醫(yī)學精品課件：2011-12-08 張令強研究生課 泛素化介導的蛋白質降解 含NCB和NM文章 分子生物學課

泛素化介導的蛋白質降解LingqiangZhang,Ph.DProfessor,PrincipalInvestigatorDept.Proteomics&Genomics(P&G)BeijingInst.RadiationMedicine(BIRM)Tel:66931216Email:zhanglq@

TheNobelPrizeinChemistry,2004“forthediscoveryofubiquitin-mediatedproteindegradation”AvramHershkoIrwinRoseAaronCiechanoverWolf,D.H.“Proteasomes:TheWorldofRegulatoryProteolysis”細胞周期和細胞分裂、分化發(fā)育的調節(jié)細胞對外環(huán)境的應激反應的調節(jié)神經網絡的形態(tài)發(fā)生細胞表面受體和離子通道的下調DNA修復免疫和感染反應的調節(jié)細胞器的生物生成……FunctionsofUbiquitination-mediatedproteindegradationCellCyclebyUbiquitinationE2FMSG1G2DK4/6EK2p21E2FAK2BCDC2p53securinSLBPRbDifferentmodificationstatesofp53andMDM2DanielaHoelleretal.Ubiquitinandubiquitin-likeproteinsincancerpathogenesis.Nat.Rev.Cancer,2006,6,776NF-κBsignallingpathwayscanonicalpathwayNon-canonicalpathwayUbiquitinHumanUb:MQIFVKTLTGKTITLEVEPNDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLADYNIQKESTLHLVLRLRGGYeastUb:MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGLys-48Gly-76

Ubiquitin-proteasomesystem(UPS)DanielaHoelleretal.Nat.Rev.Cancer,2006,6,776ProteinUbiquitinationRINGE3HECTE3FunctionsoftheUbsystemNon-proteolytic(mono-UborK63Ubchain) DNArepair,transcription,kinaseactivation.Proteolytic

(K48orK29linkedUbchains) 1)concentrationcontrol:keyregulators(e.g.p53,cyclins,HIF)

2)

proteinqualitycontrol:

misfoldedordamagedproteins(e.g.CFTR,

prion,huntingtin)DifferenttypesofLysinemodifications(Ubiquitinationandubiquitin-likemodifications)DanielaHoelleretal.Nat.Rev.Cancer,2006,6,776Ubiquitinandubiquitin-likeproteinsUbiquitin-mediatedProteolysisE1Ub-S-E3

cellcycle

signaltransduction

antigenprocessing

tumorsuppressionmisfolded

proteinscyclinsCDKinhibitorsIkBatranscriptionc-junc-fosp53b-cateninoncogenesmyc?virusesbacterialmanyE2Ub-S-substratesUbUbUbUbUb.....................Lys-48CterminusCombinatorialnatureofUPSAdaptors(afew)Rad23E3s:nextwaveofdrugtargetsProteasomeinhibitorVelcade:multiplemyelomaUPS:targetsinanti-cancertherapyE3s:determinesubstratespecificity,bettertargetsCancer,Huntington’sdisease,spinocerebellarataxias,Paget’sdiseaseofbone,AIDS-relatedcancer,musclewasting,cysticfibrosis,Alzheimer’sdisease,Parkinson’sdisease,hypertension,etc.TwomajorscientificissuescentraltoourunderstandingsofE3ligaseMechanismscontrollingtheassemblyandactivityofE3ligasesMolecularnatureofE3ligasesandtheirsubstratesTwofamiliesofE3ubiquitinligases-S-E2substrateE3??UbUbUbUbUbUbHECTfamilyE3(~40)E6APE2substrateUbUbUbUbUbUbHECTRINGfamilyE3(1000s)CullinROCE2adaptorsubstrateUbUbUbUbUbUbMDM2E2p53UbUbUbUbUbUbRINGRING:ReallyInteresting

