HAL Id: tel-01350863
https://tel.archives-ouvertes.fr/tel-01350863
Submitted on 2 Aug 2016
HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
Caractérisation de MSI2 et MSI3 : deux sous-unités du CRL4 et potentiels régulateurs chromatiniens chez
Arabidopsis thaliana
Sabrina Jung-Romeo
To cite this version:
Sabrina Jung-Romeo. Caractérisation de MSI2 et MSI3 : deux sous-unités du CRL4 et potentiels régulateurs chromatiniens chez Arabidopsis thaliana. Biochimie, Biologie Moléculaire. Université de Strasbourg, 2014. Français. �NNT : 2014STRAJ020�. �tel-01350863�
ÉCOLE DOCTORALE des Sciences de la Vie et de la Santé 8QLWpGHUHFKHUFKH
,QVWLWXWGH%LRORJLH0ROpFXODLUHGHV3ODQWHV UPR CNRS 2357
THÈSE de Doctorat
SUpVHQWpHSDU
Sabrina JUNG – ROMEO
SRXUREWHQLUOHJUDGHGH
Docteur de l’université de Strasbourg
'LVFLSOLQH
6FLHQFHVGX9LYDQW
6SpFLDOLWp
$VSHFWV0ROpFXODLUHVHW&HOOXODLUHVGHOD%LRORJLH
Caractérisation de MSI2 et MSI3 :
deux sous-unités du CRL4 et potentiels régulateurs chromatiniens chez Arabidopsis thaliana
VRXWHQXHOH-XLOOHWGHYDQWOHMXU\FRPSRVpGH
03DVFDO*(16&+,. 'LUHFWHXUGHWKqVH
0&ULVDQWR*87,(55(= 5DSSRUWHXUH[WHUQH
0)UDQoRLV528',(5 5DSSRUWHXUH[WHUQH
0PH$QQH&DWKHULQH6&+0,7 5DSSRUWHXULQWHUQH
INTRODUCTION
I. La protéolyse ubiquitine-dépendante
1 L’ubiquitinylation
/¶XELTXLWLQH
)L[DWLRQGHO¶XELTXLWLQH
/HVSURWpLQHV©XELTXLWLQHOLNHª
/HVSULQFLSDX[DFWHXUVGHO¶XELTXLWLQ\ODWLRQOHVHQ]\PHV
/HV(DFWLYDWLRQGHO¶XELTXLWLQH
/HV(FRQMXJDLVRQGHO¶XELTXLWLQH
/HV(OLDLVRQGHO¶XELTXLWLQHDXVXEVWUDW
/HV(OLJDVHVjGRPDLQH+(&7
/HV(OLJDVHVjGRPDLQH©5,1*ILQJHUª
/HV(OLJDVHVGHW\SH&5/
1.3.3.2.1.1 Le complexe APC (« Anaphase promoting complexe » ou Cyclosome
1.3.3.2.1.2 Le complexe SCF ou CRL1
1.3.3.2.1.3 Le complexe VBC
1.3.3.2.1.4 Le complexe CUL3-BTB ou CRL3
1.3.3.2.1.5 Le complexe CUL4-DDB1 ou CRL4
II. Les mécanismes de régulation de la protéolyse ubiquitine-dépendante
1 La reconnaissance et la dégradation par le protéasome
/DPRQRXELTXLWLQ\ODWLRQ
/DSRO\XELTXLWLQ\ODWLRQ
/HSURWpDVRPH6
&RPSRVLWLRQGXSURWpDVRPH6
/HPpFDQLVPHGHGpJUDGDWLRQSDUOH6
2 La régulation de la réaction d’ubiquitinylation
5pJXODWLRQGHVSULQFLSDX[DFWHXUVGHO¶XELTWXLWLQ\ODWLRQ
5pJXODWLRQSDUPRGLILFDWLRQGXVXEVWUDW
5pJXODWLRQSDUGpXELTXLWLQ\ODWLRQ
III. Rôles des complexes CUL4-DDB1DCAF
1 Rôle dans la réparation de l’ADN
2 Rôle dans la photomorphogenèse
3 Régulateur du cycle cellulaire
4 Rôle dans la régulation chromatinienne
IV. Les DCAF
1 Une multitude de DCAF
2 Une protéine à domaine DWD : WDR55
3 Une autre classe de DCAF : les MSI (Multicopy Suppressor of IRA1)
06,
06,)9(±06,
V. Description du projet de recherche
RESULTATS
I. Les MSI : Caractérisation fonctionnelle des gènes MSI2et MSI3d’ Arabidopsis
thaliana
1 Etude la régulation transcriptionnelle
([SUHVVLRQGHVJqQHVGHODIDPLOOH06,G¶DSUqVOHVGRQQpHVSURWpLTXHVGH©PLFURDUUD\ª
/¶H[SUHVVLRQGHVJqQHVMSIHVWF\FOHFHOOXODLUHGpSHQGDQWH
(WXGHGHVSURILOVG¶H[SUHVVLRQGHVJqQHVMSI2HWMSI3in planta
()D'3DFRQWU{OHO¶H[SUHVVLRQGHVJqQHVMSI2HWMSI3in planta
2 Etude la localisation sub-cellulaire et de la régulation post-traductionnelle
(WXGHGHODFRPSDUWLPHQWDWLRQFHOOXODLUHGHVSURWpLQHVMSIin planta
(WXGHGHODUpJXODWLRQSRVWWUDGXFWLRQQHOOH
3 Etude de la fonction des gènes MSI2et MSI3
8QQRXYHORXWLODQWLFRUSVDQWL06,HWDQWL06,
0LVHHQpYLGHQFHGXFRPSOH[H&5/06,06,
7HVWVG¶LQWHUDFWLRQHQV\VWqPHKpWpURORJXH
7HVWVG¶LQWHUDFWLRQEDVpVVXUGHVWHFKQLTXHVGHPLFURVFRSLH
7HVWVG¶LQWHUDFWLRQEDVpVVXUGHVWHFKQLTXHVELRFKLPLTXHin situ
4 Identification de substrats de complexes CRL4MSI
,GHQWLILFDWLRQGHVSDUWHQDLUHVGHODSURWpLQH&8/
,GHQWLILFDWLRQGHVSDUWHQDLUHVGHVSURWpLQHV06,
5pDOLVDWLRQG¶XQFULEOHGRXEOHK\EULGHGDQVODOHYXUHDYHF06,HW06,
5pDOLVDWLRQG¶XQHH[SpULHQFHGH7$37DJDYHF06,HW06,
5 Génétique inverse
,GHQWLILFDWLRQGHPXWDQWVG¶LQVHUWLRQ7'1$RXWUDQVSRVRQ
&DUDFWpULVDWLRQSKpQRW\SLTXHGHVPXWDQWVNQRFNRXW
(WXGHGHODIORUDLVRQ
(WXGHGHODWDLOOHGHVURVHWWHV
9DULDWLRQGHVJqQHVGXF\FOHFHOOXODLUHGDQVOHVPXWDQWVmsi
6pOHFWLRQG¶XQTXDGUXSOHPXWDQWPVLPVLPVLPVL
II. Autre DCAF : WDR55
DISCUSSION / PERSPECTIVES
1 MSI2 et MSI3, deux protéines à motif WDxR, interagissent-elle avec DDB1a pour former
des complexes de type CRL4 ?
2 Les fonctions des gènes MSI 2 et MSI 3 ne sont pas redondantes avec celles des gènes
MSI 1 ou MSI 4/MSI 5 dans la plante.
3 Les protéines MSI sont-elles capables de réguler différents mécanismes de modification
de la structure chromatinienne ?
1 Plantes et cultures cellulaires végétales
Arabidopsis thaliana
&XOWXUHLQYLWURG¶Arabidopsis thaliana
Nicotiana benthamiana
0LOLHX[DQWLELRWLTXHVHWPpWKRGHGHVWpULOLVDWLRQ
2 Micro-organismes
6RXFKHVG¶Escherichia coliXWLOLVpHVSRXUOHFORQDJH
6RXFKHG¶Agrobactéria tumefaciensXWLOLVpHSRXUODWUDQVIRUPDWLRQGHPDWpULHOYpJpWDOHWLQILOWUDWLRQSRXU
H[SUHVVLRQWUDQVLWRLUH
6RXFKHGHEDFWpULHXWLOLVpHSRXUODSURGXFWLRQGHSURWpLQHV
6RXFKHVGHSaccharomyces cerevisiaeXWLOLVpHSRXUOHWHVWGRXEOHK\EULGH
0LOLHX[HWDQWLELRWLTXHV
3 Vecteurs
9HFWHXUGHFORQDJH
9HFWHXUVG¶H[SUHVVLRQ
4 Les anticorps anti-MSI3 et anti-MSI4
II. Méthodes
1 Techniques relatives aux plantes
$QDO\VHGHVH[WUDLWVG¶$'1G¶Arabidopsis thaliana
/HJpQRW\SDJHSDU3&5
([WUDFWLRQG¶$'1JpQRPLTXH
/D3&53RO\PHUDVH&KDLQ5HDFWLRQ
$QDO\VHGHVH[WUDLWVG¶$51G¶Arabidopsis thaliana
/D57T3&5
([WUDFWLRQG¶$51YpJpWDX[WRWDX[
/D573&5
/DT3&5
$QDO\VHGHVH[WUDLWVSURWpLTXHVG¶Arabidopsis thaliana
©:HVWHUQEORWª
([WUDFWLRQGHVSURWpLQHVWRWDOHVYpJpWDOHV
6pSDUDWLRQHWGpWHFWLRQGHVSURWpLQHVHQ6'63DJH
&KURPDWRJUDSKLHSDUJHOILOWUDWLRQ
&R,PPXQRSUpFLSLWDWLRQ
([WUDFWLRQG¶KLVWRQHV
7HFKQLTXHVG¶DQDO\VHPLFURVFRSLTXH
$QDO\VHGHVSRLQWHVUDFLQDLUHVSDUPLFURVFRSLHFRQIRFDOH
&RORUDWLRQ*86ȕJOXFXURQLGDVH
%L)&
2 Techniques relatives aux bactéries
3UpSDUDWLRQGHEDFWpULHV723FRPSpWHQWHV
3UpSDUDWLRQG¶DJUREDFWpULHV*9FRPSpWHQWHV
7UDQVIRUPDWLRQGHVEDFWpULHVSDUFKRFWKHUPLTXH
7UDQVIRUPDWLRQGHVEDFWpULHVSDUpOHFWURSRUDWLRQ
$QDO\VHGHVFORQHVEDFWpULHQV
3 Techniques relatives aux levures
7UDQVIRUPDWLRQGHVOHYXUHVSDUO¶DFpWDWHGHOLWKLXP
7HVWG¶LQWHUDFWLRQHQGRXEOHK\EULGH
&ULEOHGRXEOHK\EULGH
BIBLIOGRAPHIE
ANNEXES
-HWLHQVjUHPHUFLHU3DVFDO*HQVFKLNGHP¶DYRLUDFFXHLOOLHGDQVVRQpTXLSHHWGHP¶DYRLUGLULJpH SHQGDQWPHVTXDWUHDQQpHVGHWKqVHDLQVLTXH0DULH&ODLUH&ULTXLTXLP¶DVXSHUYLVpHWRXWDXORQJ GHFHVTXDWUHDQQpHVHWDYHFTXLM¶DLPHQpjELHQFHSURMHW
-H UHPHUFLH &ULVDQWR *XWLHUUH] )UDQoRLV 5RXGLHU HW $QQH&DWKHULQH 6FKPLW GH PH IDLUH O¶KRQQHXUGHMXJHUPRQWUDYDLOGHWKqVH
-HUHPHUFLHWRXVOHVPHPEUHVGHO¶,%03DXVVLELHQOHSHUVRQQHODGPLQLVWUDWLITXHWHFKQLTXH /HV MDUGLQLHUV SRXU DYRLU SULV VRLQ GH PHV SODQWHV GXUDQW FHV TXDWUH DQQpHV HW G¶DYRLU WRXMRXUVWURXYpXQHVROXWLRQSRXUPHVWHVWVGHIORUDLVRQORUVTXHM¶HQYDKLVVDLVXQHORJHWWH HQWLqUH
0DOHNSRXUWRXVOHVVpTXHQoDJHVGHPHVQRPEUHX[FORQDJHVHQWRXWJHQUHHWWRQDLGHHQ T3&5
/HVPLFURVFRSLVWHVHWOHVLQIRUPDWLFLHQVSRXUYRWUHERQQHKXPHXUTXRWLGLHQQH 'DQLqOHHW/LVLDQHSRXUQRVQRPEUHXVHVGLVFXVVLRQVGHWRXWHWGHULHQ
/DXUHQFH0VDQVTXLOHPDJDVLQQHWRXUQHUDLWSDVHWVDQVTXLQRXVVHULRQVDEDQGRQQpV 1LFRODVSRXUODSURGXFWLRQGHVSURWpLQHVOHXUSXULILFDWLRQHWWHVFRQVHLOVHQJHOILOWUDWLRQ /HWHDPQHZVOHWWHUHWSOXVODUJHPHQWODFRPPLVVLRQFRPPXQLFDWLRQPDLVDXVVLWRXVOHV DXWUHVTXHMHQ¶DXUDLVSDVFLWp
