原创文献-可能超越CRISPR-Cas9的新技术

ResourceAMethodfortheAcuteandRapidDegradationofEndogenousProteinsGraphicalAbstractHighlightsdTrim-AwayisawidelyapplicablemethodtodegradeendogenousproteinsdTargetproteinsdonotneedtobemodifiedbeforedegradationdProteinsaredegradedwithinminutesofapplicationdTrim-AwayallowsefficientproteindepletioninprimaryhumancellsCliftetal.,2018,Cell172,1–15January11,2018ª2017TheAuthors.PublishedbyElsevierInchttps://doi.org/10.1016/j.cell.2017.10.033AuthorsDeanClift,WilliamA.McEwan,LarisaI.Labzin,VeraKonieczny,BinyamMogessie,LeoC.James,MelinaSchuhCorrespondencedclift@mrc-lmb.cam.ac.uk(D.C.),lcj@mrc-lmb.cam.ac.uk(L.C.J.),melina.schuh@mpibpc.mpg.de(M.S.).Pleasecitethisarticleinpressas:Cliftetal.,AMethodfortheAcuteandRapidDegradationofEndogenousProteins,Cell(2018),https://doi.org/10.1016/j.cell.2017.10.033ResourceAMethodfortheAcuteandRapidDegradationofEndogenousProteinsDeanClift,1,*WilliamA.McEwan,1LarisaI.Labzin,1VeraKonieczny,2BinyamMogessie,2LeoC.James,1,*andMelinaSchuh1,2,3,*1MedicalResearchCouncil,LaboratoryofMolecularBiology,CambridgeCB20QH,UK2MaxPlanckInstituteforBiophysicalChemistry,37077Göttingen,Germany3LeadContact*Correspondence:dclift@mrc-lmb.cam.ac.uk(D.C.),lcj@mrc-lmb.cam.ac.uk(L.C.J.),melina.schuh@mpibpc.mpg.de(M.S.)https://doi.org/10.1016/j.cell.2017.10.033SUMMARYMethodsforthetargeteddisruptionofproteinfunc-tionhaverevolutionizedscienceandgreatlyexpe-ditedthesystematiccharacterizationofgenes.Twomainapproachesarecurrentlyusedtodisruptpro-teinfunction:DNAknockoutandRNAinterference,whichactatthegenomeandmRNAlevel,respec-tively.Amethodthatdirectlyaltersendogenousproteinlevelsiscurrentlynotavailable.Here,wepre-sentTrim-Away,atechniquetodegradeendogenousproteinsacutelyinmammaliancellswithoutpriormodificationofthegenomeormRNA.Trim-Awayharnessesthecellularproteindegradationmachin-erytoremoveunmodifiednativeproteinswithinminutesofapplication.Thisrapidityminimizestheriskthatphenotypesarecompensatedandthatsecondary,non-specificdefectsaccumulateovertime.BecauseTrim-Awayutilizesantibodies,itcanbeappliedtoawiderangeoftargetproteinsusingoff-the-shelfreagents.Trim-Awayallowsthestudyofproteinfunctionindiversecelltypes,includingnon-dividingprimarycellswheregenome-andRNA-targetingmethodsarelimited.INTRODUCTIONInterferingwithproteinexpressionisapowerfulstrategytoinvestigatethefunctionofaprotein.Traditionally,DNA-modi-fyingmethodshavebeenusedtoknockoutproteinsonthegenelevel(Capecchi,1989),anapproachthathashadarecentresurgencewiththeemergenceofCRISPR/Cas9technology(DoudnaandCharpentier,2014).RNA-targetingmethodssuchasRNAiarealsowidelyusedtoknockdownexpressionofapro-teinbydestroyingthemRNA(Elbashiretal.,2001).However,inbothapproaches,proteindepletionisindirectanddependentontheinherentturnoveroftheprotein.Consequently,long-livedproteinstakemoretimetodeplete,ormayberesistanttoDNA-andRNA-targetingdepletionmethodsaltogether(Jansenetal.,2007;Smoaketal.,2016).ThelongtimeframethatisrequiredforproteindepletionwiththesemethodsalsomeansthatcellsmayhaveenoughtimetoactivatecompensatoryCell172,1–15,Thisisanopenaccessarticleundmechanisms,whichmaymaskphenotypes(Damkeetal.,1995;Rossietal.,2015).Furthermore,itisoftenimpossibletodeterminewhetherornotaphenotypeisanindirectconse-quenceofanearlierdefect.Inanattempttoovercometheseproblems,severalmethodshavebeendevelopedforconditionalproteininactivation.However,thesemethodsrequirethateithertheproteinofinterestisfirstmodified(Banaszynskietal.,2006;Caussinusetal.,2011;Neklesaetal.,2011;Nishimuraetal.,2009;Robinsonetal.,2010;Tachibana-Konwalskietal.,2010)orcanonlybeappliedtoaverysmallnumberofproteins(De-shaies,2015;Fanetal.,2014).Chemicalinhibitorscanbeequallyproblematicastheyarelimitedtodruggableproteinsandpronetooff-targeteffects(Mayer,2003).Awidelyapplicableproteindepletionmethodthatactsexclusivelyattheproteinleveliscurrentlylacking.Suchamethodwouldnotonlyallowtheacutedepletionofendogenousproteins,butalsothestudyofproteinfunctioninnon-dividingprimarycellsinwhichDNA-targetingmustbedoneatthewholeanimallevelandRNA-targetingcannotdepletestableproteins.Wesoughttodevelopatrulyposttranslationalproteindeple-tionmethodbasedonproteintargetingbyantibodies.Anti-bodiesbindtoproteinswithhighaffinityandspecificity;theyarewidelyavailablecommerciallyandcanbeproducedforalmostanyproteinwithrelativeease.Antibodiesarethereforeidealasthebasisofaprotein-targetingmethod.Antibodieshavebeenusedpreviouslytointerferewithproteinfunction(MorganandRoth,1988).However,thisrequiresthattheanti-bodybindstoanepitopethatblocksthefunctionofaproteinandcaneffectivelyandstoichiometricallycompetewithendog-enousligands.Thismethodofinhibitionisthereforeonlyappli-cabletoaverylimitednumberofproteins.Instead,weaimedtodevelopauniversallyapplicablemethodthatwouldallowustotargetanyantibody-boundproteinfordegradation.Antibody-boundpathogenscanberecognizedbythecyto-solicantibodyreceptor,TRIM21(Malleryetal.,2010).TRIM21isanE3ubiquitinligasethatbindswithhighaffinitytotheFcdomainofantibodies(Jamesetal.,2007).TRIM21iswidelyex-pressedindiversecelltypesandtissues,whichisanecessaryrequirementofitsphysiologicalrole(Yoshimietal.,2009).Dur-inginfection,TRIM21recruitstheubiquitin-proteasomesystemtoantibody-boundpathogens,leadingtotheirdestruction(Mal-leryetal.,2010).TRIM21causesdegradationofdiversepatho-gensincludingRNAandDNAviruses(Watkinsonetal.,2015),bacteria(McEwanetal.,2013),andtheproteopathicagentJanuary11,2018ª2017TheAuthors.PublishedbyElsevierInc.1ertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).Figure1.AcuteDegradationofProteinsbyTrim-Away(A)SchematicofTrim-Awayapproach.(B–E)NIH3T3cellsoverexpressingmCherry-TRIM21(notshown)andfreeGFP(greys)weremi-croinjectedwithanti-GFPantibodyorcontrolIgG(BandC)ortreatedwithDMSOorMG132andmi-croinjectedwithanti-GFPantibody(DandE).Timeshowsminutes(min)fromantibodymicroinjection;0minisjustbeforeantibodymicroinjection.Dashedlineoutlinescell.Scalebars,10mm.ErrorbarsshowSD.Numberofcellsisspecifiedinbrackets.Datafromthree(CandE)independentexperiments.SeealsoFiguresS1andS3–S7andMovieS1.Pleasecitethisarticleinpressas:Cliftetal.,AMethodfortheAcuteandRapidDegradationofEndogenousProteins,Cell(2018),https://doi.org/10.1016/j.cell.2017.10.033Tau(McEwanetal.,2017).BoththeproteasomeandtheAAAATPaseVCP/p97havebeenidentifiedasimportantco-factorsfordegradation,buttheirrequirementdiffersbetweensub-strates(Hauleretal.,2012).TheE2enzymesUbe2WandUbe2N/2V2havealsobeenimplicatedinTRIM21-mediateddegradation(Fletcheretal.,2015).TheseenzymesactsequentiallyonTRIM21itself,firstmono-ubiquitinatingit(Ube2W)andthenextendingfromthisaK63-ubiquitinchain(Ube2N/2V2).TRIM21isalsomodifiedwithK48-chainsbuttheE2enzyme(s)involvedareunknown,asisthefunctionalimportanceofthismodification.TRIM21hasnotbeenreportedtomodifythepathogenorthepathogenboundantibodies,butitisunclearifsuchmodificationshaveescapeddetectionorifauto-ubiquitinationofTRIM21issufficientforpathogendestruction.TRIM21hasalsobeenreportedtointeractwithseveralotherproteinsincludingSkp2,DAXX,IRF-3,IRF-5,IRF-8,DDX41,andSQSTM1(James,2014).However,mostoftheseinteractionshavebeendetectedbasedonimmunoprecip-itation,whichcandeliverfalsepositivesbecauseofdirectbind-ingofTRIM21totheantibodiesusedintheassay.Inthisstudy,werepurposedTRIM21toestablishamethodtodegradeendogenousproteinsthatwecalledTrim-Away.Trim-Awayallowsthestudyofproteinfunctioninvariouscelltypes,includingnon-dividingprimarycellswheregenome-andRNA-targetingmethodsarenotwellsuited.Trim-Awaydegradespro-teinswithinminutesofapplication,makingitsuitabletoinvesti-gatethefunctionofaproteinlongafterithasformedandatallstagesofacell’slife,cycle,ordifferentiation.Acuteproteindisruptionassays,whichinthepasthaverequiredcomplex2Cell172,1–15,January11,2018geneticsandalotoftime,cannowbedonebyTrim-Awaywithinhours.Trim-Awaythereforeformsthebasisforthesys-tematicstage-specificanalysisofproteinfunction.RESULTSPrincipleofProteinDegradationbyTrim-AwayWereasonedthattheantibodyreceptorandubiquitinligaseTRIM21couldbeusedasatooltodrivethedegradationofendog-enousproteinsbyusinga3-stepstrategy:first,theintroductionofexogenousTRIM21;second,theintro-ductionofanantibodyagainsttheproteinofinterest;andthird,TRIM21-mediatedubiquitinationfollowedbydegradationoftheantibody-boundproteinofinterest(Figure1A).Asweoutlineindetailbelow,thisstrategy,whichwecalled‘‘Trim-Away,’’isideallysuitedfortheacuteandrapiddegradationofendogenousproteinsinbothindividualcellsaswellasbulkcellpopulations.TotesttheTrim-Awaystrategy,wefirstsetupaproofofprin-cipleexperimentinmammaliancellculture.NIH3T3cellsover-expressingmCherry-TRIM21andfreeGFPweremicroinjectedwithanti-GFPantibody(Figure1B).Remarkably,GFPwasrapidlydegradedfollowingmicroinjectionwithahalf-lifeofjust16min(Figures1B,1C,andS3A;MovieS1).mCherry-TRIM21colocalizedwithGFPduringdegradation,consistentwithTRIM21recruitmenttoGFPviatheanti-GFPantibody(FiguresS1AandS1B;MovieS1).GFPaggregatedquicklyatthesiteofantibodymicroinjection,likelybecauseofthehighlocalconcen-trationofanti-GFPantibody(Figures1BandS1A).DegradationwasspecificallyduetotargetingofGFPbyanti-GFPantibody,becausemicroinjectionofanon-specificcontrolIgGdidnotcauseGFPdegradationinmCherry-TRIM21-overexpressingcells(Figures1B,1C,andS1A–S1D).ThedegradationofGFPwasdependentonTRIM21overexpression,becauseanti-GFPantibodyfailedtotriggerGFPdegradationincellsthatoverex-pressedmCherryinsteadofmCherry-TRIM21(FiguresS1EandS1F).TheE3ubiquitinligaseactivityofTRIM21wasrequiredfordegradation,becauseatruncatedformofTRIM21lackingtheRINGE3ubiquitinligaseandB-Boxdomains(mCherry-TRIM21DRING-Box)efficientlycolocalizedwithGFPfollowingPleasecitethisarticleinpressas:Cliftetal.,AMethodfortheAcuteandRapidDegradationofEndogenousProteins,Cell(2018),https://doi.org/10.1016/j.cell.2017.10.033anti-GFPantibodymicroinjection,butfailedtocauseGFPdegra-dation(FiguresS1GandS1H).TotestifproteindegradationbyTrim-AwayismediatedbytheproteasomeconsistentwiththeknownmechanismofTRIM21function,wetreatedcellswiththeproteasomeinhibitorMG132.Strikingly,MG132