTF) or their homologues
(NTF/NTF; CTF/CTF). The Alzheimer’s disease-associated PS1 mutations Y115H
and M146L do not affect their ability to hetero- and/or homodimerize, thus
conserving their basic integrity and function(s). These results suggest that PS1
associates intra-molecularly to form higher order complexes, which may be needed
for endoproteolytic cleavage and/or γ-secretase-associated activity.
Keywords: presenilin, gamma-secretase, homodimerization, heterodimerization
Corresponding author: Dr. Georges Lévesque, Phone: 418-525-4444 ext 3752, Fax:
418-525-4195, Email: [email protected]
Abbreviations: PS1, presenilin-1; NTF, N-terminal fragment; CTF, C-terminal
fragment; FL, full-length; β-gal, β-galactosidase; His, histidine; Ade, adenine; Trp,
tryptophan; Leu, leucine; TM, transmembrane; wt, wild-type.
55
INTRODUCTION
Mutations in the presenilin-1 (PS1) gene account for most cases of early onset
familial Alzheimer’s disease. In vivo, the PS1 protein is approximately 45-50 kDa in
size and undergoes a proteolytic cleavage by ‘presenilinase’ to generate N- and C-
terminal fragments (NTF and CTF, respectively) of approximately 32-35 and 16-20
kDa, respectively (Thinakaran 2001). PS1 forms also low (~ 100-180 kDa) and high
(> 250 kDa) molecular weight complexes containing both full-length and
heterodimeric PS1 NTF and CTF fragments (Capell, Grunberg et al. 1998; Steiner,
Capell et al. 1998; Yu, Chen et al. 1998; Hébert, Godin et al. 2003). Recent work
indicates that the higher molecular weight complexes contain, among PS1 fragments,
at least three other proteins, namely Nicastrin, Aph-1, and Pen-2 (Kimberly, LaVoie
et al. 2003; Van Gassen and Annaert 2003). Accumulating evidences tend to
demonstrate that the appearance of presenilin fragments in these complexes is
intrinsic to PS1 and γ-secretase activity, is involved in the cleavage of integral
membrane proteins such as amyloid-β precursor protein (AβPP), Notch1, ErbB4.
Using the yeast two-hybrid assay and GST pull down experiments we provide
evidence that human PS1 NTF and CTF moieties can associate directly to form
specific heterodimers and that AD-associated PS1 mutations Y115H and M146L do
not affect their ability to hetero-oligomerize. Using deletion mutants coding for
truncated PS1, we demonstrate that multiple binding regions are implicated in PS1
heterodimerization and that both cytoplasmic and transmembrane regions are
necessary. We also show that PS1 may self-oligomerize giving rise to NTF/NTF and
CTF/CTF homodimers. Although inter-molecular heterodimers have previously been
reported following in situ chemical cross-linking and co-immunoprecipitation
(Thinakaran, Regard et al. 1998; Yu, Chen et al. 1998) – which are indirect methods –
, our data are the first to confirm a direct interaction between human NTFs and CTFs
and identify potential regions of contact under in vivo conditions.
56
Experimental procedures
Yeast two-hybrid assays. cDNA sequences encoding various parts of human PS1 were amplified by PCR using
the Herculase Taq polymerase (Stratagene). The following wt PS1 cDNA were
prepared: NTF (PS11-291), 1-132 (PS11-132), TM1-2 (PS1101-153), Loop 1-2 (PS1101-132),
TM2 (PS1131-153), Loop 6-7 (PS1239-409), CTF (PS1292-467), TM8 (PS1406-426), CTF-
short (PS1427-467). AD-associated PS1 short version mutants, 1-132 Y115H and TM1-
2 M146L were also generated. cDNAs were cloned both into pGBKT7 and pGADT7
vectors (Clontech) as a fusion to the Gal4-DNA binding domain (BD) and Gal4-DNA
activating domain (AD), respectively. Each construct was sequenced and shown to be
free of autonomous Gal-4 activation. Functional expression of fusion proteins in
yeast was confirmed by Western blot.
Each of the pGBKT7-construct was co-transformed into Y187 or AH109
Saccharomyces cerevisiae (Clontech) together with each pGADT7-construct.
Transformants were selected on yeast drop-out media (Clontech) that lacked both
tryptophan and leucine (TL-) and assayed for β-gal+ in the case of Y187 and His+ and
Ade+ activity in the case of AH109. Positive controls included pGBKT7-
p53/pGADT7-T antigen and pGBKT7-PS1-Loop 6-7/pGADT7-hNPRAP as
previously described (Levesque, Yu et al. 1999).
Preparation of yeast cell lysates and immunoblotting. Transformed yeasts were harvested by centrifugation and washed twice in sterile H2O.
Pellets were resuspended in ~ 300 µl of yeast extraction buffer [Tris 50 mM pH 7.5,
150 mM NaCl, 1% CHAPS, 1 mM EDTA, 1 mM PMSF and mini-complete protease
inhibitors (Roche-Diagnostic)]. Proteins were extracted using acid washed glass
beads (Sigma) as described by the manufacturer (Clontech) and loaded on a 4-20%
NuPAGE gel (Invitrogen) as described above. Blots were probed with either the anti-
c-myc (Roche Diagnostics) or anti-PS1 PSN2 (gift from Dr. Hiroshi Mori) antibodies.
57
GST pull-down assays. PS1-CTF (PS1292-467) cDNA was cloned into the pGEX-4T1 vector and fixed on
glutathione-Sepharose beads as previously described (Hébert, Godin et al. 2003).
Proteins were eluted from the beads, mixed with 2X sample buffer and ~20 µl was
loaded on gel for SDS-PAGE. Blots were probed with anti-c-myc antibodies (Roche
Diagnostics).
RESULTS
Direct association between PS1 N-terminal and C-terminal fragments. Partial PS1 cDNA constructs were produced in order to represent putative functional
domains and sites of multiple protein interactions. Western blot analysis confirmed
correct protein expression for all constructs (Figure 1B). Although certain PS1
constructs tested herein contain hydrophobic transmembrane domains, its fusion to
the Gal-4 binding and/or activating domain in the pGBKT7/pGADT7 vectors reveals
over-all protein solubility (Figure 1A and (Hébert, Godin et al. 2003)). These
constructs were shown to be functional in yeast two-hybrid assays (Levesque, Yu et
al. 1999; Godin et al. 2003; Hébert, Godin et al. 2003). First, we investigated if our
NTF (a.a. 1-291) of human PS1 could form a heterodimer with its remaining CTF
(a.a. 292-467). Specific interaction between NTF (expressed in human HEK293T
cells) and CTF is observed following GST pull-down using GST-tagged CTF (Figure
2A). This result corroborates with previous reports demonstrating NTF/CTF
heterodimerization (Thinakaran, Regard et al. 1998; Yu, Chen et al. 1998). After this
initial result, we tested if NTF/CTF heterodimerization could be reproduced in vivo in
yeast, a system lacking both endogenous PS1 and γ-secretase activity (Edbauer,
Winkler et al. 2003; Hébert, Bourdages et al. 2003). As shown in Figure 2B,
interaction between the NTF and CTF is observed following a β-gal assay using the
yeast strain Y187.
To further verify if these two-hybrid interactions were specific, and not due to non-
specific LacZ reporter activation from strain Y187, we cotransformed all constructs in
the AH109 yeast strain (Clontech). As shown in Figure 2C, colony growth on
selective medium lacking Ade, His, Trp and Leu (AHTL-) is observed, confirming all
58
β-gal assays. Negative and positive control experiments were done using empty
vectors (either pGBKT7 or pGADT7) and hNPRAP, a known CTF PS1-interacting
protein (Levesque, Yu et al. 1999). Taken together, these results confirm that PS1 N-
terminal and C-terminal fragments associate directly to form a stable complex in vivo.
Mapping of PS1-interacting domains Using various PS1 cDNA deletion constructs (as shown in Fig. 1A), we tested which
domain(s) was implicated in PS1 NTF/CTF heterodimerization. As summarized in
table 1, both the 1-132 and the TM 1-2 fragments showed interaction with the PS1-
CTF. These observations suggested that PS1 amino acids 101-132, corresponding to
the first lumenal loop (Loop 1-2), are implicated in heterodimerization with the CTF.
However, studies using solely the PS1 Loop 1-2 as bait or as target did not result in
positive interactions. As suspected, TM 2 did bind CTF, but weakly. These
observations were confirm by GST pull-down assays showing that both TM1-2 and
TM2, but not Loop 1-2 alone or mock (pGBKT7), bind GST-PS1-CTF (Fig 3A). We
suspect that heterodimerization of PS1 fragments must implicate large intermolecular
binding domains thus possibly “taken as a whole”.
Further investigation revealed that 1-132, as contrary to the TM 1-2 fragment, can
bind to the cytoplasmic Loop 6-7 (Table 1). This suggests that the first hydrophilic N-
terminus stretch before TM 1 (a.a. 1-80) may interact with the large cytoplasmic loop,
as they are both oriented to the cytoplasm. Moreover, since both the TM1-2 and 1-
132 bind to CTF but not to CTF-short our results, altogether, suggest that PS1-
NTF/CTF heterodimerization is dependent of at least four distant binding regions: i)
the first 80 amino-acid portion of PS1-NTF ii) the second lumenal loop combined
with the second transmembrane domain (TM 1-2), iii) the large cytoplasmic loop
(Loop 6-7) and iv) a 61 amino-acids portion of PS1-CTF (a.a. 406-467).
Homodimerization between PS1 NTF and CTF fragments. We have previously reported PS1 full-length/full-length homodimerization in yeast as
well as in mammalian cells (Hébert, Godin et al. 2003). Interestingly, we also
observe NTF/NTF and CTF/CTF homodimerization (Table 1). We used GST pull-
59
down assays to confirm specific fragment homodimerization using, as example, GST-
tagged PS1-CTF against expressed PS1-CTF (Fig 3B).
Crude domain mapping of NTF/NTF homodimerization led us to demonstrate that
both the 1-132 and the TM 2 region, but not Loop 1-2, could bind PS1-NTF (Table 1).
Mapping of CTF/CTF homodimerization shows that the last 61 amino-acid portion
(a.a. 406-467) of PS1-CTF, implicated in NTF/CTF heterodimerization, is required
for binding. Since TM8/TM8 (a.a. 406-426) do not bind to each other and CTF (a.a.
292-467) do not bind to CTF-short (a.a. 427-467) or Loop 6-7 (a.a. 239-409), this
suggest that the last 61 amino-acid portion of CTF is required as a whole for binding
(Table 1 and Figure 3B). Taken together, these results suggest that binding regions
implicated in NTF/CTF heterodimerization are equally important in PS1 fragments
homodimerization.
Effects of mutations on hetero and homo-dimerization capacities of PS1-NTF
and CTF fragments.
We next investigated if mutated PS1-NTF was able to heterodimerize with the wt
PS1-CTF. We constructed two PS1-NTFs containing either the Y115H or M146L
mutation (PS11-132-Y115H and PS1TM 1-2-M146L). When assayed for interaction
against the PS1-CTF, both mutants showed interaction (Table 1). Finally, we tested if
these NTF-mutants could homodimerize with its wild-type counterpart. Mutants’
oligomerization capabilities appeared similar to their wild-type counterparts (Table 1).
As expected, these PS1 AD-associated mutants do not disturb the dimerization
suggesting that PS1 fundamental physiological function is conserved.
DISCUSSION
The structure-function analysis of PS1 N- and C-terminal complexes are important if
we consider that both fragments share putative catalytic elements (within separate
transmembrane domains) needed for PS1 enzymatic-related activity(ies) (Wolfe
2001). In this report, we clearly show direct heterodimerization between NTF and
CTF fragments of PS1. Our results demonstrate that these two fragments can form
60
heterodimers in vivo. We also demonstrated that NTF and CTF are capable of self-
association, thus forming homodimers.
Previous studies aimed at understanding PS1 structure and/or maturation have
proposed a model in which PS1 molecules are first stabilized then subsequently
cleaved to generate the NTF and CTF (Ratovitski, Slunt et al. 1997; Saura, Tomita et
al. 1999). Our results support this model and are also in accordance with the
hypothetical “tetramer” model proposed by Haass and Steiner (Haass and Steiner
2002). Hence, we propose a new model (Figure 4) in which PS1 homodimerization
precedes endoproteolytic cleavage by “presenilinase”. Following endoproteolytic
cleavage, formation of a tetramer would be a crucial step for the assembly, stability
and/or substrate specificity of the high molecular weight presenilin complex necessary
for γ-secretase activity (Haass and Steiner 2002). According to our data, AD-
associated PS1-NTF Y115H and M146L mutants failed to demonstrate a disrupted
interaction with the PS1-CTF. This observation is in accordance with in vivo studies
suggesting that PS1 mutations lead to a gain of function (Davis, Naruse et al. 1998)
and not to an abrogated protein function, as seen in PS1 knock-out mice (Shen,
Bronson et al. 1997).
Nicastrin, Aph-1 and Pen-2 (presenilin partners in the γ-secretase complex), the long
sought “limiting factors” responsible for stabilization of PS1 endogenous levels, do
not seem to be required for PS1 hetero- and homo-dimerization, since no homologues
are found in yeast (Edbauer, Winkler et al. 2003). However, these proteins may be
required to saturate, stabilize, generate and/or limit endogenous levels of mature PS1
in high molecular weight complexes (Edbauer, Winkler et al. 2003; Takasugi, Tomita
et al. 2003).
In an attempt to map domains implicated in NTF/CTF oligomerization we have
demonstrated that both NTF and CTF act as independent but inter-changing binding
units capable of binding each other (NTF/CTF) or their homologues (NTF/NTF;
CTF/CTF). We propose that each PS1 fragment acts as pseudo-independent structural
61
units, namely domain A for the NTF and domain B for the CTF. Other polytopic
transmembrane proteins, such as G-protein coupled receptors (GPCR), which are
structurally similar to presenilins, do possess two distinct binding domains
(Schoneberg, Schultz et al. 1999; Gouldson, Higgs et al. 2000). À priori, hetero-
oligomerization of PS1 domain A seems dependent of its first 80 amino-acids plus its
first lumenal loop associated with TM 2. However, our results demonstrate that most,
if not all, of the domain A is necessary, perhaps at different levels, for its
intramolecular and possibly intermolecular protein-protein binding properties. Recent
findings showing that the first 1-132 portion of the NTF, and most importantly but not
exclusively, the first transmembrane domain are needed to bind APP and/or
telencephanin (Annaert, Esselens et al. 2001) are in accordance with this hypothesis.
