1、細(xì)胞凋亡的主要信號轉(zhuǎn)導(dǎo)途徑Apoptosis and the regulation - life or death decisionScience 298:526, 20022002 Nobelfor their discoveries concerning genetic regulation of organ development and programmed cell death. 75歲的布勒吶(Sydney Brenner) 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

2、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。 1962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、

3、可以冷凍儲存等，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(Sydney Brenner) 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)。布勒吶得獎原因就是他開創(chuàng)用線蟲做生物學(xué)研究材料，而不

4、是某項(xiàng)具體研究工作。 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。諾貝爾獎委員會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)胞凋亡在不同動物用的是同樣的分子，其機(jī)理也是相同的。高等動物細(xì)胞凋亡研究領(lǐng)域里，英國的W

5、iley 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. Robert Horvitz) 霍維茨不僅自己動手時(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

6、、科大畢業(yè)的薛定、和臺灣大學(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. Robert Horvitz) 60歲的薩爾斯頓 (John Sulston) 薩爾斯頓原學(xué)有機(jī)化學(xué)。他喜歡自己動手做實(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。1

7、976年，薩爾斯頓在倫敦皇家學(xué)會會刊上發(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年代中在高等動物發(fā)現(xiàn)不久的“細(xì)胞凋亡”是同樣的。Apoptosis - (Gr. falling) a process seen in multicellular organisms, by which specific cells a

8、re killed and removed for the benefit of the organism.1972. Kerr, J.F.R., Wyllie, A.H. and Currie, A.R. Br. J. Cancer 26:239.apoptosis1965 Lockshin and Williams programmed deathProgrammed cell death is a mechanism which removes damaged cells or infected cells by activation of an intrinsic suicide pr

9、ogram without eliciting an immune response or inflammatory reaction. Programmed cell death also referred to as apoptosis was coined byKerr, Wyllie and Currie in 1972 ( Brit J.Cancer 26:239) to describe a form of cell death distinct from necrosis. Its Greek meaning “falling off” emphasizes the death

10、of living matter is an integral part of the life cycle of organisms. PCD / APOPTOSISmode of cell death that occurs under NORMAL PHYSIOLOGICAL CONDITIONSplays an important role in multicellular development, differentiation, and proliferation/homeostasis and immune responsesIt is involved in deletion

11、of entire structures, sculpting of tissues, and regulates the neuron numberadaptive mechanismactive process - requires ATPAPOPTOSISnormal cell turnovertissue homeostasisembryogenesisinduction and maintenance of immune tolerancedevelopment of nervous systemendocrine dependent tissue atrophyWhen do yo

12、u see apoptosis?membrane blebbing & changesmitochondrial leakageorganelle reductioncell shrinkagenuclear fragmentationchromatin condensationAPOPTOSIS: Morphological eventsApoptosis and necrosis have aspects in common, and, although one heeds the distinction, in practice necrosis may supervene upon a

13、 cell undergoing apoptosis, & there is other overlappingAPOPTOSISmembranes intactinvites phagocytosisshrinkage remains controlledNORMALNECROSISNECROSIS vs APOPTOSISmembranes leakyspillagewhole cell dissolved largely nuclearinflammationinduced MOSTLY by physiological stimulilack of growth factorscell

14、 stressT-cell mediatedvirally-inducedchemically-inducedAPOPTOSIS NECROSISPhysiological Significanceevoked by NON-physiological stimulicomplement attacklytic virusesphysical traumahypoxia/ischemiametabolic poisonsaffects scattered individual cellsinflammation is MOSTLY absent (phagocytosis of apoptot

15、ic bodies)APOPTOSIS NECROSISHistological FeaturesUsually affects tracts of contiguous cellsinflammation is presentNECROSISAPOPTOSISshrinking of cytoplasm and condensation of nucleusmembrane blebbingformation of membrane bound vesiclesAPOPTOSIS NECROSISMorphological Featuresswelling of cytoplasm and

16、mitochondria (influx of Na+ & water)loss of membrane integrityno vesicle formationactivation of specific enzymes like caspases & Ca2+-dependent endonucleasesATP-dependentAPOPTOSIS NECROSISBiochemical Featuresloss of ion homeostasisROS, release of non-specific lytic enzymesno energy requirementMitoch

17、ondria and organelles intactFragmentation of cell into smaller bodiesChromatin condensation and marginationAPOPTOSIS NECROSISMorphological Featuresdisintegration of organelles (swelling)Complete lysis and spillage of cell contentChromatin breakdown and condensationApoptosis in Worms,Flies,MammalsApo

