基于IP联合蛋白质组学探讨ENG互作蛋白在bEnd.3细胞中的作用  

To Investigate the Role of ENG Interacting Proteins in bEnd.3 Cells Based on IP Combined with Proteomics

作　　者：陈清杰[1,2] 李奥迪 季慧敏 孙艺瑄 蒋越 叶子奇 潘荣 CHEN Qing-jie;LI Ao-di;JI Hui-min(School of Pharmacy,Xianning Medical College,Hubei University of Science and Technology,Xianning Hubei 437100,China)

机构地区：[1]湖北科技学院医学部药学院,湖北咸宁437100 [2]湖北科技学院糖尿病心脑血管病变湖北省重点实验室

出　　处：《湖北科技学院学报（医学版）》2024年第6期461-466,F0002,共7页Journal of Hubei University of Science and Technology(Medical Sciences)

基　　金：国家自然科学基金一般面上项目(82270892)。

摘　　要：目的本研究拟筛选并验证在脑微血管内皮细胞(bEnd.3)中Endoglin(ENG)的互作蛋白及其作用。方法采用蛋白质组学和生物信息学技术鉴定并富集ENG互作蛋白,对这些互作蛋白进行亚细胞定位、GO功能注释、IPR注释和蛋白互作网络等分析。将以上分析筛选出的ENG互作蛋白,采用免疫共沉淀(IP)实验、细胞免疫荧光(IF)和蛋白免疫印迹(WB)技术进行验证和对比,探究ENG互作蛋白在棕榈酸(PA)、基因沉默和过表达ENG处理bEnd.3细胞中的表达变化及作用。结果通过蛋白质组学分析,bEnd.3细胞中ENG互作蛋白在亚细胞定位分析中细胞骨架相关成分呈现出较高的占比;通过GO注释和IPR注释进一步发现,是细胞骨架中的中间纤维和微丝蛋白呈现较高的富集状态。ENG与细胞骨架蛋白互作热力图结果显示,与对照组相比,PA处理的bEnd.3细胞中微丝蛋白Filamin B(Flnb)数值显著降低。IP和IF结果显示,bEnd.3细胞中ENG与Flnb存在蛋白互作和共定位情况。WB结果显示,bEnd.3细胞在PA处理条件下,ENG和Flnb蛋白表达均下降;在ENG沉默条件下,Flnb蛋白随ENG表达的减少而降低;在ENG过表达条件下,Flnb蛋白随ENG表达的增加而升高。结论ENG互作蛋白广泛参与到多种相关的生物途径以及信号通路之中,ENG在bEnd.3细胞中与Flnb蛋白存在相互作用且二者具有共定位现象,ENG还能够调控PA处理的bEnd.3细胞Flnb蛋白表达水平。Objective The aim of this study was to screen and verify the interaction proteins of Endoglin(ENG)in brain microvascular endothelial cells(bEnd.3)and their effects.Methods Proteomics and bioinformatics techniques were used to enrich and identify ENG interacting proteins.Subcellular localization,GO functional annotation,IPR annotation and protein interaction network analysis were performed on these interacting proteins.The ENG interacting proteins screened by the above analysis were verified and compared by immunoprecipitation(IP)experiments,cellular immune-fluorescence(IF)and Western blotting(WB)to explore the expression changes and effects of ENG interacting proteins in bEnd.3 cells treated with palmitic acid(PA),gene silencing and overexpression of ENG.Results Through proteomics analysis,ENG-interacting proteins in bEnd.3 cells showed a high proportion of cytoskeleton-related components in subcellular localization analysis.Through GO annotation and IPR annotation,it was further found that the intermediate fibers and microfilament proteins in the cytoskeleton showed a high enrichment state.The interaction heat map of ENG and cytoskeletal proteins showed that the value of Filamin B(Flnb)in bEnd.3 cells treated with PA was significantly lower than that in the control group.IP and IF results showed that there were protein interaction and co-localization between ENG and Flnb in bEnd.3 cells.WB results showed that the expression of ENG and Flnb protein decreased in bEnd.3 cells under PA treatment.Under the condition of ENG silencing,Flnb protein decreased with the decrease of ENG expression.Under the condition of ENG overexpression,Flnb protein increased with the increase of ENG expression.Conclusion ENG interacting proteins are widely involved in a variety of related biological pathways and signaling pathways.ENG interacts with Flnb protein in bEnd.3 cells and these two are co-localized.ENG can also regulate the expression level of Flnb protein in PA-treated bEnd.3 cells.

关 键 词：ENG Flnb 互作蛋白 bEnd.3 棕榈酸 

分 类 号：R34[医药卫生—基础医学]