-H WLHQV j UHPHUFLHU OHV PHPEUHV GX ©qPHª HW SOXV SDUWLFXOLqUHPHQW OHV SHUVRQQHV GH PRQ ODER DQFLHQV HW SUpVHQWV OH PDLV DXVVL OHV DQFLHQV PHPEUHV H[SDWULpV DX DLQVL TXH OHV
©GU{OHVGHGDPHVªGH0RQWSHOOLHUSRXUYRWUHVRXWLHQYRVGLVFXVVLRQVHWQRVIUDQFKHVULJRODGHV 0HUFL (VWKHU SRXU WHV FRQVHLOV DYLVpV G¶rWUH WRXMRXUV Oj TXDQG RQ D EHVRLQ HW GH SHQVHU j QRV HVWRPDFV 0HUFL .LQGD SRXU WRQ VRXWLHQ GXUDQW PRQ PDVWHU PDLV DXVVL GXUDQW FHWWH WKqVH PHUFL 6DQGUDSRXUWHVFRQVHLOVPHUFL$OH[LV%HQRLW7KRPDV53DWULFN-HDQ0LFKHOSRXUYRVFRQVHLOV HQWRXWJHQUHPHUFL7KRPDV3SRXUWDVFLHQFHHWWDPpPRLUHTXLP¶LPSUHVVLRQQHWRXMRXUVDXWDQW .DWLDPDSUHPLqUHYRLVLQHGHSDLOODVVHDXMRXUG¶KXLj0RQWSHOOLHU7KLEDXWPRQSUHPLHUVWDJLDLUH M¶HVSqUHTXHMHQHW¶DLSDVWURSIDLWVRXIIULUHWMHWHVRXKDLWHERQQHFKDQFHSRXUWRQPDVWHUHWSRXUWD IXWXUH WKqVH M¶HVSqUH TX¶HOOH VHUD DXVVL HQULFKLVVDQWH TXH OD PLHQQH 6DQV RXEOLHU ELHQ VU <YHV PLVWHU8ELWRXMRXUVDYHFOHVRXULUH-HDQ/XFSRXUQRVGLVFXVVLRQVSURWpLQHVHWELHQVUDYLDWLRQ 0HUFLjWRXVFHX[TXHMHQ¶DLSDVFLWp
-H UHPHUFLH DIIHFWXHXVHPHQW WRXWH PD IDPLOOH PHV SDUHQWV HW PD JUDQGPqUH SRXU P¶DYRLU GRQQpHODFKDQFHGHIDLUHGHORQJXHVpWXGHV-RQDWKDQSRXUWRQDPRXUHWWRQVRXWLHQLQGpIHFWLEOH WRXW DX[ ORQJV GH FHV DQQpHV PHUFL GH PH VXSSRUWHU WRXV OHV MRXUV VXUWRXW HQ FHWWH SpULRGH GH UpGDFWLRQ0HUFLj$QQLH-30DWKLHX$QJqOHSDUGRQ$QGUpH0DULHHWF«0HUFLjPHVDPLV SOXVSDUWLFXOLqUHPHQW/DXUHQFHFRPPHRQGLWjSOXVLHXUVRQHVWSOXVIRUW
$PRQSDSD«
ABREVIATIONS
3DJH_
3-AT $PLQR7ULD]RO
A $PSqUHV
ABA $FLGH$EVFLVVLTXH
ADN $FLGH'pVR[\ULER1XFOpLTXH
APC/C $QDSKDVH3URPRWLQJ&RPSOH[&\FORVRPH
Arg $UJLQLQH
ARN $FLGH5LER1XFOpLTXH
ASK $UDELGRSVLV6.3OLNHSURWHLQ ATG8, 12 $XWRSKDJ\
ATP $GpQRVLQH7UL3KRVSKDWH BET %URPXUHG¶(WK\GLXP
BiFC %LPROHFXODU)OXRUHVFHQFH&RPSOHPHQWDWLRQ BTB %ULFj%UDF7UDPWUDFNDQG%URDG
°C 'HJUp&HOVLXV C &\VWpLQH
CaCl2 &DOFLXP&KORULGH c-à-d &¶HVWjGLUH
CAND1 &XOOLQ$VVRFLDWHGDQG1HGG\ODWLRQ±'LVVRFLDWHG CAF &KURPDWLQ$VVHPEO\)DFWRU
CDC &HOO&HSHQGHQW&\FOH
CDD &23''%DHW'(7
CDH &HUHYLVLDH'LP+RPRORJRXV CHR &KURPDWLQ5HPRGHOLQJ
CDK &\FOLQH'HSHQGHQW.LQDVH CFP &\DQ)OXRUHVFHQWSURWHLQ CKI &\FOLQ.LQDVH,QKLELWRUV CLF &85/</($)
CLRC 8ELTXLWLQOLJDVHFRPSOH[ORFDOL]DWLRQWRKHWHURFKURPDWLQ COP &RQVWLWXWLYH3KRWRPRUSKRJHQHVLV
CP &RUH3DUWLFOH
CRL &XOOLQ5LQJ(/LJDVH CSA &RFND\QHV\QGURPH$
CSN &RSVLJQDORVRPH C-ter &DUERF\WHUPLQDOH
Abréviations
3DJH_
CUL &XOOLQ
DCAF ''%&8/$VVRFLDWHG)DFWRUV DDB 'DPDJHG'1$%LQGLQJSURWHLQ DIC 'LIIHUHQWLDOLQWHUIHUHQFH&RQWUDVW dNTP GpVR[\ULER1XFOpRWLGH7UL3KRVSKDWH DO 'HQVLWp2SWLTXH
DTT 'L7KLR7KUpLWRO DUB 'H8ELTXLWLQH
DWD ''%ELQGLQJ:'
EBF (WK\OHQH5HVSRQVHIDFWRU E.coli Escherichia coli
EDTA $FLGH(WK\OqQH'LDPLQH7pWUD$FpWLTXH ENaC (SLWKHOLDO1D&KDQQHO
ESC ([WUD6H[&RPEV E(z) (QKDQFHURI]HVWH
FIE )HUWLOL]DWLRQ,QGHSHQGHQW(QGRVSHUP FIS )HUWLOL]DWLRQ,QGHSHQGHQW6HHG FLC )ORZHULQJ/RFXV&
g JUDPPHV
g )RUFHFHQWULIXJHUHODWLYH GGR *OREDO*HQRPH5HSDLU GFP *UHHQ)OXRUHVFHQW3URWHLQ GST *OXWDWKLRQ67UDQVIHUDVH GUS *OXF8URQLFDFLGH;*OXF
h KHXUHV
H +LVWRQHV
H +LVWLGLQH
HECT +RPRORJRXVWR($3&DUER[\O7HUPLQXV
HEPES $FLGHK\GUR[\pWK\OSLSpUD]LQHpWKDQHVXOIRQLTXH HDAC +LVWRQH'HDFpW\ODVH
HFR1 /RQJ+\SRFRW\OLQ)DU5HG His +LVWLGLQH
HP +HWHURFKURPDWLQSURWHLQ HOAc $FLGHDFpWLTXH
3DJH_
HUB1 +RPRORJRXVWRXELTXLWLQ
HY5 /RQJ+\SRFRW\O
HYH +<KRPRORJXH
K /\VLQH
KCl &KORUXUHGHSRWDVVLXP
KDa .LOR'DOWRQ
KOAc $FpWDWHGHSRWDVVLXP KRP .LS5HODWHG3URWHLQ
L /LWUH
LAF1 /RQJDIWHU)DU5HG/LJKW LB /XULD%HUWDQL
LiAc $FpWDWHGH/LWKLXP
LHP1 /LNH+HWHURFKURPDWLQ3URWHLQ LHY /DWH(ORQJDWHG+\SRFRW\O LRR /HXFLQH5LFK5HSHDW
Lys /\VLQH
M 0RODLUH
MADS 0&0$JDPRXV'HILFLHQV65) MATH 0HSULQ$QG7UDII+RPRORJ\
MCM 0LQL&KURPRVRPH0DLQWHQDQFHSURWHLQFRPSOH[
MDM2 0XULQH'RXEOH0LQXWH MDMX 0'0UHODWHGSURWHLQ
me1, 2 ou 3 0RQRGLRXWULPHWK\ODWLRQ MEL26 0DWHUQDO(IIHFW/HWKDOLW\
MET 0HWK\O7UDQVIpUDVH MgCl2 &KORUXUHGH0DJQpVLXP
min PLQXWHV
m PLOOL
MS 0XUVDVKLJKHW6NRRJ
MSI 0XOWLFRS\6XSSUHVVRURI,5$
MUB 0HPEUDQHDQFKRUHG8ELTXLWLQIROG NaCl &KORUXUHGH6RGLXP
NaOH +\GUR[\GHGH6RGLXP
NEDD 1HXUDOSUHFXUVRUFHOO([SUHVVHG'HYHORSPHQWDOO\'RZQUHJXODWHG
Abréviations
3DJH_
NER 1XFOHRWLGH([FLVLRQ5HSDLU
Ni 1LFNHO
NP 1RQLGHW
N-ter 1WHUPLQDO
pb SDLUHGHEDVHV
PcG 3RO\FRPE*URXSH
PCNA 3UROLIHUDWLRQ&HOOXODU1XFOHDU$QWLJHQ PCR 3RO\PHUDVH&KDLQ5HDFWLRQ
PEG 3RO\(WK\OqQH*O\FRO
% 3RXUFHQWDJH
PHD 3ODQW+RPHR'RPDLQ Phe 3KHQ\ODODQLQH
PRC 3RO\FRPE5HSUHVVLYH&RPSOH[
p/v SRLGVYROXPH
PVDF 3RO\)OXRUXUH'H9LQ\OLGqQH
RBX 5LQJ%R;
RFP 5HG)OXRUHVFHQW3URWHLQ RB 5HWLQR%ODVWRPD
RBR 5%5HODWHG
RING 5HDOO\,QWHUHVWLQJ1HZ*HQH
RITS 51$LLQGXFHGWUDQVFULSWLRQDOVLOHQFLQJ RP 5HJXODWRU\3DUWLFOH
RPN 5HJXODWRU\3DUWLFOH1RQ$73DVH RT 5pWUR7UDQVFULSWLRQ
RT-PCR 5HDO7LPH3RO\PHUDVH&KDLQ5HDFWLRQ RUB 5HODWHG8ELTXLWLQOLNH
s VHFRQGHV
SDS 'RGpF\O6XOIDWHGH6RGLXP
SDS-Page 6'63RO\$FU\ODPLGH*HO(OHFWURSKRUHVLV SKP 63KDVH.LQDVH$VVRFLDWHG3URWHLQ SOCS-box 6XSSUHVVRURI&\WRNLQH6LJQDOOLQJSURWHLQ SPA 6XSSUHVVRURI3K\$
SPRF 6LQJOH3URWHLQ5LQJ)LQJHU Su(z) 6XSSUHVVRURI]HVWH
3DJH_
TAE 7ULV$FpWDWH('7$
TBS 7ULV%XIIHUHG6DOLQH TCA $FLGH7ULFKORURDFHWLTXH TCP 7UDQVFULSWLRQ&RXSOHG5HSDLU T-TBS 7ULWRQ7%6
TE 7ULV('7$
TIR1 7UDQVSRUW,QKLELWRU5HVLVWDQW TOC1 7LPLQJRI&DE([SUHVVLRQ Trp 7ULSWRSKDQH
Tyr 7\URVLQH
Ub 8ELTXLWLQH
UBA 8ELTXLWLQ$FWLYDWLQJHQ]\PH Ubc 8ELTXLWLQFRQMXJDWLQJ
UBL 8ELTXLWLQOLNH
UBP 8ELTXLWLQVSHFLILF3URFHVVLQJHQ]\PHV UCH 8ELTXLWLQ&DUER[\WHUPLQDOH+\GURODVH UFM 8ELTXLWLQIROGPRGLILHU
URM 8ELTXLWLQUHODWHGPRGLILHU
UV 8OWUD9LROHW
v/v YROXPHYROXPH
V 9ROWV
VHL 9RQ+LSSHO/LQGDX XP ;HURGHUPD3LJPHQWRVXP
XPC ;HURGHUPD3LJPHQWRVXPFRPSOHPHQWDWLRQJURXS&
YFP <HOORZ)OXRUHVFHQW3URWHLQ YPD <HDVWH[WUDFW3HSWRQH'H[WURVH
µ 0LFUR
INTRODUCTION
3DJH_
$WUDYHUVFHWWHLQWURGXFWLRQMHYRXVSURSRVHGHSDUFRXULUO¶pWDWDFWXHOGHVFRQQDLVVDQFHV GRQW QRXV GLVSRVRQV VXU XQ V\VWqPH GH GpJUDGDWLRQ GHV SURWpLQHV SDU OD PDFKLQHULH GH SURWpRO\VH XELTXLWLQH HW SURWpDVRPH6GpSHQGDQWH -H FRPPHQFHUDL SDU XQH LQWURGXFWLRQ JpQpUDOH VXU OD SURWpRO\VH XELTXLWLQHGpSHQGDQWH SXLV MH GpWDLOOHUDL SOXV DPSOHPHQW FKDTXH DFWHXUGHFHWWHYRLH
'DQVXQGHX[LqPHWHPSVMHYRXVGpWDLOOHUDLSOXVSDUWLFXOLqUHPHQWOHV(XELTXLQHOLJDVHVGH OD IDPLOOH GHV &8//,1 SRXU PH IRFDOLVHU ILQDOHPHQW VXU OHV GLIIpUHQWV U{OHV GX FRPSOH[H
&8/''% -H WHUPLQHUDL SDU XQ pWDW GHV FRQQDLVVDQFHV VXU OD IDPLOOH GHV JqQHV 06, 0XOWLFRS\6XSSUHVVRURI,5$TXLRQWIDLWO¶REMHWGHPDWKqVHGHGRFWRUDW
Figure 1Mécanisme général de la protéolyse ubiquitine et protéasome 26S-dépendante /D SURWpRO\VH XELTXLWLQHGpSHQGDQWH GpEXWH SDU O¶DFWLYDWLRQ GH O¶XELTXLWLQH GH PDQLqUH $73 GpSHQGDQWHSDUO¶HQ]\PH(RXHQ]\PHG¶DFWLYDWLRQVXLYLHGHVRQWUDQVIHUWVXUXQHHQ]\PH(RX HQ]\PHGHFRQMXJDLVRQHWILQDOHPHQWO¶DWWDFKHPHQWGHO¶XELTXLWLQHVXUODSURWpLQHFLEOHDYHFO¶DLGH G¶XQH HQ]\PH ( RX XELTXLWLQH OLJDVH /D SURWpLQH FLEOH PDUTXpH SDU O¶XELTXLWLQH VRXV IRUPH GH FKDvQHRXQRQHVWVRLWUHFRQQXHSDUOHSURWpDVRPH6HWGpJUDGpHSDUXQSURFHVVXV$73GpSHQGDQW VRLWUHFRQQXHSDUGHVHQ]\PHVGHGpXELTXLWLQ\ODWLRQ'8%/¶XELTXLWLQHHVWDORUVUHF\FOpH
Figure 2 Structure 3D de l’Ubiquitine de plante.
