细胞死亡及周期阻滞基本信号通路培训课件

细胞死亡及周期阻滞基本信号通路CELLDEATHAND

CELL-CYCLECHECKPOINTPATHWAYSDURINGDNADAMAGEJIAHong-Ti（贾弘禔）DEPTBIOCHEMMOLBIOLHSC,PekingUnivjiahongti@

DNA损伤时细胞死亡和细胞周期监控点（调控的基本）信号途径2细胞死亡及周期阻滞基本信号通路1.有哪些因素可引起DNA损伤？DNA损伤的结局如何？2.什么分子可作为DNA双链断裂/损伤的标志？用什么方法测定？3.

简述MicroRNA在DDR中的作用。（听完本讲座，结合自己研究课题和文献检索）试举一例（可能）与自己工作领域相关的MicroRNA功能及其生物学或临床意义。4.写出G1、G2/Mcheckpoint基本信号通路主要分子成分。5.程序性细胞死亡（PCD）都有哪些形式？I型（凋亡）、II型（自噬）（自噬定义）PCD主要形态学特征如何？如何检测或鉴定？其基本信号通路所涉及的主要激活分子是什么？各有何意义？本讲基本要求——思考题3细胞死亡及周期阻滞基本信号通路诱导DNA损伤与细胞死亡原因*——各种射线照射/光动力学治疗（光敏反应）化疗药物/细胞毒、基因毒试剂缺血/缺氧/氧化应激血清/营养因子/氨基酸撤出营养过剩过度兴奋/刺激中毒/钙超载感染/炎症其它*常伴有或直接、间接引起DNA损伤该讲的普遍意义——多种原因相关的DNA损伤引起细胞死亡和细胞周期阻滞4细胞死亡及周期阻滞基本信号通路THEPLETHORAOFDAMAGESINDNA

(DNA损伤的多样性/复杂性）THECONSEQUENCESOFDNAINJURY(DNA损伤的结局)3.CELL-CYCLECHECKPOINTPATHWAYS(细胞周期监控点基本信号途径)4.PROGRAMMED(REGULATED)CELLDEATH(程序性细胞死亡,PCD)本讲提纲5细胞死亡及周期阻滞基本信号通路1.APLETHORAOFDAMAGESINDNAAPERPLEXINGDIVERSITYOFDNALESIONSARISESFROMTHREEMAINCAUSES

Environmentalagents(环境因素)Productsofnormalcellularmetabolisminmitochondrion（线粒体代谢产物——ROS）SomechemicalbondsinDNAspontaneouslydisintegrateunderphysiologicalconditions（DNA自发裂解）(A)EnvironmentalAgents:

ultraviolet(UV),ionizingradiation…

(环境因素)

genotoxicchemicalsvirusinfectionAlterationsinDNAstructure:AdjacentthyminedimerDNAsinglestrandedbreaks(DNASSBs)DNAdoublestrandedbreaks(DNADSBs)Cross-linking(intra-andinter-cross-linking)…DNA损伤的多样性及主要诱因6细胞死亡及周期阻滞基本信号通路(B)ProductsofNormalCellularMetabolisminMitochondrion:

(线粒体代谢产物——ROS）Reactiveoxygenspecies(ROS)derivedfromoxidativerespirationandproductsoflipidperoxidation.

OxidativemodificationsinDNA.e.g.,8-OxoGAntioxidantdefencesystemcomposedofenzymatic(superoxidedismutase/SOD,catalase,glutathioneperoxidase/GPxandperoxyredoxins/GR)andlowmolecular-massscavengers(suchasglutathione/GSH).7细胞死亡及周期阻滞基本信号通路Fig-ProductionofROSandantioxidantdefencesystemsinhippocampalneuronsunderkainate-inducedoxidativestress.(LiSYetal.FreeRadBiolMed,48:597-608,2010)ROS产生——海人酸引起的神经元过度兴奋(举例)8细胞死亡及周期阻滞基本信号通路Fig-GenotoxicchemicalexposureinducesDNADSBs.(A)IdentificationofDNADSBsbyPFGE.(B)Immunocytochemicalstaininggamma-H2AXfoci.(C)Testinggamma-H2AX(H2AX-Ser139)byWesternblotting.(YangSY,etal.BiochemPharmacol,77:433-443,2009)DNA损伤——双链断裂的(3种)鉴定（举例）9细胞死亡及周期阻滞基本信号通路Fig-Theeffectsofkanicacid-inducedROSproductionontheintegrityofthegenomeinculturedneurons.ThegenomicDNAdamagewastestedusingfluoresceinisothiocyanate(FITC)-conjugatedavidin,whichbindsto8-oxodeoxyguanosine(8-oxodG)withastructuralsimilaritytobiotin(RadiskyDCetal.Nature,2005.436:123-127)抗生物素蛋白检测DNA氧化损伤（8-O-鸟苷）鉴定（举例）彗星实验检测DSBs

