Screening and Functional Analysis of BACE1 Interacting Proteins in Alzheimer's Disease
Objective β-Site APP cleaving enzyme 1(BACE1)is a rate-limiting enzyme involved in the formation of amyloid plaques in Alzheimer's disease(AD),and its expression and activity play a crucial role in the development of AD.The interacting protein of BACE1 can directly or indirectly regulate BACE1 in the transcription,translation,modification,intracellular transport and other links of BACE1 by directly binding,indirectly binding,and participating in various cell signal transduction pathways,so as to participate in the occurrence of AD and the process of disease.This study aimed to screen and validate the interacting proteins of BACE1,providing new insights into the mechanisms of amyloid plaque formation.Methods Co-immunoprecipitation(Co-IP)and mass spectrometry(MS)were used to enrich and identify BACE1 interacting proteins in the hippocampus of wild type(WT)mice and AD model mice.For candidate BACE1 interacting proteins,GO enrichment analysis and KEGG pathway enrichment analysis were used to explore the subcellular localization,molecular function,participating biological processes and participating signaling pathways of BACE1 interacting proteins.The protein-protein interaction(PPI)network of BACE1 was further constructed to explore the potential proteins interacting with BACE1.By searching the mouse genomeinformation(MGI)website and NCBI database,the more reliable proteins among the potential BACE1 interacting proteins were screened.Co-IP assay and immunofluorescence confocal technology were used to preliminarily verify the interaction between the proteins,and the changes in protein expression levels of the interacting proteins in AD cell models were explored.Results A total of 614 differentially expressed proteins interacting with BACE1 were identified in AD group.GO enrichment analysis showed that the BACE1 interacting proteins in the AD group were mainly located in membrane organelles such as Golgi apparatus,endoplasmic reticulum,endosome,lysosome and vesicles,which had molecular functions such as ion channel regulation,protein kinase activity,transcription factor binding and passive transmembrane transporter activity.It is mainly involved in the biological processes of immune response regulation cell surface receptor signaling pathway,targeting Golgi vesicles transport,circadian rhythm regulation,Purkinje cell layer development,etc.KEGG analysis showed that BACE1 interacting proteins in AD were mainly involved in the PI3K-Akt signaling pathway,mTOR signaling pathway and other neurodegenerative disease-related pathways.The PPI network of BACE1 showed that a total of 12 proteins were identified as high confidence binding proteins,including PRNP,APOE,SYP,NSF,NUMB,SNAP91,HSP90aal,UCHL1,BIN1,SNX27,Rheb,Ap2ml,of which,NSF,NUMB,SNAP91,HSP90aal were newly identified candidate proteins.After further verification,we found that NSF not only interacts with BACE1,but also interacts with amyloid precursor protein(APP),the substrate of BACE1,and the expression level of NSF is up-regulated in the AD cell model constructed by Aβ42 induction.Conclusion BACE1 binding proteins participate in multiple AD-associated biological processes and signal pathways.NSF is a newly identified BACE1 binding protein that interacts with BACE1,and the protein expression level of NSF is up-regulated in the AD cell model.It is predicted that the interaction between NSF and BACE1 is involved in regulating the course of AD,providing a new target and direction for the study of the mechanism of AD.
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