细胞凋亡的线粒体途径.ppt
细胞凋亡的线粒体途径细胞凋亡的线粒体途径蒋舜媛 董霞 程在全2002-12-17细胞死亡损伤性死亡损伤性死亡 Necrosis程序化死亡、细胞凋亡程序化死亡、细胞凋亡Programmed Cell DeathApoptosis细胞凋亡的特征形态学特征形态学特征:染色质的凝集染色质的凝集,嗜碱性染色增强嗜碱性染色增强,细胞核崩解此时线粒体保持形态正常细胞核崩解此时线粒体保持形态正常.细胞体积缩小细胞体积缩小,一部分细胞质和核碎片进入由膜一部分细胞质和核碎片进入由膜包被的程序死亡小体包被的程序死亡小体,他们从细胞表面出芽脱落他们从细胞表面出芽脱落,并被巨噬细胞并被巨噬细胞.上皮细胞吞噬上皮细胞吞噬.生化特征生化特征:染色质降解染色质降解,核小体间连接核小体间连接DNA部位被降解部位被降解,产产生寡聚核小体生寡聚核小体DNA片段片段,即即180-200DP 整数倍整数倍的不同长度的的不同长度的DNA片断片断.Fig.1.Schematic summary of biochemical mechanisms of apoptosis.Mitochondria and Commitment to Cell Death n线粒体是真核细胞的重要细胞器，是动物细胞生成ATP的主要地点。线粒体基质的三羧酸循环酶系通过底物脱氢氧化生成NADH。NADH通过线粒体内膜呼吸链氧化。与此同时，导致跨膜质子移位形成跨膜质子梯度和/或跨膜电位。线粒体内膜上的ATP合成酶利用跨膜质子梯度能量合成ATP。合成的ATP通过线粒体内膜ADP/ATP载体与细胞质中ADP交换进入细胞质，参与细胞的各种需能过程。Mitochondrial Pathways in physiological cell death the release of caspase activators(such as cytochrome c),changes in electron transport,loss of mitochondrial transmembrane potential,altered cellular oxidation-reduction,participation of pro-and antiapoptotic Bcl-2 family proteins.Mitochondrial Pathways in physiological cell death Ifmitochondriaarepivotalincontrollingcelllifeanddeath,thenhowdotheseorganelleskill?Atleastthreegeneralmechanismsareknown,andtheireffectsmaybeinterrelated,including(i)disruption of electron transport,oxidative phosphorylation,and adenosine triphosphate(ATP)production;(ii)release of proteins that trigger activation of caspase family proteases;and(iii)alteration of cellular reduction-oxidation(redox)potential Disruption of electron transport and energy metabolism ndisruptionofelectrontransporthasbeenrecognizedasanearlyfeatureofcelldeath.n-Irradiationinducesapoptosisinthymocytesandadisruptionintheelectrontransportchain,probablyatthecytochromeb-c1/cytochromec(cytoc)step.nCeramide(asecondmessengerimplicatedinapoptosissignaling)disruptselectrontransportatthesamestepincellsaswellasinisolatedmitochondria.nLigationofFasalsoleadstoadisruptionincytocfunctioninelectrontransport.Disruption of electron transport and energy metabolism nOne consequence of the loss of electron transport should be a drop in ATP production.Although such a drop has been observed during apoptosis,it often occurs relatively late in the process(14).nIndeed,ATP appears to be required for downstream events in apoptosis(15).nThus,although loss of mitochondrial ATP production can kill a cell,it is unlikely that this is a mechanism for induction of apoptosis.Disruption of electron transport and energy metabolism n线粒体跨膜电位的耗散与细胞凋亡有密切关系线粒体跨膜电位的耗散与细胞凋亡有密切关系n近年来陆续有报道说明线粒体跨膜电位的耗散早于核酸酶的激活，也早于磷酯酰丝氨酸暴露于细胞表面。而一旦线粒体跨膜电位耗散，细胞就会进入不可逆的凋亡过程。线粒体解联的呼吸链会产生大量活性氧，氧化线粒体内膜上的心磷脂。实验证明，用解偶联剂mClCCP会导致淋巴细胞凋亡。而如果能稳定线粒体跨膜电位就能防止细胞凋亡。Release of caspase-activating proteins nTheimportanceofmitochondriainapoptosiswassuggestedbystudieswithacell-freesysteminwhichspontaneous,nBcl-2-inhibitablenuclearcondensationandDNAfragmentationwerefoundtobedependentonthepresenceofmitochondria(16).nSubsequently,studiesinanothercell-freesystemshowedthatinductionofcaspaseactivationbyadditionofdeoxyadenosinetriphosphatedependedonthepresenceofcytocreleasedfrommitochondriaduringextractpreparation(17).Duringapoptosis(invitroandinvivo)cytocisreleasedfrommitochondriaandthisisinhibitedbythepresenceofBcl-2ontheseorganelles(18,19).nCytosoliccytocformsanessentialpartofthevertebrateapoptosome,whichiscomposedofcytoc,Apaf-1,andprocaspase-9(20).Theresultisactivationofcaspase-9,whichthenprocessesandactivatesothercaspasestoorchestratethebiochemicalexecutionofcells.Release of caspase-activating proteins nSignificantly,caspaseinhibitorsdonotpreventcytocreleaseinducedbyseveralapoptogenicagents,includingUVirradiation,staurosporine,andoverexpressionofBax(14,21,22).AnexceptioniscytocreleasefrommitochondriainducedbythetumornecrosisfactorreceptorfamilymemberFas,inwhichcytocreleaseispreventedbyinhibitionofcaspases(primarilycaspase-8)recruitedtothecytosolicdomainofligatedFas(21).Nevertheless,cytocreleasecansometimescontributetoFas-mediatedapoptosisbyamplifyingtheeffectsofcaspase-8onactivationofdownstreamcaspases(23).nTheemergentviewisthatoncecytocisreleased,thiscommitsthecelltodiebyeitherarapidapoptoticmechanisminvolvingApaf-1-mediatedcaspaseactivationoraslowernecroticprocessduetocollapseofelectrontransport,whichoccurswhencytocisdepletedfrommitochondria,resultinginavarietyofdeleterioussequelaeincludinggenerationofoxygenfreeradicalsanddecreasedproductionofATPReactive