细胞凋亡的主要信号转导途径课件

1、细胞凋亡的主要信号转导途径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岁的布勒呐(Sydney Brenner) 1953年4月，在J. Waston和F. Crick著名的DNA结构模型文章发表前不仅，布勒呐在剑桥大学见到他们。以后在英国剑桥大学的卡文迪许实验室。和克里

2、克有长期活跃的讨论和合作。 布勒呐对分子生物学有多项重要贡献，是分子生物学开创者之一。他发现mRNA，是一项可获诺贝尔奖的工作。他研究遗传密码也有几个重要贡献。1961年，他在自然杂志有两篇论文，一篇关于mRNA，一篇关于遗传密码。 1962年起，他和Crick讨论了几年，认为分子生物学的框架已经有了，要开创新领域。布勒呐想做发育和神经系统。他的想法是用简单的动物，依靠遗传学，来理解发育和神经。1963年，布勒呐提出用一种叫C. briggsiae的线虫研究发育和神经，他不断读文献和采集样本，到1965年找到叫C. elegans的线虫。这种线虫在研究上有许多优势：结构简单、透明、生活周期短、

3、可以冷冻储存等，还证明可以用遗传学手段研究线虫，1967年，他们获得第一个线虫遗传突变体。到1974年，布勒呐在遗传学杂志上发表了第一篇有关线虫的论文。75岁的布勒呐(Sydney Brenner) 部分因布勒呐喜欢不同学科的人，部分因学生物的有许多人笑话用线虫做研究，布勒呐初期招的学生常出身于数学、工程和化学。他认为背景不多的人更少成见敢于冒险。70年代加入线虫研究的人冒很大的危险，因不容易判断线虫能有多大用处。80年代中，线虫研究在生物学界成为一个“热门”，现在，世界上有许多研究线虫的实验室和科学家，在发育生物学和神经生物学都有重要发现。布勒呐得奖原因就是他开创用线虫做生物学研究材料，而不

4、是某项具体研究工作。 主要用线虫做了包括发育生物学和神经生物学的许多方面。一个重点是细胞凋亡的分子机理，他用遗传突变的方法找到关键的调节细胞凋亡的基因。诺贝尔奖委员会引用的是他的女研究生爱丽思（Ellis）和他于1986年在细胞杂志发表的论文。其后他实验室还发现其它控制细胞凋亡的基因。这些基因被霍维茨实验室的研究生克隆和进一步研究，主要是袁均英(1977年上海高考第一名进复旦)和Hengartner等。霍维茨实验室的研究，推动人们理解细胞凋亡的分子机理，他们的结果和其他研究哺乳动物细胞凋亡的结果对比，发现细胞凋亡在不同动物用的是同样的分子，其机理也是相同的。高等动物细胞凋亡研究领域里，英国的W

5、iley 和Kerr、澳大利亚的Vaux和Cory、美国的Korsmeyer等也有重要发现。细胞凋亡的生物化学机理研究的最重要突破来自于目前在得州大学西南医学中心的王晓东院士的实验室。55岁的霍维茨(H. Robert Horvitz) 霍维茨不仅自己动手时做研究杰出，他从1978年以来领导的实验室，一直是生命科学界最高产的实验室之一。他有多方面的重要工作、论文很多，他实验室开创的对小RNA和对嗅觉机理的初期研究，都有在将来达到诺贝尔高度的可能。霍维茨培养了许多出色的科学家，他的直接和间接学生遍布美国主要大学和研究机构，也有在欧洲和台湾的，其中华裔的包括北大毕业的韩珉和金亦石、复旦毕业的袁钧英

6、、科大毕业的薛定、和台湾大学的吴怡春等（其中袁钧英和薛定研究细胞凋亡）。 55岁的霍维茨(H. Robert Horvitz) 60岁的萨尔斯顿 (John Sulston) 萨尔斯顿原学有机化学。他喜欢自己动手做实验，加入布勒呐小组后，他主要工作是分析线虫的细胞谱系。和人的家谱一样，机体的每一个细胞也有谱系关系，从单个受精卵，不断分裂产生更多的细胞，最后形成身体全部许多不同的细胞。在复杂动物细胞谱系关系都是不明确的，迄今我们还不清楚拇指任何细胞和小指任何细胞的谱系关系。萨尔斯顿初期是单独，以后和霍维茨和其他少数几个人合作，用显微镜观察线虫细胞谱系。这里，线虫透明和细胞数有限的特点比较重要。1

7、976年，萨尔斯顿在伦敦皇家学会会刊上发表论文，报道线虫神经系统内部分细胞谱系的分析结果。这是第一次在任何动物有这样好的细胞谱系分析，同时，他发现，有一些特定的细胞生出后必定死亡，称为“程序性死亡”。这在当时是奇怪的现象，为什么有些细胞在每个个体的发育过程中都会出生，但是在不同的个体中又在同样的发育阶段要死去？好像生出来就是为死一样。“程序性死亡”以后证明和70年代中在高等动物发现不久的“细胞凋亡”是同样的。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. (死亡受体途径)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. (线粒体途径)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! 王晓东教授 美国科学院院士！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