Of particular importance, other reports have associated the first two transmembrane
domains as crucial for PS1 stability, endoproteolysis and cellular maturation
(Thinakaran 2001; Leem, Saura et al. 2002).
Like its N-terminal counterpart, most of the PS1-CTF fragment (domain B) seems
implicated in heterodimerization and/or homodimerization. Our results are in
agreement with previous reports which, in sum, pinpoint different portions located at
the C-terminus of PS1-CTF implicated in PS1 maturation and/or γ-secretase-
associated activity (Tomita, Takikawa et al. 1999; Thinakaran 2001; Tomita,
Watabiki et al. 2001).
Taken together, our results clearly demonstrate that presenilin 1 and its
endoproteolytic fragments may undergo self-dimerization in vivo. Because PS1 NTF
and CTF generated fragments represent important AD therapeutic targets, their
physiological existence as homo- or hetero-oligomers could have important
implications for the development and screening of new drugs.
62
ACKNOWLEDGMENTS:
We are grateful to Dr Madeleine Carreau for the revision of the manuscript. This
work was supported by grants from Canadian Institutes of Health Research (IRSC),
Fonds de la Recherche en Santé du Québec (FRSQ) and Alzheimer Society of
Canada.
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Selkoe, D.J. (2003) Proc Natl Acad Sci U S A 100, 6382-6387.
[7] Van Gassen, G. and Annaert, W. (2003) Neuroscientist 9, 117-26.
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[10] Hébert, S.S., Bourdages, V., Hiroshi, M., Levesque, G. (2003) Neurobiol. of
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[11] Godin, C., Auclair, A., Ferland, M., Hébert, S.S., Carreau, M., Levesque, G.
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(2003) Nat Cell Biol 5, 486-8.
[13] Wolfe, M.S. (2001) J Mol Neurosci 17, 199-204.
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Borchelt, D.R. (1997) J Biol Chem 272, 24536-41.
[15] Saura, C.A., Tomita, T., Davenport, F., Harris, C.L., Iwatsubo, T. and
Thinakaran, G. (1999) J Biol Chem 274, 13818-23.
[16] Haass, C. and Steiner, H. (2002) Trends Cell Biol 12, 556-62.
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[17] Davis, J.A. et al. (1998) Neuron 20, 603-9.
[18] Shen, J., Bronson, R.T., Chen, D.F., Xia, W., Selkoe, D.J. and Tonegawa, S.
(1997) Cell 89, 629-39.
[19] Takasugi, N., Tomita, T., Hayashi, I., Tsuruoka, M., Niimura, M., Takahashi,
Y., Thinakaran, G. and Iwatsubo, T. (2003) Nature 422, 438-41.
[20] Gouldson, P.R., Higgs, C., Smith, R.E., Dean, M.K., Gkoutos, G.V. and
Reynolds, C.A. (2000) Neuropsychopharmacology 23, S60-77.
[21] Schoneberg, T., Schultz, G. and Gudermann, T. (1999) Mol Cell Endocrinol
151, 181-93.
[22] Annaert, W.G., Esselens, C., Baert, V., Boeve, C., Snellings, G., Cupers, P.,
Craessaerts, K. and De Strooper, B. (2001) Neuron 32, 579-89.
[23] Leem, J.Y. et al. (2002) Neurobiol Dis 11, 64-82.
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T.C., Maruyama, K. and Iwatsubo, T. (1999) J Neurosci 19, 10627-34.
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64
FIGURE LEGENDS:
Figure 1. Schematic representation of PS1 fusion proteins. (A) Illustration (not to scale) of fusion proteins coding for PS1 generated fragments.
Black squares represent hydrophobic regions corresponding to putative
transmembrane domains. Numbers represent the amino acid positions. Kyte-Doolittle
hydrophilicity plots for each construct cloned in fusion with Gal-4 binding and/or
activating domains are presented. Value smaller than 1.1 indicates an insoluble
protein. (B) PS1 engineered fragments cloned in fusion with Gal-4 binding domain
were expressed in yeast strain Y187. Equal amount of 1% CHAPS protein extracts
were run on 4-20% NuPage, blotted, and probed with anti-myc antibodies. Note:
pGBKT7 empty vector (mock) containing the DNA Binding Domain (DNA-BD)
tagged with c-myc epitope runs at approximately 25 kDa. Similar results were
obtained with PS1 engineered fragments cloned in fusion with Gal-4 activating
domain (data not shown).
Figure 2. PS1 NTF/CTF heterodimerization. (A) 5’HA-PS1-NTF was expressed in HEK293T cells and equal amounts of 1%
CHAPS protein extracts were incubated with either GST-PS1 CTF or GST alone.
Bound material was separated on 4-20% NuPAGE, blotted, and probed with anti-
PSN2. (B) Y187 yeast co-transformed colonies were assayed for β-galactosidase
activity according to Clontech procedures. Positive interactions were measured by the
blue coloration intensity (++++ represented the highest level and (-) represented
absence of coloration). Positive controls include pGBKT7-p53/pGADT7-T antigen
and pGBKT7-PS1- Loop 6-7/pGADT7-hNPRAP. (C) AH109 yeast co-transformed
colonies were assayed for His+ and Ade+ activity according to Clontech procedures.
Growth on SD/AHTL- indicates positive interactions between fusion partners. Growth
is indicated by positive (+) signs, where +++ represent the fastest (i.e. early growth
observed between 2-4 days) rate. Negative (-) sign indicate absence of growth after 10
days incubation. Results shown represent at least 3 independent experiences.
Figure 3. GST pull-down analysis. (A) pGBKT7-PS1-TM1-2, pGBKT7-PS1-Loop 1-2, pGBKT7-PS1-TM2 and Mock
(pGBKT7) were expressed in strain Y187, and equal amounts of 1% CHAPS yeast
65
protein extracts were incubated with GST-PS1-CTF. Bound material was separated
on 4-20% NuPAGE, blotted, and probed with anti-myc. (B) pGBKT7-PS1-Loop 6-7,
pGBKT7-PS1-CTF and Mock (pGBKT7) were expressed in strain Y187, and equal
amounts of 1% CHAPS yeast protein extracts were incubated with GST-PS1 CTF.
Bound material was separated on 4-20% NuPage, blotted, and probed with anti-myc.
Figure 4. Hypothetical model of presenilin-1 maturation. Nascent presenilin-1 (PS1) is expressed as a 45-50 kDa holoprotein. Following full-
length/full-length homodimerization, PS1 is cleaved by “presenilinase” to generate
stable fragment hetero- and homodimers (tetramers). Either pre- or post- PS1
endoproteolytic cleavage, PS1 fragments associate with Nicastrin, Aph-1 and Pen-2 to
form a functionally active high molecular weight γ-secretase complex.
66
67
68
69
70
CHAPITRE V
La préséniline-1 se lie directement au « β-site APP cleaving
enzyme » (BACE1)
Note : Ce chapitre, présenté sous la forme d’un manuscrit publié dans la revue Neurobiology of Disease (Neurobiol Dis. 2003 Aug;13(3):238-45.), résume une partie des travaux effectués dans le cadre de mon projet de doctorat. Plus spécifiquement, ce travail contient des expériences visant à atteindre mon deuxième objectif de travail (décrit dans le chapitre 2 à la page 24). Comme ma contribution dans la conception (approches expérimentales), la réalisation (constructions plasmidiques et manipulations) et la rédaction (incluant le montage des figures) de ce travail est majoritaire, mon nom apparaît comme premier auteur sur le manuscrit. Il est important de souligner l’apport fortement apprécié de mon directeur de recherche, Dr Georges Lévesque, dans la rédaction de ce travail (environ 50%). Aussi, il est important de souligner l’apport très apprécié de mes consoeurs de laboratoire, Mme Valérie Bourdages (confirmation des interactions PS1 et BACE en double-hybride), Mme Chantal Godin (construction de pGBKT7-APP) et Mme Mélissa Ferland (confirmation de certains contrôles négatifs en double-hybride) dans la réalisation technique de ce travail. De plus, nous tenons à remercier Dre Madeleine Carreau pour ses conseils et son support matériel (anticorps anti-FITC, anti-PE, le DAPI et le microscope). Cet article est disponible sur Internet en version originale anglaise à l’adresse électronique suivante : http://dx.doi.org/10.1016/S0969-9961(03)00035-4
72
Résumé
Une des principales lésions neuropathologiques observées chez le cerveau des patients
atteints de la maladie d’Alzheimer sont les plaques amyloïdes. Les plaques amyloïdes
(aussi appelées plaques séniles) sont composées principalement d’un court peptide
neurotoxique, nommé peptide Aβ42. Aβ42 est généré de la protéine précurseur AβPP
suite à deux clivages par la β-secrétase (nouvelle aspartyl protéase appelée BACE) et
le complexe γ-secrétase comprenant préséniline-1 (PS1). Cette étude découle
d’expériences effectuées en levure qui avaient pour but de développer un essai
fonctionnel pour la γ-secrétase in vivo. Nous démontrons, par technique de double
hybride en levure, une interaction directe entre BACE et PS1 sauvage. Cette
observation a été confirmée en culture de cellules avec des expériences de co-
immunoprécipitation et de co-localisation dans des cellules humaines HEK293T. Nos
résultats préliminaires montrent que la PS1 se lie préférentiellement à la forme
immature (c'est-à-dire, le zymogène) de BACE. En conclusion, l’ensemble de ces
résultats suggèrent que la PS1 peut jouer un rôle dans la maturation et/ou l’activité de
BACE in vivo.
73
Presenilin-1 interacts directly with the beta-site APP cleaving enzyme
(BACE1).
Sébastien S. Hébert1, Valerie Bourdages1, Chantal Godin1, Mélissa Ferland1
Madeleine Carreau1 and Georges Lévesque1, 2
1Molecular and Human Genetics Unit, CHUQ-Pavillon St-François d'Assise, 10 rue
de l'Espinay, Québec, Canada G1L 3L5, 2 Medical Biology Department, Laval
University, Quebec, Canada, G1K 7P4
Address correspondence to: Dr. Georges Lévesque, Molecular and Human Genetics
Unit, CHUQ-Pavillon St-François d'Assise, 10 rue de l' Espinay, Québec, Canada
G1L 3L5.
Tel: 418-525-4444 ext 3752
Fax: 418-525-4195
Email: [email protected]
Running title: Presenilin-1 interacts with BACE1
Keywords: presenilin, beta-site APP cleaving enzyme (BACE1), amyloid, γ-
secretase, Alzheimer disease.
74
ABSTRACT
A neuropathological hallmark of Alzheimer disease is the presence of amyloid
plaques. The major constituent of these plaques, occurring largely in brain areas
important for memory and cognition, is the 40-42 amyloid residues (Aβ). Aβ is
derived from the amyloid protein precursor (APP) after cleavage by the recently
identified β-secretase (BACE1) and the putative γ-secretase complex containing the
presenilin 1 (PS1). In an attempt to develop a functional secretase enzymatic assay in
yeast we demonstrate a direct binding between BACE1 and PS1. This interaction was
confirmed in vivo using co-immunoprecipitation and co-localization studies in human
cultured cells. Our results show that PS1 preferably binds immature BACE1, thus
possibly acting as a functional regulator of BACE1 maturation and/or activity.
INTRODUCTION
Amyloid-beta (Aβ) deposition in brain of Alzheimer disease (AD) patients is
considered as a central event in the pathogenesis of AD in both genetic and non-
genetic forms (Hardy, Duff et al. 1998; Torreilles and Touchon 2002). Aβ42 fragment
is the major component of senile plaques and probable cause of dementia in
Alzheimer’s disease. Aβ42, which accumulates in the brain plaques, derives from an
amyloid protein precursor (APP) after a series of two endoproteolytic cleavages
(Selkoe 2001). The first cleavage involves the recently identified aspartyl protease β-
secretase, BACE1, which generate an APP C-terminal fragment, namely C99 (Vassar,
Bennett et al. 1999). The putative γ-secretase complex containing at least presenilin-1
(PS1) and nicastrin then cleaves the C99 fragment (Yu, Nishimura et al. 2000).
Together, these cleavages give rise to a series of peptides that contain the 40-42
amino acids Aβ. Both BACE1 and PS1 possess, either directly or indirectly secretase-
associated activities.
BACE1 is a type I integral membrane aspartyl protease highly expressed in neuron
(Vassar, Bennett et al. 1999). Maturation of BACE1 involves a series of post-
translation modifications during its intracellular trafficking through the trans-Golgi
75
network. One of these modifications occurs in the Golgi and consists in the BACE1
pro-domain cleavage by furin (Bennett, Denis et al. 2000). The resulting BACE1
starts at residue Glu-46 and localizes within intracellular compartments of the
secretory pathway, in particular Golgi and endosomes where it co-localized with APP
(Yan, Han et al. 2001). Contrary to PS1, no mutation in BACE1 has been associated
with Alzheimer’s disease or with an increased production of Aβ (Murphy, Yip et al.
2001). Although BACE1 catalytic activity is well documented, little is known about
the cell biology of this enzyme.
The second enzyme implicated in the Aβ cleavage is named γ-secretase. Identity of
the γ-secretase is actually unknown, although biochemical studies have shown that the
γ-secretase is a multimer enzymatic complex containing at least PS1, Nicastrin, Aph-1
and Pen-2 (Haass and De Strooper 1999; Yu, Nishimura et al. 2000; Francis, McGrath
et al. 2002; Leem, Vijayan et al. 2002). Interestingly, AD-associated mutations in PS1
lead to an increase in Aβ formation (Citron, Westaway et al. 1997). PS1 is a polytopic
transmembrane protein located primarily in the endoplasmic reticulum (ER), the early
Golgi and, to a lesser extent, at the cell surface and nuclear membrane (Sherrington,
Rogaev et al. 1995; Li, Xu et al. 1997; Kaether, Lammich et al. 2002). The precise
role of PS1 is not fully understood as it is associated with diverse biological functions
such as protein folding and trafficking, cleavage of type-1 membrane proteins,
intracellular adhesion, GSK-3 and Tau metabolism, signal transduction and neuronal
apoptosis (Sato, Imaizumi et al. 2001; Lee, Jung et al. 2002; Marambaud, Shioi et al.
