細(xì)胞死亡及周期阻滯基本信號(hào)通路培訓(xùn)課件

細(xì)胞死亡及周期阻滯基本信號(hào)通路CELLDEATHAND

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

DNA損傷時(shí)細(xì)胞死亡和細(xì)胞周期監(jiān)控點(diǎn)（調(diào)控的基本）信號(hào)途徑2細(xì)胞死亡及周期阻滯基本信號(hào)通路1.有哪些因素可引起DNA損傷？DNA損傷的結(jié)局如何？2.什么分子可作為DNA雙鏈斷裂/損傷的標(biāo)志？用什么方法測(cè)定？3.

簡(jiǎn)述MicroRNA在DDR中的作用。（聽(tīng)完本講座，結(jié)合自己研究課題和文獻(xiàn)檢索）試舉一例（可能）與自己工作領(lǐng)域相關(guān)的MicroRNA功能及其生物學(xué)或臨床意義。4.寫(xiě)出G1、G2/Mcheckpoint基本信號(hào)通路主要分子成分。5.程序性細(xì)胞死亡（PCD）都有哪些形式？I型（凋亡）、II型（自噬）（自噬定義）PCD主要形態(tài)學(xué)特征如何？如何檢測(cè)或鑒定？其基本信號(hào)通路所涉及的主要激活分子是什么？各有何意義？本講基本要求——思考題3細(xì)胞死亡及周期阻滯基本信號(hào)通路誘導(dǎo)DNA損傷與細(xì)胞死亡原因*——各種射線照射/光動(dòng)力學(xué)治療（光敏反應(yīng)）化療藥物/細(xì)胞毒、基因毒試劑缺血/缺氧/氧化應(yīng)激血清/營(yíng)養(yǎng)因子/氨基酸撤出營(yíng)養(yǎng)過(guò)剩過(guò)度興奮/刺激中毒/鈣超載感染/炎癥其它*常伴有或直接、間接引起DNA損傷該講的普遍意義——多種原因相關(guān)的DNA損傷引起細(xì)胞死亡和細(xì)胞周期阻滯4細(xì)胞死亡及周期阻滯基本信號(hào)通路THEPLETHORAOFDAMAGESINDNA

(DNA損傷的多樣性/復(fù)雜性）THECONSEQUENCESOFDNAINJURY(DNA損傷的結(jié)局)3.CELL-CYCLECHECKPOINTPATHWAYS(細(xì)胞周期監(jiān)控點(diǎn)基本信號(hào)途徑)4.PROGRAMMED(REGULATED)CELLDEATH(程序性細(xì)胞死亡,PCD)本講提綱5細(xì)胞死亡及周期阻滯基本信號(hào)通路1.APLETHORAOFDAMAGESINDNAAPERPLEXINGDIVERSITYOFDNALESIONSARISESFROMTHREEMAINCAUSES

Environmentalagents(環(huán)境因素)Productsofnormalcellularmetabolisminmitochondrion（線粒體代謝產(chǎn)物——ROS）SomechemicalbondsinDNAspontaneouslydisintegrateunderphysiologicalconditions（DNA自發(fā)裂解）(A)EnvironmentalAgents:

ultraviolet(UV),ionizingradiation…

(環(huán)境因素)

genotoxicchemicalsvirusinfectionAlterationsinDNAstructure:AdjacentthyminedimerDNAsinglestrandedbreaks(DNASSBs)DNAdoublestrandedbreaks(DNADSBs)Cross-linking(intra-andinter-cross-linking)…DNA損傷的多樣性及主要誘因6細(xì)胞死亡及周期阻滯基本信號(hào)通路(B)ProductsofNormalCellularMetabolisminMitochondrion:

(線粒體代謝產(chǎn)物——ROS）Reactiveoxygenspecies(ROS)derivedfromoxidativerespirationandproductsoflipidperoxidation.

