芘所致DNA损伤修复中的作用和交互作用蛋白分析

华中科技大学博士学位论文热休克蛋白70在苯并（a）芘所致DNA损伤修复中的作用和交互作用蛋白分析姓名：段燕英申请学位级别：博士专业：劳动卫生与环境卫生学指导教师：邬堂春20080519I70aDNA()(heatshockresponseHSR)(heatshockproteins,HSPs)HSPs()HSPsSDS-PAGEHSPsHSPs(≥100KD)HSP90(81-99KD)HSP70(65-80KD)HSP60(55-64KD)HSP40(35-54KD),HSPs(≤34KD)HSP70Hsp701)Hsp702)Hsp70DNA,3)Hsp70()4)Hsp70(a)(BaP)DNABaP(ROS)ROSIIDNAHsp70BaPDNAHsp70BaPDNAHsp70Hsp70DNADNADNADNADNAHsp70DNAHsp70BaPHsp70--DNAHsp70Hsp70Hsp70Hsp70BaPHsp70Hsp7016HBEpcDNA3.1/pcDNA3.1-hsp70G418Hsp70(16HBE/hsp70)pcDNA3.116HBE(16HBE/pcDNA)Hsp70Hsp70RNAHsp70(QCT)IIIHsp70Hsp70(50100150200µM)16HBE6hWestern-blot16HBEHsp7016HBEHsp7050µMHsp70(P0.01)200µM150µM90%QCT150µM87%WesternBlot100µM6hHsp700.01%DMSO(16HBE/DMSO)RNARNAiRNARNAmRNARNAiScience20012002RNAishRNARNA48Hsp70Hsp70Western-blotHsp70RNAi16HBE/RNAi16HBEHsp7016HBE/hsp7016HBEWestern-blot16HBE16HBE/RNAiHsp7042%(P0.01)16HBE/hsp70Hsp7078%(P0.01)16HBE/DMSO16HBE/HK16HBE/pcDNAHsp70(P0.05)100µMHsp7053%Hsp70RNAIVHsp70B(a)PBPDEDNAB(a)PHsp7016HBEDNA16µMBaP16HBE2h(024824h)Hsp70DNAControl16HBE/HK16HBE/RNAi16HBE/hsp7016HBE/pcDNADMSOS9NC(normalcultured)80%DMSOS9NC90%OTMsDNA2h2h8h24hOTMsNCnormalculturedOTMsHsp702hOTMsHsp70Hsp70OTMsP0.01Hsp702hOTMsP0.012hHsp70BaPDNAHsp70Hsp70BPDEBaPDNABPDE-DNANERHsp70HSPs,DNAHsp70DNA(BER)(MMR)NERVHsp70BPDE(HCR)BPDE10203040µMpCMVluc40hDNADNAHsp7010µMP0.0120µMP0.05Hsp7010µMP0.05BaPHsp70Hsp7016HBEBaPBPDEDNAHsp70Hsp70Hsp70DNA16µMB(a)P16HBE2h4hHsp70HPLCESIMS/MS73084swiss-protdatabase()841314%12%DNA6%7%74%6%5%5%6%2%1%27%Hsp70DNAHsp70BaPHsp70VIBaPHsp70CKIIHsp70CKIIHsp70DNABasesHsp70Hsp70APEpolyβHsp70DnaKIICKIIHsp70CKII/XRCC1XRCC4APECKIIXPBHsp70CKIIDNANERBERBERAPEHsp70CKIIHsp70CKIIHsp70CKIICKIIHsp7016HBEBaPDNAHsp70CKIIBaPHsp70CKIIHsp70CKIIP0.05Hsp70CKII(1)100µM16HBEHsp70hsp70cDNAHsp70VII(2)Hsp70BaPBPDEDNA(3)B(a)PHsp70DNA(4)Hsp70CKIIHsp70CKII(1)Hsp70Hsp70Hsp70(2)Hsp70DNA(1)Hsp70CKIIHsp70DNA(2)DNADNA70DNABaP--VIIITThheerroolleeooffHHsspp7700iinntthheerreeppaaiirrooffBBaaPP--iinndduucceeddddaammaaggeessaannddiittssiinntteerraaccttiinnggpprrootteeiinnssCandidateforPh.D.:YanyingDuanSupervisor:ProfessorTangchunWuABSTRACTHeatshockproteins(Hsps)arehighlyconservedproteinswhicharetriggeredinallorganismsexposedtoenvironmentalstressorssuchaselevatedtemperature,nicotinamide,carbonmonoxide,heavymetals,ionization,ischemiaandhypoxia.Basedupontheirapparentmolecularweight,HSPsaredividedintomanygroupssuchashigh-molecular-massHSPs(≥100kD),HSP90(81to99kD),HSP70(65to80kD),HSP60(55to64kD),HSP40(35to54kD),andsmallHSPs(≤34kD).TheHSP70familyhavebeenextensivelystudiedwhicharefoundtofunctionasmolecularchaperones,assistingnascentpolypeptidesproperconfigurationandfacilitatingthemisfoldingpeptidesdegradation.Theiroverexpressionsgreatlychangethetoleranceandsensitivityoforganismtophysical,chemicalandbiologicalharmfulstimuli.Hsp70wasmainlyincytoplasmunderphysiologicalconditionbutmovedtonucleuswhenorganismwasunderstress,besides,twopreviousresearchinourlabfoundthatHsp70levelwasinverselycorrelatedtoresidualDNAdamage,whichbothhintthatHsp70maybeinvolvedinDNArepair.However,manyrelatedresearchwasbaseonarelationbetweenHsp70andDNArepairandthatwhetherHsp70playsaroleintheDNArepairremainsunknowed.ConsideringthemolecularchaperoneessenceofHsp70,theremaybesomeproteinsubstrates,throughwhichHsp70modulatetheDNArepairprocess.Inthisstudy,weinvestigatedthepossiblerolesofHsp70inDNArepairin16HBEIXcellsbyeitherknocking-downoroverexpressingHsp70expressionlevelunderBaPtreatment.TherepairofBaP-induceddamage,assessedbyresidualDNAdamage,wasmeasuredbycometassayandtherepairofDNAadductswasassessedbyhostcellreactivationassay.Later,immunoprecipitation(IP)andhighperformanceliquidchromatographyelectrosprayionisationtandemmassspectrometry(HPLCESIMS/MS)werefurtherappliedtodetecttheHsp70-interactingproteins,amongwhichcaseinkinaseII(CKII),aSer/Thrproteinkinase,wasanimportantproteininvolvedinDNArepair.IPassay,confocalmicroscopyanalysisandautoradiographywerefurtherperformedtocharacterizetheinteractionbetweenHsp70andCKII.PartEstablishmentof16HBEcellmodelswithover-expressedandknocked-downHsp70levels16HBEcellsweretransfectedwithrecombinantplasmidpcDNA3.1/hsp70.Andthepositiveclonesappearedafteraselectionfor2weeksby800µg/mlG418(neomycin).PositivecloneswereexpandedandanalyzedwiththeexpressionofHsp70.ThecontrolgroupwastransfectedwithpcDNA3.1plasmidscontainingtheneomycinresistancegenebutnothsp70cDNA.Wedevelopedstablytransfected16HBEcelllineswithoverexpressedHsp70(16HBE/hsp70)orwithneomycinresistancegenebutnothsp70cDNA(16HBE/pcDNA).ToinhibittheexpressionofHsp70,16HBEcellsweretreatedwithdifferentconcentrationsofquercetin(QCT)(50,100,150,200µM)for6h,thenwaterbathingfor1hat42°C.ThelevesofHsp70indifferentdosegroupswereassessedbywesternblot.Comparedwiththecontrolgroup,therewasasignificantdecreaseofHsp70in50µMgroup(P0.01)andinthefollowingdosegroups(P0.01).Asforcellsurvivalrate,itwasabove90%whentheconcentrationofQCTwaslessthan150µManditdecreasedto87%whentheconcentrationofQCTwas150µM.AccordingtobothcellsurvivalrateandHsp70levelscausedbydifferentconcen