2002; Zhou, Zhang et al. 2002). The important role of both BACE1 and PS1 in the
brain Aβ build-up has made them excellent therapeutic targets. In an attempt to
develop a functional in vivo γ-secretase assay in yeast (manuscript in preparation) we
observed a direct interaction between BACE1 and PS1. Co-immunoprecipitation,
GST-pull down assay and co-localization studies in cultured human cells confirm this
interaction in vivo. Our results demonstrate that PS1 may have a regulatory effect on
BACE1 activity/maturation.
76
METHODS
1) Yeast two-hybrid assay. cDNA sequences encoding human PS11-467 wt and BACE11-501 (gift from Dr. Christian
Haass) were cloned both into pGBKT7 and pGADT7 vectors (Clontech) as a fusion to
the Gal4-DNA binding domain and Gal4-DNA activating domain, respectively. Each
clone was sequenced and shown to be free of autonomous Gal-4 activation. Each of
the pGBKT7-construct (pGBKT7-PS1 or pGBKT7-BACE1) was co-transformed into
AH109 Saccharomyces cerevisiae (Clontech) together with each pGADT7-construct
(pGADT7-PS1 or pGADT7-BACE1). Transformants were selected on yeast drop-out
media (Clontech) that lacked both tryptophan and leucine (TL-) and assayed for
histidine (His +) and adenine (Ade +) reporter activation. Positive control included
pGBKT7-p53/pGADT7-T antigen.
2) Cell culture and transfection.
Human embryonic kidney 293T cells (HEK293T) were cultured in Dulbecco's
modified Eagle's medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine
serum (Biomedia) and Geneticin ® antibiotics (Invitrogen). Stock cultures were
maintained at 37 °C in a humidified atmosphere consisting of 5% CO2, 95% air.
HEK293T cells were transiently transfected using Lipofectamine reagent (Invitrogen)
with cDNAs corresponding to full length human BACE1 and human PS1 wt with or
without myc-tagged at the N-terminus. The empty pcDNA3 vector (Invitrogen) was
used as control.
3) Preparation of cell lysates, immunoprecipitation and immunoblotting.
HEK 293T native and transfected cells were rinsed twice with ice-cold phosphate
buffer saline (PBS) then lysed for 30 min on ice in cell lysis buffer [25 mM HEPES,
150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM PMSF and mini-complete
protease inhibitors (Roche-Diagnostic)]. Insoluble material was removed by
centrifugation at 13,000 X g for 15 min at 4°C. Proteins were quantified using a
standard Bradford assay (Pierce). One mg of total protein was subjected to overnight
immunoprecipitation at 4°C with anti c-myc or HA antibodies. Protein G-plus
Agarose (VWR-Canlab) was subsequently added to the antibody-antigen complex and
77
incubated for 2 h at 4°C. The beads were washed four times with washing buffer
(0,2% Nonidet P-40, 50 mM Tris-HCl (pH 7,6), 150 mM NaCl, and 2 mM EDTA).
Equal amounts of immunoprecipitated proteins were mixed with Novex ® 2X
reducing sample buffer (0.5 M Tris-HCl pH 6.8, glycerol 20%, SDS 10%,
bromophenol blue 0.1% and 5% β-mercaptoethanol), boiled for 3 min, then subjected
to SDS-PAGE. Equal amounts of crude protein lysates (50 µg) were fractionated as
above, however, samples were kept at 37°C for 5 min before loading onto the gel.
After migration, proteins were transferred onto PVDF membranes (Amersham)
according to the manufacturer protocol. Western blots were probed with anti-PS1
antibodies, either Ab-14 (gift from Dr P. Fraser) or PSN2 (gift from Dr H. Mori), and
BACE1 specific antibody (Oncogene). Blots were revealed using chemiluminescence
detection system, ECL+ (Amersham).
4) Production of recombinant proteins and in vitro GST-binding assay.
cDNA encoding BACE1 was subcloned into pGEX4T-1 (Pharmacia) and transformed
into E.coli BL21 strain. BACE1-GST fusion protein was induced 6 h with 1 mM
IPTG and released by sonication in PBS-1% Triton X-100. Bacterial protein extracts
were incubated with 50µl of GST 4B sepharose either in suspension or in a mini-
column (Pharmacia) for 1h at 4°C with continuous rocking. After washing with PBS,
1 mg of protein extracted from non-transfected HEK293T cells were added to
BACE1-GST fusion protein linked to GST 4B sepharose and the mixture was
incubated for 2h at 4°C with continuous rocking. Samples were washed four times
with GST washing buffer (20 mM TRIS, pH 8.0, 100 mM NaCl, 0.5 % NP-40, 0.5
mM EDTA), eluted with 50 µl of GST elution buffer (Pharmacia) and mixed with 50
µl of 2X SDS-PAGE sample buffer (0.5 M Tris-HCl, pH 6.8, glycerol 20%, SDS
10%, DTT 1 mM, bromophenol blue 0.1%). Samples were incubated at 100°C for 3
min and subjected to SDS-PAGE as described above. Western blots were probed
with appropriate antibodies.
5) Immunofluorescence
Native HEK293T cells were grown on glass coverslips to subcellular confluence (i.e.
75-90% confluence). Cells were washed twice in cold PBS, fixed and permeabilized
78
in acetone/methanol (7:3), then incubated for 30 min at -20ºC. After incubation, cells
were rehydrated in PBS at room temperature for 10 min. Nonspecific binding was
blocked with 1% bovine serum albumin. Cells were probed with primary antibody,
either PSN2 or BACE1 hyperimmune serum (Oncogene). Appropriate fluorochrome
FITC- (DAKO) or PE- (Pharmingen) conjugated antibodies were used as secondary
antibodies. Cell nuclei were stained with DAPI (1ug/ml) (Sigma) and visualized using
an inverted fluorescence microscope (Nikon TE300, Nikon Canada) equiped with a
CCD camera (Coolsnap, Roper Scientific)
RESULTS BACE1 and PS1 interact in yeast. In an attempt to develop a functional secretase enzymatic assay in yeast we
demonstrated a direct binding between BACE1 and PS1. This direct interaction was
first analyzed by the yeast two-hybrid interaction trap assay. The yeast two-hybrid
assay is a robust method designed to study direct interaction between two biochemical
partners. For this assay we have constructed a series of yeast expression vectors with
cDNAs representing the open reading frame of PS1, BACE1, APP and lamin C. All
cDNAs were cloned in fusion to either the Gal-4 DNA binding domain (BD) in
pGBKT7 vector or the Gal-4 DNA activating domain (AD) in pGADT7 vector. PS1,
BACE1, and APP expression in yeast was confirmed by western blotting (data not
shown). In yeast, in contrast to mammalian cells, the BACE1 pro-domain is not
processed by furin (Hébert et al., manuscript in preparation). This particularity allows
us to use the BD or AD-BACE1 fusion protein in a yeast two-hybrid assay. Co-
transformation of PS1, BACE1, or APP constructs in yeast AH109 resulted in colonies
that were able to grow on tryptophan and leucine deficient media (-TL). Interaction
between two partners allows colonies to grow on selective media lacking tryptophan,
leucine, histidine, adenine (-TLHA). As shown in figure 1, we observed an interaction
between BACE1 and APP (Fig. 1a) as well as BACE1 and PS1 (Fig. 1c). The latter
interaction was confirmed using both BD-PS1 with AD-BACE1 (Fig. 1c) and BD-
BACE1 with AD-PS1 (Fig. 1d). BACE1-APP interaction was expected since BACE1
has been shown to directly cleave APP in vitro. Interestingly, no interaction was
observed between APP and PS1 using the yeast-2-hybrid assay, which tested for
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direct interaction between 2 non-conserved proteins in yeast (Fig. 1b). This result
suggests that association between PS1 and APP is likely to be indirect. Other groups
have also reported absence of interaction between APP and PS1 (Kim, Choi et al.
1997; Thinakaran, Regard et al. 1998; Ray, Yao et al. 1999). Papers reporting APP-
PS1 interaction have based their results only from co-immunoprecipitation or GST-
pull down methods using total cell protein extracts which are both indirect methods.
Using these indirect methods it cannot be excluded that another protein acts as a
bridge between APP and PS1. Such tripartite APP-nicastrin-PS1 interaction has
already been proposed (Chen, Yu et al. 2001). Absence of direct APP-PS1 interaction
may also reflect purified PS1 inability to process APP-C99 in vitro (G. Levesque,
personal observation) contrarily to BACE, which can process APP in a test tube
(Vassar, Bennett et al. 1999). A more plausible explanation is that PS1 acts as a
regulating co-factor of the γ-secretase complex. In this case, absence of PS1 may not
completely abolish γ-secretase activity as reported recently (Armogida, Petit et al.
2001; Wilson, Doms et al. 2002).
To evaluate the specificity of our yeast two-hybrid assays, we tested our constructs
against a non-specific protein and empty vectors. We found no interaction either
between PS1, BACE1 or APP and Lamin (Fig. 1-e, f, g respectively) or empty vectors
(Fig. 1-j, h, l respectively). These control experiments showed that BACE1 does not
lead to non-specific interactions in a yeast 2-hybrid assay. The two-hybrid assay was
validated using two known biochemical partners, anti-T and p53 (Fig. 1-n).
Altogether, our results demonstrate that interaction between BACE1 and PS1 or APP
is direct and specific and not due to autonomous or non-specific activation of the
yeast reporter genes.
BACE1 and PS1 immunoprecipitation. Although the yeast 2-hybrid assay is a robust method to identify direct protein-protein
interactions, we have undertaken additional experiments designed to determine
whether BACE1-PS1 interaction occurs in mammalian cells. These experiments are
related to immunoprecipitation from double transfected HEK293T cells. Prior to
immunoprecipitation experiments, expression of both 5’myc-tagged PS1 and BACE1
was evaluated in the doubly transfected HEK293T cells. We found that both proteins
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are highly expressed after transfection (Fig. 2A, lane 2 and 2B, lane 2). We also
demonstrated that anti-myc immunoprecipitation is specific for PS1 (Fig. 2A, lane 4).
We then analysed if PS1 can co-immunoprecipitate BACE1. Our results clearly show
that PS1 and BACE1 can be co-immunoprecipitated from lysate of cells doubly
transfected with both 5’myc PS1 and BACE1 (Fig. 2B, lane 4). In contrast, co-
immunoprecipitation did not occur from lysate of empty vector transfected cells (Fig.
2A, lane 3; B, lane 3). Non-specific immunoprecipitation using anti-HA did not pull
down PS1 or BACE1 (Fig. 2A, lane 5; B, lane 5). This anti-HA immunoprecipitation
is used as a negative control to show that PS1 nor BACE1 bind non-specifically to the
protein G-plus agarose. To demonstrate the specificity of PS1-BACE1 interaction, we
designed another set of experiments in which we used anti-BACE1 to co-
immunoprecipitate non-tagged PS1. We found that PS1 can be co-
immunoprecipitated with anti-BACE1 antibodies (Fig. 2C, lane 2). Moreover, the PS1
immunoreactive band could be abolished by preabsorption of the BACE1 antibody
with the cognate peptide (Fig. 2C, lane 3). Cumulatively, these experiments showed
that BACE1 and PS1 can be reciprocally co-immunoprecipitated in mammalian cells.
These results also confirmed their in vivo interaction.
BACE1 and PS1 GST-pull down. To exclude the remote possibility that BACE1 and PS1 interaction observed in both
yeast two-hybrid and co-immunoprecipitation studies is an artifact arising from
protein overexpression we used a GST-pull down assay to confirm BACE1
interaction with endogenous PS1 protein. Recombinant BACE1-GST fusion was
produced in E. coli BL21 strain. Expression of GST alone or BACE1-GST fusion
protein was confirmed by acrylamide gel electrophoresis after Coomassie blue
coloration (Fig. 2E, lane 1 and 2 respectively). Consistent with the yeast two-hybrid
and co-immunoprecipitation results, endogenous PS1 was pulled down with GST-
BACE1 immobilized on glutathione sepharose using either suspension or mini-
column methods (Fig. 2D, lane 2). In contrast, no PS1 was pulled down in the GST
alone fraction (Fig. 2D, lane 1). Interestingly, we observed that endogenous PS1 N-
terminal fragments could be pulled down by BACE1 (Fig. 2D, lane 2). These results
suggest that BACE1 interaction with PS1 may occur via the N-ternimal end of PS1.
Fine mapping of BACE1-PS1 interaction sites is ongoing in our laboratory.
81
BACE1 and PS1 co-localize in the cell. To further confirm BACE1- PS1 interaction results obtained from yeast two-hybrid
and immunoprecipitation experiments we studied the cellular localization of these two
biochemical partners. We demonstrate that both proteins are expressed in the same
adjacent/contiguous subcellular compartments. In non-transfected cells,
immunofluorescence study shows that PS1 and BACE1 have overlapping intracellular
distributions. Thus, PS1 has a predominantly ER/Golgi and plasma membrane
distribution (as previously demonstrated) contiguous with that of BACE1 (Fig. 3 A-
C). Co-localization of BACE1 and PS1 suggests that PS1 may regulate the maturation
or transport of BACE1 in the secretory pathway. Also, their presence at the plasma
membrane correlates with their role in APP cleavage.
DISCUSSION
Alzheimer disease is caracterized by Aβ-peptides accumulation and deposition in the
brain. The role of Aβ as a trigger of neuronal death is not clear but the amyloid
cascade hypothesis is actually very attractive and there are strong arguments
supporting this (Sinha 2002). The formation of Aβ requires the activity of both β-
secretase (BACE1) and γ-secretase complex which includes PS1. These two enzymes
are extremely important as therapeutic targets. However, little is clearly known about
their physiological functions or the biological regulation of their activities.
