弓形虫感染后宿主细胞泛素化蛋白谱变化的特征分析  

Characterization of ubiquitinated protein profile change in host cells caused by Toxoplasma gondii infection

作　　者：廖文中 徐李清 姚礼捷 陈敏 彭鸿娟 LIAO Wen-zhong;XU Li-qing;YAO Li-jie;CHEN Min;PENG Hong-juan(Department of Pathogen Biology,Guangdong Provincial Key Laboratory of Tropical Disease Research,School of Public Health,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]南方医科大学公共卫生学院病原生物学系,广东省热带病研究重点实验室,广州510515

出　　处：《中国寄生虫学与寄生虫病杂志》2021年第4期487-493,共7页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家重点研发项目(2017YFD0500400);国家自然科学基金面上项目(81772217);国家自然科学基金面上项目(201828006);国家自然科学基金面上项目(81971954);广东省科技计划项目(2018A050506038);广州市科学研究计划重点项目(201904020011)。

摘　　要：目的探究刚地弓形虫不同毒力虫株感染后宿主细胞泛素化蛋白谱变化的特点。方法将人外周血单核细胞诱导成巨噬细胞后分为3组,分别为未感染组、RH感染组、ME49感染组。2个感染组分别用弓形虫不同毒力株(RH株与ME49株)速殖子感染4 h后,收集3组细胞的总蛋白,应用泛素抗体富集泛素化蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离蛋白后进行蛋白定性质谱鉴定。检测完成后,搜索UniProt数据库中的人类数据库,利用Mascot鉴定蛋白质种类。蛋白质免疫印迹(Western blotting)分析蛋白CDC42的泛素化水平。利用DAVID数据库对感染组与对照组、不同毒力株感染组之间的显著差异性泛素化蛋白(DUP)进行基因本体(GO)注释和富集分析,利用京都基因与基因组百科全书(KEGG)在线网站对DUP进行信号通路富集分析,利用STRING 11.0在线网站对DUP进行蛋白-蛋白相互作用网络分析。结果质谱筛选结果显示,RH感染组的特异性泛素化蛋白有194种,ME49感染组的有47种,两个感染组均筛选到的宿主泛素化蛋白有31种。Western blotting检测结果显示,宿主CDC42在弓形虫感染后泛素化水平显著升高。GO聚类分析显示,细胞组分聚类中,RH感染组的DUP主要涉及能量代谢以及蛋白质合成、加工的细胞器,有67个蛋白;ME49感染组的DUP则主要涉及细胞骨架结构,有11个蛋白;在生物进程聚类中两组差异不大,不同的是,ME49感染组的DUP富集到肝配蛋白受体信号通路(3个)和网格蛋白介导的内吞作用蛋白(2个);在分子功能聚类中,RH感染组的DUP主要富集在与RNA转录/加工、GTPase活性、泛素连接酶相关的聚类项,分别有47、7和11个蛋白;ME49感染组的DUP则主要富集在肌动蛋白丝结合(3个)等GO项上。KEGG信号通路富集显示,RH感染组的DUP主要富集在核糖体(12个)、泛素介导的蛋白水解(8个)、吞噬体(7个)、核苷酸切除修复(4个)、致病性�Objective To explore the changes of ubiquitinated protein profile in host cells infected with Toxoplasma gondii.Methods After induced into macrophages,the human acute monocytic leukemia cell line(THP-1)cells were assigned into three groups:uninfected group,RH infected group,and ME49 infected group.Cells in the infection groups were infected with strain RH and strain ME49 T.gondii tachyzoites with difference virulence,respectively,for 4 h.Then total proteins were extracted from the cells,and the ubiquitinated proteins were enriched using anti-ubiquitin antibody FK-2,followed by protein separation by sodium lauryl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)for qualitative mass spectrometry identification.Human source information was searched from UniProt databse,and the protein species were identified using Mascot.The ubiquitination level of CDC42 was verified by Western blotting.The proteins were digested into peptides and underwent liquid chromatography-tandem mass spectrometry(LC-MS/MS).Using the DAVID Bioinformatics Resources 6.8,Gene Ontology(GO)annotation and enrichment analysis was performed for significantly differentially ubiquitinated proteins(DUPs)between the infection and control groups and between the infection groups with different virulence.The pathway enrichment of DUPs was analyzed with KEGG online tool.The protein-protein interaction(PPI)network was analyzed with online STRING 11.1.Results LC-MS/MS results showed that compared with the control group,there were 194 DUPs in the RH infected group,47 DUPs in the ME49 infected group,and 31 DUPs in both groups.Western blotting assay confirmed that the ubiquitination level of host CDC42 was increased significantly after T.gondii infection.GO clustering analysis showed that the DUPs in the RH infected group were mainly involved in energy metabolism and organelles of protein synthesis and processing(67 proteins),while the DUPs in the ME49 infected group were mainly involved in cytoskeleton structure(11 proteins).With regard to biological process c

关 键 词：刚地弓形虫 泛素 细胞骨架 免疫逃避 

分 类 号：R382.5[医药卫生—医学寄生虫学]