研究生課 細(xì)胞凋亡的主要信號轉(zhuǎn)導(dǎo)途徑 分子生物學(xué)課課件

細(xì)胞凋亡的主要信號轉(zhuǎn)導(dǎo)途徑LingqiangZhang,Ph.DProfessor,PrincipalInvestigatorDept.Proteomics&Genomics(P&G)BeijingInst.RadiationMedicine(BIRM)Tel:66931216Email:zhanglq@細(xì)胞凋亡的主要信號轉(zhuǎn)導(dǎo)途徑LingqiangZhang,1Apoptosisandtheregulation---lifeordeathdecisionApoptosisandtheregulation2Science298:526,20022002Nobelfortheirdiscoveriesconcerninggeneticregulationoforgandevelopmentandprogrammedcelldeath.Science298:526,20022002Nob375歲的布勒吶(SydneyBrenner)1953年4月，在J.Waston和F.Crick著名的DNA結(jié)構(gòu)模型文章發(fā)表前不僅，布勒吶在劍橋大學(xué)見到他們。以后在英國劍橋大學(xué)的卡文迪許實(shí)驗(yàn)室。和克里克有長期活躍的討論和合作。布勒吶對分子生物學(xué)有多項(xiàng)重要貢獻(xiàn)，是分子生物學(xué)開創(chuàng)者之一。他發(fā)現(xiàn)mRNA，是一項(xiàng)可獲諾貝爾獎(jiǎng)的工作。他研究遺傳密碼也有幾個(gè)重要貢獻(xiàn)。1961年，他在《自然》雜志有兩篇論文，一篇關(guān)于mRNA，一篇關(guān)于遺傳密碼。75歲的布勒吶(SydneyBrenner)1953年441962年起，他和Crick討論了幾年，認(rèn)為分子生物學(xué)的框架已經(jīng)有了，要開創(chuàng)新領(lǐng)域。布勒吶想做發(fā)育和神經(jīng)系統(tǒng)。他的想法是用簡單的動(dòng)物，依靠遺傳學(xué)，來理解發(fā)育和神經(jīng)。1963年，布勒吶提出用一種叫C.briggsiae的線蟲研究發(fā)育和神經(jīng)，他不斷讀文獻(xiàn)和采集樣本，到1965年找到叫C.elegans的線蟲。這種線蟲在研究上有許多優(yōu)勢：結(jié)構(gòu)簡單、透明、生活周期短、可以冷凍儲(chǔ)存等，還證明可以用遺傳學(xué)手段研究線蟲，1967年，他們獲得第一個(gè)線蟲遺傳突變體。到1974年，布勒吶在《遺傳學(xué)》雜志上發(fā)表了第一篇有關(guān)線蟲的論文。75歲的布勒吶(SydneyBrenner)部分因布勒吶喜歡不同學(xué)科的人，部分因?qū)W生物的有許多人笑話用線蟲做研究，布勒吶初期招的學(xué)生常出身于數(shù)學(xué)、工程和化學(xué)。他認(rèn)為背景不多的人更少成見敢于冒險(xiǎn)。70年代加入線蟲研究的人冒很大的危險(xiǎn)，因不容易判斷線蟲能有多大用處。80年代中，線蟲研究在生物學(xué)界成為一個(gè)“熱門”，現(xiàn)在，世界上有許多研究線蟲的實(shí)驗(yàn)室和科學(xué)家，在發(fā)育生物學(xué)和神經(jīng)生物學(xué)都有重要發(fā)現(xiàn)。布勒吶得獎(jiǎng)原因就是他開創(chuàng)用線蟲做生物學(xué)研究材料，而不是某項(xiàng)具體研究工作。

