Smac

Smac,aMitochondrialProteinthatPromotesCytochromec–DependentCaspaseActivationbyEliminatingIAPInhibitionChunyingDu,MinFang,YuchengLi,LilyLi,andXiaodongWang*Cell,Vol.102,33–42,July7,2000,Copyrightã2000byCellPressIAPIAPscontainonetothreewell-conserveddomains,thesocalledbaculovirusIAPrepeats(BIR).ViaitsBIRs,Xchromo–somelinkedIAP(XIAP)Apoptosomecytochromecfrommitochondriatocytosol,whereitbindstoApaf-1toformaprocaspase-9-activatingheptamericproteincomplexnamedapoptosome.SummaryWereportheretheidentificationofanovelprotein,Smac,whichpromotescaspaseactivationinthecytochromec/Apaf-1/caspase-9pathway.Smacpromotescaspase-9activationbybindingtoinhibitorofapoptosisproteins,IAPs,andremovingtheirinhibitoryactivity.Smacisthesecondmitochondrialprotein,alongwithcytochromec,thatpromotesapoptosisbyactivatingcaspases.cytochromec/Apaf-1/caspase-9procaspase-3caspase-3IdentificationofSmac.thedetergentsolublized“membrane”extract(SME)didnothavecaspase-3activatingactivitybyitself(lane4),butitsignificantlystimulatedcaspase-3activationwhenaddedtotheS-100fraction(lanes2and5).ResultsFig.1Fig.1Bnocaspase-3activationwasobservedwhenweusedanyofthesedepletedextracts(lanes4,6,and8).Fig.1PurificationofSmacFig2MolecularCloningofSmacFig.1BFig3AFig3BTotestthetissuedistributionofSmac,NorthernblotanalysiswasperformedusingmRNAblotsfrommultiplehumanadulttissues(Figure3B)SmacStimulatesCaspaseActivationbyRemovingtheInhibitionofIAPsFig4Tenmicroliteraliquotsoftheelutedmaterials(P)aswellas30laliquotsofthesupernatantgenerated(S)Fig4addingSmactothereconstitutedcaspase-3activatingsystemhadminimaleffectscomparedtowhatwasobservedintheS-100(lanes2and3).However,ifweaddedarecombinanthumanc-IAP1proteintothereaction,caspase-3activationwascompletelyinhibited(lane4),andtheinhibitionwaseliminatedinthepresenceofSmac(lane5).Fig5DiscussionSmacPromotesProcaspase-9ActivationbyCounteringIAPsThedatainthispaperdemonstratethatSmac,anovelmitochondrialprotein,isreleasedfrommitochondriaduringapoptosis,whereuponitneutralizestheinhibitoryactivityofIAPs.SmacseemstofunctionasageneralIAPneutralizerbybindingtotheseproteins(Figure4andVerhagenetal.,2000[thisissueofCell]).MultipleIAPmolecules,includingc-IAP1,c-IAP2,XIAP,andsurvivinareabletobindSmac.SincetheseIAPsaretheonlyonesthatwechecked,itispossiblethatSmacbindsotherIAPsaswell.ThebindingofSmactoIAPscouldpresumablypreventtheirinteractionswithcaspasesandthereforereleasetheirinhibitiononcaspaseactivationandcaspaseactivities.Thankyou