国家人类基因组北方研究中心课件

ProteinComplexand

Protein-proteinInteraction国家人类基因组北方研究中心Email:Centraldogma:thestoryoflifeRNADNAProteinProteinisthefinalplayerincelllifeProteinsfunctioninassociationwithotherproteinsorbiomolecules,butnotinisolationGoalsofProteomicstodiscoverproteinfunctiontounderstandcellularprocessestounderstanddiseasestatestodiscoverdrugtargettoidentifybiomarkerTypesofProteomicsExpressionProteomicsQuantitativestudyofproteinexpressionandtheirchangesbetweensamplesthatdiffersbysomevariableFunctionalProteomicsTostudyprotein-proteininteraction,3-Dstructures,cellularlocalizationandPTMsinordertounderstandthephysiologicalfunctionofthewholesetofproteome.ApproachesGenetic:yeasttwo-hybridphagedisplayBiochemical:BluenativePAGEFarWesternPull-downCoimmunoprecipitationTAPCrosslinkingBioinformatic:Co-occurrenceNeighborhoodSurfacepatchBiophysical:MassSpectrometrySPRFRETBlueNativePAGEdetergentCBB6-ACA_+BlueNativePAGESamplePreparationBlueNativePAGESDSSolubilizationwithnonionicdetergent(laurylmaltoside,TX-100,CHAPS,Mega9,octylglucoside,Brij35,etc),supplementedwith6-aminocaproicacidSeparationgel:6-13%gradientCathodebuffercontains0.02%CoomassieblueG250SeparationofmembersofmultiproteincomplexBlueNativePAGEofchloroplastthylakoidmembranesBBRC1999,259:569-575BNofsolubilizedchloroplastthylakoidmembranes(a)followedbySDS–PAGEintheseconddimension(b).CF0F1ATPsynthasewasindicated.BlueNativePAGEofmultiproteincomplexfromwholecellularlysateMCP3:176-182,2004DialysispermitstheanalysisofmultiproteincomplexesofwholecellularlysatesbyBN.IdentificationandanalysisofdistinctproteasomesbyWCL2DBN/SDSA,WCLofHEK293cellswasseparatedby2DBN/SDS(5.5–14and10%,respectively),andimmunoblottingwasperformedwithspecificantibodiesrecognizingeithersubunitsofthe20Scorecomplex(Mcp21and2),orasubunitofthe19Scapofthe26Sproteasome(S4ATPase),orasubunitofthePA28regulatorysubunit(PA28).B,Anidenticalsamplewasboiledin1%SDS,resolvedby2DBN/SDS,andimmunoblottedasdescribedinA.MCP3:176-182,2004BlueNativePAGEVisualizationofMPCsona2DWCLBN/SDSgelB,WCLofHEK293cellswasboiledwith1%SDSbeforeseparationandstaining.A,WCLofHEK293cellswaspreparedusingTritonX-100andseparatedby2DBN/SDS(5.5–17and10%,respectively).MCP3:176-182,2004BlueNativePAGEMax:functionalcloningofaMyc-bindingproteinA.

