人睾丸组织TRIM69蛋白具有催化多聚泛素链形成的活性  

The human testis TRIM69 protein possesses an activity of catalyzing formation of muti-ubiquitination

作　　者：韩永卿[1] 李容[1] 高晋兰[1] 缪时英[1] 王琳芳[1] 

机构地区：[1]中国医学科学院北京协和医学院基础医学研究所医学分子生物学国家重点实验室,北京100005

出　　处：《基础医学与临床》2013年第1期77-81,共5页Basic and Clinical Medicine

基　　金：国家重大研究计划项目(2011CB944302)

摘　　要：目的验证人源TRIM69蛋白是否具有催化多聚泛素链形成的活性。方法把人源TRIM69基因构建到原核细胞表达载体中,经过转化、诱导、表达和纯化获得TRIM69纯化蛋白,通过细胞外泛素化实验分析其是否具有催化多聚泛素链形成的活性;另外构建了TRIM69基因的全长、RING结构域的点突变、以及RING结构域截短序列的真核细胞表达载体,瞬时转染293T细胞或稳定表达泛素的HeLa细胞株,通过免疫沉淀结合免疫印迹方法检测其是否具有催化多聚泛素链形成的活性。结果泛素化分析实验表明TRIM69具有催化多聚泛素链形成的活性并且其活性依赖于它的RING结构域。结论成功鉴定了人源TRIM69蛋白具有催化多聚泛素链形成的活性,为进一步证明其是一个新的E3泛素连接酶提供依据。Objective To identify whether human TRIM69 may catalyze multiubiquitinylation.Methods The gene of human TRIM69 was cloned into a procaryotic expression vector,which was then transformed into E.coli.Purified protein of TRIM69 was obtained through the process of induction,expression,and purification,which was used in the following experiment of ＇in vitro ubiquitination assay＇ to detect whether TRIM69 could catalyze formation of multiubiquitinylated products.In addition,we constructed TRIM69 gene full length or a point mutagenesis of RING domain sequence or a deleted RING domain sequence into a eukaryotic expression vector.Then the corresponding vectors were transiently transfected into HEK293T cells or HeLa cells of stably expressed ubiquitination.Immunoprecipitation combined with immunoblotting was performed to detect whether TRIM69 catalyzed formation of multiubiquitinylated products in cells.Results The ubiquitination assay results demonstrated that TRIM69 catalyzed formation of multiubiquitinylated products,which was dependent on its RING domain.Conclusions The enzyme activity of TRIM69 in catalyzing the formation of muti-uiquitinylated products was well corrfirmed,which builds up a basis for further characterizing TRIM69 as a novel E3 ubiquitin ligase.

关 键 词：精子发生 E3泛素连接酶 TRIM RING FINGER 

分 类 号：R339.21[医药卫生—人体生理学]