酵母双杂交技术鉴定FAT10新的结合蛋白:eEF1A1

Identification of a novel FAT10-binding protein,eEF1A1,by a yeast two-hybrid system

目的:利用酵母双杂交技术筛选类泛素蛋白FAT10(HLA-F adjacent transcript 10)在肝癌细胞Hep3B中相互作用的蛋白,为探讨FAT10蛋白在肝癌发生中的作用机制提供依据。方法:利用Clontech GAL4酵母双杂交系统筛选人肝癌细胞系Hep3B的cDNA文库,以获得与FAT10相互作用的蛋白分子;通过回交实验、免疫共沉淀和激光共聚焦细胞内共定位实验验证两者之间的相互作用;通过RNA干扰降低FAT10表达观察真核翻译延伸因子1α1(eEF1A1)的表达情况。结果:酵母双杂交筛选得到相互作用蛋白eEF1A1,激光共聚焦细胞内共定位确定FAT10和eEF1A1均位于细胞质内,存在共定位;回交实验和免疫共沉淀实验再次确认eEF1A1能够与FAT10相互作用;FAT10表达降低伴随着eEF1A1的表达下调。结论:FAT10能够与eEF1A1相互作用;FAT10可能通过eEF1A1来实现其部分功能。

AIM： To investigate the role of HLA - F adjacent transcript 10 （FAT10） in tumorigenesis. METHODS： GAL4 yeast two- hybrid assay was performed to screen the human Hep3B hepatoma cell cDNA library for obtaining the host cell protein molecules which interact with FAT10. The immunofluoresence co - localization and co - im- munoprecipitation assay were applied to confirm the interaction of the identified proteins. FAT10 was knockdown by siRNA to investigate the changes of eukaryotic translation elongation factor I alpha 1 （eEFIA1） expression. RESULTS： The eEFIA1 was selected from the host ceils using yeast two - hybrid assay. The results of co - localization and co - immuno- precipitation assays further confirmed the interaction between FAT10 and eEF1A1. Interestingly, when FAT10 was down - regulated by siRNA in Hep3B ceils, the protein expression of endogenous eEF1A1 was also found to be significantly re- pressed. Furthermore, the expression of FAT10 was reduced by siRNA knockdown, thus resulting in the down - regulation of eEFIAI expression at both mRNA and protein levels in MHCC97H cells. CONCLUSION： We propose a model in which eEF1A1 serves as a substrate of FAT10 to accomplish, in part, its function in regulating the biological behaviors of tumor cells.