OxidativemodificationsinDNA.e.g.,8-OxoGAntioxidantdefencesystemcomposedofenzymatic(superoxidedismutase/SOD,catalase,glutathioneperoxidase/GPxandperoxyredoxins/GR)andlowmolecular-massscavengers(suchasglutathione/GSH).7細(xì)胞死亡及周期阻滯基本信號(hào)通路Fig-ProductionofROSandantioxidantdefencesystemsinhippocampalneuronsunderkainate-inducedoxidativestress.(LiSYetal.FreeRadBiolMed,48:597-608,2010)ROS產(chǎn)生——海人酸引起的神經(jīng)元過(guò)度興奮(舉例)8細(xì)胞死亡及周期阻滯基本信號(hào)通路Fig-GenotoxicchemicalexposureinducesDNADSBs.(A)IdentificationofDNADSBsbyPFGE.(B)Immunocytochemicalstaininggamma-H2AXfoci.(C)Testinggamma-H2AX(H2AX-Ser139)byWesternblotting.(YangSY,etal.BiochemPharmacol,77:433-443,2009)DNA損傷——雙鏈斷裂的(3種)鑒定（舉例）9細(xì)胞死亡及周期阻滯基本信號(hào)通路Fig-Theeffectsofkanicacid-inducedROSproductionontheintegrityofthegenomeinculturedneurons.ThegenomicDNAdamagewastestedusingfluoresceinisothiocyanate(FITC)-conjugatedavidin,whichbindsto8-oxodeoxyguanosine(8-oxodG)withastructuralsimilaritytobiotin(RadiskyDCetal.Nature,2005.436:123-127)抗生物素蛋白檢測(cè)DNA氧化損傷（8-O-鳥(niǎo)苷）鑒定（舉例）彗星實(shí)驗(yàn)檢測(cè)DSBs

（舉例）Fig-Cometassay.

ToscorethepercentageofDNAinthetail,theimageanalysissystemwasused.Thepercentageofcomettailarea(DNAtailarea/totalDNAarea)andthecomettaillength(fromthecenteroftheDNAheadtotheendoftheDNAtail)wereanalyzedin50cellsforoneslide.(AndersonDetal.MutatRes1994;307:261-271)Con0.2μM2μM10μM10細(xì)胞死亡及周期阻滯基本信號(hào)通路(C)

SomeChemicalBondsinDNASpontaneouslyDisintegrateunderPhysiologicalConditions（DNA自發(fā)裂解）:Hydrolysisofnucleotideresiduesleavesnon-instructiveabasicsites.Deamination