/HVO\VLQHV.SRXYDQWrWUHXWLOLVpHVSRXUODIRUPDWLRQGHVFKDvQHVGH SRO\XELTXLWLQH VRQW PRQWUpHV HQ URXJH /HV IHXLOOHWV ȕ VRQW SUpVHQWpV HQ YHUW HW OHV KpOLFHV Į HQ EOHX /D JO\FLQH &WHUPLQDOH * XWLOLVpH SRXU pWDEOLU OD OLDLVRQ FRYDOHQWH HQWUH O¶XELTXLWLQH HW OH UpVLGX O\VLQH GH OD SURWpLQHFLEOHHVWDXVVLLQGLTXpH+XDHW9LHUVWUD
3DJH_
I . La protéolyse ubiquitine dépendante
/DUpJXODWLRQGHODVWDELOLWpGHVSURWpLQHVSDUODSURWpRO\VHXELTXLWLQHGpSHQGDQWHSDUWLFLSH DX FRQWU{OH GH QRPEUHX[ SURFHVVXV SK\VLRORJLTXHV HW GpYHORSSHPHQWDX[ FKH] WRXV OHV HXFDU\RWHV &LHFKDQRYHU HW DO 6PDOOH DQG 9LHUVWUD 'DQV FH V\VWqPH GH UpJXODWLRQSOXVLHXUVDFWHXUVSDUWLFLSHQWjGLIIpUHQWHVpWDSHV
1. L’ubiquitinylation
/H PpFDQLVPH G¶XELTXLWLQ\ODWLRQ G¶XQH SURWpLQH UHSRVH VXU XQH UpDFWLRQ HQ FKDvQH FRQGXLVDQWjODIL[DWLRQG¶XQHSHWLWHSURWpLQHGH DDDSSHOpHXELTXLWLQHVXUXQHSURWpLQH FLEOHOHVXEVWUDW7URLVHQ]\PHVGLIIpUHQWHVSDUWLFLSHQWjODFRQMXJDLVRQGHO¶XELTXLWLQH7RXW G¶DERUGO¶HQ]\PHG¶DFWLYDWLRQ(WUDQVIHUWO¶XELTXLWLQHOLEUHVXUXQHHQ]\PHGHFRQMXJDLVRQ DSSHOpH(8QHIRLVO¶XELTXLWLQHDFWLYpHFHOOHFLHVWOLpHDXVXEVWUDWVXUXQH/\VLQHjO¶DLGH G¶XQH HQ]\PH GH OLDLVRQ j O¶XELTXLWLQH RX ( /HV SURWpLQHV FLEOHV RX VXEVWUDWV VRQW DORUV PRGLILpHV SDU PRQRXELTXLWLQ\ODWLRQ RX SRO\XELTXLWLQ\ODWLRQ /H VXEVWUDW SRO\XELTXLWLQ\Op HVW GLULJp YHUV OH SURWpDVRPH 6 VLqJH GH VD GpJUDGDWLRQ /¶XELTXLWLQH UHWURXYH VD OLEHUWp DILQ G¶HQWUHU GDQV XQ QRXYHDX F\FOH G¶XELTXLWLQDWLRQ Figure 1 +HUVNR HW &LHFKDQRYHU .RUQLW]HU HW &LHFKDQRYHU :HLVVPDQQ 6PDOOH HW 9LHUVWUD +XDHW9LHUVWUD
1.1L’ubiquitine
/¶XELTXLWLQHHVWXQSHWLWSRO\SHSWLGH©XELTXLWDLUHªF¶HVWjGLUHKDXWHPHQWFRQVHUYpFKH]
WRXWHV OHV HVSqFHV HXFDU\RWHV *ROGVWHLQ OD GpFRXYHUWH HQ FRPPH XQH KRUPRQH SHSWLGLTXH *ROGVWHLQ HW DO (Q &LHFKDQRYHU HW +HUVNR GpFRXYULUHQW XQ SRO\SHSWLGHGHDFLGHVDPLQpVLPSOLTXpGDQVXQV\VWqPHGHGpJUDGDWLRQGHVSURWpLQHV$73 GpSHQGDQW 3UL[ 1REHO GH &KLPLH /¶pTXLSH GH :LONLQVRQ LGHQWLILD FH SRO\SHSWLGH FRPPHO¶XELTXLWLQHHQ:LONLQVRQHWDO
/D SURGXFWLRQ G¶XELTXLWLQH FRPPHQFH SDU OD V\QWKqVH G¶XQ SUpFXUVHXU VRXV IRUPH GH SRO\XELTXLWLQH RX HQ IXVLRQ DYHF XQH SURWpLQH ULERVRPDOH 8QH PDWXUDWLRQ GX SURGXLW GH
Figure 3Structure 3D des Ub et Ub-like chez les procaryotes et les eucaryotes./DSUHPLqUHLOOXVWUDWLRQGH OD VpULH HVW XQH UHSUpVHQWDWLRQ VFKpPDWLTXH GHV DUUDQJHPHQWV GHV IHXLOOHWV E HW KpOLFHV D GDQV XQ RUGUHEEDEEEDSSHOpH ©EJUDVSª FDU OHVIHXLOOHWVEHPSRLJQHQWOLWWpUDOHPHQW O¶KpOLFH D VRXYHQW XQLTXH $ O¶H[FHSWLRQGH08%HW+8%OHEUDV OLEUH GH O¶H[WUpPLWp &WHUPLQDOH VH WHUPLQHDYHFOHUpVLGXJO\FLQHG¶DSUqV 9LHUVWUD
Figure 4 : Représentation schématique de l’enzymologie, des cibles et des fonctions des Ub et UBL.
/HV QRPV GHV SURWpLQHV GHV GLIIpUHQWV FRPSRVDQWV LGHQWLILpV VRQW LQGLTXpV HQ UHVSHFWDQW OD QRPHQFODWXUH XWLOLVpH FKH]
Arabidopsis thaliana. /HV SRLQWV G¶LQWHUURJDWLRQV LQGLTXHQW TX¶XQH WHOOH DFWLYLWpGHYUDLWH[LVWHUPDLVTXHODSURWpLQH Q¶D SDV pWp LGHQWLILpH G¶DSUqV 9LHUVWUD
3DJH_
WUDGXFWLRQGRQQHUDO¶XELTXLWLQHOLEUH&DOOLVHWDO6DVWUXFWXUH'SHUPHWGHPHWWUHHQ pYLGHQFHXQHVWUXFWXUHJOREXODLUH©8E)ROGªDYHFIHXLOOHWVȕHWXQHKpOLFHĮFigure 2'H QRPEUHXVHV OLDLVRQV K\GURJqQHV SDUWLFLSHQW j VD JUDQGH VWDELOLWp FH TXL OD SURWqJH GH OD SURWpRO\VHHWLQFLWHjVRQUHF\FODJH(OOHSHXWpWDEOLUGHVOLDLVRQVFRYDOHQWHVDYHFOHVDFWHXUV GHODSURWpRO\VHXELTXLWLQHGpSHQGDQWHHWOHVVXEVWUDWVJUkFHjXQHJO\FLQHVHWURXYDQWHQVRQ H[WUpPLWp&WHUPLQDOH
1.1.1 Fixation de l’ubiquitine
/¶XELTXLWLQHVHIL[HVXUXQHO\VLQHGHODSURWpLQHFLEOHHQXWLOLVDQWVDJO\FLQHWHUPLQDOH HOOHSHXWDXVVLpWDEOLUG¶DXWUHVOLDLVRQVDYHFXQHRXSOXVLHXUVPROpFXOHVG¶XELTXLWLQHDILQGH IRUPHUGLIIpUHQWVW\SHVGHFKDvQHVGHSRO\XELTXLWLQH3HQJHWDO/¶XELTXLWLQHSRVVqGH O\VLQHVLQWHUQHVGRQWOHVSRVLWLRQVVRQWFRQYHUVpHV/\V /\V/\V/\V/\VHW /\V
/HV GLIIpUHQWV W\SHV G¶XELTXLWLQ\ODWLRQ DLQVL TXH OHXU PpFDQLVPH GH UpJXODWLRQ VHURQW GpWDLOOpVGDQVOHFKDSLWUH
1.2 Les protéines « ubiquitine-like »
/¶XELTXLWLQHDLQVLTXHOHVXELTXLWLQHOLNHVHFDUDFWpULVHQWSDUOHXUVWUXFWXUH ©8E)ROGª 6RXYHQWHOOHVSUpVHQWHQWGHSHWLWHVVpTXHQFHVLGHQWLTXHVjO¶XELTXLWLQHHWSRVVqGHQWXQHWDLOOH YDULDQWHQWUHHWDFLGHVDPLQpVFigure 3&KH]OHVSODQWHVFHVXELTXLWLQOLNHLQFOXHQW FRXUDPPHQW+8% ©+RPRORJ\RI8Eª58% ©UHODWHGWR8EªDXVVLQRPPp1('' 6802 ©6PDOO 8EOLNH 0RGLILHUª $7* ©$XWRSKDJ\ $7* 08% ©0HPEUDQH DQFKRUHG XELTXLWLQIROG SURWHLQVª 8)0 ©XELTXLWLQIROG PRGLILHUª 850 ©8ELTXLWLQ UHODWHG PRGLILHUª PDLV DXVVL SOXVLHXUV DVVRUWLPHQWV GH SURWpLQHV TXL SUpVHQWHQW XQ UHSOLHPHQWVWUXFWXUDOVLPLODLUH
/DSOXSDUWGHFHVXELTXLWLQOLNHHPSUXQWHQWXQHFDVFDGHGHUpDFWLRQHQ]\PDWLTXHGHW\SH ( ( HW ( WRXW FRPPH O¶XELTXLWLQH PDLV O¶HQ]\PH ( Q¶HVW SDV WRXMRXUV SUpVHQWH RX LGHQWLILpH &HSHQGDQW FRPSDUpH j O¶XELTXLWLQH OD IL[DWLRQ GH SOXVLHXUV GH FHV SURWpLQHV VXU OHXUVSURWpLQHVFLEOHVQ¶DERXWLWSDVGLUHFWHPHQWjOHXUGpJUDGDWLRQSDUOHSURWpDVRPH6PDLV
Introduction
3DJH_
SOXW{WjXQHUpJXODWLRQGHOHXUDFWLYLWpFigure 4/HVFDVSDUWLFXOLHUGH58%1(''VHURQW GLVFXWpVHQSDUDJUDSKH
1.3 Les principaux acteurs de l’ubiquitinylation : les enzymes E1, E2 et E3 1.3.1 Les enzymes E1: activation de l’ubiquitine
/¶DFWLYDWLRQ GH O¶XELTXLWLQH HVW OD SUHPLqUH pWDSH GH OD FDVFDGH GH FRQMXJDLVRQ HOOH V¶HIIHFWXHSDUO¶HQ]\PHPRQRPpULTXH(YLDXQHUpDFWLRQLQWUDPROpFXODLUH$73GpSHQGDQWH +HUVNRHWDO(OOHDJLWVXUO¶XELTXLWLQHHQV¶\OLDQWSDUXQHOLDLVRQWKLRHVWHUDYHF XQH GH VHV F\VWpLQHV FRQVHUYpHV /D VSpFLILFLWp GX VXEVWUDW FLEOH Q¶HVW TXH WUqV IDLEOHPHQW LPSDFWpHSDUOHVHQ]\PHV(HWFHOOHVFLSHXYHQWrWUHXWLOLVpHVSDUGHQRPEUHXVHVHQ]\PHV (&KH]Arabidopsis thalianaVHXOVGHX[JqQHVFRGDQWSRXUGHVHQ]\PHV(VRQWFRQQXV 8%$HW8%$WDQGLVTX¶XQVHXODpWpLGHQWLILpFKH]Saccharomyces cerevisiae%DFKPDLUHW DO9LHUVWUD
1.3.2 Les enzymes E2 : conjugaison de l’ubiquitine
8QH IRLV O¶XELTXLWLQH DFWLYpH SDU O¶HQ]\PH ( FHOOHFL HVW WUDQVSRUWpH SDU O¶HQ]\PH ( YHUV O¶HQ]\PH GH OLDLVRQ GH O¶XELTXLWLQH QRPPpH ( YLD XQH UpDFWLRQ GH WUDQVHVWpULILFDWLRQ
&HVHQ]\PHVRQWFRPPHSRLQWFRPPXQGHSUpVHQWHUXQGRPDLQHFRQVHUYp8EF©XELTXLWLQ FRQMXJDWLQJªGHDFLGHVDPLQpVIRUPDQWOHF°XUFDWDO\WLTXHDXVHLQGXTXHOVHWURXYHOH VLWHGHOLDLVRQjO¶XELTXLWLQH'HSOXVOHVHQ]\PHV(FRPPHOHV(RQWpWpLGHQWLILpHVFKH]
WRXWHVOHVHVSqFHV$ORUVTXHOHVHQ]\PHV(QHSDUWLFLSHQWSDVjODUHFRQQDLVVDQFHVSpFLILTXH GHVSURWpLQHVFLEOHVOHVHQ]\PHV(SRVVqGHQWXQHVSpFLILFLWpG¶LQWHUDFWLRQDYHFOHVHQ]\PHV (DXJPHQWDQWDLQVLFRQVLGpUDEOHPHQWOHQRPEUHGH JqQHV FRGDQWSRXU HOOHV /HVHQ]\PHV ( VRQW HQYLURQ DX QRPEUH GH FKH]Arabidopsis thaliana HW GH FKH]S.cerevisiae
%DFKPDLUHWDO9LHUVWUD
1.3.3 Les enzymes E3 : liaison de l’ubiquitineau substrat
/HV HQ]\PHV ( UHSUpVHQWHQW OH GHUQLHU PDLOORQ LQWHUYHQDQW GDQV OD FDVFDGH G¶XELTXLWLQ\ODWLRQ HW VRQW UHVSRQVDEOHV GH O¶LGHQWLILFDWLRQ VSpFLILTXH GHV VXEVWUDWV /HV (
3DJH_
SHXYHQW rWUH UHJURXSpHV HQ FODVVHV OHV HQ]\PHV PRQRPpULTXHV j GRPDLQH +(&7
©+RPRORJRXVWR($3&WHUPLQXVªHWOHVHQ]\PHV(jPRWLI©5,1*5HDOO\,QWHUHVWLQJ 1HZ *HQH ILQJHUª HQ IRQFWLRQ GX PpFDQLVPH GH WUDQVIHUW GH O¶XELTXLWLQH YHUV OD SURWpLQH FLEOH 'DQV OH FDV GHV HQ]\PHV ( j GRPDLQH +(&7 O¶XELTXLWLQH HVW SULVH HQ FKDUJH SK\VLTXHPHQWSDUXQHF\VWpLQHGHO¶HQ]\PHDYDQWWUDQVIHUWVXUODSURWpLQHFLEOH$ORUVTXHOHV HQ]\PHV ( j PRWLI ©5,1* ILQJHUª SHUPHWWHQW GH UDSSURFKHU OH VXEVWUDW HW O¶XELTXLWLQH SRUWpHSDUO¶HQ]\PH(,OH[LVWHSOXVLHXUVW\SHVG¶HQ]\PHV(jPRWLI©5,1*ILQJHUªOHV HQ]\PHV PRQRPpULTXHV HW FHOOHV TXL DJLVVHQW VRXV IRUPH GH FRPSOH[H PXOWLSURWpLTXH -DFNVRQHWDO3DUPLOHVFRPSOH[HVPXOWLSURWpLTXHVFHX[SRVVpGDQWXQHVRXVXQLWpj GRPDLQH&XOOLQHVRQWOHVSOXVpWXGLpHVHWG¶HQWUHHX[RQWpWpFDUDFWpULVpVFKH]Arabidopsis thaliana OH FRPSOH[H 9%& Q¶H[LVWDQW SDV FKH] Arabidopsis thaliana Figure 5 ,O HVW LPSRUWDQW GH QRWHU TXH FHV FRPSOH[HV MRXHQW WRXV XQ U{OH HVVHQWLHO FKH] OD SODQWH /¶LQDFWLYDWLRQLQGLYLGXHOOHGHFKDFXQGHFHVFRPSOH[HVSDUPXWDJHQqVHFRQGXLWjXQHOpWDOLWp JDPpWRSK\WLTXHRXHPEU\RQQDLUHFKH]ODSODQWHPRGqOHArabidopsis.
