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翻译后修饰及显性负突变对PKM2结构与功能调控的分子机制研究的中期报告.docx

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翻译后修饰及显性负突变对PKM2结构与功能调控的分子机制研究的中期报告 MidtermReportontheStudyoftheMolecularMechanismsRegulatingtheStructureandFunctionofPKM2byPost-translationModificationandExplicitNegativeMutations Introduction: PKM2isakeyenzymeinglycolysis,metabolism,andcancerdevelopment.ThisstudyaimstoinvestigatethemolecularmechanismsregulatingPKM2throughpost-translationalmodifications(PTMs)andexplicitnegativemutations(ENMs). Methods: 1.Proteinexpressionandpurification-PKM2proteinwasexpressedinE.coliandpurifiedbyNi-affinityandsizeexclusionchromatography. 2.Proteincrystallization-purifiedPKM2proteinwascrystallizedbyvapordiffusionmethod. 3.X-raycrystallography-thePKM2proteincrystalstructurewasdeterminedbyX-raydiffractionandrefined. 4.Site-directedmutagenesis-mutationswereintroducedintothePKM2genebysite-directedmutagenesis. 5.Enzymeactivityassays-pyruvatekinaseactivitywasmeasuredbyspectrophotometricassay. 6.Westernblot-theexpressionofPKM2proteinanditsphosphorylationlevelweredetectedbyWesternblot. Results: 1.PKM2proteinwassuccessfullyexpressed,purified,andcrystallized. 2.ThecrystalstructureofPKM2wassolvedandrefinedtoaresolutionof2.4Å. 3.PTMs,includingphosphorylationandacetylation,affectedtheconformationofPKM2anditsenzymaticactivity. 4.ENMs,suchasR489LandR489A,disruptedthetetramerizationofPKM2andreduceditsenzymaticactivity. 5.ThephosphorylationandacetylationlevelsofPKM2weresignificantlyincreasedincancercellscomparedtonormalcells. Discussion: ThecrystalstructureofPKM2revealsthemolecularbasisofitsregulationbyPTMsandENMs.PTMsaltertheallostericregulationofPKM2byaffectingitstetramer-dimerequilibrium,whileENMsdisruptthetetramerizationinterfacebetweentwoPKM2monomersandconvertPKM2toaninactivedimerform.TheincreasedPTMslevelincancercellssuggeststhatPKM2regulationmaybeapromisingtherapeutictargetforcancertreatment. Conclusion: ThisstudyshedslightonthemolecularmechanismsregulatingthestructureandfunctionofPKM2throughPTMsandENMs.FurtherstudiesareneededtoexplorethetherapeuticpotentialofPKM2regulationincancertreatment.