SARS冠状病毒PLpro蛋白酶对泛素样分子的DUB活性  

Deubiquitinase Activities of Papain-like Protease from Severe Acute Respiratory Syndrome (SARS) Coronavirus against Ubiquitin-like Proteins

作　　者：陈晓娟[1] 杨宇东[1] 孙莉[1] 胡日查[1] 邢雅玲[1] 陈忠斌[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《中国生物化学与分子生物学报》2011年第7期664-670,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.30972761;No.30870536);国家科技重大专项(No.2008ZX10004-015);北京市自然科学基金项目(No.7092075);教育部留学回国人员科研启动基金项目(2009)资助~~

摘　　要：SARS冠状病毒基因组中非结构基因nsp3编码的木瓜样蛋白酶(PLpro)在病毒基因组复制及逃避宿主天然免疫中发挥重要作用,是研发抗病毒药物的重要靶标.SARS冠状病毒PLpro是一种病毒编码的去泛素化酶(DUB).为深入研究SARS冠状病毒PLpro对泛素样分子(ubiquitin-likeprotein,UBL)的DUB特性,本研究构建缺失PLpro N末端泛素样结构域(Ubl)和下游跨膜结构域(TM)的PLpro构建体(constructs),并构建3种缺失蛋白酶催化活性的突变体,检测PLpro对泛素样分子干扰素刺激基因15(ISG15)及SUMO-1的作用.实验结果表明,PLpro和PLpro-TM在细胞内具有很强的去ISG(DeISGylation)活性;缺失PLpro N末端泛素样结构域(Ubl)对PLpro的去ISG15活性没有影响;对PLpro蛋白酶活性位点C1651和H1812突变后,PLpro-TM的去ISG15活性消失,而对D1826位点突变后不影响此活性.PLpro不具有去SUMO(DeSUMOylation)活性,而PLpro-TM具有一定的去SUMO活性;PLpro催化活性相关的3个关键氨基酸残基Cys-His-Asp突变后对去SUMO活性有一定的影响.研究结果提示,SARS PLpro除了具有DUB的活性,还具有体内去ISG活性和去SUMO活性;PLpro蛋白酶活性与其去ISG活性之间有一定相关性;PLpro去SUMO-1活性具有TM依赖性.SARS冠状病毒PLpro对泛素样分子作用特性的研究为阐明病毒逃避宿主天然免疫机制和开发新型抗病毒药物提供重要的理论依据.SARS coronavirus papain-like protease（PLpro） is responsible for the proteolytic processing of pp1a（1ab） amino terminus to release nsp1,nsp2 and nsp3,and plays an important regulatory role in the assembling of SARS coronavirus replicase complex and the escape from host innate immunity.SARS PLpro has been reported of in vitro deubiquitinase（DUB） activity,but the characteristics and functions of the DUB activity are poorly understood.In this study,we first constructed several PLpro mutation constructs with N-terminal Ubl domain or C-terminal transmembrane（TM） domain deletion and the PLpro mutants at its catalytic sites（Cys-His-Asp）,then assayed for the DeISGylation and DeSUMOylating activites.We found that Ubl-deleted PLpro remained same DeISGylation activity in vivo as the wildtype suggesting the Ubl domain was dispensable.The DeISGylation of SARS-CoV PLpro was dependent on Cys-His catalytic residues,but little affected in the Asp mutation of the catalytic triad（Cys-His-Asp）.The mutation of SARS PLpro core domain did not affect the SUMO-conjugated cellular proteins,but the transmembrane form of PLpro（PLpro-TM） induced dramatic reduction of SUMO-conjugated cellular proteins.Such DeSUMOylating activity relied on intact catalytic residues,indicating that the DeSUMOylation activity of SARS PLpro required both TM-and catalytic residues.These results demonstrated that SARS PLpro might have both DeISGylating and DeSUMOylating activities in vivo.

关 键 词：SARS冠状病毒 木瓜样蛋白酶(PLpro) 去泛素化酶 干扰素刺激基因15(ISG15) SUMO-1 

分 类 号：Q5[生物学—生物化学] Q93