（举例）Fig-Cometassay.

ToscorethepercentageofDNAinthetail,theimageanalysissystemwasused.Thepercentageofcomettailarea(DNAtailarea/totalDNAarea)andthecomettaillength(fromthecenteroftheDNAheadtotheendoftheDNAtail)wereanalyzedin50cellsforoneslide.(AndersonDetal.MutatRes1994;307:261-271)Con0.2μM2μM10μM10细胞死亡及周期阻滞基本信号通路(C)

SomeChemicalBondsinDNASpontaneouslyDisintegrateunderPhysiologicalConditions（DNA自发裂解）:Hydrolysisofnucleotideresiduesleavesnon-instructiveabasicsites.Deamination

ofcytosine,adenine,guanineor5-methylcytosineconvertsthesebasestothemiscodinguracil,hypoxanthine,xanthineandthymine,respectively.

Wholechromosomalinstability(W-CIN);segmentalchromosomalinstability(S-CIN);genomicinstability(GIN)/基因组不稳定性（Geigl,J.B.etal.(2008)Defining‘chromosomalinstability’.TrendsGenet.24,64–69RickeRMetal.(2008)Wholechromosomeinstabilityandcancer:acomplexrelationship.TrendsGenet24:475-466）11细胞死亡及周期阻滞基本信号通路诺贝尔化学奖评审委员会2015年10月7日在瑞典皇家科学院宣布，2015年度诺贝尔化学奖由英国弗朗西斯·克里克研究所名誉教授/瑞典人托马斯·林达尔、美国科学家/杜克大学医学院教授保罗·莫德里和拥有美国和土耳其双重国籍的科学家/美国北卡罗来纳大学医学院教授阿齐兹·桑贾尔分享，以表彰他们在DNA损伤修复机制——BER、NER和MMR方面的研究成果；他们的发现揭示了细胞是如何维持基因组DNA稳定性的，在以DNA损伤为基础的肿瘤治疗以及在衰老中的作用。此前与DNA损伤及结局相关研究而获奖NobelPrize的有DNADamage&Mutation-HermannMuller(NobelPrize,1946)showedthattherateofmutationwasproportionaltothedoseofirradiation.GeorgeBeadle(NobelPrize,1958)showedthattheeffectofX-irradiationonmetabolismwasduetomutationsofgenes.EdwardTatum(NobelPrize,1958)furthershowedthatamutationofasinglegeneresultedonlyinasinglechemicalreaction,whichgaveevidencetotheconceptof"onegene,oneenzyme".CellCycleCheckpointSignaling-LalandHartwell,TimothyHuntandPaulNursewereawardedNobelPrizein2001fortheircontributionsinelucidatingthecellcycleregulation.Apoptosis-SydneyBrenner,RobertHorvitzandJohnESulstonwereawardedNobelPrizein2002fortheircontributionsinapoptosismechanisms.2015年度诺贝尔化学奖与DNA损伤修复12细胞死亡及周期阻滞基本信号通路TomasLindahl发现E.coliUracilDNAglycosidase及碱基切除修复(BEP)机制托马斯•林达尔(LindahlT.AnN-glycosidasefromEscherichiacolithatreleasesfreeuracilfromDNAcontainingdeaminatedcytosineresidues.ProcNatlAcadSciUSA1974;71(9):3649-3653BarnersDE,LindahlT.RepairandgeneticconsequencesofendogenousDNAbasedamageinmammaliancells.AnnRevGenet2004;38:445-476)2015年度诺贝尔化学奖——Lindahl与BEP13细胞死亡及周期阻滞基本信号通路AzizSancar利用纯化的UvrA、UvrB、UvrC重建了核苷酸切除修复(NER)系统，揭示了NER的分子机制。该机制可帮助细胞修复UV引起的DNA损伤。(SancarA.RuppWD.Anovelrepairenzyme:UVRABCexcisionnucleaseofEscherichiacolicutsaDNAstrandonbothsidesofthedamagedregion.Cell1983;33(1):249-260SancarA,RupertCS.CloningofthephrgeneandamplificationofphotolyaseinEscherichiacoli.Gene1978;4(4):295-308SancarA.Regulationofthemammaliancircadianclockbycryptochrome.JBiolChem2004;279(33):34079-34082)阿齐兹•桑加尔2015年度诺贝尔化学奖——Sancar与NEP14细胞死亡及周期阻滞基本信号通路PaulModrich利用重建错配修复(MMR)体外系统，探索从大肠杆菌到哺乳动物细胞的错配修复机制——细胞如何纠正DNA复制错误，使DNA复制出错频率减少103倍。保罗•莫德里(MuD,TursunM,DuckettDR,MmondJT,ModrichP.

