用酵母双杂交系统筛选ATF5相互作用蛋白

Screening the Interacting Proteins of Activating Transcription Factor 5 by Yeast Two-hybrid System

转录激活因子5(activating transcription factor5,ATF5)最初是从人T淋巴细胞中分离出来,并在肿瘤发展中起重要作用.为研究ATF5的作用机理,以其C末端区域为诱饵蛋白,用酵母双杂交技术从人心脏cDNA文库中筛选出了包括热休克蛋白27和核糖体蛋白L18a等与肿瘤转移、预后密切相关的蛋白.免疫共沉淀实验表明,热休克蛋白27能够特异结合在ATF5C末端区域.由此推论,ATF5通过细胞内一些与肿瘤发展、转移及预后有关的蛋白相互作用,进而影响肿瘤的进展,为ATF5的功能研究提供了重要的线索.

Activating transcription factor 5 （ATF5） was initially isolated in human T-lymphocyte and played an important role in the progression of cancers. To screen the interacting protein of ATF5, a bait plasmid pDBLeu-hATF5 was constructed by subcloning the 3＇-terminal region of human ATF5 cDNA fragment into the yeast two-hybrid vector pDBLeu. The bait pDBLeu-hATF5 together with a heart cDNA library in pPC86 vectors were then cotransformed into MaV2O3 yeast competent cells. Seventeen proteins that interact with ATF5 were identified by yeast two-hybrid screening. Four of them had homologies with heat shock 27 kD protein 1, human ribosomal protein L18a, cytochrome C oxidase subunit and human hypothetical protein FLJ10814, respectively. The specific interactions of ATF5 with heat shock 27kD protein 1 was performed in mammalian cell COS-7. The result suggested that such interactions were mediated by the bZIP domain of ATF5 at the C- terminus. These data revealed that ATF5 was able to interact directly with the proteins associated with tumor aggression, and therefore maybe linked to chemoresistance in tumor therapies.