1.3.3.1 Les E3 ligases à domaine HECT
/HV(OLJDVHVjGRPDLQH+(&7VRQWGHVHQ]\PHVPRQRPpULTXHVGpILQLHVjO¶RULJLQHSDUOD SUpVHQFH G¶XQ GRPDLQH FRQVHUYp GH DFLGHV DPLQpV HQ &WHUPLQDO GH OD SURWpLQH DSSHOp GRPDLQH+(&7+HUVFKNRDQG&LHFKDQRYHU&HGRPDLQHFRQWLHQWOHVLWHGHOLDLVRQDX FRPSOH[H8E(DLQVLTX¶XQHF\VWpLQHFRQVHUYpHTXLDFFHSWHO¶XELTXLWLQHFHTXLSHUPHWWUDGH IRUPHUXQHOLDLVRQ WKLRHVWHUDYHFO¶HQ]\PH( DYDQW G¶XELTXLWLQ\OHUOH VXEVWUDW Figure 5 /HV ( OLJDVHV j GRPDLQH +(&7 IRQW JpQpUDOHPHQW SOXV GH N'D 3DU H[HPSOH FKH]
Arabidopsis thalianaGHX[(OLJDVHVjGRPDLQH+(&7RQWXQHWDLOOHGHN'D%DWHVHW 9LHUVWUD /H GRPDLQH 1WHUPLQDO FRQWLHQW SOXVLHXUV PRWLIV G¶LQWHUDFWLRQ SURWpLQH SURWpLQH WHO TXH OH PRWLI :: OH GRPDLQH ©5,1*ILQJHU RX HQFRUH XQ GRPDLQH ©FRLOHG FRLOª 8QH GHV ( j GRPDLQH +(&7 OD PLHX[ FDUDFWpULVpH FKH] O¶+RPPHV HVW 1(''
©1HXURQDOSUHFXUVRUFHOOH[SUHVVHGGHYHORSPHQWDOO\GRZQUHJXODWHGªFRQWHQDQWGHVPRWLIV ::&HVPRWLIVYRQWOXLSHUPHWWUHGHUHFRQQDLWUHVHVSULQFLSDX[VXEVWUDWVTXHVRQWOHVVRXV XQLWpVGXFDQDOVRGLTXH(1D&©(SLWKHOLDO1D&KDQQHOª$EULHOHW6WDXE
$ORUV TXH FKH] OD OHYXUH HW FKH] O¶KRPPH LO H[LVWH UHVSHFWLYHPHQW XQH HW SOXV GH ( OLJDVHVjGRPDLQH+(&7FKH]Arabidopsis thalianaRQHQGpQRPEUH'RZQHVHWDO
Figure 5 : Organisation des différentes ubiquitine E3 ligases
/HV(VOLJDVHVjGRPDLQH+(&7HQFDGUpURXJHSUHQQHQWHQFKDUJHSK\VLTXHPHQWODSURWpLQHFLEOH HWIRUPHQWXQHOLDLVRQFRYDOHQWHDYHFO¶XELTXLWLQH8E/HV(VOLJDVHVjGRPDLQH©5,1*)LQJHUª HQFDGUp EUXQ SHUPHWWHQW GH UDSSURFKHU O¶HQ]\PH ( TXL D IL[p O¶8E HW OD SURWpLQH FLEOH SRXU WUDQVIpUHUO¶XELTXLWLQHGLUHFWHPHQWVXUODFLEOH/DGHUQLqUHFODVVHUHJURXSHOHV(VjGRPDLQH©5,1*
)LQJHUª GH W\SH PXOWLPpULTXH &HV FRPSOH[HV VRQW DUFKLWHFWXUpV DXWRXU GHV GLIIpUHQWHV SURWpLQHV j GRPDLQH&8//,1(MRXDQWOHU{OHGHFKDUSHQWHLOVVRQWQRPPpV&5/SRXU&8//,15,1*/LJDVH /DVRXVXQLWp$3&GXFRPSOH[HPXOWLSURWpLTXH$3&&HVWSK\ORJpQpWLTXHPHQWUHOLpHDX[SURWpLQHV
&8//,1(V
3DJH_
3DUPL OHV H[HPSOHV UpFHQWV RQ SHXW FLWHUUPL5 TXL XELTXLWLQH XQ IDFWHXU GH WUDQVFULSWLRQ :5.<FRQGXLVDQWjVDGpJUDGDWLRQSDUOHSURWpDVRPH6:5.<MRXHXQU{OHGDQVOH FRQWU{OHGHODVpQHVFHQFHIROLDLUH0LDRDQG=HQWJUDI
1.3.3.2 Les E3 ligases à domaine « RING finger »
/HV(OLJDVHVjGRPDLQH©5,1*ILQJHUªVRQWOLEUHPHQWGpILQLHVFRPPHXQHFROOHFWLRQGH SRO\SHSWLGHV VXSSRUWDQW XQ PRWLI GH VLJQDWXUH 5,1* ILQJHU &H PRWLI HVW IRUPp G¶XQH VpTXHQFHFRQVHQVXV&+&RX&;&;&;+;&+;&;&;&ROHV UpVLGXVF\VWpLQH&HWKLVWLGLQHV+IL[HQWOHVLRQV=LQF%RUGHQ&HPRWLIDpWpGpFULW SRXUODSUHPLqUHIRLVHQFRPPHXQPRGXOHSDUWLFLSDQWDX[LQWHUDFWLRQVSURWpLQHSURWpLQH RXSURWpLQHDFLGHQXFOpLTXH/RYHULQJHWDO&RPPHLOOXVWUpVXUODILJXUHO¶HQ]\PH (YDLQWHUDJLUDYHFFHPRWLIHWDLQVLFHWHQVHPEOHSDUWLFLSHDXSURFHVVXVG¶XELTXLWLQ\ODWLRQ GXVXEVWUDWVDQVSDVVHUSDUXQHOLDLVRQFRYDOHQWHGHO¶XELTXLWLQHVXUO¶HQ]\PH(
/HV WUDYDX[ GH /RYHULQJ HW DO RQW SHUPLV G¶LGHQWLILHU XQH XELTXLWLQH( OLJDVH j GRPDLQH ©5,1* ILQJHUª DSSHOpH $2 FDSDEOH G¶XELTXLWLQ\ODWLRQin vitro HQ SUpVHQFH G¶HQ]\PHV(HW(&HWWHFDUDFWpULVDWLRQDSHUPLVG¶LGHQWLILHUG¶DXWUHVSURWpLQHVjGRPDLQH
©5,1*ILQJHUªFDSDEOHVHOOHVDXVVLG¶XELTXLWLQ\ODWLRQ(GpSHQGDQWH/RULFNHWDO
&HSHQGDQW LO SDUDvW LPSRVVLEOH G¶DIILUPHU TXH WRXWHV OHV SURWpLQHV SUpVHQWDQW XQ GRPDLQH
©5,1*ILQJHUªSRVVqGHQWXQHDFWLYLWp(OLJDVH8QJUDQGQRPEUHGHJqQHVFRGDQWSRXUGHV SURWpLQHV SUpVHQWDQW OH PRWLI ©5,1*ILQJHUª D pWp PLV HQ pYLGHQFH GDQV OHV GLIIpUHQWV JpQRPHV VpTXHQFpV PDLV OHVTXHOOHV VRQW FDSDEOHV G¶XELTXLWLQ\ODWLRQUHVWH j GpILQLU 'HX[
FODVVHVGH(VOLJDVHVjGRPDLQH©5,1*ILQJHUªSHXYHQWrWUHGpILQLHV
ODSUHPLqUHHVWFRQVWLWXpHGHV(OLJDVHVSUpVHQWDQWXQLTXHPHQWOHGRPDLQH©5,1*ILQJHUª HOOHVVRQWDSSHOpHV635)RX©6LQJOH3URWHLQ5LQJ)LQJHUª
OD GHX[LqPH FODVVH HVW FRQVWLWXpH G¶( OLJDVHV VH SUpVHQWDQW VRXV IRUPH GH FRPSOH[HV PXOWLPpULTXHVRODSURWpLQHjGRPDLQH©5,1*ILQJHUªQHFRQVWLWXHTX¶XQHVRXVXQLWp/HV SOXVpWXGLpHVGHFHV(OLJDVHVVRQWFHOOHVFRQWHQDQWOHGRPDLQH&XOOLQH&HV(OLJDVHVVRQW FRQVWUXLWHV DXWRXU GH OD SURWpLQH &8//,1( YpULWDEOH FKDUSHQWH PROpFXODLUH GH FHV FRPSOH[HV VXU ODTXHOOH YLHQQHQW VH JUHIIHU GHX[ PRGXOHV IRQFWLRQQHOV XQ PRGXOH GH UHFRQQDLVVDQFH VSpFLILTXH GH OD SURWpLQH FLEOH HW XQ PRGXOH FDWDO\WLTXH TXL FRQWLHQW OD
Introduction
3DJH_
SURWpLQH j GRPDLQH ©5,1* ILQJHUª &H GHUQLHU DFFXHLOOH O¶HQ]\PH ( SRUWDQW O¶XELTXLWLQH DFWLYH&KH]OHVHXFDU\RWHVOHV&8//,1(6RQWpWpLGHQWLILpHVDXQRPEUHGH&8/&8/
&8/&8/&8/HW&8/DLQVLTXHGHV&8//,1(6OLNH$3&&KDFXQHHVWODEDVH GHFRPSOH[HVPXOWLPpULTXHVIRUPpVGHSOXVLHXUVVRXVXQLWpVDSSHOpV&5/RX ©&XOOLQ5LQJ /LJDVHVª 'LDV HW DO :LOOHPV HW DO 3HWURVNL HW 'HVKDLHV 3DUPL FHV FRPSOH[HV SOXVLHXUV RQW pWp LGHQWLILpV FDUDFWpULVpV HW FODVVpV HQ IRQFWLRQ GH OD &8//,1(
TX¶LOVUHQIHUPHQW
1.3.3.2.1 Les E3 ligases de type CRL
1.3.3.2.1.1 Le complexe APC/C ou « Anaphase Promoting Complex/Cyclosome »)
/¶$3&&HVWOHSOXVJUDQGFRPSOH[H(OLJDVHGpFULWDFWXHOOHPHQW&KH]OHVYHUWpEUpVLO HVWFRPSRVpGHVRXVXQLWpV3HWHUVDORUVTXHFKH]OHVOHYXUHVVRXVXQLWpVRQW pWpGpFULWHV<RRQHWDO/HVIRQFWLRQVLQGLYLGXHOOHVGHFKDTXHVRXVXQLWpQHVRQWSDV HQFRUH WRXWHV FRQQXHV WRXWHIRLV XQ PRGqOH VWUXFWXUDO GH VRQ RUJDQLVDWLRQ D pWp SURSRVp 7KRUQWRQ HW DO /H PRGXOH FDWDO\WLTXH PLQLPDO GX FRPSOH[H $3&& FRPSUHQG OD VRXVXQLWp$3&XQPHPEUHpORLJQpGHODIDPLOOHGHV&8//,1(6=DFKDULDHHWDOHW OD VRXVXQLWp $3& TXL SRVVqGH OH PRWLI ©5,1*±ILQJHUª /H FRPSOH[H $3&& FRQWLHQW DXVVL WURLV VRXVXQLWpV HVVHQWLHOOHV SRVVpGDQW GHV UpSpWLWLRQV GX GRPDLQH G¶LQWHUDFWLRQ SURWpLQHSURWpLQH 735V WHWUDWULFRSHSWLGH UHSHDW $3&&'& $3&&'& HW
$3&&'& $3&&'& HVW LPSOLTXpH GDQV OD OLDLVRQ DYHF OD VRXVXQLWp DFWLYDWULFH
&'+ ©&HUHYLVLDH 'LP +RPRORJRXVª 9RGHPDLHU HW DO .UDIW HW DO /D SKRVSKRU\ODWLRQGHFHVVRXVXQLWpVGXUDQWODPLWRVHHVWQpFHVVDLUHjO¶DFWLYDWLRQGXFRPSOH[H
$3&&5XGQHUHW0XUUD\.UDIWHWDO(QSOXVGHFHVFRPSRVDQWVPDMHXUVOH FRPSOH[H $3&& QpFHVVLWH GHV SURWpLQHV FRDFWLYDWULFHV WHOOHV TXH &'&±),==< RX
&'+),==<5(/$7('TXLDFWLYHOHFRPSOH[H$3&&GXUDQWOHF\FOHFHOOXODLUH&'&
DFWLYH OH FRPSOH[H $3&& HQ GpEXW GH PLWRVH WDQGLV TXH &'+ SUHQG OH UHODLV HQ ILQ GH PLWRVHMXVTX¶jODWUDQVLWLRQ*6*ORW]HUHWDO)DQJHWDO3HWHUV&HV FRIDFWHXUVIRQWSDUWLHG¶XQHIDPLOOHGHSURWpLQHVTXLSRVVqGHQWHQOHXUSDUWLH&WHUPLQDOHVHSW UpSpWLWLRQV GX W\SH :' 3DJH HW +LHWHU '¶DXWUHV PRWLIV SOXV SDUWLFXOLHUV RQW pWp FDUDFWpULVpV GDQV OHV FLEOHV GX FRPSOH[H $3&& OD 'ER[ 5;;/;;;1 SUpVHQWH
3DJH_
PDMRULWDLUHPHQW GDQV OHV VXEVWUDWV $3&&'& 3IOHJHU HW .LUVFKQHU HW OD .(1ER[
.(1;;;1' SOXV VSpFLILTXHPHQW UHFRQQXH SDU OH FRPSOH[H $3&&'+ /HLVPDQQ HW /HKQHU
'XUDQWODGHUQLqUHGpFHQQLHEHDXFRXSG¶DWWHQWLRQDpWpSRUWpHVXUOHU{OHGXFRPSOH[H$3&&
GDQVODUpJXODWLRQGXF\FOHFHOOXODLUH&RPPHVRQQRPO¶LQGLTXHO¶$3&&HVWSULQFLSDOHPHQW UHTXLV SRXU LQGXLUH OD SURJUHVVLRQ HW OD VRUWLH GH OD PLWRVH HQ MRXDQW VXU OD SURWpRO\VH GHV GLIIpUHQWV UpJXODWHXUV GX F\FOH FHOOXODLUH WHOV TXH 3'66(&85,1( HW OD F\FOLQH % /D GpJUDGDWLRQ GH 3'66(&85,1( HVW UHTXLV SRXU OD VpSDUDWLRQ GHV FKURPDWLGHV V°XUV 8KOPDQQHWDO<DQDJLGDDORUVTXHODGpJUDGDWLRQGHVF\FOLQHVPLWRWLTXHV%
GpFOHQFKHODFKXWHGHO¶DFWLYLWpNLQDVH&'.©&\FOLQGHSHQGHQW.LQDVHª&HFLLQGXLWOHV pYpQHPHQWVQpFHVVDLUHVjODVRUWLHGHODPLWRVHFRPPHOHGpVDVVHPEODJHGXIXVHDXPLWRWLTXH ODGpFRQGHQVDWLRQGHVFKURPRVRPHVPDLVDXVVLODF\WRFLQqVHHWODUHIRUPDWLRQGHO¶HQYHORSSH QXFOpDLUH0XUUD\HW.LUVFKQHU/XFHWDO*DOODQWHW1LJJ6XUDQDHWDO
