阿尔茨海默病中BACE1相互作用蛋白筛选及功能分析  

Screening and Functional Analysis of BACE1 Interacting Proteins in Alzheimer’s Disease

作　　者：刘聪聪 王亚琦 王培昌[1] LIU Cong-Cong;WANG Ya-Qi;WANG Pei-Chang(Clinical Laboratory,Xuanwu Hospital Capital Medical University,Beijing 100053,China)

机构地区：[1]首都医科大学宣武医院检验科,北京100053

出　　处：《生物化学与生物物理进展》2024年第8期1904-1919,共16页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(82030064,82102487);北京市“青苗”人才计划(QML20230812)资助项目。

摘　　要：目的β淀粉样蛋白前体蛋白裂解酶1(β-site APP cleaving enzyme 1,BACE1)是阿尔茨海默病(Alzheimer’s disease,AD)中淀粉样斑块形成的限速酶,其表达水平与活性在AD发生发展中起到关键作用。BACE1的相互作用蛋白可以通过直接结合、间接结合、参与各种细胞信号转导通路等方式,直接或间接在BACE1的转录、翻译、修饰、胞内运输等各个环节对BACE1进行调节,从而参与AD的发生与疾病的进程。本研究拟筛选并验证BACE1的相互作用蛋白,为进一步深入阐明淀粉样斑块形成的机制提供新的依据。方法采用免疫共沉淀(co-immunoprecipitation,Co-IP)与质谱联合应用的技术富集并鉴定AD模型小鼠海马组织中BACE1的相互作用蛋白,通过生物信息学分析筛选与BACE1有潜在相互作用的蛋白质,采用Co-IP实验及免疫荧光共聚焦技术进行初步验证,并探究BACE1相互作用蛋白在AD细胞模型中蛋白质表达水平变化。结果本研究在AD组中共鉴定到与BACE1相互作用的差异表达蛋白614个。基因本体(GO)富集分析显示,AD组BACE1相互作用蛋白主要参与细胞内信号转导、靶向高尔基体囊泡的转运、浦肯野细胞层发育等生物学过程;京都基因与基因组百科全书(KEGG)分析显示,AD中BACE1相互作用蛋白主要参与了PI3K-Akt信号通路、mTOR信号通路等神经退行性疾病相关通路。通过构建BACE1相互作用蛋白网络,筛选了12个可信度较高的相互作用蛋白,其中NSF、NUMB、HSP90aa1、SNAP91为首次发现的BACE1相互作用蛋白。经进一步验证,发现NSF与BACE1存在相互作用,并且在AD细胞模型中NSF表达水平上调。结论BACE1相互作用蛋白参与多种AD相关生物途径及信号通路,NSF为新鉴定到的BACE1相互作用蛋白,与BACE1存在相互作用。NSF在AD细胞模型中表达水平上调,预测NSF与BACE1蛋白间相互作用参与调节AD疾病的进程,为疾病的机制研究提供新的靶点和方向。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 Coimmunoprecipitation(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 r

关 键 词：β淀粉样蛋白前体蛋白裂解酶1 免疫共沉淀 质谱法 神经退行性疾病 阿尔茨海默病 

分 类 号：R34[医药卫生—基础医学] R74