The Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang 330006, China.
Dig Dis Sci. 2012 Sep;57(9):2347-54. doi: 10.1007/s10620-012-2189-1. Epub 2012 May 9.
FAT10 is known to execute its functions mainly through conjugation to different substrates, and these known functions include cytokine responses, apoptosis, mitosis, and tumorigenesis. Nonetheless, the known binding proteins of FAT10 cannot explain all its known functions. As such, the aim of this study was to identify unidentified conjugation proteins of FAT10.
The yeast two-hybrid system was employed in this study. FAT10 was used as the bait protein for screening of a cDNA library from a human hepatocellular carcinoma cell line, Hep3B. Protein interactions were confirmed based on localization studies and co-immunoprecipitation assays. The expression of mRNA and protein was determined using real-time polymerase chain reaction and western blot analyses, respectively.
In this study, we identified eukaryotic elongation factor 1A1 (eEF1A1) as a FAT10-specific binding protein. The binding between FAT10 and eEF1A1 was confirmed both in vivo and in vitro. We also found that, when the expression of FAT10 was reduced by siRNA knockdown, this resulted in downregulation of eEF1A1 expression at both the mRNA and protein levels in human hepatocellular carcinoma cells.
We propose a model in which eEF1A1 serves as a substrate of FAT10 to accomplish, in part, its functions in regulating the biological behavior of tumor cells. Since both eEF1A1 and FAT10 are important for tumorigenesis and development, comprehending the mechanisms of this interaction can provide clues for identification of novel strategic targets for drug screening and molecular typing, and possibly in the development of new effective therapeutic strategies against hepatocellular carcinoma.
已知 FAT10 主要通过与不同底物的缀合来执行其功能，这些已知功能包括细胞因子反应、细胞凋亡、有丝分裂和肿瘤发生。尽管如此，已知的 FAT10 结合蛋白并不能解释其所有已知的功能。因此，本研究的目的是鉴定 FAT10 未识别的缀合蛋白。
本研究采用酵母双杂交系统。将 FAT10 用作诱饵蛋白，从人肝癌细胞系 Hep3B 中筛选 cDNA 文库。根据定位研究和共免疫沉淀实验证实蛋白相互作用。使用实时聚合酶链反应和 Western blot 分析分别确定 mRNA 和蛋白质的表达。
在本研究中，我们鉴定了真核延伸因子 1A1（eEF1A1）为 FAT10 特异性结合蛋白。FAT10 与 eEF1A1 之间的结合在体内和体外均得到证实。我们还发现，当通过 siRNA 敲低降低 FAT10 的表达时，这导致人肝癌细胞中 eEF1A1 的表达在 mRNA 和蛋白水平均下调。
我们提出了一个模型，其中 eEF1A1 作为 FAT10 的底物，部分完成其调节肿瘤细胞生物学行为的功能。由于 eEF1A1 和 FAT10 对肿瘤发生和发展都很重要，理解这种相互作用的机制可以为鉴定新的药物筛选和分子分型的战略靶标提供线索，并可能为开发针对肝癌的新的有效治疗策略提供线索。
