Posttranslational Modifications, Localization, and Protein Interactions of Optineurin, the Product of a Glaucoma Gene 英文参考文献.docVIP

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Posttranslational Modifications, Localization, and Protein Interactions of Optineurin, the Product of a Glaucoma Gene 英文参考文献.doc

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Posttranslational Modifications, Localization, and Protein Interactions of Optineurin, the Product of a Glaucoma Gene 英文参考文献

PosttranslationalModifications,Localization,andProtein InteractionsofOptineurin,theProductofaGlaucoma Gene HongyuYing,XiangShen,BumChanPark,BeatriceY.J.T.Yue* DepartmentofOphthalmologyandVisualSciences,UniversityofIllinoisatChicagoCollegeofMedicine,Chicago,Illinois,UnitedStatesofAmerica Abstract Background:Glaucomaisamajorblindingdisease.Themostcommonformofthisdisease,primaryopenangleglaucoma (POAG), is genetically heterogeneous. One of the candidate genes, optineurin, is linked principally to normal tension glaucoma,asubtypeofPOAG.Thepresentstudywasundertakentoillustratethebasiccharacteristicsofoptineurin. Methodology/PrincipalFindings:LysatesfromratretinalganglionRGC5cellsweresubjectedtoN-orO-deglycosylationor membraneproteinextraction.Thephosphorylationstatuswasevaluatedafterimmunoprecipitation.Itwasfoundthatwhile phosphorylated,optineurinwasneitherN-norO-glycosylated,andwasbyitselfnotamembraneprotein.RGC5andhuman retinal pigment epithelial cells were double stained with anti-optineurin and anti-GM130. The endogenous optineurin exhibited a diffuse, cytoplasmic distribution, but a population of the protein was associated with the Golgi apparatus. Turnoverexperimentsshowedthattheendogenousoptineurinwasrelativelyshort-lived,withahalf-lifeofapproximately 8hours.Nativebluegelelectrophoresisrevealedthattheendogenousoptineurinformedhomohexamers.Optineurinalso interactedwithmoleculesincludingRab8,myosinVI,andtransferrinreceptortoassembleintosupermolecularcomplexes. Whenoverexpressed,optineurin–greenfluorescenceprotein(GFP)fusionproteinformedpunctatestructurestermed‘‘foci’’ intheperinuclearregion.Treatmentofnocadazoleresultedindispersionoftheoptineurinfoci.Inaddition,tetracycline- regulatedoptineurin-GFPsexpressingRGC5stablecelllineswereestablishedforthefirsttime. Conclusions/Significance: Thepresentstudy providesnewinformationregardingbasic characteristics of optineurinthat areimportantforfutureeffortsindefiningpreciselyhowoptineurinfunctionsnormallyandhowmutationsma