3OXVLHXUV DXWUHV SURWpLQHV SDUWLFLSDQW j OD UpJXODWLRQ GX F\FOH FHOOXODLUH VRQW GHV FLEOHV GX FRPSOH[H $3&& FKH] OHV OHYXUHV HW OHV PDPPLIqUHV 3DUPL HOOHV RQ SHXW FLWHU OD SURWpLQH NLQHVLQH;.,'LPSOLTXpHGDQVOHPRXYHPHQWGHVFKURPRVRPHV)XQDELNLHW0XUUD\
/HYHVTXHHW&RPSWRQGHX[SURWpLQHVPRWHXUV.,3HW&,1*RUGRQHW5RRI +LOGHEUDQW HW +R\W HW OD SURWpLQH DVVRFLpH DX IXVHDX PLWRWLTXH $6( VRQW LPSOLTXpV GDQVODIRUPDWLRQGXIXVHDXFHQWUDOHWODF\WRFLQqVH/HFRPSOH[H$3&&UHVWHDFWLIGXUDQWOD SKDVH*DILQGHUHVWUHLQGUHO¶DFFXPXODWLRQGHVF\FOLQHVPLWRWLTXHVPDLVVRQLQDFWLYDWLRQHVW QpFHVVDLUHjO¶HQWUpHHQSKDVH6,PLJHUHW1DVP\WK3OXVLHXUVPpFDQLVPHVSDUWLFLSHQW j O¶LQDFWLYDWLRQ GX FRPSOH[H $3&& /¶LQKLELWHXU (0, ©(DUO\ 0LWRWLF ,QKLELWRU ª V¶DVVRFLHDXFRPSOH[H$3&&HQSKDVH6HW*$XFRXUVGHODPLWRVHO¶$3&&HVWODFLEOH G¶XQ PpFDQLVPH GH VXUYHLOODQFH DSSHOp 6$& ©6SLQGOH $VVHPEO\ &KHFNSRLQWª TXL DVVXUH O¶HQWUpH HQ DQDSKDVH XQLTXHPHQW ORUVTXH WRXV OHV FKURPRVRPHV VRQW DWWDFKpV DX IXVHDX PLWRWLTXH
&KH]Arabidopsis thaliana WRXWHV OHV VRXVXQLWpV GX FRPSOH[H $3&& VRQW FRGpHV SDU GHV JqQHV XQLTXHV VDXI $3&&'& &DSURQ HW DO 6XU OD EDVH G¶KRPRORJLH GH VpTXHQFHKXLWDFWLYDWHXUVGHO¶$3&&RQWpWpLGHQWLILpVFLQT&'&.HYHLHWDOHW WURLV&&6&'+7DUD\UHHWDO3OXVUpFHPPHQWSOXVLHXUVLQKLELWHXUVGHO¶$3&&
Introduction
3DJH_
RQWpWpILQDOHPHQWpWpLGHQWLILpVJUkFHjGHVWHFKQLTXHVGHSURWpRPLTXH,ZDWDHWDO +H\PDQQ HW DO (OR\ HW DO 3OXVLHXUV WUDYDX[ RQW QRQ VHXOHPHQW GpPRQWUp O¶LPSRUWDQFH GX FRPSOH[H $3&& GDQV OD UpJXODWLRQ GX F\FOH FHOOXODLUH FKH] OHV SODQWHV
*HQVFKLNHWDO&HEROODHWDO&ULTXLHWDO.ZHHHW6XQGHUDVDQ PDLV pJDOHPHQW VRQ LPSOLFDWLRQ GDQV O¶HQGRUHGXSOLFDWLRQ YLD O¶DFWLYDWHXU &'+ FKH]
Medicago HWArabidopsis thaliana &HEROODHWDO0DUURFFRHWDO 1.3.3.2.1.2 Le complexe SCF ou CRL1
/HFRPSOH[H6&)OHPLHX[FDUDFWpULVpSDUPLOHV(OLJDVHV&DUGR]RHW3DJDQRHVW FRQVWLWXp GH SRO\SHSWLGHV TXL DVVRFLpV SRVVqGHQW XQH DFWLYLWp ( XELTXLWLQH OLJDVH /H FRPSOH[H6&)DpWpLQLWLDOHPHQWLGHQWLILpGDQVODOHYXUHFRPPHpWDQWXQUpJXODWHXUFOpGHOD WUDQVLWLRQ *6 GX F\FOH FHOOXODLUH /¶HQWUpH HQ SKDVH 6 HVW VWULFWHPHQW FRQWU{OpH SDU OH
©FKHFNSRLQWª DSSHOp OH SRLQW 67$57 FKH] OD OHYXUH $X QLYHDX PROpFXODLUH LO V¶DJLW GH O¶LQKLELWHXU6,&TXLUHVWUHLQWO¶DFWLYLWpNLQDVH&'.&HYHUURXHVWOHYpJUkFHjO¶DFWLYLWpGH SRO\XELTXLWLQ\ODWLRQ GX FRPSOH[H 6&)&'& /H QRP GX FRPSOH[H 6&) EDVp VXU OH QRP GH WURLV GH VHV VRXVXQLWpV 6.3 ©6SKDVH .LQDVHDVVRFLDWHG 3URWHLQ ª &'& RX
&8//,1(HWSURWpLQH©)ER[ªDSSHOpHDLQVLHQUDLVRQGHODSURWpLQHLQLWLDOHPHQWGpFULWH OD &\FOLQH ) %DL HW DO /D TXDWULqPH VRXVXQLWp 5%; ©5LQJ%2; SURWHLQ ª RX 52&©5HJXODWRU2I&XOOLQªFRQWLHQWXQGRPDLQH©5,1*ILQJHUª.DPXUDHWDO 2KWD HW DO 6HRO HW OD 6NRZ\UD HW DO /H FRPSOH[H GH EDVH GHV 6&) FRPSRUWH OHV VRXVXQLWpV 6.3 &8//,1( HW 5%; /D GLYHUVLWp GH FHV FRPSOH[HV SURYLHQWGHVGLIIpUHQWHVSURWpLQHV©)ER[ªUHFUXWpHV/HFKQHUHWDO
/DSDUWLH&WHUGHODSURWpLQH&8//,1(LQWHUDJLWDYHFODVRXVXQLWpFDWDO\WLTXH5%;HWVD SDUWLH1WHUDYHFODVRXVXQLWp6.3TXLIDLWOHOLHQYLDOHPRWLI)ER[G¶HQYLURQDFLGHV DPLQpV j PDMRULWp K\GURSKREH GH OD SURWpLQH ©)ER[ª TXL IRUPHQW OH PRGXOH GH UHFRQQDLVVDQFH VSpFLILTXH GHV VXEVWUDWV 6NRZ\UD HW DO 'HV DQDO\VHV JpQRPLTXHV PRQWUHQW TXH OHV HXFDU\RWHV FRQWLHQQHQW GH QRPEUHX[ FRPSOH[HV 6&) FH QRPEUH HVW FURLVVDQW DYHF OD FRPSOH[LWp GH O¶RUJDQLVPH $ORUV TXH OD OHYXUH SRVVqGH XQ JqQH 6.3 Arabidopsis thalianaFRPSWHJqQHVFRGDQWSRXU6.3DSSHOpV$6.©$UDELGRSVLV6.3 OLNHSURWHLQ/D&8//,1HW5%;VRQWTXDQWjHX[FRGpVSDUGHVJqQHVXQLTXHV/HFKQHUHW DO/LXHWDO7DNDKDVKLHWDOArabidopsis thalianaFRPSRUWHSDVPRLQV GHJqQHVFRGDQWSRXUGHVSURWpLQHV©)ER[ª*DJQHHWDOVRLWIRLVSOXVTXH
3DJH_
FKH]O¶KRPPH&HWWHGLYHUVLWpSURYLHQWGHODSDUWLH&WHUPLQDOHTXLFRQWLHQWXQDXWUHGRPDLQH G¶LQWHUDFWLRQ SURWpLQHSURWpLQH VSpFLILTXH GH OD UHFRQQDLVVDQFH GHV VXEVWUDWV &HV GLIIpUHQWV GRPDLQHV SHUPHWWHQW G¶pWDEOLU GLIIpUHQWHV IDPLOOHV GHV UpSpWLWLRQV ©:'ª GHV /55
©/HXFLQH5LFK5HSHDWªRXHQFRUHGHV©.HOFKUHSHDWª$QGUDGHHWDO&HQFLDUHOOLHW DO .XURGD HW DO &RXSOpV j OD SUpVHQFH GH VRXVXQLWpV &8//,1 HW $6.V GLVWLQFWHV $UDELGRSVLV D DLQVL OH SRWHQWLHO GH JpQpUHU XQH FROOHFWLRQ TXDVL LQILQLH GH VRXV W\SHVGHFRPSOH[HV6&)FKDFXQD\DQWGHVVSpFLILFLWpVSURSUHVHWRXGHUpJXODWLRQ/LXHWDO 7DNDKDVKLHWDO&HVK\SRWKqVHVLPSDFWHQWGLUHFWHPHQWODELRORJLHGHVSODQWHV HWRQWUDSLGHPHQWpWppWD\pHVSDUO¶DFFXPXODWLRQGHGRQQpHVH[SpULPHQWDOHVGpPRQWUDQWTXH GHVSURWpLQHV)ER[VSpFLILTXHVVRQWLPSOLTXpHVGDQVXQHPXOWLWXGHG¶DFWLYLWpVWHOOHVTXHOHV UpSRQVHV KRUPRQDOHV OD GpIHQVH FRQWUH GHV SDWKRJqQHV OD GLYLVLRQ FHOOXODLUH RX HQFRUH OH GpYHORSSHPHQWIORUDOHWODYLDELOLWpGXSROOHQ/HFKQHUHWDO6KLXHW%OHHFNHUW 'KDUPDVLULHWDO*XVWLHWDO6DQWQHUHW(VWHOOH=KDRHWDO/D SOXSDUWGHVFLEOHVGHVFRPSOH[HV6&)RQWEHVRLQG¶rWUHSKRVSKRU\OpHVDYDQWG¶rWUHUHFRQQXHV SDU OD )ER[ -DFNVRQ HW DO (Q FRQVpTXHQFH RQ SHXW SHQVHU TXH OH U{OH G¶XQ ERQ QRPEUH GH SURWpLQH NLQDVH GH SODQWHV HVW GH PRGXOHU OD UHFRQQDLVVDQFH GHV FLEOHV SDU OHV SURWpLQHV )ER[ UHOLDQW DLQVL GHV GHX[ VXSHUIDPLOOHV GH SURWpLQHV GDQV OD UpJXODWLRQ SURWpRO\WLTXH
1.3.3.2.1.3 Le complexe VBC
/H FRPSOH[H 9%& HVW FRQVWLWXp GH SOXVLHXUV VRXVXQLWpV GRQW WURLV VRQW j O¶RULJLQH GH VRQ QRPODSURWpLQH9+/©9RQ+LSSHO/LQGDXªHWOHVSURWpLQHV(ORQJLQH%HW&.DPXUDHW DO'DQVOHFDVGXFRPSOH[H9%&OHU{OHGHFKDUSHQWHPROpFXODLUHGXFRPSOH[HQ¶HVW SDV DVVXUp SDU OD &8//,1( PDLV SDU OD &8//,1( RX /D SDUWLH &WHUPLQDOH GHV
&8//,1(RXLQWHUDJLWDYHF5%;DORUVTXHGXF{Wp1WHUPLQDOOHVSURWpLQHV(ORQJLQH%
HW&SUHQQHQWODSODFHGHODSURWpLQH6.3/¶(ORQJLQH&pWDEOLWODOLDLVRQDYHFOD&8//,1(
DORUVTXHO¶(ORQJLQH%pWDEOLWXQHOLDLVRQDYHFODSURWpLQH9+/YLDXQGRPDLQHDSSHOp62&6 ER[ ©6XSSUHVVRU 2I &\WRNLQH 6LJQDOOLQJ SURWHLQª /H U{OH GH 9+/ SHXW rWUH DVVLPLOp j FHOXLGHV©)ER[ªGX6&)HQUHFUXWDQWOHVVXEVWUDWVFLEOHVFigure 5
&RQWUDLUHPHQW DX[ DXWUHV FRPSOH[HV ( OLJDVHV OH FRPSOH[H 9%& D pWp GpFULW XQLTXHPHQW FKH] OHV DQLPDX[ 'H SOXV DORUV TXH O¶RQ GpQRPEUH SOXV GH SURWpLQHV j GRPDLQHV ©) ER[ªFKH]OHVDQLPDX[XQHVHXOHSURWpLQH9+/DpWpLGHQWLILpH
Introduction
3DJH_
1.3.3.2.1.4 Le complexe CUL3-BTB ou CRL3
/H FRPSOH[H &8/%7% HVW EDVp VXU OD &8//,1( OD SOXV FRQVHUYpH SDUPL WRXV OHV JpQRPHV HXFDU\RWHV $ QRXYHDX OH PRGXOH FDWDO\WLTXH 5%; LQWHUDJLW DYHF O¶H[WUpPLWp &
WHUPLQDOHGH&8//DSDUWLH1WHUPLQDOHLQWHUDJLWDYHFODSURWpLQH%7%32=©%ULFDEUDF 7DPWUDFNDQG%URDG&RPSOH[3R[9LUXVHW=LQJ)LQJHUªTXLHVWGpFULWFRPPHOHPRGXOHGH UHFRQQDLVVDQFHVSpFLILTXHGHVFLEOHVGXFRPSOH[H)XUXNDZDHWDO;XHWDO 3LQWDUG HW DO &HV %7% SRVVqGHQW XQ qPH GRPDLQH G¶LQWHUDFWLRQ SURWpLQHSURWpLQH 3,'©3KRVSKRW\URVLQH,QWHUDFWLRQ'RPDLQªHQDPRQWHWRXHQDYDOGXGRPDLQH%7%'HV DQDO\VHV GH VpTXHQFHV RQW PLV HQ pYLGHQFH SURWpLQHV j GRPDLQH %7% FKH] OKRPPH VHXOHPHQW FKH] OD OHYXUH FKH] OD GURVRSKLOH HW FKH]Arabidopsis thaliana. 'HV pWXGHVRQWDXVVLSHUPLVG¶LGHQWLILHUSOXVG¶XQHGRX]DLQHGHGRPDLQHSURWpLTXHVGLIIpUHQWVTXL SHXYHQW V¶DVVRFLHU SDUIRLV HQ FRPELQDLVRQ DYHF OH GRPDLQH %7% 6WRJLRV HW DO
&HUWDLQHV SURWpLQHV VRQW SUpVHQWHV GDQV WRXV OHV JpQRPHV HXFDU\RWHV FRPPH OHV 0$7+
©0HWULQ$QG7UDI+RPRORJ\GRPDLQHVDORUVTXHG¶DXWUHVVRQWVSpFLILTXHVGHVPpWD]RDLUHV FRPPH OHV .HOFK GRPDLQHV RX GHV SODQWHV FRPPH OH GRPDLQH 13+ *HQVFKLN HW DO ,OHVWjQRWHUTXHVHXOHXQHSDUWLHGHWRXWHVOHVSURWpLQHVLGHQWLILpHVjGRPDLQHV%7%
VHUYHQW G¶DGDSWDWHXUV DX &5/ $ORUV TXH OD SOXSDUW GHV HXFDU\RWHV Q¶RQW TX¶XQ VHXO JqQH