1962年起，他和Crick討論了幾年，認(rèn)為分子生物學(xué)的框架5主要用線蟲做了包括發(fā)育生物學(xué)和神經(jīng)生物學(xué)的許多方面。一個(gè)重點(diǎn)是細(xì)胞凋亡的分子機(jī)理，他用遺傳突變的方法找到關(guān)鍵的調(diào)節(jié)細(xì)胞凋亡的基因。諾貝爾獎(jiǎng)委員會(huì)引用的是他的女研究生愛麗思（Ellis）和他于1986年在《細(xì)胞》雜志發(fā)表的論文。其后他實(shí)驗(yàn)室還發(fā)現(xiàn)其它控制細(xì)胞凋亡的基因。這些基因被霍維茨實(shí)驗(yàn)室的研究生克隆和進(jìn)一步研究，主要是袁均英(1977年上海高考第一名進(jìn)復(fù)旦)和Hengartner等。霍維茨實(shí)驗(yàn)室的研究，推動(dòng)人們理解細(xì)胞凋亡的分子機(jī)理，他們的結(jié)果和其他研究哺乳動(dòng)物細(xì)胞凋亡的結(jié)果對比，發(fā)現(xiàn)細(xì)胞凋亡在不同動(dòng)物用的是同樣的分子，其機(jī)理也是相同的。高等動(dòng)物細(xì)胞凋亡研究領(lǐng)域里，英國的Wiley和Kerr、澳大利亞的Vaux和Cory、美國的Korsmeyer等也有重要發(fā)現(xiàn)。細(xì)胞凋亡的生物化學(xué)機(jī)理研究的最重要突破來自于目前在得州大學(xué)西南醫(yī)學(xué)中心的王曉東院士的實(shí)驗(yàn)室。55歲的霍維茨(H.RobertHorvitz)主要用線蟲做了包括發(fā)育生物學(xué)和神經(jīng)生物學(xué)的許多方面。一個(gè)重點(diǎn)6霍維茨不僅自己動(dòng)手時(shí)做研究杰出，他從1978年以來領(lǐng)導(dǎo)的實(shí)驗(yàn)室，一直是生命科學(xué)界最高產(chǎn)的實(shí)驗(yàn)室之一。他有多方面的重要工作、論文很多，他實(shí)驗(yàn)室開創(chuàng)的對小RNA和對嗅覺機(jī)理的初期研究，都有在將來達(dá)到諾貝爾高度的可能?；艟S茨培養(yǎng)了許多出色的科學(xué)家，他的直接和間接學(xué)生遍布美國主要大學(xué)和研究機(jī)構(gòu)，也有在歐洲和臺(tái)灣的，其中華裔的包括北大畢業(yè)的韓珉和金亦石、復(fù)旦畢業(yè)的袁鈞英、科大畢業(yè)的薛定、和臺(tái)灣大學(xué)的吳怡春等（其中袁鈞英和薛定研究細(xì)胞凋亡）。55歲的霍維茨(H.RobertHorvitz)霍維茨不僅自己動(dòng)手時(shí)做研究杰出，他從1978年以來領(lǐng)導(dǎo)的實(shí)驗(yàn)760歲的薩爾斯頓(JohnSulston)薩爾斯頓原學(xué)有機(jī)化學(xué)。他喜歡自己動(dòng)手做實(shí)驗(yàn)，加入布勒吶小組后，他主要工作是分析線蟲的細(xì)胞譜系。和人的家譜一樣，機(jī)體的每一個(gè)細(xì)胞也有譜系關(guān)系，從單個(gè)受精卵，不斷分裂產(chǎn)生更多的細(xì)胞，最后形成身體全部許多不同的細(xì)胞。在復(fù)雜動(dòng)物細(xì)胞譜系關(guān)系都是不明確的，迄今我們還不清楚拇指任何細(xì)胞和小指任何細(xì)胞的譜系關(guān)系。薩爾斯頓初期是單獨(dú)，以后和霍維茨和其他少數(shù)幾個(gè)人合作，用顯微鏡觀察線蟲細(xì)胞譜系。這里，線蟲透明和細(xì)胞數(shù)有限的特點(diǎn)比較重要。1976年，薩爾斯頓在倫敦《皇家學(xué)會(huì)會(huì)刊》上發(fā)表論文，報(bào)道線蟲神經(jīng)系統(tǒng)內(nèi)部分細(xì)胞譜系的分析結(jié)果。這是第一次在任何動(dòng)物有這樣好的細(xì)胞譜系分析，同時(shí)，他發(fā)現(xiàn)，有一些特定的細(xì)胞生出后必定死亡，稱為“程序性死亡”。這在當(dāng)時(shí)是奇怪的現(xiàn)象，為什么有些細(xì)胞在每個(gè)個(gè)體的發(fā)育過程中都會(huì)出生，但是在不同的個(gè)體中又在同樣的發(fā)育階段要死去？好像生出來就是為死一樣?！俺绦蛐运劳觥币院笞C明和70年代中在高等動(dòng)物發(fā)現(xiàn)不久的“細(xì)胞凋亡”是同樣的。60歲的薩爾斯頓(JohnSulston)薩爾斯頓原學(xué)8Apoptosis-(Gr."falling")aprocessseeninmulticellularorganisms,bywhichspecificcellsarekilledandremovedforthebenefitoftheorganism.1972.Kerr,J.F.R.,Wyllie,A.H.andCurrie,A.R.Br.J.Cancer26:239.apoptosis1965LockshinandWilliams–"programmeddeath"Apoptosis-(Gr."falling")a9研究生課細(xì)胞凋亡的主要信號轉(zhuǎn)導(dǎo)途徑分子生物學(xué)課課件10Programmedcelldeathisamechanismwhichremovesdamagedcellsorinfectedcellsbyactivationofanintrinsicsuicideprogramwithoutelicitingan

immuneresponseorinflammatoryreaction.Programmedcelldeathalso

referredtoasapoptosiswascoinedby

Kerr,WyllieandCurriein1972(BritJ.Cancer26:239)todescribeaformofcelldeathdistinctfromnecrosis.ItsGreekmeaning“fallingoff”emphasizesthedeathoflivingmatterisanintegralpartofthelifecycleoforganisms.

PCD/APOPTOSISProgrammedcelldeathisamec11modeofcelldeaththatoccursunderNORMALPHYSIOLOGICALCONDITIONSplaysanimportantroleinmulticellulardevelopment,differentiation,andproliferation/homeostasis

andimmuneresponsesItisinvolvedindeletionofentirestructures,sculptingoftissues,andregulatestheneuronnumberadaptivemechanismactiveprocess-requiresATPAPOPTOSISmodeofcelldeaththatoccurs12normalcellturnovertissuehomeostasisembryogenesisinductionandmaintenanceofimmunetolerancedevelopmentofnervoussystemendocrinedependenttissueatrophyWhendoyouseeapoptosis?normalcellturnoverWhendoyo13membraneblebbing&changesmitochondrialleakageorganellereductioncellshrinkagenuclearfragmentationchromatincondensationAPOPTOSIS:Morphologicaleventsmembraneblebbing&changesmit14Apoptosisandnecrosishaveaspectsincommon,and,althoughoneheedsthedistinction,inpracticenecrosismaysuperveneuponacellundergoingapoptosis,&thereisotheroverlappingAPOPTOSISmembranesintactinvitesphagocytosisshrinkage