NewGeneRINGFingerGenesRINGFingerGenesYeast: 39Worm: 154Fly: 135Human: 350Arabidopsis: 490HowManyE3Ligases?Proteases: 550Kinases: 520Phosphatses: 150DNApolymerases: 16E1: 16E2: 53E3:

Cullin-ROCcomplexes 300-500 RINGproteins 450 HECTproteins ~40 U-boxproteins 10-20

Ublligases ?>1000Single-subunitRINGtypeE3:Mdm2p53bindingZn119102305322438478491RINGNESNLSNoLS181-185191-205464-471Ubc940-59Smurf175-114p300102-222ARF210-244acidic221274L5/L11/L26284-374RB273-321Gankyrin412-437Mdm2/MdmX438-491Mdm2***AktS166/S186ATMS395MDM2structureandinteractingproteinsMDM2ubiquitinatesp53Hondaetal.(1997)FEBSLetter420:25p53canbeactivatedbymultiplestressesp53MDM2OncogenicinsultsARFDNAdamageKinases(ATM,Chk1/2)NormalcellTumorcellMultiple-subunitsRINGtypeE3:

Cullin-Roc-basedligase(CRL)complexE3UbiquitinLigaseUb-S-E2substrateE3UbUbUbUbE3UbiquitinLigaseUb-S-E2substrateE3??UbUbUbUbE3UbiquitinLigaseUb-S-E2substrateE3??UbUbUbUbRINGCullinbindsROCCullin1ROCHumanCullinsandCullin-relatedproteinsKipreosetal.(1996)Cell85:829CUL5780APC2822Nd8CUL1776CUL2745CUL3768CUL4A759CUL4B717ROCPARC,CUL7……APC:anaphase-promotingcomplexfunctiontomediatemitosisprogressionresponsiblefordegradationofvariousmitoticproteins,includingcylinsandseurinsAPC11isaRINGfingerproteinRING:ReallyInterestingNewGeneSkp1bringsF-boxproteintoCul1Baietal.(1996)Cell86:263-274Cullin1Skp1F-boxROC1E2UbCullin1Skp1F-boxROC1E2UbsubstrateNdF-boxproteins:substratereceptorsF-boxproteinsWLOCDC4FE2UbCullin1Roc1Skp1UbUbUbUbUbUbUbiquitinationofCDKinhibitorSIC1bySCFCDC4-ROCligasePPSIC1SCF-ROCE3ubiquitinligaseZhengetal.(2002)Nature416:703SCF-ROCligasesE2UbCullin1Roc1UbUbUbUbUbUbUbUbUbUbUbUbUbUbUbUbUbUb……Skp1Skp2p27FSkp1

Fbp4Substrate4FSkp1

Fbp5Substrate5FSkp1

Fbp6Substrate6FSkp1

Fbp7Substrate7FSkp1

Fbp63Substrate50Fb-TrcPIkBaF

Fbp3Substrate3FHumanCullinsandCullin-relatedproteinsKipreosetal.(1996)Cell85:829CUL5780APC2822Nd8CUL1SKP1776CUL2745CUL3768CUL4A759CUL4B717??????ROCUbUbUbUbUbUbUbUbUbUbUbUbBCR(BTB-CUL3-ROC)E3ligasesCullin3Roc1E2UbUbUbUbUbUbUb……BTB4Substrate4BTBBTB5Substrate5BTBBTB6Substrate6BTBBTB5Substrate5BTBBTB6Substrate6BTBBTB200Substrate200BTBMEI-1MEL26BTBNRF2Keap1BTBSubstrate3BTB3BTBCullin4:DDB1-DCAF-ROCligasesE2UbCullin4Roc1UbUbUbUbUbUbUbUbUbUbUbUbUbUbUbUbUbUb……DDB1DDB2XPCWDDDB1