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

Wholechromosomalinstability(W-CIN);segmentalchromosomalinstability(S-CIN);genomicinstability(GIN)/基因組不穩(wěn)定性（Geigl,J.B.etal.(2008)Defining‘chromosomalinstability’.TrendsGenet.24,64–69RickeRMetal.(2008)Wholechromosomeinstabilityandcancer:acomplexrelationship.TrendsGenet24:475-466）11細(xì)胞死亡及周期阻滯基本信號(hào)通路諾貝爾化學(xué)獎(jiǎng)評(píng)審委員會(huì)2015年10月7日在瑞典皇家科學(xué)院宣布，2015年度諾貝爾化學(xué)獎(jiǎng)由英國(guó)弗朗西斯·克里克研究所名譽(yù)教授/瑞典人托馬斯·林達(dá)爾、美國(guó)科學(xué)家/杜克大學(xué)醫(yī)學(xué)院教授保羅·莫德里和擁有美國(guó)和土耳其雙重國(guó)籍的科學(xué)家/美國(guó)北卡羅來(lái)納大學(xué)醫(yī)學(xué)院教授阿齊茲·桑賈爾分享，以表彰他們?cè)贒NA損傷修復(fù)機(jī)制——BER、NER和MMR方面的研究成果；他們的發(fā)現(xiàn)揭示了細(xì)胞是如何維持基因組DNA穩(wěn)定性的，在以DNA損傷為基礎(chǔ)的腫瘤治療以及在衰老中的作用。此前與DNA損傷及結(jié)局相關(guān)研究而獲獎(jiǎng)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年度諾貝爾化學(xué)獎(jiǎng)與DNA損傷修復(fù)12細(xì)胞死亡及周期阻滯基本信號(hào)通路TomasLindahl發(fā)現(xiàn)E.coliUracilDNAglycosidase及堿基切除修復(fù)(BEP)機(jī)制托馬斯?林達(dá)爾(LindahlT.AnN-glycosidasefromEscherichiacolithatreleasesfreeuracilfromDNAcontainingdeaminatedcytosineresidues.ProcNatlAcadSciUSA1974;71(9):3649-3653BarnersDE,LindahlT.RepairandgeneticconsequencesofendogenousDNAbasedamageinmammaliancells.AnnRevGenet2004;38:445-476)2015年度諾貝爾化學(xué)獎(jiǎng)——Lindahl與BEP13細(xì)胞死亡及周期阻滯基本信號(hào)通路AzizSancar利用純化的UvrA、UvrB、UvrC重建了核苷酸切除修復(fù)(NER)系統(tǒng)，揭示了NER的分子機(jī)制。該機(jī)制可幫助細(xì)胞修復(fù)UV引起的DNA損傷。(SancarA.RuppWD.Anovelrepairenzyme:UVRABCexcisionnucleaseofEscherichiacolicutsaDNAstrandonbothsidesofthedamagedregion.Cell1983;33(1):249-260SancarA,RupertCS.CloningofthephrgeneandamplificationofphotolyaseinEscherichiacoli.Gene1978;4(4):295-308SancarA.Regulationofthemammaliancircadianclockbycryptochrome.JBiolChem2004;279(33):34079-34082)阿齊茲?桑加爾2015年度諾貝爾化學(xué)獎(jiǎng)——Sancar與NEP14細(xì)胞死亡及周期阻滯基本信號(hào)通路PaulModrich利用重建錯(cuò)配修復(fù)(MMR)體外系統(tǒng)，探索從大腸桿菌到哺乳動(dòng)物細(xì)胞的錯(cuò)配修復(fù)機(jī)制——細(xì)胞如何糾正DNA復(fù)制錯(cuò)誤，使DNA復(fù)制出錯(cuò)頻率減少103倍。保羅?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年度諾貝爾化學(xué)獎(jiǎng)——Modrich與MMR15細(xì)胞死亡及周期阻滯基本信號(hào)通路TheoutcomeofDNAdamageisdiverseandgenerallyadverse2.THECONSEQUENCESOFDNAINJURYDNA損傷后果的復(fù)雜及多樣性(Nature,411:366,2001)16細(xì)胞死亡及周期阻滯基本信號(hào)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損傷后果——急性效應(yīng)（影響DNA、RNA、蛋白質(zhì)代謝、細(xì)胞周期和凋亡）

長(zhǎng)期效應(yīng)（突變、腫瘤、衰老及疾病）17細(xì)胞死亡及周期阻滯基本信號(hào)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損傷后果——抑制DNA合成（舉例）18細(xì)胞死亡及周期阻滯基本信號(hào)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)影響ribosomalRNA生物合成及蛋白質(zhì)合成19細(xì)胞死亡及周期阻滯基本信號(hào)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細(xì)胞死亡及周期阻滯基本信號(hào)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——轉(zhuǎn)錄與加工21細(xì)胞死亡及周期阻滯基本信號(hào)通路MicroRNA作用機(jī)制MiRNAeffectormechanisms.Threepost-transcriptionalmechanismsoftargetgenedown-regulationendonucleolyticalcleavageofmRNAs