In this paper, we have clearly demonstrated using 3 different approaches that BACE1
and PS1 interact directly. Our co-localization study support previous findings in
neurons where BACE1, APP and PS1 were shown to be in the same cellular
membrane compartments (Kamal, Almenar-Queralt et al. 2001). The physiological
role of BACE1-PS1 interaction is not clear. By binding directly to each other, they
may contribute either mutually or sequentially to Aβ42 production. Our BACE1 and
PS1 co-immunoprecipitation studies have demonstrated that PS1 binds preferentially
to the glycosylated immature BACE1. This observation raises the possibility that PS1
is implicated in the proper maturation of BACE1. PS1 modulation of BACE1 activity
may explain the following observations about the PS1functional role in APP
82
processing. Firstly, it has been shown by Yu et al. (Yu, Nishimura et al. 2000) that
deletion of nicastrin (a PS1 partner in the γ-secretase complex) DYIGS conserved
domain reduces Aβ production probably by destabilization of the presenilin-nicastrin
interaction in the γ-secretase complex. Surprisingly, this Aβ reduction is not
associated with the expected increased level of APP C99, resulting from BACE1
cleavage. In this case, it seems that not only the γ-secretase activity is reduced but also
the β-secretase. Destabilization of PS1 may have an effect on BACE1 activity.
Secondly, it has been reported that mice lacking both presenilin genes, PS1 and PS2
accumulate APP C83, resulting from an α-secretase cleavage, with almost no
detectable APP C99 (Zhang, Nadeau et al. 2000). Again, this observation suggests
that presenilins may regulate BACE1 activity. Thirdly, Armogida et al. (Armogida,
Petit et al. 2001) have reported that mouse fibroblasts deficient in both presenilin 1
and 2 accumulate an endogenous APP C-terminal fragment not labeled by a specific
antibody, FCA18 , which recognizes only genuine β-secretase-derived peptides. As
discussed by the authors, this C-terminal fragment probably corresponds to APP C83.
Finally, Beher et al. (Beher, Wrigley et al. 2002) have recently demonstrated that the
production of all truncated amyloid-beta peptides, except those released by the action
of the nonamyloidogenic alpha-secretase enzyme, depends on gamma-secretase
activity. These authors suggested that none of these peptides are generated by a
separate enzyme entity and a specific inhibitor of the gamma-secretase enzyme should
have the potential to block the generation of all amyloidogenic-peptides. Our results
also support the hypothesis that both BACE1 and PS1 are working together in the
amyloid cascade.
An argument against PS1 role in modulation of BACE1 activity may be the fact that
purified BACE1 can cleave APP in a test tube in the absence of PS1. However, in
vitro testing of BACE1 activity does not reflect multiple in vivo cellular processes
leading to protein maturation and localization. We argue that PS1 probably has a role
in BACE1 transport/trafficking to ensure its maturation and precise cell compartment
localization.
83
During the preparation of this manuscript, a report by Hattori et al. (Hattori, Asai et
al. 2002) described an interaction between BACE1 and nicastrin. In their work, they
use co-immunoprecipitation methods to demonstrate the interaction. In this case, it is
possible that BACE1 and nicastrin does not bind directly to each other and PS1 is
probably the linker. Consistent with this finding, our results from yeast experiments
clearly demonstrated a direct interaction between BACE1 and PS1. The physiological
significance of the BACE1-PS1 interaction is actually being explored as well as the
effects of PS1 mutations on this interaction and on BACE1 activity. Interestingly, PS1
mutations in AD brain were shown to affect not only γ-secretase activity but β-
secretase as well (Russo, Schettini et al. 2000). It is well documented that PS1
mutations increase Aβ42 production (Citron, Westaway et al. 1997; Citron, Eckman
et al. 1998; Petanceska, Seeger et al. 2000). Since the γ-secretase substrate leading to
Aβ42 production is produced following BACE1 cleavage, modulation of BACE1
activity may also modulate the availability of the γ-secretase substrate. One
observation supporting this hypothesis is that the APP Swedish mutation leads both to
an increase of BACE1 cleavage and Aβ42 production (Sinha and Lieberburg 1999)
(Steinhilb, Turner et al. 2002). It does not seem that the γ-secretase activity is the
limiting factor in this enzymatic process.
Taken together, our results suggest that PS1 through a direct binding may be
implicated in the control of BACE1 activity. Our findings are also in agreement with
a PS1 role in the ER-Golgi transport/maturation of BACE1. While the functional
implications of this interaction are not immediately apparent, it is of note that these
two proteins are working together during Aβ formation. Nevertheless, more work is
needed to clarify the functional role of this interaction.
ACKNOWLEDGMENTS
This work was supported by grants from Canadian Institutes of Health Research
(IRSC) and Fonds de la Recherche en Santé du Québec (FRSQ).
84
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FIGURE LEGENDS
FIG 1. Two-hybrid assay demonstrating BACE-PS1 interaction. Representative Saccharomyces cerevisiae AH109 strain colony growth. Positive co-
transformation with both pGBKT7 and pGADT7 constructs was confirmed by colony
growth on TL- . Activation of His and Ade reporter genes was used to verify
interaction between two proteins. Colony formation after 4 days of incubation on
SD/TLHA- was considered as positive interaction between fusion partners. Absence
of colony after 4 days was considered as an absence of interaction. However, plates
with negative results were incubated 10 days to confirm absence of growth. Each
hybrid assay was performed at least 3 times.
FIG 2. In vivo immunoprecipitation of BACE with PS1. Immunoprecipitation and GST pull down assays confirm that BACE and PS1 interact
in vivo. A: detection of 5’myc-tagged PS1 following immunoprecipitation (IP) with
anti-myc(9E10.2). Myc-tagged PS1 can be detected in IP products from double
transfected cells (lane 4) but not in mock transfected cells (lane 3) or in non-specific
anti-HA immunoprecipitated products (lane 5). Lane 1 and 2 are lysates from mock
and double transfected cells, respectively. Western blots were probed with anti-PS1,
PSN2. B: detection of BACE in co-immunoprecipitates following IP with anti-myc
antibody. BACE can be detected in IP products from double transfected cells (lane 4)
but not in mock transfected cells (lane 3) or in non-specific anti-HA
immunoprecipitated products (lane 5). Lane 1 and 2 are lysates from mock and double
transfected cells, respectively. Western blots were probed with anti-BACE (Ab-1). C:
detection of PS1 in co-immunoprecipitates following IP with anti-BACE antibody.
PS1 can be detected in IP products from double transfected cells (lane 2) but not in
mock transfected cells (lane 1). PS1 cannot be immunoprecipitated with anti-BACE in
the presence of competitive BACE cognate peptide (lane 3). Western blots were probe
with PSN2. D: Western blot analysis of endogenous PS1 in GST-BACE precipitates
following either column or suspension GST chromatographic extraction. PS1 N-
terminal fragment from native HEK293T lysate can be detected as coeluants with
BACE using anti-PS1 antibody, Ab-14 (lane 2). Similar results were obtained using
88
anti-PS1 antibody, PSN2 (data not shown). PS1 does not coelute in extracts from GST
only fractions (lane 1). E: Coomassie gel staining showing GST or GST-BACE
fusion protein expression level (lane 1 and 2 respectively).
FIG 3. Intracellular localization of endogenous BACE and PS1. Native HEK293T cells were labeled with appropriate FITC- or PE- conjugated
fluorochrome and observed by fluorescence microscopy. Panel A: labeled with PS1
antibody PSN2 and FITC-conjugated secondary antibody. Panel B: labeled with
BACE antibody Ab-1 and PE-conjugated secondary antibody. Panel C: Merging
images showing co-localization (yellow) predominantly in the ER/Golgi structures
and at the plasma membrane. Panel D: cell nuclei stained with DAPI. Panel E:
control stained with PE-conjugated secondary antibody alone. Panel F: control
stained with FITC-conjugated secondary antibody alone.
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90
91
CHAPITRE VI
La forme immature de BACE1 régule le métabolisme de
l’holoprotéine PS1
Note : Ce chapitre, présenté sous la forme d’un manuscrit prêt à la soumission pour une revue scientifique, résume la suite des travaux effectués dans le cadre de mon projet de doctorat. Plus spécifiquement, ce travail visait à compléter mon deuxième objectif de travail (décrit dans le chapitre 2 à la page 24). Comme ma contribution dans la conception (approches expérimentales), la réalisation (constructions plasmidiques et manipulations) et la rédaction (incluant le montage des figures) de ce travail est majoritaire, mon nom apparaît comme premier auteur sur le manuscrit.
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Résumé
Les activités β- et γ-secrétases sont responsables du clivage de la protéine précurseur
AβPP et par conséquent la production des peptides amyloïdes. Dans le cerveau des
patients atteints de la maladie d’Alzheimer, ces peptides se déposent en plaques et
sont considérés par plusieurs la principale cause de démence. La β-secrétase est une
enzyme de type aspartyl protéase, la « β-site AβPP cleaving enzyme » (BACE).
Préséniline-1 (PS1), une protéine multi-transmembranaire de type serpentine, semble
constituée la portion catalytique da la γ-secrétase. Nous avons récemment démontré
une interaction directe entre BACE et PS1, suggérant une collaboration intime dans la
production des peptides amyloïdes. Dans cette étude, nous démontrons, dans un
premier temps, une interaction entre la forme immature de BACE et l’holoprotéine
PS1, et ce, à un niveau endogène dans les cellules HEK293T. Ensuite, il a été
possible de démontrer une interaction entre la BACE et différents mutants PS1
associés à une forme familiale de la maladie d’Alzheimer. Des résultats préliminaires
suggèrent que la BACE régule l’endoprotéolyse de la PS1 in vivo. L’effet de BACE
sur PS1 est indépendant de l’activité γ-secrétase et celle du proteasome. En
conclusion, l’ensemble des résultats suggèrent que BACE joue un rôle dans la
régulation du métabolisme de la PS1 et possiblement de l’activité γ-secrétase.
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BACE1 immature species regulate PS1 holoprotein levels
Sébastien S. Hébert1 and Georges Lévesque1, 2
1Molecular and Human Genetics Unit, CHUQ-Pavillon St-François d'Assise, 10 rue
de l'Espinay, Québec, Canada G1L 3L5, 2 Medical Biology Department, Laval
University, Quebec, Canada, G1K 7P4.
Address correspondence to: Dr. Georges Lévesque, Molecular and Human Genetics
Unit, CHUQ-Pavillon St-François d'Assise, 10 rue de l' Espinay, Québec, Canada
G1L 3L5.
Tel: 418-525-4444 ext 3752
Fax: 418-525-4195
Email: [email protected]
Running title: BACE1 regulates PS1 holoprotein levels
Keywords: β-site APP cleaving enzyme (BACE), presenilin, amyloid, γ-secretase, β-
secretase, Alzheimer disease.
ABSTRACT
Both β- and γ- secretases cleave amyloid beta precursor protein (APP) to produce β-
amyloid peptides. In Alzheimer's disease brain, these peptides are deposited in
amyloid plaques and considered by many as the principal cause of dementia. The
identity of β-secretase has been shown to be the transmembrane aspartic protease,
beta-site APP cleaving enzyme 1 (BACE1). Presenilin-1 (PS1), a polytopic multi-
transmembrane protein, appears to constitute the catalytic component of γ-secretase.
We have recently demonstrated an interaction between BACE1 and wild-type PS-1
thus suggesting a direct, intimate collaboration in β-amyloid peptide production. We
now report that immature BACE1 preferentially interacts with full-length
95
(holoprotein) PS1 at an endogenous level. Various AD and non-AD associated PS1
mutations do not abrogate binding with BACE1. Preliminary data suggest that
immature BACE1 protein levels regulate PS1 endoproteolysis. In conclusion, our
results suggest that BACE1 binding to PS1 may lead to altered β-amyloid production
(by reciprocal co-modulation of β- and γ-secretases).
INTRODUCTION
The main pathological hallmark of Alzheimer’s disease (AD) is the presence of
amyloid plaques in the brain. These plaques are mainly composed of short N- and C-
terminally truncated peptides derived from the amyloid beta precursor protein (APP),
a type 1 transmembrane protein with uncertain function (Van Gassen and Annaert
2003). Aβ42, a 42 amino-acid-long cleavage product of APP, is the principle peptide
found in plaques. Aβ42 is “carved” from APP by two coordinated cuts by β- and γ-
secretases (Selkoe 2001). β-secretase has shown to be the type 1 transmembrane
aspartic proteases, named beta-site APP cleaving enzyme 1 (BACE1, also known as
Asp-2 or memapsin 1) (Vassar, Bennett et al. 1999; Vassar 2002). APP cleavage by
BACE1 generates a membrane-tethered 99 amino-acid NH2-terminal fragment
(namely C99). Either subsequently or mutually, C99 is cleaved by γ-secretase within
its transmembrane domain to generate multiple-length (Aβ38, Aβ39, Aβ40, Aβ42,
Aβ43 and Aβ49) peptides (Tekirian 2001). The most abundant species found in vivo
are Aβ40 and Aβ42, Aβ42 being the more amyloidogenic form (Ling, Morgan et al.
2003). Whether this process is a normal, physiological task is not clear. Until now,
only sialyltransferase ST6Gal I, Phospholipid Scramblase I are other known BACE1
partners (Kitazume, Tachida et al. 2001; Kametaka, Shibata et al. 2003). γ-Secretase
activity, on the other hand, is related to a matured, multi-protein complex containing
presenilin-1 (PS1), Nicastrin, Aph-1 and Pen-2 (De Strooper 2003). Also, γ-secretase
action is not restricted to APP, as multiple substrates, such as Notch, Erb-B4,
Syndecan, CD44, E-Cadherin and Ire-1, are cleaved by this unusual “enzyme” (De
Strooper 2003). This proteolytic mechanism (i.e. transmembrane cleavage) is highly
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related to the intramembrane-cleaving proteases’ (I-CLIPs) family, including, SPP,
SP2 and Rhomboid (Xia and Wolfe 2003).
BACE1 is expressed as a 501 amino-acid protein and undergoes multiple post-
translational modifications, including pro-peptide cleavage, phosphorylation,
disulphide bridge formation, palmitoylation and complex N-glycosylation (Dingwall
2001; Vassar 2001). These modifications occur throughout the secretory pathway and
have differential effects on BACE1 maturation. The mature enzyme starts at position
Glu46 and presents complex (Endo-H resistant) N-glycosylation (Bennett, Denis et al.