DCAF4Substrate4WDDDB1

DCAF5Substrate5WDDDB1

DCAF6Substrate6WDDDB1

DCAF7Substrate7WDDDB1

DCAF60Substrate60WDCSACSBWD

DCAF3Substrate3WDDDB1:UV-damagedDNAbindingprotein1DCAFs:DDB1-CUL4associatedfactorsHowmanycullinligases?CUL1: ~63F-boxproteins(Skp1asadaptor)CUL2/5:~40VHL/SOCSproteins(ElonginB/Casadaptor)CUL3: ~205BTBproteins(BTBthemselvesasadaptors)CUL4A:~60DWD/WD40/DCAFproteins(DDB1asadaptor)CUL4B:?CUL7: ?PARC: ?Cullin:scaffoldproteinsROC:RINGfinger,linkcullinwithE2Skp1,ElonginB/C,BTB,DDB1:adaptor,linkcullinwithsubstratereceptorF-box,VHL,SOCS,BTB,DWDproteins:substratereceptors,linkadaptortosubstrateSubstrate:E3targets(largelyunknown,sofar,only~20havebeenidentified)PatternofCullin-ROCligasesassemblyandsubstraterecruitmentCullin-ROCligasesvs.built-inRINGligasesMDM2E2Ubp53RINGUbUbUbUbUbCUL3ROCE2UbBTBsubstrateUbUbUbUbUbCUL1ROCE2UbSKP1FPPsubstrateUbUbUbUbUbHECTdomaintypeE3FamilyofHECTtypeE3ubiquitinligasesHERCfamily:6members,HERC1-6Nedd4family:9membersSingleHECTE3s:E6AP,EDD,ARF-BP1/HECTH9HERC:HECTandRCC1-likedomainRLD:RCC1-likedomainNedd4:neuralprecursorcells-expresseddevelopmentallydownregulated4HugeMW(80~500kDa),difficulttostudyMorethan500kDa100~120kDaARF-BP1/MuleNedd4familyE3sFunctionofthecatalyticactivitiesofHECTE3sMostcomefromNedd4familyCancerMetastasisRev2007,26(3-4):587-604.KnownfunctionsandsubstratesofNedd4family調控蛋白質運輸、跨膜受體的信號轉導、病毒芽生、骨發(fā)育及骨重塑、胚胎發(fā)育等過程膜受體：如TGF-βRI、Notch、EGFR、VEGFR2、IGF-1R、CXCR4等內吞機器組分：如Cbl、Endophilin、Eps15、Hrs等轉錄因子：如Smads、KLF5、Runx2、AP-1、p53/p63/p73等其他底物：如PTEN、MEKK2、RhoA、RNApolII、Dvl-1等RegulationofthecatalyticactivitiesofHECTE3sBBRC,2007,354,329Mol.Cell,2005,19,297PNAS,2006,103,1717Cell,2007,130,651C2HECTE2CCUbWWsMH2NTDSmad7PYC2HECTWWsDE2recruitmentbySmad7Phosphorylation-mediatedreliefofauto-inhibitionC2-HECTauto-inhibitionTargetingofWWdomainslinkerofHECTtypeE3ligaseSmurf1foractivationbyCKIP-1HumanNedd4familyofE3ubiquitin-proteinligasesBioEssays2006,28,617Ca2+&p-lipidbindingSubcellularlocalizationSubstratebinding&specificityE2binding(N-lobe)Ubiquitinbinding(C-lobe)C2HECTWWsSmurf1Smurf1specificallysuppressesosteoblastactivityandadultboneformationSmurf1:Smadubiquitinationregulatoryfactor1NATURE,1999,400,687JBC,2004,279,12854Cell,2005,121,87–99.Physiological(blue)andpharmacological(orange)stimulatorsandinhibitorsofboneformationandresorptionarelisted.Therelativeimpact,whereknown,isrepresentedbythethicknessofthearrows.Solidlinesarecurrenttherapiesanddottedlinesputativeones.DeterminantsofskeletalhomeostasisandbonemassOsteoblast成骨細胞Osteoclast破骨細胞RegulationofthecatalyticactivitiesofHECTE3sBBRC,2007,354,329Mol.Cell,2005,19,297PNAS,2006,103,1717Cell,2007,130,651C2HECTE2CCUbWWsMH2NTDSmad7PYC2HECTWWsDE2recruitmentbySmad7Phosphorylation-mediatedreliefofauto-inhibitionC2-HECTauto-inhibitionQ:WhetherandhowSmurf1ligaseactivityisregulatedremainsunclear.CKIP-1isanovelSmurf1interactingpartnerScience2005,307,1621PhysiologicalfunctionofCKIP-1isunknown