remainscontrolledNORMALNECROSISNECROSISvsAPOPTOSISmembranesleakyspillagewholecelldissolved

largely‘nuclear’inflammationApoptosisandnecrosishaveas15inducedMOSTLYbyphysiologicalstimulilackofgrowthfactorscellstressT-cellmediatedvirally-inducedchemically-inducedAPOPTOSISNECROSISPhysiologicalSignificanceevokedbyNON-physiologicalstimulicomplementattacklyticvirusesphysicaltraumahypoxia/ischemiametabolicpoisonsinducedMOSTLYbyphysiologica16affectsscatteredindividualcellsinflammationisMOSTLYabsent(phagocytosisofapoptoticbodies)APOPTOSISNECROSISHistologicalFeaturesUsuallyaffectstractsofcontiguouscellsinflammationispresentaffectsscatteredindividualc17NECROSISAPOPTOSISNECROSISAPOPTOSIS18shrinkingofcytoplasmandcondensationofnucleusmembraneblebbingformationofmembraneboundvesiclesAPOPTOSISNECROSISMorphologicalFeaturesswellingofcytoplasmandmitochondria(influxofNa+&water)lossofmembraneintegritynovesicleformationshrinkingofcytoplasmandcon19activationofspecificenzymeslikecaspases&Ca2+-dependentendonucleasesATP-dependentAPOPTOSISNECROSISBiochemicalFeatureslossofionhomeostasisROS,releaseofnon-specificlyticenzymesnoenergyrequirementactivationofspecificenzymes20MitochondriaandorganellesintactFragmentationofcellintosmallerbodiesChromatincondensationandmarginationAPOPTOSISNECROSISMorphologicalFeaturesdisintegrationoforganelles(swelling)CompletelysisandspillageofcellcontentChromatinbreakdownandcondensationMitochondriaandorganellesin21ApoptosisinWorms,Flies,MammalsApoptosisinWorms,Flies,Mamma22ApoptosisCluesfromCaenorhabditiselegansDuringdevelopmentexactly131cellsdie,while959survivecontrolledbycedgenes,encodingCEDproteinsdeathsignalcausesCED-4tobindtoinactiveCED-3,activatingit,leadingtoapoptosisCED-9bindstoCED-4,preventsactivationofCED-3HomologuesfoundinhumansandotheranimalsMammaliansystemmorecomplexApoptosisCluesfromCaenorhabd23ThreeclassesofproteinsfunctionintheapoptoticpathwayThreeclassesofproteinsfunc24研究生課細(xì)胞凋亡的主要信號轉(zhuǎn)導(dǎo)途徑分子生物學(xué)課課件25研究生課細(xì)胞凋亡的主要信號轉(zhuǎn)導(dǎo)途徑分子生物學(xué)課課件26(Bcl-2)(Apaf-1)(caspase-9)(Bcl-2)(Apaf-1)(caspase-9)27ApoptosisSignalingPathwayApoptosisSignalingPathway28Therearetwomajorapoptoticpathwaysinmammaliancells.Thedeathreceptorpathway,exemplifiedbyFasLbindingtoanextracellularreceptor,causestheformationoftheDISCthatresultsintheactivationofcaspase-8.(死亡受體途徑)Themitochondrialpathwayisactivatedbymostcellularstresses.Aresultingsignalorintracellularchangecausesthereleaseofcytochromecintothecytosol.CytochromecbindstoApaf-1andprocaspase-9toformtheapoptosomeandcatalyzestheactivationofcaspase-9.(線粒體途徑)Therearetwomajorapoptotic29MitochondrialpathwayRoleofmitochondriaimportantinnecrosisimportantroleinactivatingapoptosissomedownstreameventsATPdependentcertaincaspasesandBcl-2familymemberspresentinmitochondriapermeabilitytransitionreleasescytochromeccytochromecbindstoapaf-1andcaspase-9(apoptosome)MitochondrialpathwayRoleofm30Pleaseremembernameofthemitochondrialapoptosispathwayfounder:WangXiaodong!王曉東教授美國科學(xué)院院士！Pleaseremembernameofthemi31TwocanonicalapoptosispathwaysinmammaliancellsFasL:Fasligand;FADD:Fasassociateddeathdomain-containingproteinDD:deathdomain;DED:deatheffectordomainDISC:death-inducedsignalingcomplexCARD:caspaserecruitmentdomainApaf-1:apoptosisactivatingfactor-1IAP:inhibitorofapoptosiscrosstalkDISCInitiatorcaspaseEffectorcaspaseExtrinsicpathwayIntrinsicpathwayTwocanonicalapoptosispathwa32TNF-TNFRapoptosispathway(two-complexhypothesis)TNF:tumornecrosisfactorTNFR:TNFreceptorTRADD:TNFRassociateddeathdomain-containingTRAF:TNFRassociatedfactorRIP1:receptorinteractingpartner(kinase)NFkB:nuclearfactorkappaBJNK:c-JunN-terminalkinaseC-FLIP:cellularFLICE(caspase-8)inhibitorproteintBid:truncateBidjBid:JNK-mediatedBidfragment生死TNF-TNFRapoptosispathway(tw33ApoptosisRegulationApoptosisRegulation34Positiveregulation:inductionoractivationNegativeregulation:inhibitionPositiveregulation:induction35p53ApoptosiseventsseelastslideInitiatorcaspases 6,8,9,12ActivatorsofinitiatorenzymesApoptoticsignalsExecutioncaspases 2,3,7APOPTOSIS:Signaling&ControlpathwaysIExternallydrivenInternallydrivenCytochromecExternallydrivenActivationmitochondrionp53Apoptosiseventsseelasts36p53ExternalInternalApoptosiseventsInitiatorcaspases 6,8,9,12ActivatorsofinitiatorenzymesApoptoticsignalsExecutioncaspases 2,3,7InhibitorsofapoptosisAPOPTOSIS:Signaling&ControlpathwaysIIInhibitorsExternallydrivenInternallydrivenCytochromecExternallydrivenSurvivalfactorsBcl2Inhibitionp53ExternalInternalApoptosise37REGULATIONOFAPOPTOSISREGULATIONOFAPOPTOSIS38SignalingFactors&InitiaionRegulatorormodulatorsExecutionoreffectorsClearanceSignalingFactors&Initiaion39ApoptosisInitiatingstimuliphysiologicsignalsdevelopment,tissueinvolutioncellmediatedimmunologicprocessesdrugsandtoxinsirradiation,mildhyperthermiawithdrawalofgrowthfactorshypoxia!ApoptosisInitiatingstimuli40APOPTOSIS:SignalingfactorsExternalInternalActivatorsofinitiatorenzymesInhibitorsofapoptosisInhibitorsExternallydrivenSurvivalfactorsGrowthfactorsTumorNecrosisFactor-aApoptosiseventsInitiatorcaspasesApoptoticsignalsExecutioncaspasesExternallydrivenInternallydrivenCytochromecDNAdamageStressresponsesFasligandAPOPTOSIS:Signalingfactors41APOPTOSIS:SignalingfactorsIIExternalInternalApoptosiseventsInitiatorcaspasesActivatorsofinitiatorenzymesApoptoticsignalsExecutioncaspasesInhibitorsofapoptosisInhibitorsCytochromecSurvivalfactorsGrowthfactorsAttachmenttobasallaminaForsomeepithelialcells,detachmentfromthebasallaminatriggersapoptosis,usingintegrinsignaling-inthisinstance,termedANOIKISHenceBLattachmentisasurvivalfactorAPOPTOSIS:Signalingfactors42ApoptoticPathwaysEffectors&ModulatorsCaspasefamilyBcl-2familyAdaptors,FADD,TRADDApaf-1,SmacCytochromecEndonuclease,DFF45,EndoGIAPsothersApoptoticPathwaysEffectors&43IntroductionofcaspaseCASPASEcysteinyl-aspartate-specificproteinasefamilyofcysteineproteasesspecificityforaspartateresiduespresentasproenzymes(zymogens)activatedbyproteolyticcleavage,oftenbyothercaspasesautoactivationviaaggregationhence,acascadeofactivationIntroductionofcaspaseCASPASE44ClassificationofcaspasesCaspasesApoptoticandinflammatorycaspasesinitiatorandexecutionercaspasescaspase-3animportantexecutionercaspasefinalcommonpathwayactivatedbycaspases-8,-9,-10ClassificationofcaspasesCasp45Caspasesandsubstrateslargefamily-14membersfoundinmammaliansystems(human12)actonavarietyofsubstratesregulatory&structuralproteinscytoskeletalproteinsnuclearlaminsKinasesOtherimportantmoleculesConsensussequenceinsubstratesex.DXXD,casp3;(V/I/L)EXD,casp6Caspasesandsubstrates46caspasesInitiatorcaspasesEffectorcaspasescaspasesInitiatorcaspasesEffe47ActivationofcaspasesActivationofcaspases48ProposedInteractionBetweenDifferentCaspases