RecognitionandrepairofcompoundDNAlesions(basedamageandmismatch)byhumanmismatchrepairandexcisionrepairsystems.MolCellBiol1997;17(2):760-769ModrichP.Strand-specificmismatchrepairinmammaliancells.JBiolChem1997;272:24727-24730ModrichP,LahueR.Mismatchrepairinreplicationfidelity,geneticrecombination,andcancerbiology.AnnRevBiochem1996;65:101-133)2015年度诺贝尔化学奖——Modrich与MMR15细胞死亡及周期阻滞基本信号通路TheoutcomeofDNAdamageisdiverseandgenerallyadverse2.THECONSEQUENCESOFDNAINJURYDNA损伤后果的复杂及多样性(Nature,411:366,2001)16细胞死亡及周期阻滞基本信号通路AcuteeffectsDNAmetabolismtriggerscell-cyclearrestorcelldeath.Longtermeffects

resultfromirreversiblemutationscontributingtooncogenesis.

(A)Acuteeffects:●

Interferenceof

DNAReplication:ReplicationalstressDNApolymerasesζtoκ

(translesionpolymerases)takeovertemporarilyfromtheblockedreplicativeDNApolymerase-δ/ε,andpossiblyfrompolαandmayovercomedamage-inducedreplicationalstress,protectingthegenome.highererrorrate

pointmutations

oncogenesis●

BlockingofTranscription：Anoutcomedirectlyrelatedtogenelength.Transcription-coupledrepair(TCR),adedicatedrepairsystem,assureshighpriorityrepair.Transcriptionalstress,arisingfrompersistentblockageofRNAsynthesis,constitutesanefficienttriggerforp53-dependentapoptosis(anti-cancermechanism).DNA损伤后果——急性效应（影响DNA、RNA、蛋白质代谢、细胞周期和凋亡）

长期效应（突变、肿瘤、衰老及疾病）17细胞死亡及周期阻滞基本信号通路Fig.Genotoxicagent…exposureinhibitsBrdUincorporationintoDNA.Thecellswereexposedtogenotoxicagent…for12–72handsubsequentlypulsedwith10mMBrdUfor30minat37ºCpriortoharvest.Afterfixation/permeabilization,cellsweretreatedwithDNaseandstainedwithFITC-conjugatedanti-BrdUantibody.DNAcontentswereanalyzedbyflowcytometrywithFACSDivasoftware.(A)FlowcytometricanalysisofBrdU-positivecells.ThecelluntreatedwithBrdUisusedasblank.(B)HistogramsshowingthepercentageofBrdU-positive…cells.(YangS-Yetal.BiochemPharmacol2009;77:433–443)DNA损伤后果——抑制DNA合成（举例）18细胞死亡及周期阻滞基本信号通路Fig-IncreasednucleolarE2F1indicatesribosomalstress.(A)TheorganizationoftherRNAgenesandrRNAtranscriptionandprocessinginmammaliancells.(B)InhibitionofrRNAtranscriptioninitiationbyADR.InthepresenceorabsenceofcaffeineorKU55933pretreatment,…RNAwasextractedandsubjectedtonorthernblothybridization,usingITS-1andITS-2asprobes.28Sand18SrRNAstainedbymethyleneblueasloadingcontrol.(C)InhibitionofrRNAtranscriptioninitiationbyADRin