2000). Ultimately, BACE1 is found in the early endosomal compartment and on the
cell surface. Until now, no genetic linkage, pathogenic mutations or allelic
associations between BACE1 and familial or sporadic AD cases have been reported
(Cruts, Dermaut et al. 2001; Nicolaou, Song et al. 2001). However, three independent
groups have recently observed higher BACE1 protein levels and activity in sporadic
AD cases (Fukumoto, Cheung et al. 2002; Holsinger, McLean et al. 2002; Tyler,
Dawbarn et al. 2002). Interestingly, BACE1 overexpression in cells does increase
C99 and Aβ42 secretion, thus suggesting that β-secretase is the limiting factor in
Aβ42 generation. Inversely, ablation of the gene encoding BACE1 completely
impairs β-secretase cleavage of APP and abolishes the generation of Aβ (Vassar,
Bennett et al. 1999; Roberds, Anderson et al. 2001).
PS1, a polytopic multi-transmembrane protein, is expressed as an approximately 45
kDa protein and undergoes rapid endoproteolysis within its principal cytoplasmic loop
(located between transmembrane domains 6 and 7) to generate N-terminal and C-
terminal fragments of approximately 28 kDa and 18 kDa in size, respectively (Tandon
and Fraser 2002). Fragment levels are highly regulated in vivo so that overexpression
of PS1 barely increases observed fragment levels, and instead leads to an
accumulation of full-length (holoprotein) protein levels. This phenomenon is not
clearly understood, but seems to be regulated in part by Pen-2 and/or Aph-1 (Lee,
Shah et al. 2002; Gu, Chen et al. 2003; Luo, Wang et al. 2003). Interestingly, PS1 N-
and C-terminal fragments (NTF and CTF, respectively) are considered by many as the
catalytic derivative of PS1 and active component of the γ-secretase complex (Wolfe
2003). Mutations of PS1 lead to the most aggressive forms of early-onset familial AD
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(Selkoe 2001). All PS1 mutations – more than one hundred have been reported –, are
linked with increased γ-secretase activity as observed indirectly by measuring Aβ42
secretion levels. Wild-type PS1 overexpression alone does not increase Aβ42
production. However, mutant presenilins do increase Aβ42 production without
increasing NTF and CTF fragment levels, thus implying a “gain of function” or
“modifying” effect (Citron, Westaway et al. 1997).
Aβ42 production plausibly involves co-regulation of both β- and γ-secretases. We
have recently demonstrated a direct interaction between ectopically expressed BACE1
and wild-type (wt) PS1 in yeast and mammalian cells (Hébert, Bourdages et al. 2003).
We now demonstrate that immature BACE1 preferentially interacts with full-length
(holoprotein) PS1 at an endogenous level. This binding is neither abrogated nor
accentuated by various AD-associated (H115Y, L392V) mutants, dominant negative
(D257A, D385A) γ-secretase mutants or the polymorphic (E318G) PS1 mutant. Our
preliminary data suggest that immature BACE1 protein levels regulate PS1
endoproteolysis. This downregulation effect on PS1 was abolished with the D257A
and D385A dominant negative mutants in PS1 -/- fibroblast cells. These results
suggest that BACE1 binding to PS1 may lead to altered PS1 metabolism and,
possibly, altered β-amyloid production.
EXPERIMENTAL PROCEDURES
1) Cell culture and transfection.
Human embryonic kidney 293T (HEK293T), murine neuroblastoma 2A (N2A), PS1
wild-type (+/+) and PS1 knockout (-/-) fibroblast cells were cultured in Dulbecco's
modified Eagle's medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine
serum (Biomedia) and Geneticin ® antibiotics (Invitrogen). Stock cultures were
maintained at 37 °C in a humidified atmosphere consisting of 5% CO2, 95% air.
HEK293T and PS1 -/- cells were transiently transfected using Lipofectamine reagent
(Invitrogen) with cDNAs corresponding to full length human BACE1, human PS1 and
human HES-1. The empty pcDNA3 vector (Invitrogen) was used as control (mock).
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2) Preparation of cell lysates, immunoprecipitation and immunoblotting.
Native and transfected cells were rinsed twice with ice-cold phosphate buffer saline
(PBS) then lysed for 30 min on ice in cell lysis buffer [25 mM HEPES, 150 mM
NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM PMSF and mini-complete protease
inhibitors (Roche-Diagnostic)]. Insoluble material was removed by centrifugation at
13,000 X g for 15 min at 4°C. Proteins were quantified using a standard Bradford
assay (Pierce). One mg of total protein was subjected to overnight
immunoprecipitation at 4°C with appropriate antibodies. Protein G-plus Agarose
(VWR-Canlab) was subsequently added to the antibody-antigen complex and
incubated for 2 h at 4°C. The beads were washed four times with washing buffer
(0,2% Nonidet P-40, 50 mM Tris-HCl (pH 7,6), 150 mM NaCl, and 2 mM EDTA).
Equal amounts of immunoprecipitated proteins were mixed with Novex ® 2X
reducing sample buffer (0.5 M Tris-HCl pH 6.8, glycerol 20%, SDS 10%,
bromophenol blue 0.1% and 5% β-mercaptoethanol), boiled for 3 min, then subjected
to SDS-PAGE. Equal amounts of crude protein lysates (50 µg) were fractionated as
above, however, PS1 samples were kept at 37°C for 5 min before loading onto the
gel. After migration, proteins were transferred onto PVDF membranes (Amersham)
according to the manufacturer protocol. Western blots were probed with anti-PS1
antibodies, either Ab-14 (gift from Dr P. Fraser), N-19 (Oncogene) or PSN2 (gift
from Dr H. Mori), and BACE1 specific antibodies, either Ab-1 (a.a. 45-60)
(Oncogene), BACE C-terminal (a.a. 485-501) (Oncogene) or anti-V5 (Sigma) (in the
case of BACE1-V5), and anti-HA (in the case of HES-1-HA). Blots were revealed
using chemiluminescence detection system, ECL+ (Amersham).
3) Yeast two-hybrid assays.
cDNA sequences encoding various parts of human PS1 were amplified by PCR using
the Herculase Taq polymerase (Stratagene). The following wt PS1 cDNAs were
prepared: NTF (PS11-291), Loop 6-7 (PS1239-409) and CTF (PS1292-467). Wild-type PS1
and BACE1(1-501) (gift from Dr. Christian Haass) cDNAs were cloned both into
pGBKT7 and pGADT7 vectors (Clontech) as a fusion to the Gal4-DNA binding
domain (BD) and Gal4-DNA activating domain (AD), respectively. Each construct
was sequenced and shown to be free of autonomous Gal-4 activation. Functional
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expression of fusion proteins in yeast was confirmed by Western blot. Each of the
pGBKT7-construcst (pGBKT7-PS1 NTF, pGBKT7-CTF or pGBKT7-Loop 6-7) were
co-transformed into AH109 Saccharomyces cerevisiae (Clontech) together with each
pGADT7-constructs (pGADT7-BACE1, pGADT7-Lamin or pGADT7-hNPRAP).
Transformants were selected on yeast drop-out media (Clontech) that lacked both
tryptophan and leucine (TL-) (not shown) and assayed for histidine (His +) and
adenine (Ade +) reporter activation. Positive control included pGBKT7-PS1 Loop 6-
7/pGADT7-hNPRAP and pGBKT7-p53/pGADT7-T antigen (not shown).
4) Production of recombinant proteins and in vitro GST-binding assay.
cDNA encoding human BACE1 was subcloned into pGEX4T-1 (Pharmacia) and
transformed into E.coli BL21 strain. BACE1-GST fusion protein was induced 6 h
with 1 mM IPTG and released by sonication in PBS-1% Triton X-100. Bacterial
protein extracts were incubated with 50µl of GST 4B sepharose either in suspension
or in a mini-column (Pharmacia) for 1h at 4°C with continuous rocking. After
washing with PBS, 1 mg of protein extracted from PS1 wt, PS1 L392V and PS1
D257A-transfected HEK293T cells were added to BACE1-GST fusion protein linked
to GST 4B sepharose and the mixture was incubated for 2h at 4°C with continuous
rocking. Samples were washed four times with GST washing buffer (20 mM TRIS,
pH 8.0, 100 mM NaCl, 0.5 % NP-40, 0.5 mM EDTA), eluted with 50 µl of GST
elution buffer (Pharmacia) and mixed with 50 µl of 2X SDS-PAGE sample buffer
(0.5 M Tris-HCl, pH 6.8, glycerol 20%, SDS 10%, DTT 1 mM, bromophenol blue
0.1%). Samples were incubated at 100°C for 3 min and subjected to SDS-PAGE as
described above. Western blots were probed with anti-PSN2 (PS1).
5) Endoglycosidase H digestions.
50 µg of crude HEK293T cell protein extracts were treated with 0.5 units/ml of
ENDO H (Sigma) as specified by manufactures’ protocol. Controls were treated with
H2O. After overnight incubation at 37°C, protein samples were mixed with Novex ®
2X reducing sample buffer (0.5 M Tris-HCl pH 6.8, glycerol 20%, SDS 10%,
bromophenol blue 0.1% and 5% β-mercaptoethanol), boiled for 3 min, then subjected
to SDS-PAGE. After migration, proteins were transferred onto PVDF membranes
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(Amersham) according to the manufacturer protocol. Western blots were probed with
the anti-BACE1 Ab-1 antibody. Blots were revealed using chemiluminescence
detection system, ECL+ (Amersham).
RESULTS AND DISCUSSION
Immature BACE1 binds full-length (holoprotein) PS1 at an endogenous level.
We have reported previously (Hébert, Bourdages et al. 2003) that in cells
overexpressing both wild-type presenilin-1 (PS1) and BACE1, each protein can be
reciprocally co-immunoprecipitated. To discern whether endogenous PS1 interacts
directly with BACE1, reciprocal co-immunoprecipitation studies were performed in
native HEK293T cells. Immunoprecipitation with pre-immune serum (PIS) failed to
bring down PS1 or BACE1 (Fig. 1Aa, lane 1). Curiously, Ab-14 antibody, which
preferentially recognizes PS1 N-terminal fragments (Fig. 1Aa, lane 3 and (Yu, Chen
et al. 1998)), do not immunoprecipitate BACE1. Additionally, PS1 C-terminal
antibody α-loop (Yu, Chen et al. 1998), which preferentially recognise ~18 kDa C-
terminal fragments, did not pull down BACE1 (data not shown). Only after extensive
film overexposure did we observe N-terminal fragments bound to BACE1 (which
were stoichiometry much less than in lane 2 [i.e. PS1 full-length vs. NTF ratio]) (not
shown). However, immunoprecipitation using N-19 antibody, which recognizes PS1
holoprotein at an endogenous level (Hébert, Godin et al. 2003), conjointly pull down
full-length PS1 (Fig 1Aa, lane 2, upper panel) and immature BACE1 (Fig 1Aa, lane 2,
middle panel). Inversely, BACE C-terminal antibodies, but not PIS, did bring down
endogenous full-length PS1 when probed with N-19 (Fig 1Ab, lane 2). Of particular
importance, co-immunoprecipitation of partners at an endogenous level could not be
observed in Triton X-100 extraction buffer (data not shown) (only in 0.5-1%
CHAPS).
These results led us to consider that immature BACE1 may preferably bind to full-
length PS1. To confirm this hypothesis, crude fine mapping of BACE1-PS1 binding
was assessed by yeast-two hybrid. We have previously demonstrated direct binding
between full-length PS1 and BACE1 by two-hybrid (Hébert, Bourdages et al. 2003).
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As shown in Fig 1B, PS1 loop 6-7, which contains the “intact” PS1 endoproteolytic
cleavage site, preferentially binds DNA-BD-BACE1 fusion protein. Western blot
analysis of yeast protein extracts expressing DNA-BD-BACE1 confirmed the
“immature” state (i.e. zymogen) of BACE1 in yeast (Hébert S.S., personal
observation). Taken together, these results suggest that the non-cleaved PS1 species
(i.e. the ‘intact’ holoprotein) preferentially binds immature BACE1 at an endogenous
level.
PS1 γ-secretase mutants bind BACE1.
All PS1 AD-associated mutations elevate γ-secretase activity measured indirectly by
enhanced Aβ42 secretion (Borchelt, Thinakaran et al. 1996; Qian, Jiang et al. 1998).
Since β-secretase cleavage is a prerequisite step in Aβ42 generation, it is tempting to
speculate that PS1 mutants also regulate β-secretase activity. Interestingly, two recent
papers have reported γ-secretase-dependent modulation of BACE1 activity (Fluhrer,
Multhaup et al. 2003; Shi, Tugusheva et al. 2003). How PS1 influences BACE1
activity is actually not known. We tested if various PS1 mutations, as compared to
wild-type PS1, could modulate BACE1 binding. First, we tested if exogenously
expressed PS1 variants could bind GST-tagged BACE1. As shown in figure 2A,
upper panel, full-length PS1 wild-type, AD-associated L392V and D257A γ-secretase
dead mutants bound GST-BACE1. Western blot analysis of crude protein extracts
confirmed PS1 expression (Fig 2A, middle panel). Correct GST-BACE1 fusion
protein expression and purification is visualized by Coomassie blue staining (Fig 2A,
lower panel) of “bait” protein extracts. The ~75 kDa molecular mass of GST-BACE1
corresponds to the calculated molecular mass of the non-modified (non-glycolysated)
~50 kDa BACE1 molecule tagged in fusion to the ~25 kDa GST protein.
These initial experiments prompted us to confirm our observations in mammalian
cells. For this, we used HEK293T cells exogenously expressing both non-tagged PS1
and BACE1-V5. Western blot analysis of anti-V5 immunoprecipitates confirmed
specific BACE1-V5 immunoprecipitations (Fig. 2B, upper panel). In parallel, anti-V5
immunoprecipitations could immonoprecipitate PS1 AD-associated L392V, H115Y
and ∆EX10 mutants (middle panel, lanes 3-5), as well as D257A and D385A γ-
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secretase negative mutants (middle panel, lanes 6 and 7). Furthermore, the PS1 AD
polymorphic E318G mutant (Aldudo, Bullido et al. 1998) also binds BACE1-V5 (Fig.
2B, upper panel, lane 8). Protein expression levels for BACE1-V5 in crude cell
lysates were verified by Western blot (data not shown), as well as for PS1 (Fig. 2B,
lower panel, lanes 1-8). Similar co-immunoprecipitation results were observed in
mouse neuroblastoma N2A cells (data not shown). Cumulatively, these results
suggest that BACE1-PS1 binding is independent of γ-secretase-related mutations, and
that all PS1 mutants tested herein can equally bind to immature BACE1.
Immature BACE1 regulates PS1 holoprotein levels.
