泛素调节的蛋白质降解

20042004(AvramHershko)(AaronCiechanover)(IrwinRose)()(forthediscoveryofubiquitin-mediatedproteindegradation)1600030000,2050,1977GoldbergATPAaronCiechanover,AvramHershko,IrwinRose7080,19922,76,8.45ku,1975,,(ubique,everywhere),333,(ubiquitin-activatingenzyme,E1)(ubiquitin-conjugatingenzyme,E2)(ubiquitin-proteinligatingenzyme,E3)3E1,E2E2E2,E3E3E2E3,E2,(5),4(,),30000,1979Goldberg:20S26S,26S20S19S,26S,(20S),79,(lock)(19S),,,(ATP),,E35(1)E1,ATP(2)E2,E2(3)E3E2E3,E2()(4)E3(5),(6)79652360E3APC(anaphase-promotingcomples,)APC121E3!p53,p5350%p53p53E3Mdm2p53DNAp53Mdm2Mdm2p53p53p53DNAp53DNADNAE3E6-APp53,p53p53p53DNADNA__________________________________[1]200412[2]2004200411[3]200420052232[4]:Aproteomicsmethodhasbeendevelopedtopurifyandidentifythespecificproteinsmodifiedbyubiquitin(Ub)fromhumancells.Inpurifiedsamples,Uband21otherproteinswereidentifiedbyliquidchromatography-tandemmassspectrometry(LC-MS/MS)spectrausingSEQUEST®TheseproteinsincludedseveraloftheexpectedcarriersofUbincludingUb-conjugatingenzymesandhistoneproteins.Toperformtheseexperiments,acelllinecoexpressingepitopetaggedHis(6X)-Ubandgreenfluorescentprotein(GFP)wasgeneratedbystablytransfectingHEK293cells.Ubiquitinatedproteinswerepurifiedusingnickel-affinitychromatographyanddigestedinsolutionwithtrypsin.ComplexmixturesofpeptideswereseparatedbyreversedphasechromatographyandanalyzedbynanoLC-MS/MSusingtheLCQquadrupoleion-trapmassspectrometer.ProteinsidentifiedfromHis(6X)-Ub-GFPtransfectedcellswerecomparedtoalistofproteinsfromHEK293cells,whichassociatewithnickel-nitrilotriaceticacid(Ni-NTA)agaroseintheabsenceofHis-taggedUb.Inaproofofprincipleexperiment,His(6X)-Ub-GFPtransfectedcellsweretreatedwithAs(111)(10lam,24h)inanattempttoidentifysubstratesincreasinglymodifiedbyUb.Inthisexperiment,proliferatingcellnuclearantigen,aDNArepairproteinandknownubiquitinsubstrate,wasconfidentlyidentified.Thisproteomicsmethod,developedfortheanalysisofubiquitinatedproteins,isasteptowardslarge-scalecharacterizationofUb-proteinconjugatesinnumerousphysiologicalandpathologicalstates..Hernadez-MunozI,LundAH,vanderStoopP,etal.StableXchromosomeinactivationinvolvesthePRC1PolycombcomplexandrequireshistoneMACROH2A1andtheCULLIN3/SPOPubiquitinE3ligaseAbstract:XinactivationinvolvesthestablesilencingofoneofthetwoXchromosomesinXXfemalemammals.InitiationofthisprocessoccursduringearlydevelopmentandinvolvesXist(X-inactive-specifictranscript)RNAcoatingandtherecruitmentofPolycombrepressivecomplex(PRC)2andPRC1proteins.ThisrecruitmentresultsinaninactivestatethatisinitiallylabilebutisfurtherlockedinbyepigeneticmarkssuchasDNAmethylation,histonehypoacetylation,andMACROH2Adeposition.Here,wereportthattheE3ubiquitinligaseconsistingofSPOPandCULLIN3isabletoubiquitinatethePolycombgroupproteinBMI1andthevarianthistoneMACROH2A.WefindthatinadditiontoMACROH2A,PRC1isrecruitedtotheinactivatedXchromosomeinsomaticcellsinahighlydynamic,cellcycle-regulatedmanner.Importantly,RNAi-mediatedknock-downofCULLIN3orSPOPresultsinlossofMACROH2A1fromtheinactivatedXchromosome(Xi),leadingtoreactivationoftheXiinthepresenceofinhibitorsofDNAmethylationandhistonedeacetylation.Likewise,XireactivationisalsoseenonMacroH2A1RNAiundertheseconditions.Hence,weproposethatthePRC1complexisinvolvedinthemaintenanceofXchromosomeinactivationinsomaticcells.WefurtherdemonstratethatMACROH2A1depositionisregulatedbytheCULLIN3/SPOPligasecomplexandisactivelyinvolvedinstableXinactivation,likelythroughtheformationofanadditionallayerofepigeneticsilencing..TagoK,ChioccaS,SherrCJSumoylationinducedbytheArftumorsuppressor:Ap53-independentfunctionAbstract:Themousep19Arfproteinhasbothp53-dependentandp53-independenttumor-suppressiveactivities.Arftriggerssumoylationofmanycellularproteins,includingMdm2andnucleophosmin(NPM/B23),withwhichp19Arfphysicallyinteractsinvivo,andthisoccursequallywellincellsexpressingorlackingfunctionalp53.InanArf-nullNIH3T3cellderivative(MT-Arfcells)engineeredtoreexpressanArftransgenedrivenbyazinc-induciblemetallothioneinpromoter,sumoylationofendogenousMdm2andNPMproteinswasinitiatedasp19Arfwasinducedandwasobservedbeforep53-dependentcellcyclearrest.Predominatelynucleoplasmicmoleculesvisualizedbyimmunofluorescencewithantibodiestosmallubiquitin-likemodifier(SUMO)1localizedtonucleoliasp19Arfaccumulatedthere.TwoArfmutants,oneofwhichbindstoMdm2andNPMbutisexcludedfromnucleoliandtheotherofwhichentersnucleolibutishandicappedinbindingtoMdm2andNPM,weredefectiveininducingsumoylationofthesetwotargetproteinsanddidnotlocalizebulksumoylatedmoleculestonucleoli.TheCELOadenovirusprotein,Gam1,whichinhibitstheSUMOactivatingenzyme(E1)andleadstodown-regulationoftheSUMOconjugatingenzyme(E2/Ubc9),hadnooverteffectontheabilityofp19Arftoactivatep53orthep53-responsivegenesencodingMdm2andp21Cip1,despitethefactthatArf-inducedsumoylationofMdm2wasblocked.ReductionofUbc9levelswithshorthairpinRNAsrenderedsimi