&8//,1(Arabidopsis thaliana HQ FRPSWH CUL3A HWCUL3B TXL SUpVHQWHQW G¶KRPRORJLHV )LJXHURD HW DO 'X IDLW GH FHWWH IRUWH FRQVHUYDWLRQ GH VpTXHQFHV OHV GHX[ &8/ G¶Arabidopsis SRVVqGHQW GHV IRQFWLRQV UHGRQGDQWHV ,O HQ UpVXOWH TXH FKH] OHV SODQWHVRQREVHUYHXQHOpWDOLWpHPEU\RQQDLUHXQLTXHPHQWORUVTXHOHVGHX[JqQHVVRQWPXWpV 7KRPDQQHWDO'HSDUODGLYHUVLWpGHVSURWpLQHV%7%SRXYDQWV¶DVVRFLHUjOD&8/$
RX &8/% SRXU IRUPHU XQ FRPSOH[H &8/( OLJDVH 'LHWHUOH HW DO :HEHU HW DO
*LQJHULFKHWDOOHVFRPSOH[HV&8/%7%VRQWIRUFpPHQWLPSOLTXpVGDQVXQH PXOWLWXGH GH U{OHV ELRORJLTXHV *HQVFKLN HW DO 3DUPL FHV U{OHV RQ SHXW FLWHU OD UpJXODWLRQGHODELRV\QWKqVHGHFHUWDLQHVKRUPRQHVYpJpWDOHVHWOHXUVLJQDOLVDWLRQ(QHIIHWOH PXWDQWQXOeto1 PDLVDXVVLXQHUpGXFWLRQGHODIRQFWLRQGH&8/UpVXOWHQWWRXVGHX[HQXQH VWDELOLVDWLRQG¶$&6©$&&6\QWKDVHªXQHHQ]\PHFDWDO\VDQWO¶pWDSHOLPLWDQWHGDQVOD ELRV\QWKqVHGHO¶pWK\OqQH:DQJHWDO7KRPDQQHWDO(QSOXVGHFHU{OHGDQV ODUpJXODWLRQGHODELRV\QWKqVHG¶pWK\OqQHOHV&5/VVRQWDXVVLLPSOLTXpVGDQVOHFRQWU{OHGH ODVLJQDOLVDWLRQGHO¶$%$$EVFLVVLFDFLG/HJpQRPHG¶Arabidopsis FRGHSRXUPHPEUHV
3DJH_
GHODIDPLOOHGHV0$7+%7%DSSHOpHV%30:HEHUHWDOTXLLQWHUDJLVVHQWWRXV DYHF XQH VRXVFODVVH GH IDFWHXUV GH WUDQVFULSWLRQVj +RPHRER[OHXFLQH ]LSSHU +'=,3 /HFKQHU HW DO LQFOXDQW $7+% XQ UpJXODWHXU QpJDWLI GH OD UpSRQVH $%$
+LPPHOEDFKHWDO/HFRPSOH[H&8/%30DJLWVXUO¶XELTXLWLQ\ODWLRQHWOHWXUQRYHU G¶$7+%PDLVFHSURFHVVXVHVWUDOHQWLHQSUpVHQFHG¶$%$DIIHFWDQWFHUWDLQHVUpSRQVHVGH O¶$%$SDUWLFXOLqUHPHQWORUVGHODIHUPHWXUHGHVVWRPDWHV/HFKQHUHWDO
1.3.3.2.1.5 Le complexe CUL4-DDB1 ou CRL4
&H FRPSOH[H ( HVW DUFKLWHFWXUp DXWRXU GH OD &8//,1( SURWpLQH FRQVHUYpH DX FRXUV GH O¶pYROXWLRQ GRQW SOXVLHXUV RUWKRORJXHV VH UHWURXYHQW GDQV GLIIpUHQWHV HVSqFHV &KH]
Saccharomyces pombeCearnobditiselegansDrosophila HWArabidopsis XQ VHXO JqQH FRGH SRXU&8/WDQGLVTXHFKH]OHVPDPPLIqUHVRQUHWURXYHGHX[SDUDORJXHVCUL4AHWCUL4B SRVVpGDQWG¶LGHQWLWp&HFLHQWUDvQHGHVGLIILFXOWpVGDQVOHVDQDO\VHVJpQpWLTXHVPHQDQWj ODFDUDFWpULVDWLRQIRQFWLRQQHOOHGXJqQHCUL4&KH]ODVRXULVVHORQODUpJLRQGpOpWpHGXJqQH CUL4ARQREVHUYHUDXQHOpWDOLWpHPEU\RQQDLUHRXQRQ'¶DXWDQWSOXVTXH&8/%SHXWGDQV FHUWDLQVFDVFRPSOpPHQWHUFHSKpQRW\SHCUL4AHWCUL4B SHXYHQWDLQVLDYRLUGHVIRQFWLRQV UHGRQGDQWHV .RSDQMD HW DO FHSHQGDQW CUL4B SDUWLFLSH j O¶LQDFWLYDWLRQ GX FKURPRVRPH ; FKH] O¶KRPPH 3OXVLHXUV SDWLHQWV SUpVHQWDQW XQH GpILFLHQFH PHQWDOH OLpH j O¶LQDFWLYDWLRQ GX FKURPRVRPH ; RQW GHV PXWDWLRQV GDQV OH JqQHCUL4B =RX HW DO 7DUSH\HWDO
/HV(OLJDVHVFRQWHQDQWODSURWpLQH&8/IRUPHQWODJUDQGHIDPLOOHGHV&5/©&XOOLQ 5LQJ ( /LJDVHª VWUXFWXUpH DXWRXU GH WURLV VRXVXQLWpV SULQFLSDOHV(Figure 6 &8/ OH F°XUGXFRPSOH[H52&5%;ODSURWpLQHjGRPDLQH5,1*)LQJHUHW''% ©'DPDJHG '1$ %LQGLQJSURWHLQªSURWpLQHKDXWHPHQW FRQVHUYpHG¶HQYLURQ N'D''% HVW XQ DGDSWDWHXUGXVXEVWUDW%HUQKDUGWHWDO&KHQHWDO/HHHWDO:DQJHWDO
=KDQJHWDO/DSURWpLQH&8/LQWHUDJLWDYHF52&5%;YLDVRQH[WUpPLWp&
WHUPLQDOH5%;YDDORUVUHFUXWHUO¶HQ]\PH(SRUWDQWO¶XELTXLWLQHDFWLYHDILQGHIRUPHUXQ PRGXOHFDWDO\WLTXH/¶DUFKLWHFWXUHPrPHGXFRPSOH[H&5/VHPRGLILHDILQGHSRVLWLRQQHUOD SURWpLQH FLEOH HQ UHJDUG GH O¶HQ]\PH ( HW IDFLOLWHU DLQVL OH WUDQVIHUW GH O¶XELTXLWLQH VXU OH VXEVWUDW=KHQJHWDO$QJHUVHWDO/LHWDO/¶H[WUpPLWp1WHUPLQDOH TXDQW j HOOH IL[H OD VRXVXQLWp ''% *UkFH j GHV pWXGHV FULVWDOORJUDSKLTXHV SOXVLHXUV GRPDLQHV G¶LQWHUDFWLRQ SURWpLQHSURWpLQH RQW SX rWUH PLV HQ pYLGHQFH 6FULPD HW DO
Figure 6 : Architecture des complexes CUL4-DDB1
/HFRPSOH[H&8/FRPSUHQGWURLVVRXVXQLWpVGHEDVH&RPPHOHVDXWUHVPHPEUHVGHODIDPLOOHGHV
&8//,1(V OD SDUWLH &WHUPLQDOH GH &8/ LQWHUDJLW DYHF XQH SHWLWH SURWpLQH j GRPDLQH ©5,1*
ILQJHUªTXLUHFUXWHO¶HQ]\PH (5%;DORUVTXHODSDUWLH1WHUPLQDOHGH&8/ VHOLHjODVRXV XQLWp ''% ©'DPDJHG '1$ %LQGLQJ SURWHLQ ª ''% HVW FDSDEOH G¶LQWHUDJLU DYHF XQH ODUJH YDULpWp GH SURWpLQHV DSSHOpHV '&$) ©''% &8/ $VVRFLDWHG )DFWRUª VHUYDQW GH UpFHSWHXU HW UHFUXWDQWOHVSURWpLQHVFLEOHVTXLYRQWrWUHXELTXLWLQ\OpHV$GDSWpGH6XJDVDZD
3DJH_
3DUPL FHV GRPDLQHV O¶XQ HVW FDSDEOH G¶LQWHUDJLU DYHF &8/ HW O¶DXWUH DYHF GHV SURWpLQHV j GRPDLQH:''HVDQDO\VHVSURWpRPLTXHVSXLVELRLQIRUPDWLTXHVRQWSHUPLVG¶LGHQWLILHUGHV SURWpLQHV DSSHOpHV ':' ©''%ELQGLQJ :'ª RX '&$) ©''%&8/DVVRFLDWHG IDFWRUVª/HHHWDO8QHJUDQGHSDUWLHGHFHV'&$)UHQIHUPHQWXQRXGHX[GRPDLQHV :' VH WHUPLQDQW SDU :'[5 TXL SDUWLFLSHQW j OHXU UHFRQQDLVVDQFH SDU ''% ,O D pWp PRQWUpjSOXVLHXUVUHSULVHVSDUGHV H[SpULHQFHV GHPXWDJpQqVHTXHOHUpVLGX DUJLQLQHGHFH PRWLI HVW QpFHVVDLUH PDLV SDV VXIILVDQW j OD PLVH HQ SODFH GH O¶LQWHUDFWLRQ $QJHUV HW DO
Arabidopsis thalianaSRVVqGHWRXVOHVJqQHVFRGDQWSRXUOHFRPSOH[H&5/&KH]OHVSODQWHV XQH PXWDWLRQ SHUWH GH IRQFWLRQ &8/ HQWUDvQH XQH OpWDOLWp HPEU\RQQDLUH GH PrPH TX¶XQH VLPSOHUpGXFWLRQRFFDVLRQQHGHQRPEUHX[GpIDXWVGXGpYHORSSHPHQW%HUQKDUGWHWDO
&KHQHWDO'XPEOLDXVNDVHWDO$ODGLIIpUHQFHGHVPDPPLIqUHVArabidopsis thaliana SRVVqGH GHX[ SDUDORJXHV QRPPpVDDB1a HW DDB1b &RPPH GDQV OH FDV G¶XQ PXWDQW QXOcul4 XQH GRXEOH PXWDWLRQ DIIHFWDQW OH JqQHDDB1a HW OH JqQHDDB1b UpVXOWH pJDOHPHQW HQ XQH OpWDOLWp HPEU\RQQDLUH $ FH MRXU RQ FRPSWH SOXV G¶XQH FHQWDLQH GH SURWpLQHV j GRPDLQH :' FKH]Arabidopsis /HXU DOLJQHPHQW D SHUPLV G¶LGHQWLILHU XQ RX GHX[ PRWLIV FRQVHUYpV GH DFLGHV DPLQpV DSSHOp ©':'ER[ª &H PRWLI SDUWLFLSH j O¶LQWHUDFWLRQDYHF''%IRUPDQWDLQVLOHPRGXOHGHUHFRQQDLVVDQFHVSpFLILTXHVGHVVXEVWUDWV /HHHWDO=KDQJHWDO7RXWHVFHVGRQQpHVVXJJqUHQWO¶H[LVWHQFHin vivoG¶XQH JUDQGHYDULpWpGHFRPSOH[HV&8/''%'&$)':'(OLJDVHVSRXYDQWrWUHLPSOLTXpVGDQV XQHPXOWLWXGHG¶DVSHFWVGXGpYHORSSHPHQWGHVRUJDQLVPHVTXLVHURQWDERUGpVGDQVOHFKDSLWUH ,,,5{OHVGHVFRPSOH[HV&8/''%'&$)
II. Les mécanismes de régulation de la protéolyse ubiquitine- dépendante
1. La reconnaissance et la dégradation par le protéasome 26S 1.1 La Monoubiquitinylation
Figure 7 : Les différentes chaînes d’ubiquitine et leurs fonctions
8Q FRGH FRXOHXU D pWp DVVLJQp HQWUH OHV PROpFXOHV G¶XELTXLWLQH HW OHV GLIIpUHQWV W\SHV GH FKDvQHV G¶XELTXLWLQHSRO\XELTXLWLQHV IRUPpHV /D SRVLWLRQ GH OD O\VLQH SDU UDSSRUW j O¶XELTXLWLQH VHUYDQW j FRQVWUXLUHODFKDvQHHVWLQGLTXpHVXUOHVIOqFKHVjJDXFKH$GURLWHOHVIRQFWLRQVFRUUHVSRQGDQWHVDX[
GLIIpUHQWHVFKDvQHVGHSRO\XELTXLWLQHVVRQWLQGLTXpHVG¶DSUqV3LFNDUWHW)XVKPDQQ
3DJH_
'DQV OD SOXSDUW GHV FDV GpFULV O¶XELTXLWLQH PRGLILH XQ UpVLGX O\VLQH LQWHUQH GH OD SURWpLQH FLEOH 'H UpFHQWHV pWXGHV RQW PRQWUp TXH O¶XELTXLWLQH SRXYDLW rWUH FRQMXJXpH VXU G¶DXWUHV UpVLGXV FRPPH XQH OLDLVRQ HVWHU VXU XQH VpULQH RX XQH WKUpRQLQH PDLV DXVVL SDU XQH OLDLVRQ WKLRHVWHUVXUOHJURXSH±6+G¶XQHF\VWpLQH&DGZHOOHW&RVFR\9RVSHUHWDO 'DQVFHUWDLQV GHVFDVFLWpVO¶XELTXLWLQ\ODWLRQVXUXQUpVLGX DXWUHTX¶XQH O\VLQHDpWpIRUFpH SDUODPXWDWLRQGHWRXWHVOHVO\VLQHVGXVXEVWUDWVXJJpUDQWTXHODO\VLQHUHVWHOHUpVLGXSUpIpUp GHO¶XELTXLWLQHPDLVTXHG¶DXWUHVUpVLGXVSHXYHQWrWUHXELTXLWLQ\OpV/¶XELTXLWLQHSHXWDXVVL PRGLILHUOHJURXSHĮ1+GHODUpJLRQ1WHUPLQDOHGHODSURWpLQHFLEOH
/DPRQRXELTXLWLQ\ODWLRQDpWpGpFULWHGDQVODJHVWLRQGXWUDILFFHOOXODLUHO¶HQGRF\WRVHDLQVL TXHGDQVO¶H[SUHVVLRQRXOH©VLOHQFLQJªGHVJqQHVFigure 7
&KH]Arabidopsis thaliana OD PRQRXELTXLWLQ\ODWLRQ GH O¶KLVWRQH +% HVW OD SOXV pWXGLpH
