国立成功大学生物化学研究所硕士论文

IdentificationoftheResiduesofHumanCytochromecInvolvedinActivatingCaspasesICApaf-1apoptosomeCApaf-1WD40procaspase9caspase3CApaf-1transducin(2TRC)Tolb(1CRZ)Apaf-1WD40(7)WD40(6)CLysineApaf-1WD40AsparticacidCK72,K73,K86,K87andK88AlaLysApaf-1Y67CcaspasesY67Y67WY67HY67AY67FCK562caspase3caspase3Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin(DEVD-AFC)CcaspasesK86K87K88AlaCaspase3Apaf-1IIApoptosisisanimportantformofprogrammedcelldeathrequiredfortheembryonicdevelopmentandtissuehomeostasisofmulticellularorganisms.Theextrinsicdeathreceptors-andintrinsicmitochrondria-dependentpathwaysfollowingtheapoptoticstimulusleadingtocaspasesactivationhavebeenwellcharacterized.Cytochromecreleasedfrommitochondriafunctionsasatriggerfortheformationofapoptosomeintheintrinsicapoptoticpathway.Theapoptosomeisaheptamericcomplexcomprisedofapoptoticproteaseactivatingfactor-1(Apaf-1)andcytochromec.Theapoptosomebindsandactivatesprocaspase-9,resultinginactivationoffurthercaspases,suchascaspase-3,whichorchestratethefinalpackagingoftheapoptoticcell.Apaf-1isa130-kDaproteinconsistingofacaspaserecruitmentdomain(CARD),anarmdomain,andtwoWD-40repeats.ItwasshownthattheWD-40repeatsactasarecognitiondomainformitochondrialdamagethroughbindingtocytochromec,allowingApaf-1tooligomerizeandinteractwithprocaspase-9throughtheCARD-CARDinteraction.However,littleisknownabouthowhumancytochromecinteractswithApaf-1.Using3Dstructureofbovinetransducinβ/γcomplex(2TRC)andE.coliTolbprotein(1CRZ)asstructuraltemplates,3DmodelstructuresoftwoWD-40repeats(WD-40(7)andWD-40(6))wasgeneratedusinghomologymodeling.BasedonthestructuralanalysesofhumancytochromecdockingintotwoWD-40repeatsofApaf-1model,wehypothesizedthatthebasicresiduesK72,K73,K86,K87andK88ofcytochromecmaybeinvolvedintheinteractionwiththeacidicDresiduesoftwoWD-40domainsofApaf-1.ItwasreportedthatthenitrationofY67residuepromotedaconformationalchange,resultinginaffectingactivationofcaspasesbycytochromec.ToidentifytheresiduesofIIIcytochromecinvolvedinactivationofcaspases,weusedsite-directedmutagenesisonhumancytochromecandcell-freecaspaseactivationassaytocarryoutthestudy.Inthisstudywehaveexpressedsevencytochromecmutants(K72A/K73A,K72A/K86A,K86A/K87A/K88A,Y67A,Y67F,Y67H,andY67W)andpurifiedthemtohomogeneitywiththeyieldsof5-15mg/L.ToidentifytheinteractionbetweenhumancytochromecandApaf-1,wedeterminedthebiologicalactivityofrecombinantcytochromecbyusingacell-freecaspaseactivationassay.ThismethodindirectlymeasurestheirabilitytobindApaf-1,andthefluorogenicAsp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin(DEVD-AFC)wasusedasthecaspase-3substrate.ThemutationsofK72,K86,K87,andK88toalaninescausedthedecreaseinactivatingcaspase3,suggestingthattheseresiduesmaybeinvolvedinApaf-1binding.Theseresultsmaybeimportantnotonlyforidentifyingthebindingresiduesofcytochromectoapaf-1,butalsoforunderstandingcellapoptosis.IV勲VIIIIVVVIIVIIIXXIXII11-111-1-1.11-1-2.C31-1-3.酶61-29112-1112-1-1C112-1-2cytochromec192-1-3cytohromec20VI2-1-4SDS-PAGE222-2cytochromecCaspases262-2-1K562262-2-2Caspases3272-3酶292-3-1酶Ac-MP-11292-3-2酶酶(peroxidase)32343-1C343-1-1C343-1-2C353-2-1C363-2-2酶373-2-3酶酶(peroxidase)38394-1C394-2C414-3酶4344455561VII1552553(primer)564C575C586YeastC597YeastC60VIII1C612(apoptosis)C613humancytochromecyeastcytochromechemelyase(cyc-3)pET-21a(+)624635yeastiso-1-cytochromeccytochromeccytochromec646CY67K72K73K86K87K88657C(A)SPSepharose(B)P-30GelA280668Tricine-SDS-PAGEcytochromec669CK72A-K73A/K86A-K88AHPLCA4156710C6811K562celllysateCCell-freecaspaseactivationassay69IX12K562celllysateCCHorseYeastCell-freecaspaseactivationassay7013K562celllysateCell-freecaspaseactivationassay7014C7115YeastC(A)酶Ac-MP-11(B)MP-11MP-87216CMP-11Ac-MP-117317MP-11酶7318YeastSaccaromycescerevisiaeiso-1-cyto-chromec(cyc-1)cytochromechemelyase(cyc-3)pET-21a(+)7419YeastcytochromecA2807520Tricine-SDS-PAGEYeastcytochromecWH167521C-WH16PepsinY-MP11(CWHC)7622酶酶77Xhemehemoproteinbufferelutionbufferchromatographygelfiltrationchromatographylyophilizationmassspectrahighperformanceliquidchromatography(HPLC)spectrophotometercuvettecellpathlengthextinctioncoefficientopticalspectracytochromecPCRmachineXIAmpAmpicillinBisacrylamideN,N’-Methylenebisacrylamidebpbasepaird.d.H2OdoubledistilledwaterdNTPdeoxyribonucleosidetriphosphateE.ColiEscherichiaColiEDTAEthylenediaminetetra-aceticacidHPLChighperformanceliquidchromatographyIPTGIsopropyl-β-D-thiogalactosidekbkilobasekDakilodaltonLBLuria-BertaniO.DOpticaldensityPAGEPolyacrylamidegelelectrophoresisPCRPolymerasechainreactionrpmrotationperminutesSDSSodiumdodecylsulfateTEMEDN,N,N′,N′-tetramethylethylenediamineTFAtrifluoroaceticacidTOCSYTotalcorrelationspectroscopyTrisTris(hydroxymethyl)-aminomethaneXII1.SIGMA-201CENTRIFUGE5415CHITACHIhimacCR21SORVALLINSTRUMENTSRC5C2.ADVANTECLAB-Thermo-shakerTS-20HOTECHOrbitalshakerbath-9023.Bio-RADMini-proteanIIHoferMupid-2Mini-gelHoferSemi-drytransferunitsTE774.PHARMACIAElectrophoresisSupplyEPS300PHARMACIAElectrophoresisSupplyEPS600PHARMACIAElectrophoresisSupplyEPS3500XL5.OHTAKEFrenchPress6.pHWPApHmeterCD-720CORNINGpH/ionmete