translationalinhibition

destabilizationofmRNAs22細(xì)胞死亡及周期阻滯基本信號(hào)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損傷影響MicroRNA基因轉(zhuǎn)錄激活和加工促進(jìn)miR生物合成(HeLetal.MicroRNAsjointhep53network—anotherpieceinthetumour-suppressionpuzzle.NatRevCancer2007;7:819–822)23細(xì)胞死亡及周期阻滯基本信號(hào)通路RegulationofMicroRNAsresponsibleforrepair,checkpointandapoptosisinDNAdamageresponse(DDR):miRNA影響DNA損傷修復(fù)、細(xì)胞周期和凋亡(HuH&GattiR,2010)24細(xì)胞死亡及周期阻滯基本信號(hào)通路(CaoJ-Xetal.,CellDeathDis,2014)InductionofmicroRNAsandinhibitionofDNAsynthesis:Forinstance,miR-630targetsCDC7,therebyinhibitingCDC7-mediatedinitiationofDNAsynthesis…DNA損傷后果——誘導(dǎo)miRNA（舉例）25細(xì)胞死亡及周期阻滯基本信號(hào)通路Identificationofcell-cyclearrestbyflowcytometryDNA損傷的后果——細(xì)胞周期阻滯(及檢測(cè)方法)●Cell-cycleArrest:Thecell-cyclemachinerysomehowsensesgenomeinjuryandarrestsatspecificcheckpointsinG1,S,G2andMtoallowrepairoflesionsbeforetheyareconvertedintopermanentmutations.26細(xì)胞死亡及周期阻滯基本信號(hào)通路●ApoptoticCellDeath:Whendamageistoosignificant,acellmayoptfortheultimatemodeofrescuebyinitiatingapoptosisattheexpenseofawholecellDNA損傷的后果——細(xì)胞凋亡(及檢測(cè)方法)27細(xì)胞死亡及周期阻滯基本信號(hào)通路●

Catastrophe:

DNAdamagetriggersChk2-dependentcentrosomeinactivation,whichinducesdefectsinspindleassemblyandchromosomesegregationandmitoticcatastrophe(Cell,113:87-99,2003)DNA損傷的后果——有絲分裂災(zāi)難(RoninsonIBetal.Ifnotapoptosis,thenwhat?Treatment-inducedsenescenceandmitoticcatastropheintumorcells.DrugResistUpdat2001;4:303-13)28細(xì)胞死亡及周期阻滯基本信號(hào)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損傷的后果——腫瘤29細(xì)胞死亡及周期阻滯基本信號(hào)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損傷后果——染色體分裂異常(及檢測(cè)方法)（舉例）30細(xì)胞死亡及周期阻滯基本信號(hào)通路Fig-GenotoxicchemicalexposureinducespolyploidyinthecellsDNA損傷后果——產(chǎn)生多倍體或非整倍體(及檢測(cè)方法)（舉例）31細(xì)胞死亡及周期阻滯基本信號(hào)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損傷的后果——細(xì)胞衰老53BP1,MDC1andNBS132細(xì)胞死亡及周期阻滯基本信號(hào)通路DeterminationofsenescenceComparingPKH2fluorescenceprofilesofthewild-type,p21-/-andp53-/-HCT116celllinessixdaysafterexposuretodoxorubin(adriamycin,ananti-cancerchemotherapydrugandisclassifiedasananthracyclineantiobiotic.Doxorubicinisusedtotreatmanycancers).Theinhibitionorknockoutofp53orp21decreasedbutdidnotabolishdrug-orradiation-inducedsenescence,indicatingpartialrequirementforp53andp21intreatment-inducedsenescence.(DrugResistanceUpdate,4:303,2001）DNA損傷的后果——細(xì)胞衰老(及檢測(cè)方法)(舉例)33細(xì)胞死亡及周期阻滯基本信號(hào)通路(TinaRich,etal,Nature2000,407:777-783)3.CELL-CYCLECHECKPOINTPATHWAYS細(xì)胞周期檢驗(yàn)點(diǎn)信號(hào)途徑34細(xì)胞死亡及周期阻滯基本信號(hào)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-期檢驗(yàn)點(diǎn)基本信號(hào)途徑35細(xì)胞死亡及周期阻滯基本信號(hào)通路●