18、ptosisClues from Caenorhabditis elegansDuring development exactly 131 cells die, while 959 survivecontrolled by ced genes, encoding CED proteinsdeath signal causes CED-4 to bind to inactive CED-3, activating it, leading to apoptosisCED-9 binds to CED-4, prevents activation of CED-3Homologues found i

19、n humans and other animalsMammalian system more complexThree classes of proteins function in the apoptotic pathway(Bcl-2)(Apaf-1)(caspase-9)Apoptosis Signaling PathwayThere are two major apoptotic pathways in mammalian cells.The death receptor pathway, exemplified by FasL binding to an extracellular

20、 receptor, causes the formation of the DISC that results in the activation of caspase-8. (死亡受體途徑)The mitochondrial pathway is activated by most cellular stresses. A resulting signal or intracellular change causes the release of cytochrome c into the cytosol. Cytochrome c binds to Apaf-1 and procaspa

21、se-9 to form the apoptosome and catalyzes the activation of caspase-9. (線粒體途徑)Mitochondrial pathwayRole of mitochondriaimportant in necrosisimportant role in activating apoptosissome downstream events ATP dependentcertain caspases and Bcl-2 family members present in mitochondriapermeability transiti

22、on releases cytochrome ccytochrome c binds to apaf-1 and caspase-9 (apoptosome)Please remember name of the mitochondrial apoptosis pathway founder: Wang Xiaodong! 王曉東教授 美國科學(xué)院院士！Two canonical apoptosis pathways in mammalian cellsFasL: Fas ligand; FADD: Fas associated death domain-containing protein D

23、D: death domain; DED: death effector domain DISC: death-induced signaling complexCARD: caspase recruitment domain Apaf-1: apoptosis activating factor-1 IAP: inhibitor of apoptosiscrosstalkDISCInitiator caspaseEffector caspaseExtrinsic pathwayIntrinsic pathwayTNF-TNFR apoptosis pathway (two-complex h

24、ypothesis)TNF: tumor necrosis factorTNFR: TNF receptorTRADD: TNFR associated death domain-containingTRAF: TNFR associated factorRIP1: receptor interacting partner (kinase)NFkB: nuclear factor kappa BJNK: c-Jun N-terminal kinaseC-FLIP: cellular FLICE (caspase-8) inhibitor proteintBid: truncate BidjBi

25、d: JNK-mediated Bid fragment生死Apoptosis RegulationPositive regulation: induction or activationNegative regulation: inhibitionp53Apoptosis events see last slideInitiator caspases 6, 8, 9,12Activators of initiator enzymesApoptotic signalsExecution caspases 2, 3, 7APOPTOSIS: Signaling & Control pathway

26、s IExternally drivenInternally drivenCytochrome cExternally drivenActivationmitochondrionp53ExternalInternalApoptosis eventsInitiator caspases 6, 8, 9,12Activators of initiator enzymesApoptotic signalsExecution caspases 2, 3, 7Inhibitors of apoptosisAPOPTOSIS: Signaling & Control pathways IIInhibito

27、rsExternally drivenInternally drivenCytochrome cExternally drivenSurvival factorsBcl2InhibitionREGULATION OF APOPTOSISSignaling Factors & Initiaion Regulator or modulatorsExecution or effectorsClearance ApoptosisInitiating stimuliphysiologic signalsdevelopment, tissue involutioncell mediated immunol

28、ogic processesdrugs and toxinsirradiation, mild hyperthermiawithdrawal of growth factorshypoxia!APOPTOSIS: Signaling factorsExternalInternalActivators of initiator enzymesInhibitors of apoptosisInhibitorsExternally drivenSurvival factorsGrowth factorsTumor Necrosis Factor-aApoptosis eventsInitiator

29、caspasesApoptotic signalsExecution caspasesExternally drivenInternally drivenCytochrome cDNA damage Stress responsesFas ligandAPOPTOSIS: Signaling factors IIExternalInternalApoptosis eventsInitiator caspasesActivators of initiator enzymesApoptotic signalsExecution caspasesInhibitors of apoptosisInhi

30、bitorsCytochrome cSurvival factorsGrowth factorsAttachment to basal laminaFor some epithelial cells, detachment from the basal lamina triggers apoptosis, using integrin signaling - in this instance, termed ANOIKISHence BL attachment is a survival factorApoptotic Pathways Effectors & ModulatorsCaspas

31、e familyBcl-2 familyAdaptors, FADD, TRADDApaf-1, SmacCytochrome cEndonuclease, DFF45, EndoGIAPsothersIntroduction of caspaseCASPASE cysteinyl-aspartate-specific proteinasefamily of cysteine proteasesspecificity for aspartate residuespresent as proenzymes (zymogens)activated by proteolytic cleavage,