CKII,caseinkinaseIIphosphorylationsite;BR,basicregion;HLH,helix-loop-helix;LZ,leucinezipper.B.Plaquesthatexpressbeta-galactosidasefusionprteinswerescreenedfortheirabilitytoreactwith125I-labeldGST-MycC92.Topleft,secondaryplatingoffiveputativepositivedemonstratesthereactivityoftwooftheprimaryplaques,Max11andMax14.Topright,asanegativecontrol,GSTwaslabeledtoasimilarspecificactivityandcomparedwithGST-MycC92forbidningtoMax14plaques.Bottom,bindingofGST-MycC92toMzx14plaqueswasassayedwithorwithoutaffinitypurifiedcarboxylterminal-specificanti-Myc(Ab)orpeptideimmunogen(peptide).MycC92Science251:1211-7,1991FarWesternAssociationofRbwithHIP1HeLanulearextract(~100ug)(lane1,2)andHIP1(~200ng)purifiedfromHeLa(lane3,4)wereelectrophoresed,blotted,andrenaturedinsitu.Adjacentstripswerecutfromthefiltersandprobedwith32P-GST-RB(379-928)(lane1,3)or32P-GST-RB(379-928;706F)(lane2,4)Cell70:351-364,1992FarWesternGSTPulldownGST-mFasfusionproteinsGSTPulldown1491662042933061306194194194194194194292283276268221194306306221GST-mFas-associatedpolypeptidesfrom32S-labeledHeLa,L929,andJurkatcelllysatesGSTPulldownPreclearation:25ugGST/50ulGSH-Seph.Incubation:10ugGST/GST-mFas-(194-306)Wash:0.5%NP-40,20mMTris,pH8.0,200mMNaClElution:50ul20mMGSHin50mMTrisGST-mFas-associatedpolypeptidesarestabletohighsaltconcentrationsGSTPulldownHeLacelllysateswerescreenedwitheitherGSTorGST-mFas-(194–306)asdescribedaboveexceptthattheSepharose-proteincomplexeswerewashedwithLysisBuffercontainingdifferentsaltconcentrations(asindicated).Theelutedmaterialwassubjectedto12%SDSandfluorography.DifferentialassociationwithmutantformsofGST-mFasGSTPulldownHeLaL929292283276268221SchematicrepresentationofthemouseFasantigenanditsbindingproteinsGSTPulldownCo-ImmunoprecipitationAntibodyIdentificationTheproteinagainstwhichtheantibodywasraisedshouldbeprecipitatedfromcelllysate.(1)Independentantibodiesraisedagainstthesameproteinrecognizethesamepolypeptide;(2)Targetproteinshouldnotbeidentifiedwithantibodiesfromcelllineswithouttargetprotein;FalsepositiveandcontrolCo-Immunoprecipitation1.Antibodycontrol MonoclonalAb:anotherMoAbagainstsimilarprotein Antiserum:serumbeforeimmunizationfromthesameanimal PolyclonalAb:purifiedPoAbagainstanotherprotein2.Multipleantibodies differentAbsagainstdifferentepitopes; theepitopemaybethesiteforassociationwithotherproteins;3.Celllinesdepletedoftargetprotein Controlexperimentshouldbepractisedindepletedcelllines4.Inactivebiologicalmutant5.Interactionverificationbeforeandaftercelllysis unphysiologicalinteractionReductionofnonspecificproteinbackgroundCo-Immunoprecipitation1.toincreaseionicstrengthinwashbuffer;2.toreducetheamountofprimaryAb;3.topreclearcelllysatewithcontrolAb.

