ProjectSummary Manyhumandiseasesarecausedwhenaproteinorenzymeismutatedormisfolded.Forexample,thedysfunctionof the HECT (Homologous to E6AP Carboxyl Terminus) E3 ubiquitin ligases has been linked to neurodevelopmental syndromes (i.e. Angelman, Prader-?Willi and Wolfram), Huntington?s, cancer (i.e. breast, lung, prostate, and Wilms? tumors), and viral infections (i.e. HIV, HPV, Ebola, and Influenza A/B). It is paramount that we understand how these enzymesworktoaidinthefuturedevelopmentoftherapiestotreatHECTE3ubiquitinligasedysfunctionandenhance humanhealth.TheHECTE3ubiquitinligasesareenzymesinvolvedintheubiquitylation-?signalingpathwaythatcoordinate theposttranslationalattachmentofthe8.5kDasignalingproteinubiquitintotheirspecifictargetproteinsinthecell. TheobjectiveofthisprojectistounderstandthestructuralandbiochemicalbasisforHECT-?dependentubiquitylation.We willelucidatetheuniquemechanismsthatthe28humanmembersoftheHECTE3ubiquitinligasefamilyusetoattach ubiquitin to their intracellular substrates. All 28 HECT E3 ubiquitin ligases encoded in the human genome contain the characteristic HECT domain, consisting of an N-?terminal lobe and a C-?terminal lobe, that is responsible for catalyzing ubiquitinchainattachmenttoatargetprotein.Currentlythemechanismthateachofthese28uniqueenzymesusesand the identities of specific residues in and around the active site required for catalysis remain unclear. The long term scientificgoalofthePIistofullyinvestigatethe3DstructuresandunderlyingenzymologyfortheC-?terminallobesofall28 humanHECTE3ubiquitinligasestolearnhowthisregionofeachenzymecontrolspolyubiquitinchainassemblyandlinkage specificity. The major foci of this proposal will be to determine the role of dynamics and conformational flexibility in HECT-? dependentubiquitylation(Aim1),andtodecipherthecatalyticmechanismsandtheroleofdimerizationinregulating HECT E3 ligase activity (Aim 2). Our preliminary studies using NMR spectroscopy and other biochemical approaches suggest that some of the HECT E3 ubiquitin ligases use novel mechanisms found exclusively in their HECT domain C-? terminallobesthatcontaintheabsolutelyconservedcatalyticcysteine.Theseinherentdifferencesprovideanenticing opportunitytoexpandourcurrentunderstandingofHECT-?dependentubiquitylation.Ourfindingswilloffernewinsight intothemolecularmechanismsusedbytheHECTE3ubiquitinligasesandhelpuslearnhowandwhyHECTE3ubiquitin ligasedysfunctionoccursandcanpossiblybecontrolled.Undergraduatestudentswillbeanessentialpartofthesuccess of this R15 AREA research project. The contribution from Biochemistry and Molecular Biology (BCMB) undergraduate studentsatClarkUniversitywillbeintegraltothecompletionoftheproposedworkand,asaresultoftheirimportancein this research program, they will receive extensive guidance from the PI and will share in manuscript preparation and publicationauthorship.