肠致病性大肠杆菌效应因子E2348C-1040泛素连接酶活性及泛素链连接方式的鉴定  

Identification of ubiquitin ligase activity and ubiquitin ligation mode of enteropathogenic Escherichia coli effector E2348C-1040

作　　者：黄炎玉 李崭 陈芳红 李涛[1] 王慧 HUANG Yan-yu;LI Zhan;CHEN Fang-hong;LI Tao;WANG Hui(State Key Laboratory of Pathogen and Biosecurity,Institute of Microbiology and Epidemiology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China)

机构地区：[1]军事科学院军事医学研究院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《军事医学》2023年第3期182-187,共6页Military Medical Sciences

基　　金：国家自然科学基金(82072251)。

摘　　要：目的鉴定肠致病性大肠杆菌(EPEC)效应因子E2348C-1040泛素连接酶活性,检测其催化形成的泛素链的连接方式。方法构建原核表达质粒,转化感受态细胞,表达纯化E2348C-1040蛋白质。通过体外泛素化实验鉴定其泛素连接酶活性,利用泛素赖氨酸位点突变蛋白结合Western印迹实验检测该分子催化形成的泛素链的连接位点。结果成功构建原核表达质粒,获得纯度大于85%、浓度约为0.5 mg/ml的E2348C-1040蛋白质。分子水平的酶活实验结果表明E2348C-1040分子具有泛素连接酶活性,能催化形成不依赖于赖氨酸的泛素链。通过构建真核表达质粒转染HEK-293T细胞,发现E2348C-1040分子可在宿主细胞中催化形成甲硫氨酸(methionine 1,M1)连接的线性泛素链。结论EPEC效应因子E2348C-1040具有泛素连接酶活性,能催化泛素形成M1连接的线性泛素链。这一发现将为揭示效应因子E2348C-1040在宿主细胞内的作用机制提供研究线索。Objective To identify the ubiquitin ligase activity of enteropathogenic Escherichia coli(EPEC)effector E2348C-1040,and detect the ubiquitin chain type catalyzed by the effector.Methods The E2348C-1040 gene was cloned into pET-22b(+)and transformed into E.coli BL21(DE3),which was used to express and purify E2348C-1040 protein.The ubiquitin ligase activity was identified by in vitro ubiquitination assay.The ubiquitin mutants and Western blotting assay were used to detect the ubiquitin chain binding site.Results The prokaryotic expression plasmid pET-22b(+)-E2348C-1040 was constructed.The purity of the obtained E2348C-1040 protein was more than 85%,and the concentration was about 0.5 mg/ml.The results of in vitro ubiquitination assay showed that E2348C-1040 could catalyze the formation of lysine-independent ubiquitin chain.Then,an eukaryotic expression plasmid pcDNA3.1(+)-E2348C-1040was constructed and used to transfect HEK-293T cells.The results showed that E2348C-1040 could catalyze the formation of methionine 1(M1)-linked linear ubiquitin chain in host cells.Conclusion The EPEC effector E2348C-1040 is an E3 ubiquitin ligase,which can catalyze ubiquitin to form M1 linked linear ubiquitin chain.This discovery will provide clues to the mechanism of effector E2348C-1040 in host cells.

关 键 词：肠致病性大肠杆菌 效应因子 泛素化 泛素连接酶 

分 类 号：R378.2[医药卫生—病原生物学]