Metabolic Regulation of Protein N-Alpha-Acetylatio

MetabolicRegulationofProteinN-Alpha-AcetylationbyBcl-xLPromotesCellSurvivalCarolineH.Yi,1HelingPan,1,11JanSeebacher,1,11Il-HoJang,4,11SvenG.Hyberts,2GregoryJ.Heffron,2MatthewG.VanderHeiden,3,7,8RenliangYang,9FupengLi,9JasonW.Locasale,3HadarSharfi,3BoZhai,1RicardRodriguez-Mias,2HarryLuithardt,10LewisC.Cantley,3,5,6GeorgeQ.Daley,4JohnM.Asara,5,6StevenP.Gygi,1GerhardWagner,2Chuan-FaLiu,9andJunyingYuan1,*1DepartmentofCellBiology2DepartmentofBiologicalChemistryandMolecularPharmacology3DepartmentofSystemsBiology4Children’sHospitalBoston5DivisionofSignalTransductionBethIsraelDeaconessMedicalCenter,Boston,MA02115,USA6DepartmentofMedicine7DanaFarberCancerInstituteHarvardMedicalSchool,Boston,MA02115,USA8KochInstitute,MassachusettsInstituteofTechnology,Cambridge,MA02142,USA9DivisionofChemicalBiologyandBiotechnology,SchoolofBiologicalSciences,NanyangTechnologicalUniversity,60NanyangDrive,Singapore637551,RepublicofSingapore10SolutionsLabs,400TechnologySquare,Cambridge,MA02139,USA11Theseauthorscontributedequallytothiswork*Correspondence:[e-mail]10.1016/j.cell.2011.06.050SUMMARYPreviousexperimentssuggestaconnectionbetweentheN-alpha-acetylationofproteinsandsensitivityofcellstoapoptoticsignals.Here,wedescribeabiochemicalassaytodetecttheacetylationstatusofproteinsanddemonstratethatproteinN-alpha-acetylationisregulatedbytheavailabilityofacetyl-CoA.BecausetheantiapoptoticproteinBcl-xLisknowntoinfluencemitochondrialmetabolism,wereasonedthatBcl-xLmayprovidealinkbetweenproteinN-alpha-acetylationandapoptosis.Indeed,Bcl-xLoverexpressionleadstoareductioninlevelsofacetyl-CoAandN-alpha-acetylatedproteinsinthecell.ThiseffectisindependentofBaxandBak,theknownbindingpartnersofBcl-xL.Increasingcellularlevelsofacetyl-CoAbyadditionofacetateorcitraterestoresproteinN-alpha-acetylationinBcl-xL-expressingcellsandconferssensitivitytoapoptoticstimuli.Weproposethatacetyl-CoAservesasasignalingmoleculethatcouplesapoptoticsensi-tivitytometabolismbyregulatingproteinN-alpha-acetylation.INTRODUCTIONIncreasingevidencesuggeststhatspecificmetabolicalterationsassociatedwithcancercellsmaynotbeancillarytotheirtrans-formationbutareinstrumentaltotheirtumorigenicpotentialbymediatingcellproliferation,growth,andsurvival(VanderHeidenetal.,2009).Manyoncogenesandtumorsuppressorgenesknowntopromoteexcesscellproliferationalsoalterbiosyn-thetic(oranabolic)processes.Forexample,Aktexpressionstimulatesglucoseuptakeandglycolysis,thepentosephos-phatepathway,andfattyacidsynthesis.c-Mycexpressionpromotesglutaminemetabolismaswellaspurineandpyrimidinebiosynthesis.Furthermore,mutationsingenesencodingmeta-bolicenzymeshavebeenidentifiedbycancergeneticassocia-tionstudies(VanderHeidenetal.,2009).Howspecificmetabo-litescontributetoincreasedproliferationandapoptoticresistanceintumorcellsremainsacentralunansweredquestion.Theproto-oncogeneBcl-xLhasaprominentroleinpromotingcellsurvivalandcancerdevelopment(Boiseetal.,1993).ItiswellestablishedthatBcl-xLprotectsagainstapoptosisbydirectlybindingandinhibitingBax/Bakoligomerization-medi-atedmitochondrialpermeabilization.However,certainBcl-xLmutants,suchasF131V/D133AandG148E,thatareunabletobindtoBaxorBak,neverthelessretain70%–80%antiapoptoticactivityofWTBcl-xL(Chengetal.,1996).Curiously,Bcl-xLhasalsobeenshowntoregulatemitochondrialrespirationandmetabolism(Gottliebetal.,2000;VanderHeidenetal.,1999).WhetherthemetabolicfunctionofBcl-xLcontributestoitsroleinmediatingapoptoticresistanceisunclear.OurunexpectedidentificationofanN-terminalacetyltransfer-ase,ArrestDefective1(dARD1),inagenome-wideRNAinterfer-ence(RNAi)screeninDrosophilacellsforapoptoticregulators(Yietal.,2007)promptedustopositthatproteinN-alpha-acety-lation,amajorN-terminalmodification,linkscellmetabolismCell146,607–620,August19,2011ª2011ElsevierInc.607toapoptoticinductionincancercells.SincedARD1isepistatictodiap1,whichencodesforadirectinhibitorofcaspasesinDrosophila,andARD1isrequiredforcaspaseactivationinmammaliancells(Yietal.,2007),theroleforARD1inmediatingcaspaseactivationisevolutionarilyconserved.HowARD1regulatescaspaseactivationhasnotyetbeenillustrated.Inmammaliancells,proteinN-alpha-acetylationismediatedbythehighlyconservedN-acetyltransferaseproteincomplexes(NatA,NatB,NatC,NatD,andNatE).TheNatAcomplexconsistsofthecatalyticsubunit,ArrestDefective1(hNaa10p/ARD1),andtheauxiliarysubunit,N-acetyltransferase1(NAT1/hNaa15p/NATH),whereasNatBconsistsofN-terminalacetyl-transferase3(hNaa20p/NAT3)andmitochondrialdistributionandmorphology20(hNaa25p/Mdm20).AlthoughtheNatcom-plexesareimplicatedinregulatingcell-cycleprogression,cellproliferation,andtumorigenesis,themechanismsthatconnectN-alpha-acetylationtothecellularproteinapparatusareunknown(Ametzazurraetal.,2008;PolevodaandSherman,2003;Starheimetal.,2008,2009).RecentN-acetylomestudiesrevealincompleteacetylationstatusofproteins(Arnesenetal.,2008;Goetzeetal.,2009).AlthoughacommonlyacceptedviewisthatpartialacetylationresultsfromthedegeneratenatureofproteinN-terminalsequences,weconsideredthepossibilitythatproteinN-alpha-acetylationmightberegu