CKIP-1:caseinkinase2interactingprotein-1ImplicatedinofPI3K-regulatedmusclecelldifferentiationRegulationofAP-1activityandapoptosisRegulationofcytoskeletonreorganizationRecruitmentofnuclearATMandCK2kinasestoplasmamembraneJBC,2000JBC,2004MCB,2004EMBOJ.,2005MCB,2005Cell.Signal.,2006Cell.Signal.,2007CancerRes.,2007

PHLZCKIP-1PH:pleckstrinhomologydomainLZ:leucinezipperCKIP-1isnotasubstrateofSmurf1CKIP-1promotesSmurf1self-degradationCKIP-1promotesSmurf1auto-ubiquitinationCKIP-1enhancesthetrans-E3activityofSmurf1towardsitssubstratesSmad1/5CKIP-1enhancesthetrans-E3activityofSmurf1towardsitssubstratesSmad1/5CKIP-1specificallyregulatesE3activityofSmurf1anddegradationofBMPpathwaySmadsCKIP-1interactswithSmurf1bothinvivo&invitroMappingtheinteractingregionsNeitherSmurf2norothermembersofNedd4familyinteractswithCKIP-1Paradox:bindingWWdomainsv.s.E3-promotingeffectPhospho-lipidbindingSubstratebindingE2binding(N-lobe)Ubiquitinbinding(C-lobe)C2HECTWWsTwopossibilities:1,CKIP-1andsubstratebindtoeachoftwoWWdomains.2,CKIP-1bindstothelinkerregionbetweenthetwoWWdomains.Smurf1CKIP-1bindstothelinkerregionbetweenWWdomainsofSmurf1ThelinkerregionishighlyhomologousbutdifferentbetweenSmurf1andSmurf2.MappingthekeyaminoacidsinthelinkerAThelinkerisessentialforbinding細節(jié)決定成??！ThelinkerisessentialforCKIP-1promotingSmurf1ubiquitinationCKIP-1enhancestheaffinityofSmurf1toitssubstrate

CKIP-1enhancesSmurf1E3ligaseactivityCKIP-1targetstheWWlinkerofSmurf1CKIP-1augmentsSmurf1-SmadaffinityQ:Howaboutinvivo?Fig.S2cGenetargetinginmouseembryonicstemcellshasbecomethe‘goldstandard’fordetermininggenefunctioninmammals.Genetargetingnowliesatthecentreoffunctionalgenomicanalysis.MarioR.Capecchi,NatureReviewGenetics,2005,6,507-512discoveryofprinciplesforintroducingspecificgenemodificationsinmicebytheuseofembryonicstemcells2007NobelPrize-Physiology/MedicineEstablishmentofCKIP-1deficientmiceCKIP-1-/-micedisplayage-dependentincreasedbonemass(similartoSmurf1-/-mice)CKIP-1-/-BiggerbonesSmurf1-/-CKIP-1-/-micedisplayenhancedosteoblastbutnotosteoclastactivityCKIP-1-/-micedisplayreducedSmurf1activityCKIP-1isrequiredforefficientSmurf1functionC2HECTMH1MH2Smad1/5E2CCUbSmurf1PYWWWWintheabsenceofCKIP-1:Smurf1lowactivityC2HECTMH1MH2Smad1/5E2CCUbSmurf1PYCKIP-1WWWW