metaboliclabelingofnascentrRNA.AfterADRexposurefor6h,H1299wasincubated…for~0.5–4hinthepresence10mCi/ml32P-orthophosphate.TotalRNAwasisolatedand1.5μgtotalRNAwasseparatedona1%agaroseformaldehydegel.(D)InhibitionofrRNAtranscriptionbyADRinPyroninY(PY)-stainedcells(JinY-Qetal.CellCycle,2014;13:1627-1638)影响ribosomalRNA生物合成及蛋白质合成19细胞死亡及周期阻滞基本信号通路●

AlterationofMicroRNAs’expressioninDNAdamageresponseMicroRNAs(miRNAs/miR)areapproximately22nucleotideslong,endogenous,single-stranded,nonprotein-codingRNAmoleculesthatpost-transcriptionallyregulategeneexpression.miRNAgenomicorganization:microRNAshavetheirowntranscriptionunits(intergenic)oraretranscribedwithothergenes.IntronicandexonicmiRNAlociarefoundinproteincodinggenesandnon-codingtranscriptionunits(notdepicted).MicroRNAsareencodedontheplus-orminus-strandofDNA.MicroRNA基因20细胞死亡及周期阻滞基本信号通路MostmiRNAgenesaretranscribedbyRNApolII.AmiRNAclusteronChr19istranscribedbyRNApolIII.Thelongprimarytranscript(pri-miRNA)ofamiRNAgeneispolycistronicforclusteredmiRNAgenes.Aftertranscriptionthepri-miRNAis“cropped”byaproteincomplexcalled“microprocessor”whichcomprisestheRNaseIIIendonucleaseDroshathatcleavesbothstrandsoftheds-RNAbetweenupperandlowerstemofthepri-miRNAandtheds-RNAbindingproteinDgcr8thatrecognizesthess-RNA-ds-RNAjunctiontobringthecatalyticalcenterofDroshaintotherightposition.Croppingreleasesanapproximately70-ntlonghairpinprecursorofthemiRNA(pre-miRNA).Forfurtherprocessing,pre-miRNAsareexportedtothecytoplasmbyExportin5-RanGTP.Inthecytoplasm,anotherRNAseIIIenzymetermedDicercleavesthepre-miRNAhairpinbetweenitsstemanditsterminalloopandtherebyreleasesashortmiRNAduplexwith2-nucleotide3’-overhangsthatcontainthematuremiRNAandacomplementarymiRNA(annotated:hsa-miR-...andhsa-miR-...*).MicroRNA生物合成——转录与加工21细胞死亡及周期阻滞基本信号通路MicroRNA作用机制MiRNAeffectormechanisms.Threepost-transcriptionalmechanismsoftargetgenedown-regulationendonucleolyticalcleavageofmRNAs

translationalinhibition

destabilizationofmRNAs22细胞死亡及周期阻滞基本信号通路DNAdamageaffectsthebiogenesisofmiRNAs.(A)DNAdamageregulatesspecificmiR’sexpressionthroughtranscription,andp53isanexemplaryTFfactor;(B)DNAdamageregulatesasubsetofmiRs’bymodulatingtheprocessingandmaturationofmiRNAbiogenesis.p53mayinteractwiththeDrosha/DGCR8complexthroughp68helicasetoenhancethemiRNAs’expression;(C)whetherDNAdamageinfluencesmiRNAsexpressionbymodulatingthedegradationstepofmiRNAsneedsfurtherinvestigation.(HuH&GattiR,JMolCellBiol,2010,1–9;doi:10.1093/jmcb/mjq042)DNA损伤影响MicroRNA基因转录激活和加工促进miR生物合成(HeLetal.MicroRNAsjointhep53network—anotherpieceinthetumour-suppressionpuzzle.NatRevCancer2007;7:819–822)23细胞死亡及周期阻滞基本信号通路RegulationofMicroRNAsresponsibleforrepair,checkpointandapoptosisinDNAdamageresponse(DDR):miRNA影响DNA损伤修复、细胞周期和凋亡(HuH&GattiR,2010)24细胞死亡及周期阻滞基本信号通路(CaoJ-Xetal.,CellDeathDis,2014)InductionofmicroRNAsandinhibitionofDNAsynthesis:Forinstance,miR-630targetsCDC7,therebyinhibitingCDC7-mediatedinitiationofDNAsynthesis…DNA损伤后果——诱导miRNA（举例）25细胞死亡及周期阻滞基本信号通路Identificationofcell-cyclearrestbyflowcytometryDNA损伤的后果——细胞周期阻滞(及检测方法)●Cell-cycleArrest:Thecell-cyclemachinerysomehowsensesgenomeinjuryandarrestsatspecificcheckpointsinG1,S,G2andMtoallowrepairoflesionsbeforetheyareconvertedintopermanentmutations.26细胞死亡及周期阻滞基本信号通路●ApoptoticCellDeath:Whendamageistoosignificant,acellmayoptfortheultimatemodeofrescuebyinitiatingapoptosisattheexpenseofawholecellDNA损伤的后果——细胞凋亡(及检测方法)27细胞死亡及周期阻滞基本信号通路●

