CoIP-MS法筛选CHCHD2互作蛋白及其功能的初步分析

Identification and preliminary functional analysis of CHCHD2 interacting proteins by CoIP-MS

目的:筛选氧化应激状态下CHCHD2(coiled-coil-helix-coiled-coil-helix domain-containing 2)的互作蛋白,以期挖掘出CHCHD2保护神经细胞对抗氧化应激损伤的潜在机制。方法:通过Lipofectamine 2000分别在人神经母细胞瘤细胞系SH-SY5Y中转染含有Flag标签的对照质粒及CHCHD2过表达质粒,用100μmol/L过氧化叔丁醇(tert-butyl hydroperoxide,TBHP)或蒸馏水处理24 h后,采用免疫共沉淀(CoIP)的方法富集各组细胞中与CHCHD2相结合的蛋白,SDS-PAGE跑浓缩胶,切取条带,胶内酶解后进行液相色谱-质谱联用(LC-MS/MS)分析、数据库检索及生物信息分析,筛选与CHCHD2互作的蛋白,并对功能进行初步分析。结果:(1)CHCHD2具有保护SH-SY5Y细胞对抗TBHP诱导的氧化应激损伤作用;(2)CoIP-MS结果提示,不同于生理状态,在氧化应激状态下共有64个蛋白是与CHCHD2互作的特有差异表达蛋白(differentially expressed proteins,DEPs);(3)通过GO功能注释和KEGG富集分析,我们发现氧化应激状态下特有DEPs主要在外泌体和细胞浆中发挥作用,参与蛋白翻译及翻译起始等生物过程,在蛋白及poly(A)RNA结合方面发挥分子功能,并参与糖代谢过程;(4)DEPs还参与了负性调控活性氧生物合成过程和对过氧化氢的反应等抗氧化应激相关生物过程,其中肿瘤坏死因子受体相关蛋白1(tumor necrosis factor receptor-associated protein 1,TRAP1)和热休克蛋白家族D成员1(heat shock protein family D member1,HSPD1)是抗氧化应激过程中重要的候选蛋白;(5)通过蛋白互作网络分析,我们发现在氧化应激状态下特异性存在3个蛋白[Y盒结合蛋白1(Y-box-binding protein 1,YBX1)、含TCP1分子伴侣亚基6A(chaperonin containing TCP1 subunit 6A,CCT6A)和细胞色素C氧化酶装配因子4同源物(cytochrome C oxidase assembly factor 4 homolog,COA4)]与CHCHD2有直接相互作用,也是后续需要重点关注的蛋白。结论:利用CoIP-MS法成功筛选出生理状态及氧化应激状态下CHCHD2的互作蛋白,并挖掘出与其抗氧化应激过程密切相关的2个候选蛋白(TRAP1和HSPD1)及另外3个与其直接作用的候选蛋白(YBX1、CCT6A和COA4),为进一步深入探索CHCHD2抗氧化应激作用中的生物过程及分子机制奠定基础。

AIM:To screen the proteins interacting with coiled-coil-helix-coiled-coil-helix domain-containing2(CHCHD2)under oxidative stress,and to explore the underlying mechanism of CHCHD2 protecting against oxidative stress-induced neuronal damage.METHODS:The control plasmid and the CHCHD2 overexpression plasmid containing Flag tags were transfected into human neuroblastoma cell line SH-SY5Y using Lipofectamine 2000.After treatment with100μmol/L tert-butyl hydroperoxide(TBHP)or ddH_(2)O for 24 h,the CHCHD2-binding proteins in different groups were enriched by co-immunoprecipitation(CoIP).The proteins were concentrated by SDS-PAGE,and the bands in the gel were cut for further enzymolysis and analysis by liquid chromatography with tandem mass spectrometry(LC-MS/MS).The CHCHD2-interacting proteins were identified via database searching and bioinformatic analysis.RESULTS:(1)CHCHD2 played a role in protecting SH-SY5Y cells against THBP-induced oxidative stress injury.(2)The results of CoIP-MS showed that a total of 64 CHCHD2-interacting proteins were specifically detected under oxidative stress condition rather than physiological condition.(3)The results of GO function annotation and KEGG pathway enrichment showed that the differentially expressed proteins(DEPs)specifically detected in oxidative stress group mainly existed in the exosome and cytosol.These DEPs participated in biological processes including protein translation and initiation,protein and/or ploy(A)RNA binding,and glucose metabolism.(4)The DEPs were also involved in anti-oxidative stress-related biological processes including negative regulation of reactive oxygen species biosynthetic process,and response to hydrogen peroxide.Tumor necrosis factor receptor-associated protein 1(TRAP1)and heat shock protein family D member 1(HSPD1)were two important candidate proteins.(5)The results of protein-protein interaction network analysis showed that Y-boxbinding protein 1(YBX1),chaperonin containing TCP1 subunit 6A(CCT6A)and cytochrome C oxidase assembly factor4 homolog(COA4)directly interacted with CHCHD2 under oxidative stress condition rather than physiological condition,and were also candidate proteins for further validation.CONCLUSION:The CHCHD2-interacting proteins under physiological condition and oxidative stress condition were successfully identified by CoIP-MS,and two proteins(TRAP1 and HSPD1)closely related to the anti-oxidative stress process,as well as three proteins(YBX1,CCT6A and COA4)directly interacted with CHCHD2 under oxidative stress condition were identified as top-candidate proteins.These findings support the necessity for further exploration of the molecular mechanism and biological process of CHCHD2 in its anti-oxidative stress effect.