DuringApoptosis2ProposedInteractionBetweenD49CASPASECASCADEPlasmaMembraneCASP8APAF-1proCASP9CASP9Cyt.cCASP7CASP6CASP3AMPLIFICATIONCOMMITMENTCellularChanges/insultsApoptosisMito-chondriaCASPASECASCADEPlasmaMembrane50EffectofcaspasescleavageonsubstratesFurtherdiscussiononcaspasescanbefoundinEarnshaw,W.C.etal.1999.Annu.Rev.Biochem.

68:383.EffectofcaspasesFurtherdis51Pro-apoptoticregulators(Bad,Bax)promotecaspaseactivationPro-apoptoticregulators(Bad,52Sometrophicfactors(NGF)preventapoptosisbyinducinginactivationofapro-apoptoticregulatorSometrophicfactors(NGF)pre53ApoptoticPathwaysEffectors&ModulatorsCaspasefamilyBcl-2familyAdaptors,FADD,TRADDApaf-1,SmacCytochromecEndonuclease,DFF45,EndoGIAPsothersApoptoticPathwaysEffectors&54Bcl-2isaproto-oncogenethatwasfirstdiscoveredinB-celllymphoma.Bcl-2protein:anti-apoptoticproteininhibitsthereleaseofcytochromec,andactivationofAPAF-1.