PS1 mutations account for the majority of early-onset familial AD cases. Whether
PS1 mutations affect fragment protein levels is still a matter of debate (Thinakaran,
Borchelt et al. 1996; Lee, Borchelt et al. 1997; Okochi, Ishii et al. 1997). However,
three independent studies have reported increased BACE1 levels in sporadic AD
cases. Hence, it is possible that deregulation of BACE1 and/or PS1 protein levels is a
prerequisite and/or necessary step in AD development. To further characterize
BACE1-PS1 interaction, we tested if these partners could influence each other’s
expression and/or maturation. First, we tested if BACE1 maturation was affected in
PS1 knock-out (PS1 -/-) fibroblast cells. These γ-secretase deficient cells are known
to dramatically down-regulate and inhibit γ-secretase complex formation (reviewed in
(De Strooper 2003)). As shown in figure 3A, lane 4, upper panel, BACE1 maturation
and protein levels were not substantially affected in PS1 -/- cells. Semi-quantitative
RT-PCR of endogenous mouse BACE1 transcripts in PS1 -/- cells, as compared to
PS1 +/+ and N2A cells, were also not affected (not shown). Western blot confirmed
absence of endogenous PS1 in kock-out cells (Fig 3A, lower panel).
To confirm that BACE1 maturation was independent of PS1 (and γ-secretese
activity), we measured both immature and mature BACE1 proteins levels in
HEK293T cells co-transfected with BACE1 and the PS1 γ-secretase dominant
negative mutant D385A. As shown in Fig 3B, lane 3, the D385A mutant did not
substantially affect BACE1 expression and maturation (as observed by comparing the
immature vs. mature protein levels ratio). This observation could be more easily
visualised after ENDO H treatment of cell extracts (Fig 3B, lanes 4-6), which
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generates a lower BACE1 immature species by releasing non-complex sugars (Capell,
Steiner et al. 2000). In summary, these results suggest that BACE1 post-translational
modifications (i.e. maturation) are independent of PS1 protein levels and γ-secretase
activity.
We next verified if increased BACE1 protein levels could influence PS1 maturation
and/or protein levels in HEK293T cells. Surprisingly, when BACE1 is co-expressed
with PS1, the latter is drastically down-regulated (Fig 3C, lane 2). This down-
regulation was specific for PS1 holoprotein, as NTF levels (and CTF, data not shown)
weren’t significantly altered (Fig 3D, lanes 3-6). These observations were
reproducible in N2A cells (data not shown).
BACE1 expression levels have previously been associated with “differential” β-
secretase activity. Indeed, high BACE1 expression levels favour C89 over C99-like
APP cleavage (Liu, Doms et al. 2002). In order to verify if the effect on PS1 was an
artefact due to high BACE1 expression levels, we conducted a BACE1 dose-
dependent kinetic experiment. As shown in figure 3D, middle panel, PS1 holoprotein
down-regulation was dependent on BACE1 expression levels. This result was
specific for BACE1, as an ectopically co-expressed control protein, HES-1 (Fig 3D,
lower panel), full-length wild-type AβPP (data not shown), or empty vector (data not
shown) did not significantly affect PS1 protein levels. Surprisingly, mature BACE1
protein levels saturate while immature species accumulate in a dose-dependent
manner (Fig 3D, upper panel).
To further confirm if BACE immature species could regulate PS1 holoprotein levels,
we generated a pro-domain deletion mutant (artificial “mature” version) of BACE1
(∆pro-BACE) tagged at its N-terminal region with an HA epitope. This construct has
perturbed maturation properties as assessed by Western blot (as observed by
deficiency of “immature” [i.e. zymogene] Endo-H-sensitive BACE1, Hébert S.S.,
personal observation and [(Benjannet, Elagoz et al. 2001)]) and cellular trafficking.
In corroboration with our previous observations, ∆pro-BACE has attenuated effect on
PS1 holoprotein down-regulation (Fig 3E, lane 3). Cumulatively, these results
suggest that BACE1 immature species regulate PS1 holoprotein metabolism.
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BACE1 may regulate PS1 endoproteolysis.
We next tested if the AD-associated PS1 L392V and ∆EX10 mutants were also
susceptible to BACE1-dependent down-regulation. As shown earlier, both of these
mutants can bind BACE1. As shown in figure 4A, lane 4, and Fig 5, PS1 L392V was
equally down-regulated (when compared to wild-type PS1) when co-expressed with
BACE1 in HEK293T cells. Curiously, the EX10 mutant seemed significantly less
affected by BACE1 co-expression (Fig 4A, lane 6, and Fig 5). Again, similar results
were observed in N2A cells (data not shown). These results suggest that PS1 AD-
associated mutants are, a priori, similarly prone to BACE1-dependent down-
regulation. However, it seems that the EX10 ∆mutant, who lacks the PS1
endoproteolytic site, is slightly less prone to BACE1-dependent down-regulation in
HEK293T cells.
Although other cellular mechanisms have been shown to regulate PS1 holoprotein
levels (e.g. caspases, proteasome), probably the most biologically relevant to PS1
metabolism is its endoproteolysis. To further explore if BACE1 could directly
influence PS1 endoproteolysis, we tested if the γ-secretase dominant negative PS1
D385A mutant was also subject to BACE1-dependent down-regulation. The PS1
artificial D385A aspartate mutation was previously shown to inhibit PS1
endoproteolysis (Wolfe, Xia et al. 1999; Berezovska, Jack et al. 2000) in vivo.
Surprisingly, PS1 D385A was less prone to BACE1 down-regulation. Indeed, only
55% of PS1 D385A protein levels, as compared to 85% of PS1 wild-type and L392V
protein levels were affected by BACE1 overexpression in HEK293T cells (Fig. 4B
and C). These experiments, which were repeated four times, suggest that BACE1
regulates PS1 endoproteolysis.
To date, PS1 endoproteolysis was shown to be in part regulated by γ-secretase activity
(Beher, Wrigley et al. 2001; Campbell, Iskandar et al. 2002). However, significant
PS1 endoproteolysis is observed in PS1-transfected γ-secretase deficient PS1 -/- cells
(Fig. 4D, lanes 1 and 2) and insect cells (Pitsi, Kienlen-Campard et al. 2002). This
led us to consider that possibly other mechanism(s) may control PS1 endoproteolysis
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in vivo. To further explore this possibility, we used PS1 -/- cells to avoid any
“residual” γ-secretase activity implicated in PS1 endoproteolysis. Since BACE1
proteins levels aren’t affected in PS1 -/- cells (Fig. 3A), this “residual” PS1
endoproteolysis may be dependent of endogenous BACE1. To further explore this
possibility, we overexpressed PS1 and BACE1 in our PS1 -/- cells. As expected, co-
expression of BACE and PS1 in PS1 -/- cells had similar effects on PS1 wt and
L392V holoprotein levels (Fig 4 D, lanes 6 and 7, and Fig 4E), leaving trace amounts
of PS1 holoprotein. Lack of PS1 fragment accumulation can be attributed to absence
of “mature” γ-secretase partners in PS1 -/- cells (De Strooper 2003; Takasugi, Tomita
et al. 2003). Surprisingly, the ∆EX10 PS1 mutant, which lacks the endoproteolytic
cleavage site, is much less prone to BACE1-dependent down-regulation, as compared
to HEK293T cells. Predictably, no endoproteolysis of γ-secretase dead PS1 D257A
and D385A mutants is observed in PS1 -/- cells when co-expressed with BACE1 (Fig
4D, lanes 9 and 10, and Fig 4E). Last, we tested if exogenous γ-secretase inhibitors
could block BACE1-dependent PS1 down-regulation. As sown in Figure 5A, lane 3,
the potent γ-secretase inhibitor DAPT, did not attenuate PS1 holoprotein down-
regulation when co-expressed with BACE1. Cumulatively, these results suggest that
BACE1 regulates PS1 endoproteolysis independently of γ-secretase activity.
Furthermore, BACE1-dependent PS1 down-regulation seems to some extent
dependent of its endoproteolytic region (amino acids 292-298).
PS1 down-regulation is to a certain extent independent of the proteasome.
As mentioned above, the proteasome is additionally implicated in PS1 holoprotein
endoproteolysis (Honda, Yasutake et al. 1999; Van Gassen, De Jonghe et al. 1999).
As previously reported, treating cells with ALLN, a well-known proteasome inhibitor,
did accentuate endogenous PS1 holoprotein levels (Fig 5B, lane 2) in HEK293T cells.
As expected, ALLN treatment only partially inhibited (approximately 45%) BACE1-
dependent PS1 holoprotein down-regulation, as observed with endogenous (Fig 5B,
lane 3) and overexpressed PS1 (Fig 5B, lane7). Again, no substantial increase in PS1
fragment levels was observed when expressed with BACE1 (Fig 5B, lower panel). In
summary, these results suggest that, at least to a certain extent, BACE1-dependent
PS1-downregulation is independent of proteasome activity in vivo.
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After more than 20 years in biomedical and biochemical research, we are now starting
to understand the complex neuro-pathological mechanisms leading to Alzheimer’s
disease. With the “amyloid cascade hypothesis” gaining popularity, modern genetic
techniques have led to the identification of multiple genes implicated in amyloid
metabolism. First, identification of presenilin (PS1 and PS2) genes, and subsequently
Nicastrin, Aph-1 (Aph1a and Aph1b) and Pen-2 genes, has led to an initial
characterization of γ-secretase (De Strooper 2003). Second, BACE1 [and its
homologue BACE2 (Farzan, Schnitzler et al. 2000)] has recently been cloned and
linked to β-secretase. Major efforts are presently deployed to understand β- and γ-
secretase activities in a biological context, even more so since the recent development
of drug-based therapies (Fassbender, Masters et al. 2001; De Strooper and Woodgett
2003; Lahiri, Farlow et al. 2003). Although data concerning the enzymatic properties
of β- and γ-secretases are accumulating, less is known about the proteins’ biochemical
properties in vivo.
We have previously reported that both ectopically expressed human BACE1 and PS1
directly associate. In this study, we confirm BACE1-PS1 binding at an endogenous
level in human HEK293T cells. Most importantly, we validate our previous findings
by demonstrating preferential binding between immature BACE1 and holoprotein
PS1 (most likely in the ER/early Golgi compartments (personal observation by
immuno-localisation, Hébert, S.S.) and (Annaert and De Strooper 1999; Huse and
Doms 2000). Our initial characterization of BACE1-PS1 binding led us to
demonstrate that various PS1 point-mutations do not significantly abrogate nor
accentuate binding with BACE1. Hence, PS1 AD- and non-AD associated mutants
are capable of binding BACE1, thus conserving their basic fundamental function(s).
Nonetheless, fine-mapping of BACE1-PS1 binding is underway in our laboratory.
The results above indicate that immature BACE1 is tightly associated with
“immature” holoprotein PS1. Accumulating data tend to demonstrate that maturation
of the γ-secretase complex is strictly regulated by PS1. Therefore, it is possible to
imagine that PS1 also regulates β-secretase (BACE1) maturation. We addressed this
possibility by first evaluating BACE1 protein levels in PS1 K/O cells. We show that
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BACE1 protein levels weren’t substantially affected in PS1 K/O cells. Moreover,
BACE1 overexpression in PS1 -/- cells, as compared to PS1 +/+ cells, did not lead to
abrogated maturation (i.e. immature vs. mature levels ratio) (data not shown). Also,
no effect of γ-secretase dead PS1 D385A on BACE1 maturation, as assessed by
Western blotting (Fig 3B) and immuno-localisation of BACE1 (not shown), further
suggest that BACE1 maturation is not dependent of PS1 protein levels and γ-secretase
activity. It would be interesting to evaluate BACE1 maturation in PS1/PS2 double
knock-out cells, to avoid misleading conclusions based on possible PS2
complementation in PS1 K/O cells.
Accentuated BACE1 expression levels have recently been observed in sporadic AD
cases. To further explore the BACE1-PS1 relationship, we tested if BACE1
expressions levels could influence PS1 metabolism. Surprisingly, BACE1
overexpression led to a dramatic down-regulation of PS1 holoprotein levels without
significantly affecting endoproteolytic fragment levels (Fig 3D, lanes 3-6). This
phenomenon correlates with immature BACE1 expression levels (shown to
specifically bind PS1 holoprotein, this paper). Curiously, mature BACE1 levels
saturate when overexpressed, thus suggesting a “tightly regulated effect”, as seen with
overexpressed PS1 and Nicastrin (Arawaka, Hasegawa et al. 2002; Yang, Tandon et
al. 2002). However, when compared to BACE1 endogenous levels, mature BACE1
(and immature) species are significantly higher (data not shown), possibly implicating
different/less stringent limiting cellular factors. Also, endogenous BACE1 levels are
not submitted to the “replacement phenomenon” as seen with PS1 and Nicastrin (data
not shown). Nevertheless, further efforts are needed to better understand BACE1
maturation regulation.
Although PS1 holoprotein endoproteolysis and fragment generation is a normal,
physiological process, we are still lacking comprehensive knowledge about PS1
maturation regulation. Only recently after the discovery of the “minimal” constituents
of the γ-secretase complex (i.e. Nicastrin, APH-1 and PEN-2), are we starting to
understand the metabolism of PS1 and generation of PS1 NTF and CTF fragments.
However, both PS1 holoprotein and fragments protein levels are separately regulated
in vivo (Ratovitski, Slunt et al. 1997; Zhang, Kang et al. 1998), and accumulation of
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PS1 fragments levels in vivo require fully functional “mature” γ-secretase
components (Kim, Ikeuchi et al. 2003; Takasugi, Tomita et al. 2003).
To our surprise, BACE1-dependent down-regulation of PS1 holoprotein species was
completely inhibited by the PS1 D257A and D385A mutations, and partially inhibited
by ∆EX10, in PS1 -/- cells. Since PS1 aspartate mutations are less prone to
endoproteolysis, and PS1 ∆EX10 mutant has no endoproteolytic cleavage site, this led
us to consider that BACE1 could regulate PS1 endoproteolysis. To date, PS1
endoproteolysis was shown to be mostly regulated by γ-secretase or proteaseome-
related activities. However, the results presented in this paper suggest that BACE1-
dependent PS1 holoprotein down-regulation seems independent of these two latter
activities. Recent data demonstrate that “presenilinase” is actually an aspartyl
protease pharmacologically distinct from γ-secretase (Campbell, Reed et al. 2003) and
calpain (proteasome) activities. It is tempting to speculate that the aspartyl protease
BACE1 may itself be, or at least up-regulate, the long sought “presenilinase”. In
corroboration with this hypothesis, recent data from in vitro studies have
demonstrated direct BACE1 “presenilinase” activity toward an artificial PS1
“endoproteolytic site” octapeptide. Furthermore, we have preliminary data suggesting
that Aph-1a (but not Nicastrin), which also binds to (and stabilizes) PS1 holoproteins
(LaVoie, Fraering et al. 2003), partially inhibits BACE1-dependent down-regulation
possibly acting as a “substrate” competitor (not shown).