BindingofpVHLtoElonginBandCCo-Immunoprecipitation1.vonHippel-Lindaudiseaseisahereditarycancersyndromecharacterizedbythedevelopmentofmultipletumors;2.VHLsusceptibilitygene,mutatedinthemajorityofVHLkindreds,isatumorsuppressor;3.toelucidatethebiochemicalmechanismsunderlyingtumorsuppressionbypVHL,searchforcellularproteinsthatboundtowtpVHL,butnottotumor-derivedpVHLmutants.Science269:1444-6,1995IdentificationofVHL-associatedproteinsCo-ImmunoprecipitationLysatesfrom786-Orenalcarcinomacells,transfectedwiththeindicatedpVHLconstructs,wereimmunoprecipitatedwithanti-HA(AandB)orwithanti-VHL(C).Detectionbyautoradiography(A,C)orbyimmunoblotting(B).openarrows:exopVHLclosedarrows:VHL-APpVHL(1-115):withoutresiduesfrequentlyalteredbynaturallyoccurringVHLmutationsand,unlikepVHL(wt),doesnotsuppresstumorformationinvivo.pVHL(167W):thepredictedproductofamutantVHLallelethatiscommoninVHLfamilies.anti-VHLMappingthep14andp18bindingsiteonpVHLCo-Immunoprecipitationa-HAA.786-OcellsproducingHA-VHL(wt)orHA-VHL(1-115)werelabeledwith35S-methione,lysed,andimmunoprecipitatedwithanti-HA.Parental786-Ocellsweresimilarlylabeled,lysed,andincubatedwithGSHSepharosepreloadedwithGST-VHL(117-213)orGSTalone.BandC.786-Ocellswerelabeled,lysed,andincubatedwithGSHSephorasepreloadedwiththeindicatedGST-VHLfusionprotein.In(C),theindicatedpeptides(finalconc.~0.1,1,or10uM)wereaddedtotheGST-VHLfusionproteinbeforeincubationwiththeradiolabeledextract.ThewtpeptideisTLKERCLQWRSLVKP(underlinedresiduesaresitesofgerm-linemissensemutations).ThemutantpeptideisTLKERFLQWRSLVKP.thebindingsiteforElonginBandCinpVHLCo-ImmunoprecipitationDistributionofgerm-lineVHLmutations.TheshadedregionrepresentsthebidningsiteforElonginBandC.BindingofpVHLtoElonginBandElonginCinvivoCo-ImmunoprecipitationA.ACHN(VHL+/+),CAKI-1(VHL+/+),786-O(VHL-/-),and293(VHL+/+)cellswerelabeledwith35S-methione,lysed,andimmunoprecipitatedwithanti-VHLoracontrolantibody.Theimmunoprecipitaeswerewashedunderhigh-saltconditions.TheidentificationofpVHL(wt)(openarrow)wasconfirmedbyanti-pVHLimmunoblotanalysis.The~19kDproteinimmediatelyabovep18(*)intheACHN,CAKI-1,and293cellanti-VHLimmunoprecipitatesreactswithapolyclonalantibodytoVHL.B.ComparisonofpeptidesgeneratedbypartialproteolysisofElonginBandC,translatedinvitro,withp18andp14.TAP:tandemaffinitypurificationSequenceandstructureoftheTAPtagCBPTEVIgBDbaitTAPOverviewoftheTAPprocedureTAPSchematicrepresentationofthesplitTAPtagstrategyTAPSchematicrepresentationofthesubstractionstrategyTAPProteincompositionofTAP-purifiedU1snRNPTAPStep-by-stepanalysisoftheTAPstrategyTAPProteinspresentinthefinalTAPfraction(lanes7and8),orpresentaftereachofthesingleaffinitypurificationsteps(lanes1–4),wereanalyzed.Snu71-TAP(lanes1,3,and7)orwild-typeextracts(lanes2,4,and8)wereused.Lane5:molecularweightmarker.Lane6:anamountofTEVproteaseidenticaltotheamountusedtoeluteproteinsboundtoIgGbeads(lanes2,3,7,and8).RightarrowsindicatetheU1snRNP-specificproteinsincludingthetaggedSnu71pafterTEVcleavage;thearrowontheleftindicatestheSnu71pproteinfusedtotheTAPtagbeforeTEVcleavage.TAPinhighereucaryotesTAPQuestions:overexpressionendogenousexpressionSolutions:RNAinterferenceKnockintechniqueStrengthsandweaknessesofcommonlyusedaffinityapproachesfortheretrievalofproteincomplexesFRET:

fluorescenceresonanceenergytransferWhenwillFREToccur?1)SpectraloverlapDonoremissionspectrummustsignificantlyoverlaptheabsorptionspectrumoftheacceptor(>30%)2)Distancebetweenthedonorandacceptorisbetween2-10nm3)Favorableorientationoffluorophores2~10nmDonoremissionAcceptorabsorptionFRETE:energytransferefficiencyR0:intermoleculardistancewhenhalfofenergyistransferedr:distancebetweenfluorophoresE=R06/(R06+r6)whenr=2R0,E=1/65R0=4.9nmImagingproteinphosphorylationbyFRETtargetGFPFabCy3transfectionmicroinjectionorincubationtargetGFPFabCy3activatorlaserFRETDetectionofproteininteractionbyFRETtargetGFPFabCy3Protein1CFPProtein2YFPFRETProtein1Cy3Protein2FITCinvitrophosphorylationinvivoFRETrevealsinterleukin(IL)-1-dependentaggregationofIL-1typeIreceptorsthatcorrelateswithreceptoractivationFRETJBC270:27562-8,1995AbbreviationFRETIL-1:interleukin1IL-1RI:IL-1typeIreceptorIL-1ra:IL-1receptorantagnistCHO-mu1c:CHO-K1cellsstablytransfectedwithwild- typeIL-1receptorCHO-extn:CHO-K1cellsstablytransfectedwith cytoplasmictail-truncatedIL-1receptorM5:noncompetitiveanti-IL1RImonoclonalantibodyFITC-M5:M5labeledwithadonorprobe,FITCCy3-M5:M5labeledwithaacceptorprobe,Cy3IL-1a-dependentFRETbetweendonorFITC-M5andacceptorCy3-M5boundtoIL-1RIonthesurfaceofCHO-mu1ccellsFRETA,amixtureof5nMFITC-M5and5nMCy3-M5wasincubatedwithCHO-mu1ccells(3X106cells/ml)containingwild-typetransfectedreceptorsfor50minat22°C.IL-1aorIL-1rawasaddedatafinalconcentrationof30nMimmediatelyafterthetimepointatt=0min(arrow),andchangesintheratioofCy3-M5fluorescencetoFITC-M5fluorescenceweremonitoredovertime.Changesinthisratiowerealsomonitoredforthecontrolsampletowhichnoligandwasadded.B,normalizedfluorescenceratioforcellswithaddedIL-1aorIL-1racalculatedfromdatainA.IL-1aIL-1racontrolIL-1aIL-1raIL-1abutnotIL-1racausesaggregationbetweenIL-1RI-labeledwithFITCandCy3FabfragmentsofM5asdetectedbyFRETFRETAmixtureof20nMFITC-M5-Faband20nMCy3-M5-FabwasaddedtoCHO-mu1ccellstransfectedwithwild-typereceptorsandincubatedat22°Cfor50min.IL-1aorIL-1rawasaddedtoafinalconcentrationof10nMimmediatelyafterthetimepointat0min.ChangesinthenormalizedratioofCy3-M5FabfluorescencetoFITC-M5Fabfluorescenceweremonitoredovertimeat22°C.IL-1aIL-1raIL-1-dependentenergytransferbetweenIL-1RIistemperatureFRETAmixtureof20nMFITC-M5Faband12nMCy3-M5FabwasaddedtoCHO-mu1ccells(3X106cells/ml)withtransfectedwild-typeIL-1RIandpreincubatedateither4°C(A)or22°C(B)for50min.Immediatelyafterthebase-linedatapointatt=0min,IL-1awasadded(arrow)atafinalconcentrationof10nMtobothsamples.ChangesinthenormalizedratioofCy3-M5FabfluorescencetoFITC-M5Fabfluorescencewasmonitoredovertimeatthecorrespondingpreincubationtemperature.Att=85min,thetemperatureforsample(A)waschangedfrom4to22°C,andthetemperatureforsample(B)waschangedfrom22to4°C.Changesinthenormalizedfluorescenceratiocontinuedtobemonitoreduntilt=180min.ABIL-1a-dependentFRETcanbedetectedbetweenFITC-M5FabandCy3-M5FabboundtothecytoplasmictaildeletedmutantIL-1RIonCHO-extncellsFRETAmixtureof20nMFITC-M5Faband12nMCy3-M5Fabwasaddedtowild-typetransfectedreceptorsonCHO-mu1ccellsandincubatedat22°Cfor50min(A).Amixtureof20nMFITC-M5Faband12nMCy3-M5FabwasaddedtoCHO-extncells(cytoplasmictaildeletedmutantIL-1RI)andincubatedat22°Cfor50min(B).IL-1awasaddedtoafinalconcentrationof20nMatthearrow,andchangesinthenormalizedratioofCy3-M5FabfluorescencetoFITC-M5Fabfluorescenceweremonitoredovertimeat22°C.ABSPR:SurfacePlasmaResonanceDiagramofBIAcoreSPRInteractionsbetween

lectinsandimmobilizedglycoproteinsSPRSPRInteractionsbetween

lectinsandimmobilizedglycoproteinsAnoverlayplotofbindi