Catastrophe:

DNAdamagetriggersChk2-dependentcentrosomeinactivation,whichinducesdefectsinspindleassemblyandchromosomesegregationandmitoticcatastrophe(Cell,113:87-99,2003)DNA损伤的后果——有丝分裂灾难(RoninsonIBetal.Ifnotapoptosis,thenwhat?Treatment-inducedsenescenceandmitoticcatastropheintumorcells.DrugResistUpdat2001;4:303-13)28细胞死亡及周期阻滞基本信号通路(B)LongtermeffectsDNADSBsinducedbyx-rays,chemicalsorduringreplicationofSSBs

andpresumablyduringrepairof

interstrandcrosslinksareparticularlyrelevantfortherecombination

machinery.●

DNARecombinationandTranslocation:CellswithspecializedDNArecombinationactivities,suchasB-andT-cells,maybeverysensitivetoDSBs.

OncogenictranslocationsinleukaemiaandlymphomasInductionofcancers

e.g.,BCR/ABLfusiongeneresultstheactivationoftyrosinekinase●AberrantMitosis:DSBsalsoposeproblemsduringmitosis,asintactchromosomesareaprerequisiteforproperchromosomesegregationduringcelldivision.Chromosomalaberrations:aneuploidydeletions(lossofheterozygosity,LOH)

chromosomaltranslocationscarcinogenesis.DNA损伤的后果——肿瘤29细胞死亡及周期阻滞基本信号通路Fig-MitoticDNAdamage:InductionofE2F1andexposedofcellsto8-Cl-Adocausechromosomesegregationerrors.(A)

Tripletstainingofnuclei,γ-H2AXandp-H3-S10toshowchromosomalbridge.(B)NucleiwerestainedwithDAPI(blue),microtubuleswaslabeledwithanti-β-tubulinantibodyandFITC-conjugatedIgG(green).Thinarrowindicateschromosomalbridge;thickarrowandarrowheadindicatechromosomebreakageandlaggingchromatin,respectively.(HanY-Yetal.MolCellBiochem2013;384:187–196)DNA损伤后果——染色体分裂异常(及检测方法)（举例）30细胞死亡及周期阻滞基本信号通路Fig-GenotoxicchemicalexposureinducespolyploidyinthecellsDNA损伤后果——产生多倍体或非整倍体(及检测方法)（举例）31细胞死亡及周期阻滞基本信号通路●

Senescence：

Senescencecanbetriggeredwhentelomeres—theendsoflinearchromosomes—cannotfulfiltheirnormalprotectivefunctions.Telomere-initiatedsenescencereflectsaDNAdamagecheckpointresponsethatisactivatedwithadirectcontributionfromdysfunctionaltelomeresSenescenthumanfibroblastsdisplaymolecularmarkerscharacteristicofcellsbearingDNADSBs:SenescentmarkersnuclearfociofphosphorylatedhistoneH2AXtheirco-localizationwithDNArepairfactorsDNAdamagecheckpointfactors

activationof

theDNAdamagecheckpointkinasesCHK1/CHK2(Sphase)Chromatinimmunoprecipitation(ChIP)andwhole-genomescanningapproachesshowthatthechromosomeendsofsenescentcellsdirectlycontributetotheDNAdamageresponse,andthatuncapped