In summary, the results presented in this report provide first evidence suggesting a
physiological role for the BACE-PS1 interaction. These results may explain how
increased BACE1 proteins levels, as observed in sporadic AD, could lead to higher β-
and possibly γ-secretase (by regulating PS1 holoprotein levels, and presumably
increasing endoproteolytic “active” fragments) activities in vivo, thus gradually
accentuating Aβ peptide generation. Of course, further work is needed to better
understand the relationship between BACE1, PS1 and presumably the other
components of the γ-secretase complex, and this, in a more physiological context.
109
ACKNOWLEDGMENTS:
This work was supported by grants from Canadian Institutes of Health Research
(IRSC), Fonds de la Recherche en Santé du Québec (FRSQ) and Alzheimer Society of
Canada.
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FIGURE LEGENDS
FIG 1. Immature BACE1 binds full-length PS1 at an endogenous level. (A) a. Immature BACE1 binds full-length PS1 at an endogenous level. Detection of
co-immunoprecipitates following IP in endogenous HEK293T cell extracts.
Endogenous immature BACE1 can be immunoprecipitated with the N-19 antibody
(lane 2), but not with pre-immune serum (PIS) (lane 1) or Ab-14 (lane 3). b. Inverse
co-immunoprecipitation following PIS (lane 1) or anti-BACE C-terminal (lane 2)
immunoprecipitation. Blot is probed with N-19 antibody showing recuperated full-
length PS1 (lane 2) with immature BACE1 (not shown). (B) Two-hybrid assay
demonstrating BACE/PS1-loop 6-7 interaction. Representative Saccharomyces
cerevisiae AH109 strain colony growth. Positive co-transformation with both
pGBKT7 and pGADT7 constructs was confirmed by colony growth on TL- (not
shown). Activation of His and Ade reporter genes was used to verify interaction
between two proteins. Colony formation after 4 days of incubation on SD/TLHA- was
considered as positive interaction (+++) between fusion partners. Absence of colony
after 4 days was considered as an absence of interaction. However, plates with
negative results were incubated 10 days to confirm absence of growth. The pGBKT7-
PS1 Loop 6-7/pGADT7 BACE1 essay gave inconsistent results (+/-). Each hybrid
assay was performed at least 3 times.
FIG 2. In vivo immunoprecipitation of BACE1 with PS1 mutants. GST pull down assays and immunoprecipitation demonstrate that BACE and PS1
mutants interact in vivo. (A) Western blot analysis of transfected PS1 wt (lanes 1 and
2), PS1 L392V (lane 3) and PS1 D257A (lane 4) in GST-BACE precipitates following
suspension GST chromatographic extraction. PS1 holoproteins from transfected
HEK293T lysates can be detected as coeluants with BACE using anti-PS1 antibody,
PSN2 (lane 2-4). Similar results were obtained using anti-PS1 antibody, Ab-14 (data
not shown). PS1 does not coelute in extracts from GST only fractions (lane 1).
Western blot of crude protein extracts for PS1 is shown in middle panel. Coomassie
gel staining showing GST or GST-BACE fusion protein expression level (lower
panel). (B) Detection of various PS1 mutants in co-immunoprecipitates following IP
with anti-V5 (BACE1-V5) antibody. BACE-V5 (both immature and mature forms)
can be detected in IP products from double transfected cells but not in non-specific
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anti-myc immunoprecipitated products (upper panel). Western blots showing PS1
mutants coeluted with anti-V5 (BACE1) (middle panel, probed with anti-PSN2).
Lanes 1-8, lower panel, are crude lysates from double transfected cells, blotted with
anti-PS1 (PSN2).
FIG 3. BACE1-induced down-regulation of PS1 holoprotein levels. (A) BACE maturation is not affected in PS1 -/- cells. Endogenous cell extracts from
HEK293T, N2A, PS1 +/+ and PS1 -/- fibroblast cells were probed with either anti-
BACE C-terminal or Ab-14 (PS1) antibodies. Note that both immature and mature
protein levels of BACE are not affected by the absence of PS1. (B) γ-Secretase
activity is not implicated in BACE maturation. Either mock, PS1 wt or PS1 D385A
cDNAs were co-transfected with BACE in HEK293T cells. Proteins were extracted,
treated (lanes 1-3) or not (lanes 4-6) with ENDO-H overnight, separated by SDS-
PAGE and probed with Ab-1 (anti-BACE N-terminal) antibody. Negative controls for
ENDO-H treatment of cell extracts were conducted with H2O (not shown). (C)
BACE-dependent PS1 holoprotein down-regulation. Western blot of PS1 (anti-PSN2)
protein levels in double transfected (PS1 wt and BACE) HEK293T cells. Note the
substancial and specific PS1 holoprotein down-regulation when co-expressed with
BACE (lane 2) but not alone (lane 1) nor mock (not shown). (D) Dose-dependent PS1
holoprotein down-regulation induced by immature BACE species as observed by
Western blot in double transfected HEK293T cells. Note that PS1 N-terminal
fragment levels are not substantially affected by BACE overexpression. Negative
tranfection controls include mock (not shown) and HES-1 (lower panel). (E)
Artificially engineered “mature” BACE partially inhibits PS1-induced holoprotein
down-regulation. Western blot demonstrating partial PS1 holoprotein down-regulation
by ∆pro-BACE. Blot is probed with anti-PSN2.
FIG 4. PS1 aspartate mutations inhibit BACE-dependent down-regulation. (A) PS1 AD-associated mutants are also affected by BACE overexpression. Equal
amounts of proteins extracted from PS1 wt, PS1 L392V and PS1 EX10 double
transfected HEK293T cells were mixed with reducing SDS-PAGE sample buffer,
boiled for 2 min, then fractionated on SDS-PAGE and transferred to PVDF
membrane. The membrane was probed using PSN2 (PS1), Ab-1 (BACE) and HA
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(HES1-HA) antibodies. Negative control includes mock (not shown) and HES-1
(lanes 1, 3, 5 and 7). (B) PS1 D385A mutant is partially unaffected by BACE
overexpression in HEK293T cells. Equal amounts of proteins extracted from PS1
D385A double transfected HEK293T cells were mixed with reducing SDS-PAGE
sample buffer, boiled for 2 min, then fractionated on SDS-PAGE and transferred to
PVDF membrane. The membrane was probed using PSN2 antibody. (C) Graphic
demonstrating percentage of PS1 holoprotein down-regulation induced by BACE
overexpression (10 µg) in HEK293T cells. Results from four independent
experiences are shown. (D) γ-Secretase aspartate mutants are unaffected by BACE
overexpression in PS1 knockout cells. Equal amounts of proteins extracted from PS1
wt, PS1 L392V, PS1 EX10, PS1 D257A and PS1 D385A single (lanes 1-5) or double
(lanes 6-10) transfected PS1 -/- fibroblast cells were mixed with reducing SDS-PAGE
sample buffer, boiled for 2 min, then fractionated on SDS-PAGE and transferred to
PVDF membrane. The membrane was probed using PSN2 (PS1) antibody. (E)
Graphic demonstrating percentage of PS1 holoprotein down-regulation induced by
BACE overexpression (10 µg) in PS1 -/- cells. Results from three independent
experiences are shown.
FIG 5. BACE-dependent PS1 holoprotein down-regulation is independent of γ-
secretase and proteasome activities.
(A) BACE-dependent PS1 holoprotein down-regulation is independent of γ-secretase
activity. Proteins were extracted from double transfected (PS1 wt and BACE) (lanes
1-3) HEK293T cells. Prior to extraction, 1 petri dish was treated 16H at 37°C with 1
µM of DAPT (lane 3), a highly potent γ-secretase inhibitor. (B) BACE-dependent
PS1 holoprotein down-regulation is independent of proteasome (calpain) activity.
Either single (i.e.BACE, lane 3) or double (i.e. BACE and PS1 wt, lanes 4-7)
transfected HEK293T cells were treated with 50 µM of the highly potent proteasome
inhibitor ALLN for 16H at 37°C (lanes 2, 3, 6 and 7). Proteins were extracted,
subjected to SDS-PAGE, and probed with anti-PSN2 antibody. Note that PS1 N-
terminal (and C-terminal, not shown) fragments do not accumulate in vivo.
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CONCLUSIONS ET PERSPECTIVES
CHAPITRE VII
Ce projet avait pour but d’étudier le rôle qu’exerce la préséniline-1 (PS1) dans la
régulation de l’amyloïde bêta 42 neurotoxique. Plus spécifiquement, nous voulions
approfondir la relation qui existe entre PS1 et l’activité γ-secrétase. Mes objectifs de
travail reposaient sur l’hypothèse que, tel qu’il est observé avec d’autres protéines
multi-transmembranaires, l’oligomérisation de la PS1 pourrait jouer un rôle important
dans la régulation et/ou l’activation de la γ-secrétase.
L’ensemble des travaux présenté dans les chapitres 1 et 2 nous a permis de suggérer
un nouveau modèle de maturation post-traductionnelle pour la PS1 (Hébert, Godin et
al. 2003a; Hébert, Godin et al. 2003b). Effectivement, il a été possible de démontrer
qu’une dimérisation de la PS1 sauvage et mutante est une étape prémonitoire à la
génération des fragments NTF et CTF du complexe γ-secrétase actif (via la formation
d’un complexe de bas poids moléculaire). Analogue à ces résultats, une étude très
récente de Schroeter et al. (2003), portant sur la caractérisation des propriétés
allostériques de la γ-secrétase, suggère qu’un dimère PS1 (tétramère NTF/CTF, voir
chapitre 5) constituerait le centre catalytique de l’enzyme. De plus, ils suggèrent
qu’une dimérisation de la PS1 normale et/ou mutée est nécessaire à la reconnaissance
et à la spécificité du clivage d’un substrat donné (dans leur cas, entre AβPP et Notch).
125
D’autres études seront nécessaires afin de vérifier si l’oligomérisation de la PS1 dans
la cellule nécessite par exemple la participation de d’autres protéines. Tel que reporté
dans les chapitres 2, 3 et 4, la PS1 peut se lier aux protéines du complexe γ-secrétase.
Il est donc possible d’imaginer que la maturation et de la PS1 (et la génération du
tétramère NTF/CTF) nécessite l’apport des protéines Nicastrine, Aph-1 et Pen-2 in
vivo. En accord avec cette hypothèse, deux études récentes effectuées en culture de
cellules démontrent que Nicastrine et Aph-1 peuvent lier et stabiliser le complexe de
bas poids moléculaire (dimères) de la PS1 (Hu and Fortini 2003; LaVoie, Fraering et
al. 2003) pour ensuite favorisée la génération des fragments NTFs et CTFs (via un
processus protéolytique impliquant la Pen-2) (Francis, McGrath et al. 2002; Takasugi,
Tomita et al. 2003). Il serait donc intéressant de déterminer si l’interaction entre
Nicastrine, Aph-1 et PS1 est dépendante de la dimérisation de cette dernière. Pour
tester cette hypothèse, nous pourrions utilisé le mutant de délétion PS1 ∆TM1-2
(correspondant aux acides aminés 82-154). En effet, des études ont démontré qu’une
délétion de la portion PS1 TM1-2 a un effet dominant négatif (tel qu’observé avec les
mutations aspartates D257A et D385A) sur la maturation protéique de la PS1 et
l’activité γ-secrétase (Leem, Saura et al. 2002; Cai, Leem et al. 2003). Donc, il serait
intéressant d’évaluer si la PS1 ∆TM1-2 peut bel et bien se dimériser et/ou se lier aux
autres protéines du complexe γ-secrétase (soit par technique de double hybride en
levure ou par co-immunoprécipitation en cellules).
Le second volet de cette étude, dont les résultats sont présentés dans les chapitres 5, 6
et en annexe 1, a été orienté sur le développement d’un essai enzymatique pour la γ-
secrétase en levure. Premièrement, nos expériences ont permis de mettre en évidence
une interaction directe entre PS1 et BACE (Hébert, Bourdages et al. 2003). Des
études antérieures portant sur la caractérisation des secrétases et la génération des
peptides amyloïdes avaient suggérées une régulation à la hausse de l’activité β-
secrétase par la PS1 mutée (Ancolio, Marambaud et al. 1997; Sudoh, Kawamura et al.
1998; Komano, Sudoh et al. 1999). Il est donc possible d’imaginer qu’une interaction
directe entre la BACE (β-secrétase) et la PS1 (normale et mutée) soit responsable
d’une telle co-régulation enzymatique. Toutefois, la manière spécifique dont
126
l’activité β- (et γ-) secrétase est régulée par une PS1 mutée de façon à augmenter la
production des peptides amyloïdes Aβ42 demeure toujours du domaine de l’inconnu;
surtout si la PS1 ne semble pas posséder d’activité enzymatique intrinsèque (tel que
suggéré en annexe 1 et [(Edbauer, Winkler et al. 2003)]).
Outre un rôle « évident » de l’interaction PS1-BACE, d’autres études présentées au
chapitre 6 nous permettent de suggérer une régulation de la PS1 par la BACE. En
effet, nos résultats préliminaires démontrent que la quantité (et possiblement
l’activité) de la BACE immature régule l’holoprotéine de la PS1. De plus, un certain
nombre d’expériences réalisées parallèlement à ce travail m’ont permis de démontrer
que la régulation à la baisse de l’holoprotéine PS1 par la BACE immature était
atténuée par la co-expression de Aph-17. Cette observation laisse supposer qu’il
existe une compétition entre Aph-1 et BACE pour la PS1. Curieusement, l’ensemble
des résultats présentés dans le chapitre 6, joint aux résultats de la littérature (voir
Tableau 2), suggère que l’aspartyl protéase BACE soit une « présénilinase ». Il serait
donc intéressant de vérifier si par exemple BACE peut cliver directement PS1 in vitro
(en utilisant soit la PS1 pleine longueur ou la loupe hydrophile PS1 6-7 [acides
aminés 239-409] purifiée comme substrat pour la BACE purifiée [disponible
commercialement]). Si cette dernière hypothèse s’avère vraie, nos résultats
permettront pour la première fois d’établir un mécanisme moléculaire commun entre
les formes familiales et sporadiques de la maladie d’Alzheimer.
