油菜中—未知BnRCH蛋白的E3连接酶活性分析-豆柴文库

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油菜中—未知BnRCH蛋白的E3连接酶活性分析
Introduction
Theubiquitin-proteasomesystem(UPS)isanessentialregulatorypathwaythatplaysacrucialroleinproteinturnoveranddegradation.Thispathwayisahighlyregulatedprocessinvolvingthecovalentattachmentofubiquitintoaspecifictargetprotein.Theprocessinvolvessequentialactivationofthreeenzymes-E1,E2,andE3.TheE3ubiquitinligasesareresponsiblefortherecognitionofspecificsubstratesandpromotingtheirubiquitination.TherearevariousfamiliesofE3ligases,andtheRINGfingerproteinfamilyisthelargest.TheRINGfingerE3ligasecatalyzesubiquitintransferfromtheE2enzymetothesubstrateprotein.Inthisstudy,weanalyzedtheE3ligaseactivityofanunknownRINGfingerprotein,BnRCH,ofBrassicanapus.
MaterialsandMethods
TheBrassicanapusplantwasgrownundercontrolledconditionsinagrowthchamber.TheleaveswereharvestedandusedfortheisolationoftheBnRCHprotein.TherecombinantHis-taggedBnRCHwasexpressedandpurifiedusingNi-NTAbeads.TheubiquitinationassaywasperformedinvitrousingpurifiedE1,E2,andBnRCHproteinsuntagged.TheE1,E2,andBnRCHproteinswereincubatedwith3μgofubiquitinand5μgofbovineserumalbumin(BSA)in50μlofbuffercontaining50mMTris-HCl,pH7.5,5mMMgCl2,2mMATP,and2mMdithiothreitol(DTT)at37°Cfor3h.ReactionswerestoppedusingtheSDS-PAGEsamplebuffer.
Results
TheWesternblotanalysisoftheinvitroubiquitinationassayrevealedthatBnRCHhasE3ligaseactivity.TheBSAwasusedasthesubstrateproteinintheassay.TheubiquitinationofBSAproteinwasdetectedwhenBnRCH,E1,andE2wereallpresentinthereactionmixture.Theabsenceofanyoftheseenzymespreventedtheubiquitinationofthesubstrateprotein.TheE3ligaseactivityofBnRCHwasspecifictothesubstrateprotein,andnoubiquitinationoftheE1,E2,orBnRCHproteinitselfwasdetected.TheseresultssuggestthatBnRCHisafunctionalE3ligaseandisspecificforubiquitinatingasubstrateprotein.
Discussion
TheUPSpathwayplaysanessentialroleinregulatingcellularprocessessuchascellcycleprogression,proteinqualitycontrol,andsignaltransduction.TheE3ligasesareresponsibleforthespecificityoftheubiquitinationprocessbyrecognizingandbindingtospecificsub