SeveralmodelsarediscussedinHengartner,M.O.2000.Nature

407:770.AdditionalinformationaboutBcl-2familymemberscanbefoundinGross,A.etal.1999.Genes&Dev.13:1899.Bcl-2proteinBcl-2protein55Bcl-2familyContainBcl-2Homologydomains(BH1-4)Pro-andanti-apoptoticproteins

atleast19membersContainTMdomain(mit/nuc/ER)Sensorssomewithpro-andotherswithanti-apoptoticfunctions.TheratiobetweenthesetwotypeshelpsdeterminethefateofthecellBAX:pro-apoptoticproteinthatinducesthereleaseofcytochromecfromthemitochondria.Bcl-2familyContainBcl-2Homo56ClassificationofBcl-2familyClassificationofBcl-2family57研究生課細(xì)胞凋亡的主要信號轉(zhuǎn)導(dǎo)途徑分子生物學(xué)課課件58Bax/Bcl-2BaxBcl-2Bcl-2Bcl-2BaxBaxSurvivalApoptosisBcl-2andBaxbalanceBax/Bcl-2BaxBcl-2Bcl-2Bcl-2Bax59Bax/Bcl-2OuterMitochondrialMembraneCyt.cBaxAPAF-1ProCASP9CASP9Cyt.cAPOPTOSISBcl-2Bcl-2&BaxactoncytocreleaseBax/Bcl-2OuterMitochondrialCy60P53-GUARDIANOFTHEGENOMEp53inducesapoptosis,whenexpressedinveryhighlevels-irreversibleDNAdamagecellhascompletedS-phase(DNAreplicated)DNADamageDamageResponsep53Arrest/RepairG1/SG2ApoptosisviaBaxBax–targetofp53P53-GUARDIANOFTHEGENOMEp561DNADamage-inducedapoptosisRoleofp53andBaxDNADamage-62AnotherlevelofapoptoticregulationPro-apoptoticwhenunphosphorylatedInhibitsanti-apoptoticBcl-2familymembersSensitizesthecelltoapoptosisInactivewhenphosphorylated(ex.byAkt1;1999,Science)Bcl-2Bcl-2BaxBaxBADBADproteinoftheBcl-2familyAnotherlevelofapoptoticreg63AmodelfortheroleofBcl-2familymembersinapoptosis.AmodelfortheroleofBcl-264研究生課細(xì)胞凋亡的主要信號轉(zhuǎn)導(dǎo)途徑分子生物學(xué)課課件65ApoptoticPathwaysEffectors&ModulatorsCaspasefamilyBcl-2familyAdaptors,FADD,TRADDApaf-1,SmacCytochromecEndonuclease,DFF45,EndoGIAPsothersApoptoticPathwaysEffectors&66SignallingpathwaysinducedbyCD95Fas-associatedDeathDomainDeath-inducingsignalingcomplexSignallingpathwaysinducedby67IAPproteins(DIAP,XIAP,etc.)IAPinhibitorsReaper,Hid,Grim(fly)Smac,DIABLO(mammal)IAPproteins(DIAP,XIAP,etc.68SignalingFactors&InitiaionRegulatorormodulatorsExecutionoreffectorsClearanceSignalingFactors&Initiaion69PhagocyteApoptotic

CellRAC-1DOCK180CRKIIELMOCytoskeletal

Reorganizationfor

EngulfmentC1qReceptorBridgeC1qC1q

Binding

SitePSPhosphatidyl-

serine

ReceptorsScavenger

Receptors?OxidizedLDL-likeSiteApoptosisandPhagocytosisPhagocytesrecognize“eat-me”orcellcorpsesignalsontheapoptoticcellsurface.Thesesignalthephagocytetoactivatecellularengulfmentmachinery.Phosphatidylserineexposureonthetargetcellsurfaceandthephosphatidylserinereceptoronthephagocyteareessentialforphagocytosis.Definingotherreceptors,bridgemolecules,“eat-me”signalsandsignalingmoleculesinvolvedininitiatingthecytosolicchangesneededforengulfmentareveryactiveareasofresearch.Thearticleslistedbelowreviewcurrentknowledgeandarethesourcesforthisdiagram.Savill,J.andFadok,V.2000.Nature.407:784.Canradt,B.2002.NatureCellBiol.

4:E139.PhagocyteApoptotic

CellRAC-1DO70.EngulfmentPhagocyteApoptotic

CellWith"eatme"

signalsPhagocyteHealthy

CellPhagocyteApoptotic

CellWith"eatme"

signalsPhagocyte

PrecursorApoptotic

CellWith"eatme"

signalsPhagocyteinduces

apoptoticmachinery

inhealthycellApoptoticcellinduces

phagocyticmachinery

inphagocyteApoptoticcellinduces

maturationofprecursor

intophagocyteApoptosisandPhagocytosisThefirstpathwayshowstheengulfmentofanapoptoticcellexposing“eat-me”signals.DatafrommammaliansystemsandgeneticstudiesfromCaenorhabditiseleganshaveshownthatphagocytesandtargetcellshaveseveraltypesofinteractions.Conradthasproposedseveralmodels(2-4)toindicatethemorecomplexphagocyte-targetinteractions.Conradt,B.2002.NatureCellBiol.4:E139.Greene,D.R.andBeere,H.M.2001.Nature.412:.4.EngulfmentPhagocyteApo71IMPLICATIONSOFAPOPTOSISHYPER-ACTIVATIONneurodegenerativediseases(Parkinson’sandAlzheimer’sdisease)ischemia-reperfusioninjurydiabetes,immunodeficiency(AIDS)SUPPRESSIONcancerautoimmunedisordersIMPLICATIONSOFAPOPTOSISHYPER72SummarySourcesofCellDeath;necrosisapoptosisNeedfortheApoptosisPathwaysCausesofApoptosisMechanismsandregulationofApoptosisSummarySourcesofCellDeath;731，哺乳動(dòng)物細(xì)胞的兩條經(jīng)典的細(xì)胞凋亡途徑。2，caspase和Bcl-2家族在細(xì)胞凋亡中的作用。復(fù)習(xí)題復(fù)習(xí)題74千島湖風(fēng)光千島湖風(fēng)光75細(xì)胞凋亡的主要信號轉(zhuǎn)導(dǎo)途徑LingqiangZhang,Ph.DProfessor,PrincipalInvestigatorDept.Proteomics&Genomics(P&G)BeijingInst.RadiationMedicine(BIRM)Tel:66931216Email:zhanglq@細(xì)胞凋亡的主要信號轉(zhuǎn)導(dǎo)途徑LingqiangZhang,76Apoptosisandtheregulation---lifeordeathdecisionApoptosisandtheregulation77Science298:526,20022002Nobelfortheirdiscoveriesconcerninggeneticregulationoforgandevelopmentandprogrammedcelldeath.Science298:526,20022002Nob7875歲的布勒吶(SydneyBrenner)1953年4月，在J.Waston和F.Crick著名的DNA結(jié)構(gòu)模型文章發(fā)表前不僅，布勒吶在劍橋大學(xué)見到他們。以后在英國劍橋大學(xué)的卡文迪許實(shí)驗(yàn)室。和克里克有長期活躍的討論和合作。布勒吶對分子生物學(xué)有多項(xiàng)重要貢獻(xiàn)，是分子生物學(xué)開創(chuàng)者之一。他發(fā)現(xiàn)mRNA，是一項(xiàng)可獲諾貝爾獎(jiǎng)的工作。他研究遺傳密碼也有幾個(gè)重要貢獻(xiàn)。1961年，他在《自然》雜志有兩篇論文，一篇關(guān)于mRNA，一篇關(guān)于遺傳密碼。75歲的布勒吶(SydneyBrenner)1953年4791962年起，他和Crick討論了幾年，認(rèn)為分子生物學(xué)的框架已經(jīng)有了，要開創(chuàng)新領(lǐng)域。布勒吶想做發(fā)育和神經(jīng)系統(tǒng)。他的想法是用簡單的動(dòng)物，依靠遺傳學(xué)，來理解發(fā)育和神經(jīng)。1963年，布勒吶提出用一種叫C.briggsiae的線蟲研究發(fā)育和神經(jīng)，他不斷讀文獻(xiàn)和采集樣本，到1965年找到叫C.elegans的線蟲。這種線蟲在研究上有許多優(yōu)勢：結(jié)構(gòu)簡單、透明、生活周期短、可以冷凍儲(chǔ)存等，還證明可以用遺傳學(xué)手段研究線蟲，1967年，他們獲得第一個(gè)線蟲遺傳突變體。到1974年，布勒吶在《遺傳學(xué)》雜志上發(fā)表了第一篇有關(guān)線蟲的論文。75歲的布勒吶(SydneyBrenner)部分因布勒吶喜歡不同學(xué)科的人，部分因?qū)W生物的有許多人笑話用線蟲做研究，布勒吶初期招的學(xué)生常出身于數(shù)學(xué)、工程和化學(xué)。他認(rèn)為背景不多的人更少成見敢于冒險(xiǎn)。70年代加入線蟲研究的人冒很大的危險(xiǎn)，因不容易判斷線蟲能有多大用處。80年代中，線蟲研究在生物學(xué)界成為一個(gè)“熱門”，現(xiàn)在，世界上有許多研究線蟲的實(shí)驗(yàn)室和科學(xué)家，在發(fā)育生物學(xué)和神經(jīng)生物學(xué)都有重要發(fā)現(xiàn)。布勒吶得獎(jiǎng)原因就是他開創(chuàng)用線蟲做生物學(xué)研究材料，而不是某項(xiàng)具體研究工作。