S-phaseCheckpointPathwayATM-CHK2-CDC25A-CDC45axisformsarapidresponsesystem;BRCA1asaATMtarget;Mre11-NBS1-RAD50complexisrequiredforradioresistantDNAsynthesis(RDS);MDC1,anewlydiscoveredBRCT-repeatproteinasamediatortorecruitrepairproteins(Nature421:952;9612003);SMC(GenesDev16:560,2002);E2F1S-期檢驗(yàn)點(diǎn)基本信號(hào)途徑(Oncogene,22:5834,2003)36細(xì)胞死亡及周期阻滯基本信號(hào)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細(xì)胞死亡及周期阻滯基本信號(hào)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-期檢驗(yàn)點(diǎn)基本信號(hào)途徑(SpurgersKBetal.Identificationofcellcycleregulatorygenesasprincipaltargetsofp53-mediatedtranscriptionalrepression.JBiolChem2006;281:25134–25142.38細(xì)胞死亡及周期阻滯基本信號(hào)通路Fig-Activationofp53canpromoteandmaintainG2arrest.ThisdependsonfunctionalpRbandismediatedbyseveraltargetgenes(FlattPMet.p53regulationofG2checkpointisretinoblastomaproteindependent.MolCellBiol.2000;20:4210–4223.)G2/M期檢驗(yàn)點(diǎn)基本信號(hào)途徑擴(kuò)展（例）39細(xì)胞死亡及周期阻滯基本信號(hào)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)程序性細(xì)胞死亡——I型PCD40細(xì)胞死亡及周期阻滯基本信號(hào)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)凋亡的介導(dǎo)分子——Caspases和Bax/Bak41細(xì)胞死亡及周期阻滯基本信號(hào)通路Thenamecaspasederivesfromcysteine-dependentaspartatespecificprotease:catalysisisgovernedbyacriticalconservedCyssidechainoftheenzyme,andbyastringentspecificityforcleavingproteinsubstratescontainingAsp.

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

MtandnonMtpathway(JCTimmer&GSSalvesen,CDD14:66-72,2007)細(xì)胞凋亡的類型——CDCD/CICD42細(xì)胞死亡及周期阻滯基本信號(hào)通路Twomajorapoptoticpathwaysinmammaliancells(HengartnerMO.Nature,2000;407:770TinaRich,etal,Nature2000,407:777-783)細(xì)胞凋亡基本信號(hào)途徑——線粒體和非線粒體途徑43細(xì)胞死亡及周期阻滯基本信號(hào)通路很多分子(包括miR/未顯示)參與細(xì)胞凋亡調(diào)節(jié)44細(xì)胞死亡及周期阻滯基本信號(hào)通路TheclassIIIPI3K

complexmediatesnucleationofthephagophoremembrane,enwrappingcytosolicproteins,proteinaggregates,andorganelles(suchasMt).Bcl-2blocksthisstepbybindingandinhibitingBeclin1,acomponentinthePI3Kcomplex.Atg12–Atg5-Atg16andAtg8–PEconjugates(LC3-IIinmammalianscells)arerecruitedtothephagophore,togetherwiththetransmembraneproteinAtg9,facilitatingthephagophorexpansionstep.Uponvesiclecompletion,mostoftheAtgproteinsaredissociatedfromtheautophagosome,allowingautophagosome-lysosomefusionandcargodegradationbylysosomalproteases.自噬體/自噬溶酶體的形成過(guò)程(HeC,KlionskyDJ.Annu.Rev.Genet.2009.43:67–93)46細(xì)胞死亡及周期阻滯基本信號(hào)通路Fig-(a)Electronmicroscopicanalysisofmouseembryonicfibroblastsduringnutrientstarvation.Mitochondriaandfragmentsofendoplasmicreticulumareobservedinsideanautophagosome.(b)Accumulationofubiquitin-positiveinclusionbodiesinneuronsofAtg5-deficientmice.Dorsalrootganglionneuronsfromwild-type(left)andAtg5-deficient(right)neonateswerestainedwithamonoclonalanti-ubiquitinantibody.細(xì)胞自噬的定義及鑒定.(MizushimaN&LevineB.NatCellBiol/Review,12:823–830,2010)●TheDefinitionofAutophagyAutophagyisaprocessbywhichcytoplasmiccomponentsincludingmacromolecules(proteins,glycogens,lipidsandnucleotides)andorganelles(mitochondria,peroxisomesandendoplasmicreticulum)aredegradedbythelysosome(seeFig).47細(xì)胞死亡及周期阻滯基本信號(hào)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)細(xì)胞自噬的檢測(cè)48細(xì)胞死亡及周期阻滯基本信號(hào)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]自噬調(diào)節(jié)的（基本）信號(hào)途徑——mTOR抑制和III型PI3K激活49細(xì)胞死亡及周期阻滯基本信號(hào)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