32、often by other caspasesautoactivation via aggregationhence, a cascade of activationClassification of caspasesCaspasesApoptotic and inflammatory caspasesinitiator and executioner caspasescaspase-3 an important executioner caspasefinal common pathwayactivated by caspases -8, -9, -10Caspases and substr

33、ateslarge family - 14 members found in mammalian systems (human 12)act on a variety of substratesregulatory & structural proteinscytoskeletal proteinsnuclear laminsKinasesOther important moleculesConsensus sequence in substrates ex. DXXD, casp3; (V/I/L)EXD, casp6caspasesInitiator caspasesEffector ca

34、spasesActivation of caspasesProposed Interaction Between Different CaspasesDuring Apoptosis2CASPASE CASCADEPlasma MembraneCASP 8APAF-1proCASP 9CASP 9Cyt.cCASP 7CASP 6CASP 3AMPLIFICATIONCOMMITMENTCellularChanges/insultsApoptosisMito-chondriaEffect of caspases cleavage on substratesFurther discussion

35、on caspases can be found in Earnshaw, W.C. et al. 1999. Annu. Rev. Biochem. 68:383.Pro-apoptotic regulators (Bad, Bax) promote caspase activationSome trophic factors (NGF) prevent apoptosis by inducing inactivation of a pro-apoptotic regulatorApoptotic Pathways Effectors & ModulatorsCaspase familyBc

36、l-2 familyAdaptors, FADD, TRADDApaf-1, SmacCytochrome cEndonuclease, DFF45, EndoGIAPsothersBcl-2 is a proto-oncogene that was first discovered in B-cell lymphoma. Bcl-2 protein: anti-apoptotic protein inhibits the release of cytochrome c, and activation of APAF-1. Several models are discussed in Hen

37、gartner, M.O. 2000. Nature 407:770. Additional information about Bcl-2 family members can be found in Gross, A. et al. 1999. Genes & Dev. 13:1899.Bcl-2 proteinBcl-2 familyContain Bcl-2 Homology domains (BH 1-4)Pro- and anti-apoptotic proteins at least 19 membersContain TM domain (mit/nuc/ER)Sensorss

38、ome with pro- and others with anti-apoptotic functions. The ratio between these two types helps determine the fate of the cellBAX: pro-apoptotic protein that induces the release of cytochrome c from the mitochondria.Classification of Bcl-2 familyBax/Bcl-2BaxBcl-2Bcl-2Bcl-2BaxBaxSurvivalApoptosisBcl-

39、2 and Bax balanceBax/Bcl-2Outer MitochondrialMembraneCyt. cBaxAPAF-1ProCASP 9CASP 9Cyt. cAPOPTOSISBcl-2Bcl-2 & Bax act on cyto c releaseP53 - GUARDIAN OF THE GENOMEp53 induces apoptosis, whenexpressed in very high levels - irreversible DNA damagecell has completed S-phase (DNA replicated)DNADamageDa

40、mageResponsep53Arrest/RepairG1/SG2Apoptosisvia BaxBax target of p53DNA Damage-induced apoptosisRole of p53 and BaxAnother level of apoptotic regulationPro-apoptotic when unphosphorylatedInhibits anti-apoptotic Bcl-2 family membersSensitizes the cell to apoptosisInactive when phosphorylated (ex. by A

41、kt1; 1999, Science)Bcl-2Bcl-2BaxBaxBADBAD protein of the Bcl-2 familyA model for the role of Bcl-2 family members in apoptosis.Apoptotic Pathways Effectors & ModulatorsCaspase familyBcl-2 familyAdaptors, FADD, TRADDApaf-1, SmacCytochrome cEndonuclease, DFF45, EndoGIAPsothersSignalling pathways induc

42、ed by CD95Fas-associated Death DomainDeath-inducing signaling complexIAP proteins (DIAP, XIAP, etc.)IAP inhibitorsReaper, Hid, Grim (fly)Smac, DIABLO (mammal)Signaling Factors & Initiaion Regulator or modulatorsExecution or effectorsClearance PhagocyteApoptoticCellRAC-1DOCK 180CRKIIELMOCytoskeletalR

43、eorganization forEngulfmentC1q ReceptorBridgeC1qC1qBindingSitePSPhosphatidyl-serineReceptorsScavengerReceptors?Oxidized LDL-like SiteApoptosis and PhagocytosisPhagocytes recognize “eat-me” or cell corpse signals on the apoptotic cell surface. These signal the phagocyte to activate cellular engulfment machinery.Phosphatidylserine exposure on the target cell surface and the phosphatidylserine receptor on the phagocyte are essential for phagocytosis.Defining other receptors, bridge molecules, “eat-me” signals and signaling molecules in