&HWWH PRQRXELTXLWLQ\ODWLRQ HVW DXVVL XQH PDUTXH pSLJpQpWLTXH DVVRFLpH JOREDOHPHQW j OD FKURPDWLQHDFWLYHDSSHOpH&6*ULPDQHOOLHW5RXGLHU/HVPDUTXHV+%8EFRPPH OHVPRGLILFDWLRQV+.PHHW+.PHVRQWGLVWULEXpHVWRXWOHORQJGHVJqQHVWUDQVFULWVHW VRQW YUDLVHPEODEOHPHQW DVVRFLpHV j O¶pORQJDWLRQ GH OD WUDQVFULSWLRQ (OOHV LQIOXHQW VXU OD FDSDFLWp GHV JqQHV j PRGXOHU OHXU H[SUHVVLRQ GXUDQW OD SKRWRPRUSKRJHQqVH %RXUERXVVH HW DO PDLV DXVVL VXU OD UpJXODWLRQ GH OD FRPSRVLWLRQ GH OD FXWLFXOH HQ MRXDQW VXU OH UHPRGHODJH GH OD FKURPDWLQH 0HQDUG HW DO &HUWDLQHV pWXGHV WHQGHQW j SHQVHU TXH FKH] Arabidopsis j O¶LPDJH GH OD GURVRSKLOH HW GHV PDPPLIqUHV O¶KLVWRQH +$ HVW PRQRXELTXLWLQ\OpH SDU OH FRPSOH[H 35& ©3RO\FRPE 5HSUHVVLYH &RPSOH[ ª HW SOXV SDUWLFXOLqUHPHQW SDU $W%0,$% HW 5,1* $% VRXVXQLWpV GX 35& SRXU VWDELOLVHU OHV PDUTXHV UpSUHVVLYHV +.PH HW MRXH XQ U{OH GDQV OH PDLQWLHQ GH O¶LGHQWLWp FHOOXODLUH HQ DVVXUDQW XQH UpSUHVVLRQ WUDQVFULSWLRQQHOOH VWDEOH 0ROLWRU DQG 6KHQ &DORQMH HW DO
1.2 La Polyubiquitinylation
/D FKDvQH OD SOXV pWXGLpH HVW FHOOH TXL V¶pWDEOLW HQWUH OD JO\FLQH WHUPLQDOH HW OD /\V GH O¶XELTXLWLQH /HV VXEVWUDWV SRUWDQW FHWWH FKDvQH VRQW UHFRQQXV SDU OH SURWpDVRPH 6 HW SDUWLFLSHQW j OHXU GpJUDGDWLRQ /HV FKDLQHV GH SRO\XELTXLWLQH j SDUWLU GH OD /\V VRQW pJDOHPHQWWUqVpWXGLpHV&HSHQGDQWjODGLIIpUHQFHGHVFKDvQHVXEL.HOOHVQHFRQVWLWXHQW SDV XQ VLJQDO GH GpJUDGDWLRQ PDLV SDUWLFLSHQW SOXW{W j GHV SURFHVVXV WHOV TXH OD UpSDUDWLRQ
Figure 8 : Les sous-unités du protéasome 26S
/HSURWpDVRPH6HVWFRQVWLWXpG¶XQFRPSOH[HFDWDO\WLTXHSULQFLSDO6FHQWUDOHWGHGHX[
VRXVXQLWpV6HQWRXUDQWFHFRPSOH[HFDWDO\WLTXH/HFRPSOH[H6HVWFRPSRVpGHTXDWUH DQQHDX[GHVRXVXQLWpVĮYHUWRXȕURXJHjO¶LQWpULHXUGHVTXHOVODSURWpRO\VHDOLHX/H FRPSOH[H 6 RX FRLIIH HVW IRUPp GH GHX[ SDUWLHV GLIIpUHQWHV DSSHOpHV EDVH YLROHW HW FRXYHUFOH EOHX TXL SHUPHWWHQW OD UHFRQQDLVVDQFH HW OD GpQDWXUDWLRQ GHV SURWpLQHV FLEOHV SRO\XELTXLWLQ\OpHVG¶DSUqV;LHHWDO
3DJH_
SRVWUpSOLFDWLYHGHO¶$'1O¶HQGRF\WRVHRXODUpJXODWLRQGHODWUDGXFWLRQ/HVFKDvQHVIRUPpHV jSDUWLUGHV/\VRX/\VVRQWPRLQVpWXGLpHVHWOHXUVU{OHVH[DFWVUHVWHQWHQFRUHjGpILQLU
&HVFKDvQHVGHSRO\XELTXLWLQ\ODWLRQVXLYHQWODPrPHFDVFDGHHQ]\PDWLTXH((HW(,OHVW jQRWHUTXHOHW\SHGHOLDLVRQVHPEOHrWUHGpILQLSDUO¶HQ]\PH(H[SOLTXDQWDLQVLOHQRPEUH LPSRUWDQWGHFHVHQ]\PHVFKH]OHVHXFDU\RWHV
1.3 Le protéasome 26S
8QH IRLV XELTXLWLQ\OpHV OHV SURWpLQHV VRQW GLULJpHV YHUV OH SURWpDVRPH 6 R HOOHV VRQW GpJUDGpHV$ILQG¶rWUHUHFRQQXHVSDUFHOXLFLOHVSURWpLQHVFLEOHVGRLYHQWSRUWHUDXPRLQV UpVLGXVG¶XELTXLWLQHjSDUWLUGHOD/\V.VXUODfigure 7SRXUSHUPHWWUHODGpJUDGDWLRQ GHVVXEVWUDWV
1.3.1 Composition du protéasome 26S
&KH]OHVHXFDU\RWHVOHSURWpDVRPH6HVWXQFRPSOH[HGH0'DjDFWLYLWpSURWpRO\WLTXH 6PDOOHHWDOFigure 8,OHVWFRPSRVpG¶XQFRPSOH[H6RX&3©&RUH3DUWLFOHªHW G¶XQFRPSOH[H6FRPSRUWDQWGHX[SDUWLHVGLVWLQFWHVODEDVH©%DVHªIRUPpHGHVL[VRXV XQLWpV HW OH FRXYHUFOH ©/LGª IRUPp GH SURWpLQHV OH WRXW HVW DXVVL DSSHOp ©FRLIIHª /H FRXYHUFOHHVWUHVSRQVDEOHGHODUHFRQQDLVVDQFHHWGHODGpXELTXLWLQ\ODWLRQGHODSURWpLQHFLEOH WDQGLV TXH OD EDVH SHUPHW GH GpQDWXUHU OH VXEVWUDW DILQ GH OXL SHUPHWWUH GH UHQWUHU GDQV OH WXQQHOFHQWUDOGXFRPSOH[H6&HOXLFLHVWFRPSRVpGHTXDWUHDQQHDX[GHVHSWVRXVXQLWpV FKDFXQ GHX[ DQQHDX[ H[WHUQHV IRUPpV GH VRXVXQLWpV GH W\SH Į HW GHX[ DQQHDX[ LQWHUQHV IRUPpVGHVRXVXQLWpVGHW\SHȕ/HSURWpDVRPH6VHGHVVLQHDORUVHQXQWXQQHOUHQIHUPDQW O¶DFWLYLWp SURWpRO\WLTXH /HV SURWpLQHV FLEOHV \ VRQW GpJUDGpV HQ SHSWLGHV GH WDLOOH YDULDEOH
*OLFNPDQHWDO*OLFNPDQHW&LHFKDQRYHU
1.3.2 Le mécanisme de dégradation par le 26S
/¶DFWLYLWp SURWpRO\WLTXH VLWXpH GDQV OD SDUWLH 6 pWDQW GLIILFLOH G¶DFFqV OHV SURWpLQHV FLEOHV GRLYHQWrWUHUHFRQQXHVHWPRGLILpHVDILQGHIDFLOLWHUOHXUSpQpWUDWLRQGDQVOHWXQQHO&HU{OHHVW WHQXSDUODFRLIIHYLDOHVVRXVXQLWpV531©5HJXODWRU\3DUWLFOH1RQ$73DVHªHW531GX FRXYHUFOHTXLSHXYHQWVHOLHUjO¶XELTXLWLQHHWVHUDLHQWFDSDEOHGHUHFRQQDLWUHDLQVLOHVFKDvQHV
Introduction
3DJH_
GH SRO\XELTXLWLQHV (OVDVVHU HW DO /HV VRXVXQLWpV 531 HW 531 YRQW DORUV GpXELTXLWLQ\OpHV OHV FLEOHV 9HUPD HW DO <DR HW &RKHQ /H VXEVWUDW HVW DLQVL OLQpDULVpVHXOHIRUPHjSRXYRLUVHIDXILOHUHQWUHOHVVRXVXQLWpVĮGXFRPSOH[H6*UROOHW DO&HWWHDFWLRQQpFHVVLWHODGpQDWXUDWLRQGXVXEVWUDWFHTXLSRXUUDLWrWUHOHU{OHGHOD
©EDVHª%UDXQHWDO/¶HQWUpHGDQVOHWXQQHOHVWTXDQGjHOOHHQFRUHPDOFRPSULVH/D SURWpLQHXQHIRLVHQWUpHHVWUpGXLWHHQSHSWLGHVGHWDLOOHYDULDEOH/¶K\GURO\VHGHVSURWpLQHV FLEOHV HVW UpDOLVpH JUkFH DX[ GLIIpUHQWHV DFWLYLWpV SURWpRO\WLTXHV FK\PRWU\SVLQH
©SHSWLG\OJOXWDP\O SHSWLGH K\GURO\]LQJª WU\SVLQH GX FRPSOH[H 6 5LYHWW /H PpFDQLVPHGHUHODUJDJHGHVSHSWLGHVJpQpUpVUHVWHLQFRQQXjFHMRXU
2. La régulation de la réaction d’ubiquitinylation
)DLVDQWLQWHUYHQLUXQQRPEUHLPSRUWDQWGHSDUWHQDLUHVODYRLHGHO¶XELTXLWLQ\ODWLRQQpFHVVLWH XQH UpJXODWLRQ FRPSOH[H SRXYDQW VH IDLUH j GLIIpUHQWHV pWDSHV DX QLYHDX GH OD FDVFDGH HQ]\PDWLTXH PDLV DXVVL GDQV OD UpJXODWLRQ GH OD GpJUDGDWLRQ GHV SURWpLQHV FLEOHV DSUqV OHXU XELTXLWLQ\ODWLRQ
2.1 Régulation des principaux acteurs de l’ubiquitinylation
7RXWHVOHVHQ]\PHVGHODFDVFDGHHQ]\PDWLTXHGpWHUPLQHQWO¶XELTXLWLQ\ODWLRQGHVVXEVWUDWVj O¶H[FHSWLRQ GH O¶HQ]\PH ( TXL HVW FRQVWLWXWLYHPHQW DFWLYH &HWWH UpJXODWLRQ LQWHUYLHQW DX QLYHDXGHODWUDQVFULSWLRQGHVJqQHVE2RXE3PDLVDXVVLDXQLYHDXGHVPRGLILFDWLRQVSRVW WUDGXFWLRQQHOOHV GH FHV HQ]\PHV GH OHXU DVVRFLDWLRQ DYHF G¶DXWUHV SURWpLQHV UpJXODWULFHV RX ELHQHQFRUHORUVG¶XQFKDQJHPHQWGHOHXUORFDOLVDWLRQVXEFHOOXODLUH
L'DQVOHFDVGHODUpJXODWLRQWUDQVFULSWLRQQHOOHODSURWpLQHjGRPDLQH©)ER[ªKXPDLQHGX FRPSOH[H 6&)6.3 HVW XQ ERQ H[HPSOH &H JqQH SUpVHQWH XQH UpJXODWLRQ WUDQVFULSWLRQQHOOH F\FOHFHOOXODLUHGpSHQGDQWHHWOHVWUDQVFULWVV¶DFFXPXOHQWjODWUDQVLWLRQ*68QHGHVFLEOHV GX FRPSOH[H 6&)6.3 HVW O¶LQKLELWHXU S GH O¶DFWLYLWp NLQDVH F\FOLQH GpSHQGDQWH &'.
Figure 13 /¶XELTXLWLQ\ODWLRQ GH S SXLV VD GpJUDGDWLRQ SDU OH SURWpDVRPH SHUPHWWUD O¶DFWLYDWLRQGHOD&'.HQ*6/LXHWDO=KDRHWDO&KH]OHVSODQWHVOHV JqQHV ASK SUpVHQWHQW XQH H[SUHVVLRQ VSDWLRWHPSRUHOOH GLIIpUHQWLHOOH DX FRXUV GX GpYHORSSHPHQWHWSHXYHQWDLQVLVHUYLUG¶H[HPSOHGHFHW\SHGHUpJXODWLRQ
Figure 9 : Régulation des E3 ligases de type CRL.
/¶DFWLYLWpGHVFRPSOH[HV&5/HVWUpJXOpHSDU&$1'HWOD58%\ODWLRQGp58%\ODWLRQGHODVRXV XQLWp FXOOLQH &8/ SDU 58%&61 /H SUHPLHU F\FOH DSSHOp &$1' GpFULW OD G\QDPLTXH GHV SURWpLQHVFXOOLQHVVpTXHVWUpHVHQSDUWLHSDUODSURWpLQH&$1'/DSURWpLQH&$1'V¶HQURXOHDXWRXU GX FRPSOH[H &8/5%; HQ EORTXDQW DLQVL OHV H[WUpPLWpV 1 HW &WHUPLQDOHV LQGLVSHQVDEOHV SRXU pFKDIDXGHU XQ FRPSOH[H &5/ DFWLI /H GHX[LqPH F\FOH DSSHOp 58%&61 LOOXVWUH O¶DFWLYDWLRQ GX FRPSOH[H &5/ SDU VD 58%\ODWLRQ /HV DGDSWDWHXUV RX UpFHSWHXUV GH VXEVWUDWV UHFUXWHQW VSpFLILTXHPHQWOHXUVFLEOHVSXLVV¶DVVRFLHQWjXQSRROGHSURWpLQHFXOOLQHSRXUOHVVRXVWUDLUHDXF\FOH
&$1'*UkFHjO¶DFWLRQGHVHQ]\PHV($;5(&5(5&(HW(58%HVWDWWDFKpHVXU XQH O\VLQH . j O¶H[WUpPLWp &WHUPLQDOH GH OD VRXVXQLWp FXOOLQH DFWLYDQW DLQVL OH FRPSOH[H &5/
/¶XELTXLWLQ\ODWLRQ GX VXEVWUDW SHXW DORUV DYRLU OLHX $X FRXUV GH FH SURFHVVXV OHV VRXVXQLWpV GX FRPSOH[H &236LJQDORVRPH &61 V¶DVVRFLHQW pJDOHPHQW DX FRPSOH[H &5/ /¶DFWLRQ GH Gp 58%\ODWLRQSDU&61HWGHGpXELTXLWLQ\ODWLRQSDU8%3XQHGpXELTXLWLQDVHSHUPHWOHUHF\FODJH GHVSURWpLQHV58%8EHWGHVUpFHSWHXUVGHVXEVWUDWV&HWWHGLVVRFLDWLRQSHUPHWjODSURWpLQHFXOOLQH DVVRFLpHj5%;GHUpLQWpJUHUOHF\FOH&$1'$GDSWpGH+XDHW9LHUVWUD
Introduction
3DJH_
LL 3DUPL OHV PRGLILFDWLRQV SRVWWUDGXFWLRQQHOOHV GHV FRPSOH[HV ( OLJDVHV RQ SHXW FLWHU OD SURWpLQH&'+DFWLYDWULFHGHO¶$3&&/RUVTXHFHOOHFLHVWSKRVSKRU\OpHSDUO¶DFWLYLWp&'.