telomeresdirectlyassociatewithmany,butnotall,DNAdamageresponseproteins.（FagagnaFd’AD,etal.Nature2003;426:194-198;VenturaAetal.Nature2007;445:661-665）DNA损伤的后果——细胞衰老53BP1,MDC1andNBS132细胞死亡及周期阻滞基本信号通路DeterminationofsenescenceComparingPKH2fluorescenceprofilesofthewild-type,p21-/-andp53-/-HCT116celllinessixdaysafterexposuretodoxorubin(adriamycin,ananti-cancerchemotherapydrugandisclassifiedasananthracyclineantiobiotic.Doxorubicinisusedtotreatmanycancers).Theinhibitionorknockoutofp53orp21decreasedbutdidnotabolishdrug-orradiation-inducedsenescence,indicatingpartialrequirementforp53andp21intreatment-inducedsenescence.(DrugResistanceUpdate,4:303,2001）DNA损伤的后果——细胞衰老(及检测方法)(举例)33细胞死亡及周期阻滞基本信号通路(TinaRich,etal,Nature2000,407:777-783)3.CELL-CYCLECHECKPOINTPATHWAYS细胞周期检验点信号途径34细胞死亡及周期阻滞基本信号通路●

G1-phaseCheckpointPathway*ATM-CHK2-CDC25A-CDK2axisformsarapidresponsesystem;*ATR-CHK1-CDC25A-CDK2;CDC25A-CDK4;*ATM-CHK2/ATR-CHK1-p53-p21pathway(Oncogene,22:5834,2003)G1-期检验点基本信号途径35细胞死亡及周期阻滞基本信号通路●

S-phaseCheckpointPathwayATM-CHK2-CDC25A-CDC45axisformsarapidresponsesystem;BRCA1asaATMtarget;Mre11-NBS1-RAD50complexisrequiredforradioresistantDNAsynthesis(RDS);MDC1,anewlydiscoveredBRCT-repeatproteinasamediatortorecruitrepairproteins(Nature421:952;9612003);SMC(GenesDev16:560,2002);E2F1S-期检验点基本信号途径(Oncogene,22:5834,2003)36细胞死亡及周期阻滞基本信号通路Fig-TherolesforE2F-1inthecontrolofproliferation,apoptosisandDNArepair.(StevensC&LaThangueNB.DNARep2004;3:1071–1079)(BiswasAK&JohnsonDG.TranscriptionalandnontranscriptionalfunctionsofE2F1inresponsetoDNAdamage.CancerRes2012;72:13-7;CressWD.E2F1:AnewroleintheDNAdamageresponse.CellCycle2011;10:1718ChenJetal.E2F1promotestherecruitmentofDNArepairfactorstositesofDNAdouble-strandbreaks.CellCycle2011;10:1287-94WongJVetal.Networkcalisthenics:controlofE2Fdynamicsincellcycleentry.CellCycle2011;10:3086-94)37细胞死亡及周期阻滞基本信号通路●

G2/M-phaseCheckpointPathway(Oncogene,22:5834,2003)AkeyeffectorofG2checkpointisCDC2(CDK1):*ATM-CHK2-CDC25C-Cdc2/CDK1axis*ATR-CHK1-CDC25C-Cdc2/CDK1axisATR-CHK1-CDC25A-CDC2axisWeel-CDC2inhibition*PLK1(-)andPLK3(+)(polo-likekinasefamily)playacrucialrolesininitiationandexitfrommitosisp21-PCNA-CDC2-CyclinBcomplexexcludesCDC25C(-)G2/M-期检验点基本信号途径(SpurgersKBetal.Identificationofcellcycleregulatorygenesasprincipaltargetsofp53-mediatedtranscriptionalrepression.JBiolChem2006;281:25134–25142.38细胞死亡及周期阻滞基本信号通路Fig-Activationofp53canpromoteandmaintainG2arrest.ThisdependsonfunctionalpRbandismediatedbyseveraltargetgenes(FlattPMet.p53regulationofG2checkpointisretinoblastomaproteindependent.MolCellBiol.2000;20:4210–4223.)G2/M期检验点基本信号途径扩展（例）39细胞死亡及周期阻滞基本信号通路Programmed(regulated)celldeath(PCD)controlscellnumbersandtissuesizeduringdevelopment,andbothanti-PCDandpro-PCDmodulatorsfeatureprominentlyintheestablishmentoftissueandcellarchitecture.(A)