oxygen species and cellular redox.nMitochondriaarethemajorsourceofsuperoxideanionproductionincells.nDuringtransferofelectronstomolecularoxygen,anestimated1to5%ofelectronsintherespiratorychainlosetheirway,mostparticipatinginformationofO2.Anythingthatdecreasesthecouplingefficiencyofelectronchaintransportcanthereforeincreaseproductionofsuperoxides.Reactive oxygen species and cellular redox.nSuperoxidesandlipidperoxidationareincreasedduringapoptosisinducedbymyriadstimuli(28).nHowever,generationofROSmaybearelativelylateevent,occurringaftercellshaveembarkedonaprocessofcaspaseactivation.nInthisregard,attemptstostudyapoptosisunderconditionsofanoxiahavedemonstratedthatatleastsomeproapoptoticstimulifunctionintheabsenceornearabsenceofoxygen,whichimpliesthatROSsarenotthesinequanonofapoptosis(29,30).nHowever,ROSscanbegeneratedunderconditionsofvirtualanaerobiosis(31),andthustheirroleinapoptosiscannotbeexcludedsolelyonthisbasis.Figure 2.Model for caspase activation by mitochondria.osmotic disequilibrium leading to an expansion of the matrix space,organellar swelling,and subsequent rupture of the outer membranethe other envisions opening of channels in the outer membrane thus releasing cyto c from the intermembrane space of mitochondria into the cytosol.PT Pore nInmanyapoptosisscenarios,themitochondrialinnertransmembranepotential(m)collapses(32),indicatingtheopeningofalargeconductancechannelknownasthemitochondrialPT pore(33)(Fig.2).nitsconstituentsincludebothinnermembraneproteins,suchastheadenine nucleotide translocator(ANT),andoutermembraneproteins,suchasporin(voltage-dependent anion channel;VDAC),whichoperateinconcert,presumablyatinnerandoutermembranecontactsites,andcreateachannelthroughwhichmolecules1.5kDpass(32,34).PT Pore nOpening of this nonselective channel in the inner membrane allows for an equilibration of ions within the matrix and intermembrane space of mitochondria,thus dissipating the H+gradient across the inner membrane and uncoupling the respiratory chain.nPerhaps more importantly,PT pore opening results in a volume dysregulation of mitochondria due to the hyperosmolality of the matrix,which causes the matrix space to expand.nBecause the inner membrane with its folded cristae possesses a larger surface area than the outer membrane,this matrix volume expansion can eventually cause outer membrane rupture,releasing caspase-activating proteins located within the intermembrane space into the cytosol(Fig.1).Fig.3.The mitochondrial permeability transition.PT Pore 的性质的性质 通过一些实验室的研究，以下诸点值得指出：n线粒体内膜通透性转变既是细胞凋亡的必须条件，也是它的充足条件。nPT孔道打开后导致线粒体许多功能的致命性变化从而启动了死亡途径。nPT孔道作为许多生理效应的感受器（二价阳离子、ATP、ADP、NAD、m、pH、巯基与多肽)，整合了电生理、氧化还原与细胞代谢状态的信息。nPT孔道的组成成分ADP-ATP载体是能量代谢的重要分子，由于ADP-ATP载体是由一个基因家族的几个成员所编码，它的表达有严格的组织专一性。因此，PT孔道在不同细胞中的调节可能稍有不同。nPT孔道的作用有自放大的效应。PT诱导m耗散，而反过来 mClCCP使m去极化会导致PT。一些PT的结果例如m 耗散，活性氧的生成本身也会导致PT。这就说明PT会有正反馈，从而在细胞凋亡中有自摧毁的作用。反过来，如果能防止m的耗散，就能避免氧化还原不平衡、磷酯酰丝氨酸的暴露与蛋白酶和核酸酶的激活。PT Pore 有开放与关闭二种构象有开放与关闭二种构象nPT孔道开放导致细胞凋亡。n在PT孔道开放时线粒体释放细胞凋亡诱导因子(AIF)。AIF可能是一种蛋白水解酶，位于线粒体膜间间隙，它能被蛋白酶抑制剂如N-苄氧羰基-缬氨酰-丙氨酰-门冬氨酰氟甲基酮(N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone)所抑制。n苍术苷促进PT通道开放。苍术苷只能与ADP-ATP载体的胞液侧结合。n此外从线粒体释放的细胞色素C也是一种细胞凋亡诱导因子。n而PT孔道关闭能防止细胞凋亡。n当PT孔道与环孢菌素A(cyclosporin A)或SH，或米酵菌酸(bongkrek acid)结合时PT孔道被关闭。米酵菌酸可与ADP-ATP载体的胞液及基质二侧结合Fig.4 The cytochrome c-induced caspase activation pathway.OligomerizationRecruitCleaveActiviateFig.5.Displacement of IAPs from caspases by Smac/Diablo.Fig.6.Multiple apoptotic pathways emanate from the mitochondria.chromatin condensation and fragmentationThanks!Thanks!2002-12-17
