Enzyme-SubstrateRelationshipsintheUbiquitinSystem:ApproachesforIdentifyingSubstratesofUbiquitinLigasesHazelF.O’ConnorandJonM.Huibregtse*DepartmentofMolecularBiosciences,UniversityofTexasatAustin,Austin,TX78712AbstractProteinubiquitylationisanimportantpost-translationalmodification,regulatingaspectsofvirtuallyeverybiochemicalpathwayineukaryoticcells.Hundredsofenzymesparticipateintheconjugationanddeconjugationofubiquitin,aswellastherecognition,signalingfunctions,anddegradationofubiquitylatedproteins.RegulationofubiquitylationismostcommonlyatthelevelofrecognitionofsubstratesbyE3ubiquitinligases.CharacterizationofthenetworkofE3-substraterelationshipsisamajorgoalandchallengeinthefield,asthisitexpectedtoyieldfundamentalbiologicalinsightsandopportunitiesfordrugdevelopment.TherehasbeenremarkablesuccessinidentifyingsubstratesforsomeE3ligases,inmanyinstancesusingstandardprotein-proteininteractiontechniques(e.g.,two-hybridscreens,co-immunoprecipitationspairedwithmassspectrometry).However,someE3shaveremainedrefractorytocharacterization,whileothershavesimplynotyetbeenstudiedduetothesheernumberanddiversityofE3s.ThisreviewwilldiscusstherangeoftoolsandtechniquesthatcanbeusedforsubstrateprofilingofE3ligases.IntroductionUbiquitylationhasmanyessentialfunctionsincellcyclecontrol,DNAdamageresponses,proteintrafficking,proteinturnover,thedisposalofmisfoldedanddamagedproteins,andmanyotherbiochemicalpathways.Thestrictcontrolofthesubstrateselectionprocessisthereforeimperative.Ahierarchalsystemofenzymes,culminatingintherecognitionofsubstratesbyE3ligases,coordinatestheconjugationofubiquitintosubstrates.ThefirststeptowardconjugationisATP-dependentactivationofubiquitinbyanE1enzyme.TheE1firstcatalyzestheformationofaubiquitin-adenylateintermediate,andthentheactivesitecysteineoftheE1formsathioesterwiththeC-terminusofubiquitin,withthereleaseofAMP.UbiquitinisthentransferredfromtheE1enzymetotheactive-sitecysteineofanE2enzymeinatransthiolationreaction.E3ligasesbindubiquitin-chargedE2sandsubstratesa...
