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ResearchInterestProposalIamtheDoctordegreeapplicantXuZhiwei.FromSeptember2008toJune2013,IstudiedthelaboratorymedicineatNantongUniversity.InSeptember2013Iwasrecommendedtothegraduateschoolbytheprofessorsduetomyexcellentassessment.Throughundergraduatestudies,Icontactedsuchtheoriesasmolecularbiology,cellularbiology,immunologyandsoon.However,keenlyconsciousthatmycurrentacquisitionisfarfromenoughformetomeettheneedsofthefastdevelopingmedicine,IameagertopursuefurtherstudiesinChinesemedicalsciences.Withmyhardworking,Iknowaboutthebasicknowledgeofmedicalimmunology.Uptonow,IhaveknownalotabouttheUniversityofMacau.ThegraduateschoolofuniversityplaysaprominentroleintheacademicresearchandtransformsMacaointoaknowledge-basedsociety.IamdedicatedtopursuitingmedicalresearchandhopingtocometotheuniversityofMacauforrichexperiencesandsuccessinPhDprogram.IamnowapplyingforChineseMedicalScienceintheUniversity.Thisplannedresearchhasthefollowingaims:ThedeubiquitinatingenzymeUSP14hasbeenidentifiedandbiochemicallystudied,butitsmechanismsincancerremainstobeelucidated.ProteinglycosylationwithO-linkedN-acetylglucosamine(O-GlcNAc)isareversiblepost-translationalmodificationoccurringonserineorthreonineresidues.Intriguingly,ithasbeenobservedthatO-GlcNAcylationisparticularlyabundantoncancercells.TheaimofthisstudywastoevaluatetheO-GlcNAcylationofUSP14inpatientswithcancerandtodefineitsmechanismsincancercellproliferationandapoptosis.a.TodemonstratethatO-GlcNAcylationofUSP14.b.ToexploreInterplaybetweenUSP14O-GlcNAcylationandphosphorylationc.TodeterminehowO-GlcNAcylationregulatesmetabolicreprogrammingandSignalingincancercells.d.TotestwhetherO-GlcNAcylationregulatesproliferationandapoptosis.2.ResearchContextDeregulatingcellularenergeticsisemergingasacharacteristichallmarkofcancercells.Withinsuchcells,glucoseandglutamineareusedatanincreasedrate,resultingintheproductionofATPinamannerindependentofoxygenconcentration.Elevatedglucoseandglutaminefluxareneedednotonlytoservetheenergeticdemandsofcancercells,butalsotoprovidetheessentialcarbonandnitrogenusedinmacromoleculesynthesis,fuelingtherapidgrowthandproliferationseenintumors.Thisincreaseinglucoseandglutamineuptakecanaltermultiplemetabolicandsignalingpathwaysincancercells,includingforexamplethehexosaminebiosyntheticpathway(HBP).TheHBPreliesonglucoseandglutamineuptake,andapproximately3%–5%ofthetotalglucoseenteringacellisshuntedintothispathway.Thiscriticalmetaboliteisrequiredforthebiosynthesisofavarietyofextracellularglycopolymers,includingbothN-andO-glycans,however,italsoservesasthesubstrateforO-linkedb-N-acetlyglucosamine(O-GlcNAc)transferase(OGT).O-GlcNAcylationisdirectlyinvolvedingrowthhormone(gibberellicacid)signallinginplants,andbothSPYandSECRETAGENT(SEC)encodeO-GlcNActransferases.MutationsineitherSPYorSECcauseseveregrowthdefects;simultaneousmutationofbothgenesislethal.Unlikeplants,mammalsandinsectsseemtohaveonlyasinglegeneencodingthecatalyticsubunitoftheO-GlcNActransferase(OGT)GenedisruptioninmiceestablishedthatOGTisrequiredforembryonic-stem-cellviability20.Tissue-targeteddisruptioninmiceshowedthatO-GlcNAcylationisessentialtoseveralcelltypes.OGTdeletioncauseshyperphosphorylation,whichisfollowedbycelldeath,inducesT-cellapoptosisandcausesgrowtharrestinfibroblasts.Cre–lox-mediateddeletionofOGTinculturedfibroblastsresultsindeathaspre-existingOGTproteinlevelsdiminish.ThismodificationcanberemovedbytheglycosidehydrolaseO-GlcNAcase(OGA)thatcatalyzescleavageofO-GlcNAcfromproteins.Thismodificationcanalterproteinfunctiondirectlyor,insomecases,bycompetingwithphosphorylationsites.O-GlcNAcandO-phosphatesite-mappingstudiessuggestthatthereareatleastfourdifferenttypesofdynamicinterplaybetweenO-GlcNAcandO-phosphate.First,thereiscompetitiveoccupancyatthesamesite,forexamplethatwhichoccursinthetranscriptionfactorc-Myc25andoestrogenreceptor-β26,andontheoncoproteinSV-40largeT-antigen27andendothelialnitricoxidesynthase28.Second,competitiveandalternativeoccupancyoccuratadjacentsites,suchasthatobservedinthetumoursuppressorp53andsynapsinI.Third,thereisacomplexinterplaywherebysomeO-phosphateattachmentsitesonagivenproteinarethesameassomeO-GlcNAcsites,whereasothersareadjacentto,orevendistantfrom,eachother,suchasontheC-terminaldomainofRNApolymeraseIIandoncytokeratins32.Thefinaltypeofinterplayinvolvesproteinsinwhichthisrelationshiphasyettobeclearlydefined.TheinterplaybetweenO-GlcNAcandO-phosphateisalsounderscoredbytherecentfindingthatOGTtransientlyformscomplexescontainingthecatalyticsubunitofproteinphosphatase1(PP1c).Cancercells,however,uptakeglucoseatahigherrateandproducelacticacidratherthanmetabolizingpyruvatethroughthetricarboxylicacid(TCA)cycle.ThisadaptivemetabolicshiftistermedtheWarburgeffect,leadingtoanaerobicglycolysis,andisthoughttoprovideanevolutionaryadvantagetocancercellsbyprovidingbothincreasebioenergeticsandbiosynthesis.Manyprotooncogenes(e.g.,RasandMyc)andtumorsuppressors(e.g.,p53)influencemetabolism,andmutationsinthesegenescanupregulateglucoseuptakeincancercellsandpromoteametabolicphenotypesupportingtumorcellgrowthandpr