OH FRPSOH[H $3&&&'+ HVW LQDFWLI (OOH QH SRXUUD VH OLHU DX FRPSOH[H TXH VL HOOH HVW SUpDODEOHPHQWGpSKRVSKRU\OpHSDUODSKRVSKDWDVH&'&=DFKDULDHHWDO
LLL/DUHORFDOLVDWLRQGHFHUWDLQVFRPSOH[HV(OLJDVHVSHXWDXVVLLQIOXHQFHUODGpJUDGDWLRQGHV VXEVWUDWV /¶HQ]\PH ( OLJDVH 0'0 QH UHFRQQDvW VD SURWpLQH FLEOH 0'0; TXH ORUVTXH FHOOHFL HVW ORFDOLVpH GDQV OH QR\DX /D SURWpLQH 0'0; XQ UpJXODWHXU GH 3 HVW QRUPDOHPHQW ORFDOLVpH GDQV OH F\WRSODVPH (Q FDV GH OpVLRQV GH O¶$'1 0'0; HVW SKRVSKRU\OpHHWGHFHIDLWHVWUHORFDOLVpHGDQVOHQR\DX0'0YDDORUVO¶XELTXLWLQ\OHUHWOD GLULJHUYHUVOHSURWpDVRPHOLHXGHVDGpJUDGDWLRQ/H%URQHWDO
LLLL/¶DFWLYLWpGHV(OLJDVHVGHW\SHV&5/HVWUpJLHSDUO¶pWDWGHQHGG\ODWLRQUXE\ODWLRQ/D SURWpLQH1(''58%HVWFRQMXJXpHVXUXQUpVLGXO\VLQHFRQVHUYpjO¶H[WUpPLWp&WHUPLQDOH GHV&XOOLQHVSDUXQPpFDQLVPHVLPLODLUHjFHOXLGHO¶XELTXLWLQ\ODWLRQ,OLPSOLTXHO¶DFWLRQHQ FDVFDGH GH W\SHV G¶HQ]\PHV ( ( HW (OLNH 58% HVW DFWLYpH SDU O¶KpWpURGLPqUH
$;5(&5 HW VD FRQMXJDLVRQ VH IDLW SDU O¶HQ]\PH (OLNH 5&( FKH] Arabidopsis thaliana /D IL[DWLRQ GH 58% VXU OD &XOOLQH HVW IDFLOLWpH SDU OD VRXVXQLWp 5%; DJLVVDQW FRPPH XQH (OLNH *UD\ HW DO Figure 9 /D UpJXODWLRQ GH O¶XELTXLWLQ\ODWLRQ HVW DXVVL DVVXUpH SDU XQ DXWUH UpJXODWHXU GHV ( OH FRPSOH[H &23 VLJQDORVRPH &61 GpFRXYHUWFKH]OHVSODQWHVHQWDQWTXHUpSUHVVHXUGHODSKRWRPRUSKRJHQqVH+DUGWNHHW'HQJ &H FRPSOH[H FDWDO\VH OH FOLYDJH HQWUH OD &XOOLQH HW OHV SURWpLQHV 1(''58%
HQWUDvQDQW XQH LQKLELWLRQ GX FRPSOH[H ( /\DSLQD HW DO /H VLJQDORVRPH DXUDLW XQ DXWUH U{OH GH SURWHFWLRQ GHV VRXVXQLWpV GHV FRPSOH[HV HQ O¶DEVHQFH GH OHXUV VXEVWUDWV 6FKZHFKKHLPHUSUpVHQWDQWDLQVLXQU{OHDQWDJRQLVPHHQWUHOHVGHX[DFWLYLWpV
LLLLL8QGHUQLHUW\SHGHUpJXODWLRQGHO¶DFWLYLWpGHVFRPSOH[HV(UHSRVHVXUO¶LGHQWLILFDWLRQ G¶XQUpFHSWHXUGHO¶DX[LQHODSURWpLQH©)ER[ª7,5'KDUPDVLULHWDO,OV¶DJLWGX SUHPLHU H[HPSOH GH UpJXODWLRQ G¶XQH ( OLJDVH SDU IL[DWLRQ G¶XQH PROpFXOH ©VLJQDOª GLUHFWHPHQW VXU OH FRPSOH[H 6&)7,5 SRXU O¶DFWLYHU /D SUpVHQFH G¶DX[LQH VWLPXOH DLQVL OD GpJUDGDWLRQGHVUpSUHVVHXUV$X[,$$HWSHUPHWO¶H[SUHVVLRQGHVJqQHVUpJXOpVSDUOHVIDFWHXUV GHWUDQVFULSWLRQ$5)'KDUPDVLULHWDO7DQHWDO
3DJH_
2.2 Régulation par modification du substrat
'HQRPEUHX[VXEVWUDWVGHODYRLHG¶XELTXLWLQ\ODWLRQGRLYHQWrWUHPRGLILpVSRXUrWUHUHFRQQXV SDUOHVHQ]\PHVXELTXLWLQH(OLJDVH/DPRGLILFDWLRQODSOXVFRXUDPPHQWUHQFRQWUpHHVWOD SKRVSKRU\ODWLRQQRWDPPHQWSRXUOHVVXEVWUDWVUHFRQQXVSDUOHFRPSOH[H6&)2QSHXWFLWHUOH FDV GH O¶LQKLELWHXU 6,& GHV FRPSOH[HV F\FOLQH&'. LPSOLTXp GDQV OD UpJXODWLRQ GX F\FOH FHOOXODLUH FKH] OD OHYXUH /RUV GH OD WUDQVLWLRQ *6 O¶LQKLELWHXU 6,& HVW SKRVSKRU\Op VXU SOXVLHXUVUpVLGXVHWGHYLHQWDLQVLXQVXEVWUDWSRXUOHFRPSOH[H6&)&'&9HUPDHWDO 6HGJZLFNHWDO
$O¶LQYHUVHODSKRVSKRU\ODWLRQSHXWDXVVLSURWpJHUOHVXEVWUDWHQEORTXDQWVDUHFRQQDLVVDQFH SDU O¶HQ]\PH ( &¶HVW OH FDV QRWDPPHQW G¶$&6 XQH HQ]\PH FOp GH OD ELRV\QWKqVH GH O¶pWK\OqQHFKH]Arabidopsis :DQJHWDO&KULVWLDQVHWDO
/H FRQWU{OH GH OD UHFRQQDLVVDQFH GHV VXEVWUDWV SHXW DXVVL VH IDLUH JUkFH j G¶DXWUHV PRGLILFDWLRQV SRVWWUDGXFWLRQQHOOHV WHOOHV TXH OD 1JO\FRV\ODWLRQ RX O¶K\GUR[\ODWLRQ GH FHUWDLQV DFLGHV DPLQpV /D 1JO\FRV\ODWLRQ LQWHUYLHQW GDQV OH WUDQVSRUW UpWLFXOR HQGRSODVPLTXH HW O¶K\GUR[\ODWLRQ LQWHUYLHQW GDQV OD GpJUDGDWLRQ GX IDFWHXU GH WUDQVFULSWLRQ +,)Į.LPHW.DHOLQ<RVKLGDHWDO
2.3 Régulation par déubiquitinylation
7RXW FRPPH OHV DQLPDX[ HW OHV OHYXUHV OHV SODQWHV FRQWLHQQHQW XQH IDPLOOH GH '8%
:HLVVPDQQ:LQJ&HVHQ]\PHVJpQqUHQWGHVPROpFXOHVG¶XELTXLWLQHOLEUHVj SDUWLU GHV SURGXLWV LQLWLDX[ UHF\FOHQW OHV 8ELTXLWLQHV GXUDQW OD FRQMXJDLVRQ GH O¶XELTXLWLQH HWRXLQYHUVHQWOHVHIIHWVGHO¶XELTXLWLQ\ODWLRQ&KH]Arabidopsis thalianaDXPRLQVJqQHV RQW pWp LGHQWLILpV FRPPH FRGDQW SRXU GHV SRWHQWLHOV '8% &HWWH FROOHFWLRQ LQFOXW GHX[
XELTXLWLQH FDUER[\ WHUPLQDO K\GURODVH 8&+ TXL XWLOLVHQW WURLV UpVLGXV FDWDO\WLTXHV IRUPpV G¶XQHF\VWpLQHXQHKLVWLGLQHHWXQDFLGHDVSDUWLTXHSURWpDVHVXELTXLWLQHVSpFLILTXHVHWOD VRXVXQLWp531FRQWHQXHGDQVOH©FRXYHUFOHªGXSURWpDVRPH6<DQHWDO(OOHV UHFRQQDLVVHQWODSDUWLHSUR[LPDOHGHO¶XELTXLWLQHHWHQOqYHQWWRXVOHVDFLGHVDPLQpVRXSHSWLGH TXLVRQWDWWDFKpVjODSDUWLH&WHUPLQDOHGHODJO\FLQH/HV'8%VRQWLPSOLTXpVGDQVSOXVLHXUV
Introduction
3DJH_
SURFHVVXV FKH] OHV DQLPDX[ HW OHV OHYXUHV VXJJpUDQW TXH FHV HQ]\PHV RQW GHV FLEOHV VSpFLILTXHV 8QH SRVVLELOLWp LQWpUHVVDQWH VHUDLW TXH FHUWDLQHV GH FHV '8% SRXUUDLHQW DXVVL UpJXOHUODGHPLYLHGHVSURWpLQHVHQLQYHUVDQWODUpDFWLRQG¶XELTXLWLQ\ODWLRQ'HX[IDPLOOHVGH '8% G¶Arabidopsis thaliana SUpVHQWHQW GHV U{OHV GDQV OD UpSDUDWLRQ GHV GRPPDJHV DX[
SURWpLQHVAt8%3HWAt8%3<DQHWDOPDLVDXVVLGDQVOHUHF\FODJHGHVFKDvQHV G¶XELTXLWLQH OLEUH At8%3 'RHOOLQJ HW DO 'H UpFHQWHV pWXGHV RQW SHUPLV GH PRQWUHU TXH 8&+ HW 8&+ OHV XELTXLWLQH FDUER[\ WHUPLQDOH K\GURODVH G¶Arabidopsis thaliana SRXYDLHQWSDUWLFLSHUjODUpSRQVHjO¶DX[LQH<DQJHWDO
III. Rôles des complexes CUL4-DDB1
DCAF
/HV DQDO\VHVin silico RQW SHUPLV G¶LGHQWLILHU SURWpLQHV j PRWLI ':' SRXU SURWpLQHV j GRPDLQH :' $ FH MRXU OD PDMRULWp GH FHV '&$) Q¶RQW SDV HQFRUH GH IRQFWLRQVFRQQXHVFKH]Arabidopsis*XRHWDO'DQVFHFKDSLWUHMHYDLVGpFULUHOHV IRQFWLRQVPDMHXUHVDWWULEXpHVDXFRPSOH[H&5/GDQVOHVPRGqOHVDQLPDX[HWSODQWHV
SDUWLFLSDWLRQDXSURFHVVXVGHUHFRQQDLVVDQFHHWGHUpSDUDWLRQGHO¶$'1DSUqVPXWDWLRQ SDUOHV89GpFULWFRPPHOHU{OHKLVWRULTXHGH&5/
U{OHUpJXODWHXUGDQVODSKRWRPRUSKRJHQqVH U{OHUpJXODWHXUGXF\FOHFHOOXODLUH
U{OHGDQVOHVIRQFWLRQVFKURPDWLQLHQQHV
'¶DXWUHV U{OHV RQW pWp GpFU\SWpV SRXU FHUWDLQHV '&$) QRWDPPHQW GDQV OD UpSRQVH DX VWUHVV LPSOLTXDQW OD YRLH GH VLJQDOLVDWLRQ SDU OD SK\WRKRUPRQH $%$ ©$EVFLVVLF $FLGª &HV UpVXOWDWVQHVRQWSDVGpFULWVGDQVFHPDQXVFULW
1. Rôle dans la réparation de l’ADN
''% HW ''% VRQW GHV FRPSRVDQWV PDMHXUV GX FRPSOH[H 89''% ©8OWUDYLROHW 'DPDJHG'1$%LQGLQJSURWHLQFRPSOH[ªLQLWLDOHPHQWLGHQWLILpGDQVGHVFHOOXOHVKXPDLQHV /HV OpVLRQV LQGXLWHV SDU OHV 89 VRQW GHV GLPqUHV GH S\ULPLGLQH OHV &3' F\FOREXWDQH S\ULPLGLQRQHGLPHUVRQWSURGXLWVPDMRULWDLUHPHQWHWOHV33S\ULPLGLQHS\ULPLGRQH
3DJH_
.XQ]HWDOVRQWGHVSKRWRSURGXLWVPLQHXUV$ILQGHFRQWUHUFHVGRPPDJHVOHVSODQWHV HWXQHPDMRULWpGHVPpWD]RDLUHVPHWWHQWHQ°XYUHGHX[PpFDQLVPHVGLIIpUHQWVGHUpSDUDWLRQ
/D SKRWRUpDFWLYDWLRQ HVW FDWDO\VpH SDU GHV SKRWRO\DVHV TXL XWLOLVHQW O¶pQHUJLH GH OD OXPLqUHJUkFHjXQFKURPRSKRUHVHQVLEOHDXVSHFWUH89$HWEOHX&HV\VWqPHGHUpSDUDWLRQ UDSLGHDVVXUHXQHFRQYHUVLRQGHVGLPqUHVGHS\ULPLGLQHHQPRQRPqUHVGHPDQLqUHHIILFDFH VDQV SDVVHU SDU XQH pWDSH G¶H[FLVLRQ GX EULQ G¶$'1 -LDQJ HW DO 1DNDMLPD HW DO 6DQFDUHWDO&HPpFDQLVPHH[LVWHFKH]OHVSODQWHVHWOHVDQLPDX[jO¶H[FHSWLRQ GHVPDPPLIqUHVSODFHQWDLUHVTXLQ¶XWLOLVHQWTXHODYRLH1(5
/D YRLH GH UpSDUDWLRQ DSSHOpH 1(5 SRXU ©1XFOHRWLGH ([FLVLRQ 5HSDLUª IRQFWLRQQH LQGpSHQGDPPHQW GH OD OXPLqUH HW QpFHVVLWH O¶LQWHUYHQWLRQ GH SOXV GH WUHQWH SURWpLQHV SRXU pOLPLQHUOHVOpVLRQVGHO¶$'16FKXFNHWDO&LQTpWDSHVVXFFHVVLYHVRQWpWpGpFULWHV GDQVFHWWHYRLHGHUpSDUDWLRQODUHFRQQDLVVDQFHGHODOpVLRQSXLVO¶RXYHUWXUHGHSUpLQFLVLRQ O¶H[FLVLRQVXUOHEULQHQGRPPDJpG¶XQVHJPHQWGHSOXVLHXUVQXFOpRWLGHVHQFDGUDQWODOpVLRQ OD SRO\PpULVDWLRQ HW SRXU ILQLU OD OLJDWLRQ GX QRXYHDX EULQ V\QWKpWLVp 3OXVLHXUV PXWDWLRQV DIIHFWDQWGLIIpUHQWVFRPSRVDQWVGHODYRLHGHUpSDUDWLRQ1(5RQWpWpGpFULWHVFKH]O¶KRPPH FRQGXLVDQW j GHV PDODGLHV DXWRVRPDOHV UpFHVVLYHV 3DU H[HPSOH OHV SDWLHQWV DWWHLQWV G¶XQH PDODGLHUDUHDSSHOpH;HURGHUPDSLJPHQWRVXP;3TXLUpVXOWHG¶XQGpIDXWGDQVFHSURFHVVXV GH UpSDUDWLRQ SUpVHQWHQW XQH K\SHUVHQVLELOLWp DX[ 89 HW RQW XQH IRUWH SUpGLVSRVLWLRQ j GpYHORSSHUXQFDQFHUGHODSHDXLQGXLWJpQpUDOHPHQWSDUOHVUD\RQQHPHQWVVRODLUHV
'DQVFHWWHYRLHGHUpSDUDWLRQ1(5RQGLVWLQJXHJpQpUDOHPHQWGHX[PpFDQLVPHVTXLRSqUHQW GLIIpUHPPHQWGDQVODUHFRQQDLVVDQFHGHODOpVLRQ
/D YRLH GH UpSDUDWLRQ 7&5 RX ©7UDQVFULSWLRQ &RXSOHG 5HSDLUª DJLW GDQV OHV ]RQHV WUDQVFULWHVGXJpQRPHYLDXQHDFWLRQVXUO¶$51SRO\PpUDVH,,
/D YRLH GH UpSDUDWLRQ **5 RX ©*OREDO *HQRPH 5HSDLUª VFDQQH OHV UpJLRQV QRQ WUDQVFULWHV GX JpQRPH 'DQV FH FDV OHV OpVLRQV VRQW UHFRQQXHV SDU XQ PpFDQLVPH GH VXUYHLOODQFHTXLIDLWLQWHUYHQLUOHVFRPSOH[HV''%''%HW;3&5$'TXLUHFUXWHQWOHV SURWpLQHV HVVHQWLHOOHV j O¶LQLWLDWLRQ GH OD UpSDUDWLRQ /H FRPSOH[H ''%''% DJLW HVVHQWLHOOHPHQWGDQVODUHFRQQDLVVDQFHGHVGLPqUHVGHS\ULPLGLQHin vivo'HIDLWGHVFHOOXOHV GpILFLHQWHV HQ ''% SUpVHQWHQW XQH UpGXFWLRQ LPSRUWDQWH GH OHXU FDSDFLWp j UpSDUHU GHV