inthepresenceofCKIP-1:Smurf1highactivityAB泛素連接酶Smurf1的新的調控模式

Regulator-linkerpattern第一次揭示HECT類E3的WW連接區(qū)的重要調控功能對于理解E3活性調控機制及應用研究提供了新思路發(fā)現(xiàn)了HECT類泛素連接酶Smurf1的WW結構域連接區(qū)介導的酶活調控新機制LuK,YinXetal.,Nat.CellBiol.,2008,10(4),994-1002.通過RNAi敲低CKIP-1表達、促進骨形成進行骨質疏松治療的前期研究Nature,2003,423:316.骨重塑是骨生物學的重要科學問題之一1,骨生長如何終止的？2,礦化為什么限制于骨骼系統(tǒng)？3,骨重塑如何維持整體骨量的穩(wěn)態(tài)平衡的？骨重塑骨質疏松骨形成和骨吸收是骨重塑的兩個基本過程Nature,2003,423:349.成骨細胞破骨細胞骨質疏松藥物需要深入開展成骨細胞調控研究破骨細胞分化與活化：60多個基因成骨細胞分化與活化：約15個基因對骨形成的機制了解遠沒有骨吸收清楚骨形成是當前骨重塑研究中的重點問題我國的老年人群及骨質疏松治療用藥現(xiàn)狀中國：>60歲，1.67億人，>12%，>20億美元/年國家衛(wèi)生部：居民慢性疾病患病率，高血壓26.2‰，胃腸炎10.3‰，骨質疏松8.8‰，位列第三1994-1998，中成藥，仙靈骨葆膠囊，活性維生素D1998-，阿侖膦酸鹽，雌激素受體調節(jié)劑，雌激素替代治療，降鈣素，骨化三醇等2009-，第一個靜脈用藥雙膦酸鹽（艾本）在中國上市絕大多數(shù)靶向骨吸收，國際上目前只有甲狀旁腺激素基于靶向骨形成，但PTH用藥18個月后出現(xiàn)骨吸收增加研發(fā)新的促進骨合成代謝的新藥已成為新的重點和熱點方向（抑制破壞的同時積極推進重建）nucleuscytoplasmPMBMP-Smad1/5通路介導骨形成BMP:骨形態(tài)發(fā)生蛋白BoneformationSmad4Smad1/5PPSmad1/5PPSmad4Smad1/5PPSmad1/5PPCofactorPPPPBMPBMPRIIBMPRISmad1/5PPSmurf1促Smad1/5降解、抑制骨形成Smurf1:Smadubiquitinationregulatoryfactor1，HECT類泛素連接酶骨量上升骨密度增加骨形成能力增強Smurf1-/-nucleuscytoplasmPMBoneformationSmad4Smad1/5PPSmad1/5PPSmad4Smad1/5PPSmad1/5PPCofactorPPPPBMPBMPRIIBMPRISmad1/5PPUbUbUbSmad1/5Smurf1UbZhuHetal.,Nature,1999,400:687-93ZhaoMetal.,JBC,2004,279:12854-9YamashitaMetal.,Cell,2005,121:101-13Smurf1活性過高將導致骨質疏松骨形成骨吸收特征：骨量減少、骨組織微結構退行性改變、脆性增加危害：疼痛，骨折，致殘，并發(fā)癥危及生命人群：絕經后婦女和老年人群趨勢：社會老齡化加劇，患病率上升，患者年輕化骨平衡骨質疏松骨失衡科學問題1.Smurf1的連接酶活性是如何受到調控的？軍事醫(yī)學科學院張令強2.Smurf1的調控與骨發(fā)育、骨重塑的關系？3.可否基于Smurf1調控進行骨質疏松治療研究？C2HECTMH1MH2Smad1/5E2CCUbSmurf1PYW