treatmentpreventedthedegradationofGFPfollowinganti-GFPantibodymicroinjectionintomCherry-TRIM21-overexpressingcells(Figures1Dand1E).Therefore,proteindegradationbyTrim-Awayreliesontheubiquitin-protea-somepathway(Figure1A).Trim-AwayCanBeUsedtoDegradeProteinsinPrimaryCellsWenextaskedifTrim-Awaycouldbeappliedtopost-mitoticprimarycells.Wechosemammalianoocytesbecausetheyaretranscriptionallysilent,whichprecludesproteindisruptionbydirectgenomeediting.Moreover,RNAiisinefficientinthesecellsduetolargeamountsofstoredproteins(CliftandSchuh,2013;Pfenderetal.,2015).WefirsttestedifTRIM21overexpressionhasanyinfluenceonoocytemeiosis.Oocytesthatoverex-pressedTRIM21progressedthroughmeiosiswithsimilareffi-ciencyandtimingascontroloocytes:neithertheratenorthetimingofnuclearenvelopebreakdownoranaphasewassignifi-cantlydifferent(FiguresS6A–S6C,S6E,andS6F).AlsospindlemorphologyduringmeiosisIandmeiosisIIwasnotperturbed(FiguresS6A,S6D,andS6G).Thus,TRIM21overexpressiondoesnotperturboocytemeiosis.Wethenperformedasimilarproof-of-principleGFPdegrada-tionexperimentasdescribedaboveinisolatedmouseoocytes.Microinjectedanti-GFPantibodytriggeredrapidGFPdegrada-tioninoocytesoverexpressingmCherry-TRIM21.Incontrast,controlIgGhadnoeffectonGFPproteinlevels(Figures2Aand2B).AsinNIH3T3cells,mCherry-TRIM21colocalizedwithGFPduringGFPdegradation(FigureS2A),andGFPdegradationwasdependentonTRIM21’subiquitinligaseactivity(FiguresS2C–S2H).Beforeproteindegradation,weobservedatransientincreaseinGFPintensityatthesiteofGFP-antibodymicroinjec-tion(Figure2A).ThisislikelyduetoalocalenrichmentofGFP-antibodyatthemicroinjectionsite,whichresultsinsequestrationofGFPfromtheentireoocytevolumetothisregion.WenoticedthatmCherry-TRIM21wasdepletedconcomi-tantlywithGFPuponanti-GFPantibodymicroinjection(FiguresS2AandS2B).WesternblottingofwholecellextractsconfirmedthatTRIM21,antibodyandtargetproteinarealldegradedduringTrim-Away(FigureS2I).ThisisconsistentwiththeproposedmechanismofTRIM21functionandsuggeststhatTRIM21andantibodylevelscouldbecomelimitingduringTrim-Awayifthetargetisinsignificantmolarexcess.Toinvestigatethisfurther,wemodifiedrelativelevelsofTRIM21andGFPandfoundthatTRIM21needstobepresentinexcessofthetargetproteintofacilitatecompleteproteindegradation(FiguresS2J–S2L).Trim-AwayCanTargetDiverseCellularSubstratesAwidelyapplicableproteindepletionmethodshouldbeabletotargetdiversesubstrateswithinthecell.Toaddressthis,welocal-izedGFPtodifferentregionsofthecellandtestedtheefficiencyofdegradationbyTrim-Away.GFPcontaininganN-terminalN-myr-istoylandS-palmitoylmotif(membrane-anchoredGFP)localizedtotheplasmamembraneandvesicle-likestructuresinthecyto-plasm(Figure2C);GFPfusedtothehistoneH2B(H2B-GFP)wasefficientlyincorporatedintochromatin(Figure2E);GFPcon-taininganuclearlocalizationsignal(NLS-GFP)accumulatedinthenucleus(Figure2G).Allthreedifferentsubstrateswererapidlydegradedbyanti-GFPantibodyandTRIM21withsimilarkineticstofreecytosolicGFP(Figures2C–2H).Remarkably,thehalf-lifefordegradationbyTrim-Awaywasaslittleas9min(Figures2B,2D,2F,2HandS3A).Thisisidenticaltotherateofproteindegra-dationachievedbytheauxin-inducibledegronsystem(Hollandetal.,2012),butwiththeadvantagethatTrim-Awayrequiresnomodificationofthetargetprotein.Altogether,thesedatashowthatTrim-Awaycandegradeproteinsthatarelocalizedtodifferentregionsofthecellandincorporatedintolargerproteincomplexesandcellularstructures.WenoticedthatNLS-GFPwasdegradedinthecytoplasm,presumablybecauseNLS-GFPshuttlesinandoutofthenucleusandcanthereforebeboundbyantibodiesinthecytoplasm(Fig-ure2G).WethereforetestedifTrim-Awaycanalsodegradere-tainednuclearproteins,suchasGFP-H2Bthatisstablyassoci-atedwithchromatinwithinthenucleus.IncontrasttoNLS-GFP,GFP-H2Bwasnotdegradedwhenthechromatinwascontainedwithinanintactnucleus(FiguresS3BandS3C).WereasonedthatwemightbeabletoovercomethislimitationbyfusingtheFc-domainofanantibodytoananobody.TheFc-nanobodyfusionismuchsmallerinsizeandshouldbeabletoenterthenucleus.Strikingly,H2B-GFPwasquicklydegradedalsoinsidethenucleuswhenTRIM21andtheFc-nanobodyfusionwereco-expressed(Figures2I–2K).ThisdemonstratesthatFc-nano-bodyfusionscanbeusedtodegradeproteinsinsidethenucleus.ItalsoillustratesthatthegrowingnumberofnanobodiesiscompatiblewithTrim-AwaywhenthesenanobodiesarefusedwithanFc-domain.RescueExperimentsConfirmTrim-AwaySpecificityInprinciple,itshouldbepossibletodegradeanyendogenousproteininthecellthatcanbeaccessedbyantibodiesusingTrim-Away(Figure1A).Totestthis,wedecidedtotargetanendogenousproteininmouseoocytescalledEg5.Eg5isamicrotubulemotorproteinrequiredforproperspindleassemblyduringmitosisandmeiosis(CliftandSchuh,2015;Mayeretal.,1999;SchuhandEllenberg,2007).WhenthefunctionofEg5isdisrupted,amonopolarmicrotubuleasterformsandthespindlefailstobecomebipolar.WechosetoTrim-AwayEg5becausewecoulddirectlycomparethephenotypefollowingEg5degrada-tiontothatofEg5inhibitionwiththesmallmoleculeinhibitormonastrol(Mayeretal.,1999).Strikingly,microinjectionofanti-Eg5antibodyintooocytesoverexpressingTRIM21causedtheformationofmonopolarspindles;preciselythephenotypeexpectedifEg5isdegradedandidenticaltooocytestreatedwiththeEg5inhibitormonastrol(Figures3A–3C).NeithercontrolIgGmicroinjectionintoTRIM21-overexpressingoocytes,noranti-Eg5antibodymicroinjectionalonecausedmonopolarspindles(Figures3Band3C),con-firmingthatEg5degradationrequiresbothTRIM21andanti-Eg5antibody.Eg5proteinwascompletelydegradedasshownbyimmunoblottingwithtwodifferentanti-Eg5antibodies(Figure3D).Cell172,1–15,January11,20183(legendonnextpage)4Cell172,1–15,January11,2018Pleasecitethisarticleinpressas:Cliftetal.,AMethodfortheAcuteandRapidDegradationofEndogenousProteins,Cell(2018),https://doi.org/10.1016/j.cell.2017.10.033Pleasecitethisarticleinpressas:Cliftetal.,AMethodfortheAcuteandRapidDegradationofEndogenousProteins,Cell(2018),https://doi.org/10.1016/j.cell.2017.10.033ToconfirmthatthemonopolarspindlephenotypewasduetoEg5degradation,weperformedarescueexperimentbymicro-injectingmRNAencodingEg5-mEGFPintoEg5Trim-Awayoo-cytes(Figures3Aand3E).Eg5-mEGFPexpressiontransformedthemonopolarspindlesbacktoabipolarstate(Figures3Eand3F;MovieS2).Eg5-mEGFPcanberecognizedbyanti-Eg5antibody,butlikelyrescuesbecauseitisexpressedinexcess.Altogether,thesedatashowthatendogenousproteinscanbedegradedusingTrim-Away,andrescueassayscanbeusedtoconfirmthespecificityofaTrim-Awayphenotype.Trim-AwayIsSuitabletoDegradeLong-LivedProteinsAcutelyLong-livedintracellularproteinsaresurprisinglycommon(Toyamaetal.,2013),butstudyingtheirfunctionischallenging.ThelackofproteinturnovermeansthatdepletionbyRNAiisinef-fective.Geneknockoutsarealsooftennotsuitable,becauselong-livedproteinsarefrequentlyespeciallyimportantinnon-dividingprimarycellsandessentialforviability(Toyamaetal.,2013).WereasonedthatbecauseTrim-Awayactsexclusivelyattheproteinlevel,itshouldbesuitabletodegradeevenverylong-livedpro-teinsandtherebystudytheirfunctionlongaftertheirsynthesis.Totestthis,wetargetedthelong-livedRec8proteininmouseeggsthatwerearrestedinmetaphaseofthesecondmeioticdi-vision.Rec8ispartofthecohesinproteincomplexthatmediatessisterchromatidcohesioninoocytesfrombirthuntilovulation(Tachibana-Konwalskietal.,2010).Rec8doesnotturnover(Tachibana-Konwalskietal.,2010),butremainsstablyassoci-atedwithchromosomesformonthsinmiceandpossiblyde-cadesinhumans.WeaskedifwecoulduseTrim-AwaytodegradeendogenousRec8acutelyinmetaphaseIIarrestedeggs,longafterithasbeenincorporatedintochromosomes.Microinjectionofanti-Rec8antibody(Eijpeetal.,2003)intoeggsoverexpressingmEGFP-TRIM21triggeredtheprematureseparationofsisterchromatids(Figures4A–4C;MovieS3),producing40singlechromatids(Figure4D),whichisindicativeofRec8degradationandcompletelossofsisterchromatidcohesion(Tachibana-Konwalskietal.,2010).Noseparationwasobservedwhencon-trolIgGwasmicroinjectedintomEGFP-TRIM21-overexpressingoocytesorwhenoocytesoverexpressingantibody-binding-defi-cientTRIM21(mEGFP-TRIM21DPRYSPRY)weremicroinjectedwithanti-Rec8antibody(Figures4Band4C).ThisconfirmedthatsisterchromatidseparationwasduetoRec8degradationtriggeredbyTRIM21recruitmenttoRec8viaanti-Rec8antibody(Figure4A).PreviousfunctionalstudiesofRec8requiredcom-plexmousegeneticstoreplacetheRec8genewithaversionFigure2.Trim-AwayDegradesDiverseCellularSubstrates(A–H)OocytesoverexpressingmCherry-TRIM21(notshown)andeitherfreeGFP((GandH)weremicroinjectedwitheitheranti-GFPantibodyorcontrolIgG.Timesmicroinjection.(I)Schematicofnanobody-Fcfusionapproach.(JandK)Prophase-arrestedoocytesexpressingH2B-GFPweremicroinjectedwGFPnanobody-Fcfusionprotein.(J)Representativeexamplesand(K)quantificaWhitedashedlineoutlinesoocyte.YellowarrowshowsH2B-GFP.Yellowdashoocytesisspecifiedinbrackets.Datafromthree(B)ortwo(D,F,H,andK)indeSeealsoFiguresS2–S7.thatexpressesaTEVprotease-cleavableRec8protein(Tachi-bana-Konwalskietal.,2010).TheRec8Trim-Awayexperimentsdescribedhereprovidethefirstevidencethatendogenous,unmodifiedRec8proteinisresponsibleforsisterchromatidcohesioninmouseeggs.Remarkably,sisterchromatidsbegantoseparateonaveragejust11minafteranti-Rec8antibodymicroinjectionintomEGFP-TRIM21-overexpressingeggs(Fig-ures4Band4E),implyingTrim-Awaycandegradeevenverylong-livedproteinswithunprecedentedspeed.Trim-AwayCanBeUsedtoDegradeSpecificProteinVariantsSelectivelyGiventhatTrim-Awaycapitalizesonthehighspecificityofanti-bodies,itshouldalsobesuitabletoselectivelydegradesplicevariants,posttranslationallymodifiedordisease-causingproteinvariantswhilepreservingthehealthyprotein,aslongasanti-bodiesareavailablethatarevariant-specific.Totestthis,weaskedifTrim-Awaycanbeusedtoselectivelydegradethedisease-causingvariantoftheproteinhuntingtin,whichcausesHuntington’sdisease.Weco-expressedthenormalorthedisease-causingvariantofhuntingtinproteintogetherwithmCherry-TRIM21inNIH3T3cells.Wethenmicroinjectedthecellswithanantibody(3B5H10)whichspecificallybindstothedisease-causingvariantofhuntingtin(Milleretal.,2011)(Fig-ureS4A).Thedisease-causingvariantofhuntingtinproteinwasrapidlydegradedfollowingmicroinjectionoftheantibody,whereasnormalhuntingtinprote