1962年起，他和Crick討論了幾年，認(rèn)為分子生物學(xué)的框架80主要用線蟲做了包括發(fā)育生物學(xué)和神經(jīng)生物學(xué)的許多方面。一個(gè)重點(diǎn)是細(xì)胞凋亡的分子機(jī)理，他用遺傳突變的方法找到關(guān)鍵的調(diào)節(jié)細(xì)胞凋亡的基因。諾貝爾獎(jiǎng)委員會(huì)引用的是他的女研究生愛麗思（Ellis）和他于1986年在《細(xì)胞》雜志發(fā)表的論文。其后他實(shí)驗(yàn)室還發(fā)現(xiàn)其它控制細(xì)胞凋亡的基因。這些基因被霍維茨實(shí)驗(yàn)室的研究生克隆和進(jìn)一步研究，主要是袁均英(1977年上海高考第一名進(jìn)復(fù)旦)和Hengartner等?；艟S茨實(shí)驗(yàn)室的研究，推動(dòng)人們理解細(xì)胞凋亡的分子機(jī)理，他們的結(jié)果和其他研究哺乳動(dòng)物細(xì)胞凋亡的結(jié)果對比，發(fā)現(xiàn)細(xì)胞凋亡在不同動(dòng)物用的是同樣的分子，其機(jī)理也是相同的。高等動(dòng)物細(xì)胞凋亡研究領(lǐng)域里，英國的Wiley和Kerr、澳大利亞的Vaux和Cory、美國的Korsmeyer等也有重要發(fā)現(xiàn)。細(xì)胞凋亡的生物化學(xué)機(jī)理研究的最重要突破來自于目前在得州大學(xué)西南醫(yī)學(xué)中心的王曉東院士的實(shí)驗(yàn)室。55歲的霍維茨(H.RobertHorvitz)主要用線蟲做了包括發(fā)育生物學(xué)和神經(jīng)生物學(xué)的許多方面。一個(gè)重點(diǎn)81霍維茨不僅自己動(dòng)手時(shí)做研究杰出，他從1978年以來領(lǐng)導(dǎo)的實(shí)驗(yàn)室，一直是生命科學(xué)界最高產(chǎn)的實(shí)驗(yàn)室之一。他有多方面的重要工作、論文很多，他實(shí)驗(yàn)室開創(chuàng)的對小RNA和對嗅覺機(jī)理的初期研究，都有在將來達(dá)到諾貝爾高度的可能?；艟S茨培養(yǎng)了許多出色的科學(xué)家，他的直接和間接學(xué)生遍布美國主要大學(xué)和研究機(jī)構(gòu)，也有在歐洲和臺(tái)灣的，其中華裔的包括北大畢業(yè)的韓珉和金亦石、復(fù)旦畢業(yè)的袁鈞英、科大畢業(yè)的薛定、和臺(tái)灣大學(xué)的吳怡春等（其中袁鈞英和薛定研究細(xì)胞凋亡）。55歲的霍維茨(H.RobertHorvitz)霍維茨不僅自己動(dòng)手時(shí)做研究杰出，他從1978年以來領(lǐng)導(dǎo)的實(shí)驗(yàn)8260歲的薩爾斯頓(JohnSulston)薩爾斯頓原學(xué)有機(jī)化學(xué)。他喜歡自己動(dòng)手做實(shí)驗(yàn)，加入布勒吶小組后，他主要工作是分析線蟲的細(xì)胞譜系。和人的家譜一樣，機(jī)體的每一個(gè)細(xì)胞也有譜系關(guān)系，從單個(gè)受精卵，不斷分裂產(chǎn)生更多的細(xì)胞，最后形成身體全部許多不同的細(xì)胞。在復(fù)雜動(dòng)物細(xì)胞譜系關(guān)系都是不明確的，迄今我們還不清楚拇指任何細(xì)胞和小指任何細(xì)胞的譜系關(guān)系。薩爾斯頓初期是單獨(dú)，以后和霍維茨和其他少數(shù)幾個(gè)人合作，用顯微鏡觀察線蟲細(xì)胞譜系。這里，線蟲透明和細(xì)胞數(shù)有限的特點(diǎn)比較重要。1976年，薩爾斯頓在倫敦《皇家學(xué)會(huì)會(huì)刊》上發(fā)表論文，報(bào)道線蟲神經(jīng)系統(tǒng)內(nèi)部分細(xì)胞譜系的分析結(jié)果。這是第一次在任何動(dòng)物有這樣好的細(xì)胞譜系分析，同時(shí)，他發(fā)現(xiàn)，有一些特定的細(xì)胞生出后必定死亡，稱為“程序性死亡”。這在當(dāng)時(shí)是奇怪的現(xiàn)象，為什么有些細(xì)胞在每個(gè)個(gè)體的發(fā)育過程中都會(huì)出生，但是在不同的個(gè)體中又在同樣的發(fā)育階段要死去？好像生出來就是為死一樣。“程序性死亡”以后證明和70年代中在高等動(dòng)物發(fā)現(xiàn)不久的“細(xì)胞凋亡”是同樣的。60歲的薩爾斯頓(JohnSulston)薩爾斯頓原學(xué)83Apoptosis-(Gr."falling")aprocessseeninmulticellularorganisms,bywhichspecificcellsarekilledandremovedforthebenefitoftheorganism.1972.Kerr,J.F.R.,Wyllie,A.H.andCurrie,A.R.Br.J.Cancer26:239.apoptosis1965LockshinandWilliams–"programmeddeath"Apoptosis-(Gr."falling")a84研究生課細(xì)胞凋亡的主要信號轉(zhuǎn)導(dǎo)途徑分子生物學(xué)課課件85Programmedcelldeathisamechanismwhichremovesdamagedcellsorinfectedcellsbyactivationofanintrinsicsuicideprogramwithoutelicitingan

immuneresponseorinflammatoryreaction.Programmedcelldeathalso

referredtoasapoptosiswascoinedby

Kerr,WyllieandCurriein1972(BritJ.Cancer26:239)todescribeaformofcelldeathdistinctfromnecrosis.ItsGreekmeaning“fallingoff”emphasizesthedeathoflivingmatterisanintegralpartofthelifecycleoforganisms.

PCD/APOPTOSISProgrammedcelldeathisamec86modeofcelldeaththatoccursunderNORMALPHYSIOLOGICALCONDITIONSplaysanimportantroleinmulticellulardevelopment,differentiation,andproliferation/homeostasis

andimmuneresponsesItisinvolvedindeletionofentirestructures,sculptingoftissues,andregulatestheneuronnumberadaptivemechanismactiveprocess-requiresATPAPOPTOSISmodeofcelldeaththatoccurs87normalcellturnovertissuehomeostasisembryogenesisinductionandmaintenanceofimmunetolerancedevelopmentofnervoussystemendocrinedependenttissueatrophyWhendoyouseeapoptosis?normalcellturnoverWhendoyo88membraneblebbing&changesmitochondrialleakageorganellereductioncellshrinkagenuclearfragmentationchromatincondensationAPOPTOSIS:Morphologicaleventsmembraneblebbing&changesmit89Apoptosisandnecrosishaveaspectsincommon,and,althoughoneheedsthedistinction,inpracticenecrosismaysuperveneuponacellundergoingapoptosis,&thereisotheroverlappingAPOPTOSISmembranesintactinvitesphagocytosisshrinkage