TypeIPCD——ApoptoticCellDeathThetermofapoptosis(fromtheGreek,‘fallingaway’)wascoinedbyCurrieetaltodescribeacommontypeofPCDinvarioustissueandcelltypes(BrJCancer26:239-257,1972).Thesedyingcellssharemanymorphologicalfeatures,distinctfromthefeaturesofnecrosis.Morphology:

Chromatincondensation,fragmentation,apoptoticbodiesTriggers:

deathreceptors,trophicfactorwithdrawal,DNAdamage,viralinfections,…(BredesenDEetal.Nature2006;443:796-802;RoninsonIBetal.DrugResistUpdat2001;4:303-13)4.PROGRAMMED(REGULATED)CELLDEATH(PCD)程序性细胞死亡——I型PCD40细胞死亡及周期阻滞基本信号通路Mediators:

Caspases,BH1–3,BH3proteinsTheBclfamilyisolatedasageneinB-celllymphoma(bcl)iscomprisedofoveradozenproteins,andisclassifiedintothreegroups.ThegroupIincludingBcl-2andBcl-xLischaracterizedbyfourconservedBcl-2homology(BH)domains(BH1–BH4)andanti-apoptoticactivity,andaC-terminalhydrophobictail,whichlocalizestheproteinstotheoutersurfaceofmitochondriawiththebulkoftheproteinfacingthecytosol.ThegroupIIconsistsofBaxandBakwithpro-apoptoticactivity.MembersofthisgrouphaveasimilaroverallstructuretogroupIproteins,containingthehydrophobictailandallbuttheN-terminal,BH4domain.Theirpro-apoptoticactivityisdeterminedbyrelativelylargeregionsincludingtwolargeα-helicesthatparticipateinmembraneinsertion.GroupIIIconsistsofalargeanddiversecollectionofproteinswhoseonlycommonfeatureisthepresenceofthe~12–16-amino-acidBH3domain.Althoughsomemembers(e.g.,Bid)aredivergenthomologuesofBcl-2andBax,otherssharelittlesequence/structuralsimilaritywithgroupIandII.Inhibitors:Caspaseinhibitors,BH1–4proteinsExamples:TypeIPCD,nuclearPCD(BredesenDEetal.,Nature,443:796-802,2006;HengatnerMO,ibid,407:770-776)凋亡的介导分子——Caspases和Bax/Bak41细胞死亡及周期阻滞基本信号通路Thenamecaspasederivesfromcysteine-dependentaspartatespecificprotease:catalysisisgovernedbyacriticalconservedCyssidechainoftheenzyme,andbyastringentspecificityforcleavingproteinsubstratescontainingAsp.

*Caspase-dependentcelldeath(CDCD);Caspase-independentcelldeath(CICD)**

MtandnonMtpathway(JCTimmer&GSSalvesen,CDD14:66-72,2007)细胞凋亡的类型——CDCD/CICD42细胞死亡及周期阻滞基本信号通路Twomajorapoptoticpathwaysinmammaliancells(HengartnerMO.Nature,2000;407:770TinaRich,etal,Nature2000,407:777-783)细胞凋亡基本信号途径——线粒体和非线粒体途径43细胞死亡及周期阻滞基本信号通路很多分子(包括miR/未显示)参与细胞凋亡调节44细胞死亡及周期阻滞基本信号通路TheclassIIIPI3K