En résumé, les résultats présentés dans cette thèse ont clairement démontré que
l’oligomérisation de la PS1 joue un rôle déterminant dans la génération des fragments
NTFs et CTFs, composants actifs du complexe γ-secrétase. De plus, nous avons pu
mettre en évidence une interaction entre PS1 et BACE, les deux joueurs clés dans la
production du peptide amyloïde bêta-42 toxique, constituant majeur des plaques
amyloïdes observées chez les patients atteints de la maladie d'Alzheimer.
7 Hébert S.S. (2003) Résultats non publiés
127
Même s’il existe plus de en plus de littérature scientifique traitant de la préséniline-1
(plus de 1250 publications depuis les 7 dernières années), la fonction précise de la
PS1 demeure inconnue. Mes résultats apportent des connaissances indispensables à
l’élucidation du rôle biologique des protéines impliquées dans le métabolisme de
l’amyloïde bêta, plus spécifiquement de la PS1 et de la BACE. De plus, puisque PS1
et BACE sont des cibles thérapeutiques contre la formation de l’amyloïde (des
médicaments qui inhibent l’activité de la β- ou de la γ- secrétase sont maintenant au
stade des essais cliniques), ces résultats aideront au développement d’un outil
thérapeutique efficace pour lutter contre le développement ce la maladie d’Alzheimer.
De plus, ces nouvelles connaissances pourront avoir une répercussion sur l’étude des
causes de la forme sporadique de la maladie ainsi que sur l’étude du rôle de la PS2
Tableau 2. Ressemblances biologiques et biochimiques entre la « présénilinase » et la BACE (β-secrétase). * FFMA (Formes familiales de la maladie d’Alzheimer)
« Présénilinase »
BACE (β-secrétase)
Références
Protéine intra-membranaire Principalement localisée dans le RE Aspartyl protéase (distinct de la γ-secrétase) Pas inhibée par inhibiteurs γ-secrétase (ex : DAPT) Activité présente dans cellules PS1 -/- Clive entre les acides aminés 292-298 de la PS1 Les mutations associées aux FFMA* (à l’exception de mutant PS1 ∆EX10) n’influencent pas la production des fragments NTF/CTFs. Activité insensible aux mutations aspartates (D257A et D385A)
Protéine intra-membranaire Principalement localisée dans le RE (forme immature) Aspartyl protéase (distinct de la γ-secrétase) Pas inhibée par inhibiteurs γ-secrétase (ex : DAPT) Activité présente dans cellules PS1 -/- Clive entre les acides aminés 292-298 de la PS1 (peptide in vitro) Les mutations associées aux FFMA* (à l’exception du mutant PS1 ∆EX10) n’influencent pas la production des fragments NTF/CTFs. Activité insensible aux mutations aspartates (D257A et D385A)
(Vassar, Bennett et al. 1999; Campbell, Iskandar et al. 2002) (Capell, Steiner et al. 2000; Campbell, Reed et al. 2003) (Vassar, Bennett et al. 1999; Campbell, Reed et al. 2003) (Campbell, Iskandar et al. 2002) et chapitre 6 (Pitsi, Kienlen-Campard et al. 2002; Nyabi, Bentahir et al. 2003) et chapitre 6 (Podlisny, Citron et al. 1997) (Campbell, Iskandar et al. 2002) et chapitre 6 (Kimberly, Xia et al. 2000; Campbell, Reed et al. 2003) et chapitre 6
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145
ANNEXE 1
RÉSULTATS COMPLÉMENTAIRES
Résumé
Les résultats présentés ci-dessous apportent des éléments pertinents à l’atteinte de
mon deuxième objectif de travail qui avait pour but, entre autre, de déterminer si la
PS1 possède une activité intrinsèque γ-secrétase. Ces expériences ont été réalisées en
levure dont certains résultats sont présentés aux chapitres 5 et 6.
Introduction
Le peptide neurotoxique amyloïde-β42 est généré suite à un clivage séquentiel de
AβPP par deux enzymes, soit la β- et la γ-secrétase (Xia, W. (2001). On a
nouvellement cloné et caractérisé BACE, la β-secrétase impliquée dans le premier
clivage (Hussain, Powell et al. 1999; Sinha, Anderson et al. 1999; Vassar, Bennett et
al. 1999; Yan, Bienkowski et al. 1999). Jusqu’à présent, on n’a pu identifier avec
précision la γ-secrétase. Toutefois, il existe une forte corrélation entre l’activité γ-
secrétase et la présence de PS1. Par exemple, les souris à mutation nulle (knock-out)
pour PS1 ne présentent pas d’activité γ-secrétase et, par conséquent, pas
d’accumulation du peptide Aβ42 (Xia, W. 2001). À l’inverse, plusieurs lignées de
souris transgéniques dans lesquelles la PS1 normale ou mutante est surexprimée au
cerveau ont été créées. Toutes les lignées mutantes, contrairement aux normales,
146
présentent une augmentation de la production du peptide Aβ42 au cerveau (Citron,
M., D. Westaway, et al. 1997). Contrairement à BACE, il n’a pas été possible de
démontrer clairement une activité enzymatique intrinsèque pour PS1 in vivo ni in
vitro; faute d’essai enzymatique convenable. Pour cette raison, je vais prendre
comme modèle biologique la levure afin de développer un essai enzymatique in vivo
pour la β- et la γ-secretase. Le substrat choisi pour cette étude est la protéine AβPP.
Matériels et méthodes
Dans un premier temps, les ADNc correspondants aux protéines humaines suivantes:
1) PS1 pleine longueur normale 2) BACE et 3) AβPP ont été clonées dans les
vecteurs pGBKT7 et/ou pGADT7 (Clontech) (Figure 9). Par la suite, ces
constructions ont été co-transformées dans la souche de levure AH109 (Clontech).
Différentes combinaisons plasmidiques ont été testées (par exemple : pGBKT7/AβPP
+ pGADT7/BACE ou pGBKT7/AβPP + pGADT7/PS1). Par croissance sur un milieu
sélectif, il a été possible de cribler les colonies positives (méthodes décrites par le
manufacturier) et de vérifier, par immunobuvardage, la présence des protéines
d’intérêt8 dans un clone positif donné. Or, cette approche a permit d’évaluer si la PS1
possède une activité γ-secrétase intrinsèque. En effet, par immunobuvardage avec des
anticorps spécifiques à l’AβPP, il a été possible de vérifier si l’AβPP subit un clivage
endoprotéolytique lorsque co-exprimée avec la BACE et/ou la PS1) (Figures 10).
8 Hébert, S.S. (2002) Résultats non publiés et Figure 11
147
Figure 9 Les vecteurs « BD-matchmakers™ » utilisés dans le système « Two-Hybrid system 3 » de Clontech. Le vecteur pGBKT7 (en haut) contient le domaine de liaison à l’ADN (« DNA-BD ») qui code pour les acides aminés 1-147 de la protéine de fusion GAL4. Le vecteur pGADT7 (en bas) permet l’expression de l’autre protéine d’intérêt fusionnée aux acides aminés 768-881 du domaine d’activation GAL4 (« AD »). Le promoteur ADH1 assure une expression élevée des protéines de fusions GAL4 DNA-BD et AD (donc des protéines d’intérêt). Dépendamment du vecteur utilisé, les protéines d’intérêt sont fusionnés aux épitopes Myc (dans le cas de pGBKT7) ou HA (dans le cas de pGADT7). De plus, les sites de clonage multiple (« MCS, Multiple Cloning Site ») des vecteurs respectifs sont illustrés (à droite).
148
Figure 10. Stratégie utilisée pour étudier le métabolisme de l’AβPP dans la levure. Des études de co-expression en levure permettent de déterminer les propriétés catalytiques des protéines impliquées dans le métabolisme de l’Aβ (par immunobuvardage avec les anticorps anti-myc, anti-V5 ou anti-Aβ). Par exemple, la co-expression de l’AβPP et la PS1 (normale ou mutée) en levure permet de déterminer si PS1 peut bel et bien cliver l’AβPP, et ce, par observation des métabolites AβPP produits.
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Résultats
Figure 11. Métabolisme de la protéine AβPP dans la levure. Immunobuvardages représentatifs démontrant l’expression des protéines AβPP, PS1 et BACE (en fusion avec la protéine « DNA-BD » du vecteur pGBKT7 (Clontech) d’un poids approximatif de 23 kDa en levure AH109. Différentes combinaisons de co-transformation ont été testées (ex : pGBKT7/AβPP + pGADT7/BACE). Dans le cas d’une triple transformation (AβPP, PS1 et BACE), la sélection des transformants (clones) positifs en levure s’est effectuée par PCR (non montré). Les contrôles d’immunobuvardages incluent des extraits de protéines de levure de la souche vide (AH109) ou en présence du vecteur d’expression vide (pGBKT7). D’autres tests visant à déterminer la production des peptides amyloïdes (avec l’anti-Aβ) furent effectués mais se sont avérés négatifs (non montrés).
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Interprétations, conclusion et perspectives
Dans un premier temps, l’ensemble des résultats présentés dans ce chapitre démontre
que toutes les protéines testées (AβPP, BACE et PS1) peuvent être surexprimées en
levure (Figure 10). Toutefois, ni BACE ni PS1 ne semble subir de modifications
post-traductionnelles telles qu’observées dans la cellule (Benjannet, Elagoz et al.
2001; Thinakaran 2001). Par exemple, il n’y a aucune libération du pro-domaine de
BACE9, tel qu’observé par son poids moléculaire d’environ 90 kDa. De plus, nous
n’observons aucun clivage endoprotéolytique pour la PS1, tel qu’observé par la
présence de l’holoprotéine à environ 75 kDa.
Puisqu’il n’y a aucun homologue de la furine dans la levure (www.ncbi.nlm.nih.gov)
il n’est pas étonnant que la BACE demeure intacte. L’absence de clivage
endoprotéolytique de la PS1, tant qu’à lui, suggère qu’aucune « présénilinase »
endogène n’est présente dans la levure. De plus, cette expérience laisse supposer que
l’holoprotéine de la PS1 ne possède pas d’activité autocatalytique.
Malheureusement, il nous a été impossible d’observer clairement un clivage (soit de
type β- ou γ-) de l’AβPP en levure. Toutefois, nous croyons que ces résultats,
quoique partiels, aident à la compréhension des mécanismes protéolytiques entourant
la production des peptides amyloïdes in vivo. Par exemple, notre démonstration que
la présence du pro-domaine de BACE nuit au clivage β de l’AβPP nous suggère qu’il
existe bel et bien un rôle fonctionnel de celui-ci dans la régulation de l’enzyme. En
perspective, il serait intéressant d’évaluer les propriétés enzymatiques du mutant
BACE ∆pro-domaine (décrit au chapitre 6 et [(Benjannet, Elagoz et al. 2001; Shi,
Chen et al. 2001)]) en levure. Il serait ainsi possible d’évaluer, toujours par
immunobuvardage, si BACE « mature » peut cliver l’AβPP. Aussi, il est possible
9 Et, dans ce cas-ci, du domaine de liaison à l’ADN (« DNA-BD »).
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d’imaginer que la présence du domaine d’activation à l’ADN (« AD »), provenant du
vecteur d’expression pGADT7 (Figure 9), nuit à l’activité de la BACE. Donc, il
serait intéressant d’évaluer les propriétés biologiques et enzymatiques de la BACE
exprimée d’un vecteur ne contenant aucune protéine de fusion (ex : domaine de
liaison/activation à l’ADN et/ou épitopes).
En résumé, les résultats présentés dans ce chapitre ont clairement démontré que les
protéines impliquées dans le métabolisme de l’Aβ peuvent être exprimées en levure.
A part quelques rectifications concernant le système d’expression (vecteurs) utilisé,
nous croyons que la levure est un modèle biologique de choix pour étudier le rôle des
secrétases dans la génération des peptides amyloïdes neurotoxiques. Cette hypothèse
est fortement appuyée par la démonstration récente de la reconstitution artificielle du
clivage γ-secrétase dans la levure à partir du complexe γ-secrétase (co-expression des
protéines préséniline-1, Nicastrine, Aph-1 et Pen-210) et le fragment C99 de l’AβPP
(Edbauer, Winkler et al. 2003). De plus, le système double hybride en levure
permettra une approche pour la caractérisation future des interactions
protéines:protéines nécessaires à la production des peptides amyloïdes neurotoxiques.
10 Note : Dans cette étude, les protéines ne contenaient pas de domaine de liaison/d’activation à l’ADN.
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ANNEXE 2
Mutations de la préséniline-2. Schéma (non à l’échelle) des mutations (acides
aminés en rouge) menant à une forme familiale de la maladie d’Alzheimer dans le
gène préséniline-2 (PS2). (Image adaptée de John Hardy, National Institute on Aging,
Bethesda, MD)
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ANNEXE 3
Les acides aminés et leurs symboles.
ACIDES AMINÉS CODONS
A Ala Alanine GCA GCC GCG GCTC
C Cys Cystéine TGC TGT
D Asp Aspartate GAC GAT
E Glu Glutamate GAA GAG
F Phe Phénylalanine TTC TTT
G Gly Glycine GGA GGC GGG GGT
H His Histidine CAC CAT
I Ile Isoleucine ATA ATC ATT
K Lys Lysine AAA AAG
L Leu Leucine TTA TTG CTA CTC CTG CTT
M Met Méthionine ATG
N Asn Asparagine AAC AAT
P Pro Proline CCA CCC CCG CCT
Q Gln Glutamine CAA CAG
R Arg Arginine AGA AGG CGA CGC CGG CGT
S Ser Sérine AGC AGT TCA TCC TCG TCT
T Thr Thréonine ACA ACC ACG ACT
V Val Valine GTA GTC GTG GTT
W Trp Tryptophane TGG
Y Tyr Tyrosine TAC TAT