remainscontrolledNORMALNECROSISNECROSISvsAPOPTOSISmembranesleakyspillagewholecelldissolved

largely‘nuclear’inflammationApoptosisandnecrosishaveas90inducedMOSTLYbyphysiologicalstimulilackofgrowthfactorscellstressT-cellmediatedvirally-inducedchemically-inducedAPOPTOSISNECROSISPhysiologicalSignificanceevokedbyNON-physiologicalstimulicomplementattacklyticvirusesphysicaltraumahypoxia/ischemiametabolicpoisonsinducedMOSTLYbyphysiologica91affectsscatteredindividualcellsinflammationisMOSTLYabsent(phagocytosisofapoptoticbodies)APOPTOSISNECROSISHistologicalFeaturesUsuallyaffectstractsofcontiguouscellsinflammationispresentaffectsscatteredindividualc92NECROSISAPOPTOSISNECROSISAPOPTOSIS93shrinkingofcytoplasmandcondensationofnucleusmembraneblebbingformationofmembraneboundvesiclesAPOPTOSISNECROSISMorphologicalFeaturesswellingofcytoplasmandmitochondria(influxofNa+&water)lossofmembraneintegritynovesicleformationshrinkingofcytoplasmandcon94activationofspecificenzymeslikecaspases&Ca2+-dependentendonucleasesATP-dependentAPOPTOSISNECROSISBiochemicalFeatureslossofionhomeostasisROS,releaseofnon-specificlyticenzymesnoenergyrequirementactivationofspecificenzymes95MitochondriaandorganellesintactFragmentationofcellintosmallerbodiesChromatincondensationandmarginationAPOPTOSISNECROSISMorphologicalFeaturesdisintegrationoforganelles(swelling)CompletelysisandspillageofcellcontentChromatinbreakdownandcondensationMitochondriaandorganellesin96ApoptosisinWorms,Flies,MammalsApoptosisinWorms,Flies,Mamma97ApoptosisCluesfromCaenorhabditiselegansDuringdevelopmentexactly131cellsdie,while959survivecontrolledbycedgenes,encodingCEDproteinsdeathsignalcausesCED-4tobindtoinactiveCED-3,activatingit,leadingtoapoptosisCED-9bindstoCED-4,preventsactivationofCED-3HomologuesfoundinhumansandotheranimalsMammaliansystemmorecomplexApoptosisCluesfromCaenorhabd98ThreeclassesofproteinsfunctionintheapoptoticpathwayThreeclassesofproteinsfunc99研究生課細(xì)胞凋亡的主要信號轉(zhuǎn)導(dǎo)途徑分子生物學(xué)課課件100研究生課細(xì)胞凋亡的主要信號轉(zhuǎn)導(dǎo)途徑分子生物學(xué)課課件101(Bcl-2)(Apaf-1)(caspase-9)(Bcl-2)(Apaf-1)(caspase-9)102ApoptosisSignalingPathwayApoptosisSignalingPathway103Therearetwomajorapoptoticpathwaysinmammaliancells.Thedeathreceptorpathway,exemplifiedbyFasLbindingtoanextracellularreceptor,causestheformationoftheDISCthatresultsintheactivationofcaspase-8.(死亡受體途徑)Themitochondrialpathwayisactivatedbymostcellularstresses.Aresultingsignalorintracellularchangecausesthereleaseofcytochromecintothecytosol.CytochromecbindstoApaf-1andprocaspase-9toformtheapoptosomeandcatalyzestheactivationofcaspase-9.(線粒體途徑)Therearetwomajorapoptotic104MitochondrialpathwayRoleofmitochondriaimportantinnecrosisimportantroleinactivatingapoptosissomedownstreameventsATPdependentcertaincaspasesandBcl-2familymemberspresentinmitochondriapermeabilitytransitionreleasescytochromeccytochromecbindstoapaf-1andcaspase-9(apoptosome)MitochondrialpathwayRoleofm105Pleaseremembernameofthemitochondrialapoptosispathwayfounder:WangXiaodong!王曉東教授美國科學(xué)院院士！Pleaseremembernameofthemi106TwocanonicalapoptosispathwaysinmammaliancellsFasL:Fasligand;FADD:Fasassociateddeathdomain-containingproteinDD:deathdomain;DED:deatheffectordomainDISC:death-inducedsignalingcomplexCARD:caspaserecruitmentdomainApaf-1:apoptosisactivatingfactor-1IAP:inhibitorofapoptosiscrosstalkDISCInitiatorcaspaseEffectorcaspaseExtrinsicpathwayIntrinsicpathwayTwocanonicalapoptosispathwa107TNF-TNFRapoptosispathway(two-complexhypothesis)TNF:tumornecrosisfactorTNFR:TNFreceptorTRADD:TNFRassociateddeathdomain-containingTRAF:TNFRassociatedfactorRIP1:receptorinteractingpartner(kinase)NFkB:nuclearfactorkappaBJNK:c-JunN-terminalkinaseC-FLIP:cellularFLICE(caspase-8)inhibitorproteintBid:truncateBidjBid:JNK-mediatedBidfragment生死TNF-TNFRapoptosispathway(tw108ApoptosisRegulationApoptosisRegulation109Positiveregulation:induc