complexmediatesnucleationofthephagophoremembrane,enwrappingcytosolicproteins,proteinaggregates,andorganelles(suchasMt).Bcl-2blocksthisstepbybindingandinhibitingBeclin1,acomponentinthePI3Kcomplex.Atg12–Atg5-Atg16andAtg8–PEconjugates(LC3-IIinmammalianscells)arerecruitedtothephagophore,togetherwiththetransmembraneproteinAtg9,facilitatingthephagophorexpansionstep.Uponvesiclecompletion,mostoftheAtgproteinsaredissociatedfromtheautophagosome,allowingautophagosome-lysosomefusionandcargodegradationbylysosomalproteases.自噬体/自噬溶酶体的形成过程(HeC,KlionskyDJ.Annu.Rev.Genet.2009.43:67–93)46细胞死亡及周期阻滞基本信号通路Fig-(a)Electronmicroscopicanalysisofmouseembryonicfibroblastsduringnutrientstarvation.Mitochondriaandfragmentsofendoplasmicreticulumareobservedinsideanautophagosome.(b)Accumulationofubiquitin-positiveinclusionbodiesinneuronsofAtg5-deficientmice.Dorsalrootganglionneuronsfromwild-type(left)andAtg5-deficient(right)neonateswerestainedwithamonoclonalanti-ubiquitinantibody.细胞自噬的定义及鉴定.(MizushimaN&LevineB.NatCellBiol/Review,12:823–830,2010)●TheDefinitionofAutophagyAutophagyisaprocessbywhichcytoplasmiccomponentsincludingmacromolecules(proteins,glycogens,lipidsandnucleotides)andorganelles(mitochondria,peroxisomesandendoplasmicreticulum)aredegradedbythelysosome(seeFig).47细胞死亡及周期阻滞基本信号通路Fig-(A)TransmissionelectronmicroscopyrevealsthepresenceoflargevacuolesinanumberoftheCPT-treatedcells,suggestingtheactivationofanautophagicresponse(B)showingaggregatedmitochondrialabeledwith

MTG

(mitochondria-selectivemitotrackergreen)

(green)

andredfluorescentvacuolarstainingwithMDC(mitochondrial

diseasecriteria=specificdyemonodansylcadaverine)

accumulation

inCPT-treatedcells.Theoverlayshows

colocalizationofalargenumberofthemitochondriawiththeautophagicvacuoles.(C)Similarly,GFP-taggedLC3(microtubuleassociatedprotein1lightchain3)expressingMCF-7cellsshowsoftenoverlappingofGFP-LC3withusingmitotrackerredstainingfollowingexposuretoCPT.(AbedinMJetal.,CDD14:500-510,2007)细胞自噬的检测48细胞死亡及周期阻滞基本信号通路Fig-Autophagyisinducedbydeprivationofnutrients,hormones,andenergy.(a)Regulatorypathwaysofautophagybyaminoacids,hormones,andenergyinmammals.(b)Signalingofautophagyinyeastproteases(HeC,KlionskyDJ.Annu.Rev.Genet.2009.43:67–93)[Mammaliantargetofrapamycin(mTOR);heterodimerictuberoussclerosiscomplex(TSC)composedofTSC1andTSC2;smallGTPase(Rheb);AMP-activatedproteinkinase(AMPK);PI3KcomplexcontaininghVps34]自噬调节的（基本）信号途径——mTOR抑制和III型PI3K激活49细胞死亡及周期阻滞基本信号通路REGULATIONMECHANISMSANDSIGNALINGPATHWAYSOFAUTOPHAGY

32autophagy-related(ATG)geneshavebeenidentified.

E1enzymeAtg7activatesAtg8andtransfersittoAtg3(E2).Atg8isfinallyconjugatedtothetargetlipidPE(LC3-IIinmammalians)viaanamidebond,facilitatedbythe

E3-likeAtg12–Atg5conjugateInyeast,uponTorinhibitionbystarvationorrapamycintreatment,thekinaseactivityofAtg1isactivatedandAtg1bindstoAtg13andAtg17toformanAtg1-Atg13-Atg17

scaffoldandtorecruitmultipleAtgproteinstothePAS(phagophoreassemblysite)andtoinitiateautophagosomeformation.

Inmammals,mTORinteractswith,phosphorylates/inactivatesULKs(Unc-51-likekinase1/ULK1and-2/ULK2)andAtg13undernutrient-richconditions.UponmTORinhibitionbystarvationorrapamycin,ULK1andULK2areactivatedandphosphorylateAtg13andFIP200/Atg17,whichareessentialforautophagyactivity.

Inmulticellularorganisms,animportantfunctionofautophagy(theclearanceofcytosolicubiquitinatedsubstratesoraggregateproneproteins)(degradativeprocess)isselectiveandmediatedbymammalianproteinp62/sequestosome1(SQSTM1).

ThenucleationandassemblyoftheinitialphagophoremembranerequiretheclassIIIphosphatidylinositol3-kinase

(PtdIns3K)complex,whichiscomposedofthePtdIns3KVps34(vacuolarproteinsorting34),amyristoylatedserine/threonine