Identification And Functional Study Of Vav2 And APP Interaction And Research On Phosphorylation Modification Of APP In Nanodiscs

The amyloid precursor protein(APP),which is closely linked to the pathological process of Alzheimer’s disease,serves as the primary research object for this project.The amyloid precursor protein(APP)is a widely expressed type I transmembrane(TM)glycoprotein present at the neuronal synapse.This amyloidogenic pathway is initiated by β-secretase cleavage of APP generating the membrane-bound C99 fragment,which is subsequently cleaved by y-secretase to release Aβ peptides.The accumulation of Aβpeptides within plaques is one of the neuropathological hallmarks of AD.In this manuscript,we report that Vav2,a guanine nucleotide exchange factor(GEF)for Rho family GTPase,is a novel interaction partner of APP.The mechanism and function of the discovered interaction between the intracellular domain of APP and Vav2 protein were thoroughly investigated.The Vav2 protein was first considered as a potential target for the treatment of Alzheimer’s disease.Additionally,we examined the phosphorylation modification process and interactions of the intracellular domain of APP by mimicking the real state of the transmembrane and intracellular domain of the APP in the cell membrane environment using the C99 protein assembled in nanodiscs.The following are the key findings from the research:We uncover a novel interaction between Vav2 and APP and a regulatory role of Vav2 in APP turnover.Using ITC and NMR experiments,we found that Vav2-SH2 domain was able to bind directly to the Y682-phosphorylated intracellular tail.To understand the structural basis of this recognition specificity,we then determined the crystal structure of Vav2-SH2 domain in complex with the APP-derived phosphopeptide APP-pY682(QNG-pY-ENPT,residues 679-686 of APP695)at 2.45 A resolution.The interaction of APP and Vav2 in a full-length manner was further confirmed by GST pull-down experiments,co-immunoprecipitation and immunofluorescence staining.Moreover,we found that overexpression of Vav2 significantly increased APP protein level and promoted Aβ40 generation in 20E2 cells,an AD cell model.We further show that Vav2 overexpression inhibited APP protein degradation.This function of Vav2 requires its SH2 domain.We further highlight that Vav2 may be a potential therapeutic target for AD.APP intracellular domain interacts with cellular proteins and undergoes modification in membrane environment.In this proposal,we assembled C99,which contains the transmembrane and intracellular domain of APP,into nanodiscs,a membrane mimic that is similar to native membrane.Studies have demonstrated that C99-nanodiscs can work well with liquid NMR technology and a variety of biochemical techniques,and it can be used to study biochemical processes in a membrane environment that is close to physiological.We then investigated the effects of the membrane environment on the phosphorylation modification of APP by GSK3β and the cis-trans isomerization of APP by Pin1.The current preliminary research findings demonstrate that the APP intracellular domain is more likely to undergo cis-trans isomerization by Pin1 and not to be phosphorylated by GSK3β kinase in the membrane environment.We are still working on the precise mechanisms and quantitative analyses of catalytic effectiveness.Taken together,this study provides a way to study the APPprotein interaction on complex membrane environment and also a point of view for future work in the significance of membrane environment in phosphorylation modifications of APP intracellular domain.