Identification of a Novel Binding Protein of FAT10: Eukaryotic Translation Elongation Factor 1A1
Identification of a Novel Binding Protein of FAT10: Eukaryotic Translation Elongation Factor 1A1
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DOI：
10.1007/s10620-012-2189-1
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发表时间：
2012-05
影响因子：
3.1
通讯作者：
Xin Yu;Xiuxia Liu;Tian-de Liu;K. Hong;Jun Lei;Rongfa Yuan;J. Shao
中科院分区：
文献类型：
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作者：
Xin Yu;Xiuxia Liu;Tian-de Liu;K. Hong;Jun Lei;Rongfa Yuan;J. Shao
背景已知FAT10主要通过与不同底物的结合来执行其功能，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。然而，已知的FAT10结合蛋白不能解释其所有已知的功能。因此，本研究的目的是鉴定未知的FAT10结合蛋白。以FAT10为诱饵蛋白，筛选人肝细胞癌细胞系Hep3B的cDNA文库。基于定位研究和免疫共沉淀分析证实了蛋白质之间的相互作用。结果鉴定出真核细胞延伸因子1A1(EEF1A1)是一种FAT10特异性结合蛋白。体内和体外均证实了FAT10与eEF1A1的结合。我们还发现，当FAT10的表达被siRNA敲除后，这导致了人肝癌细胞中eEF1A1在mRNA和蛋白水平的表达下调。结论我们提出了一种以eEF1A1为底物的FAT10模型，以部分完成其调节肿瘤细胞生物学行为的功能。由于eEF1A1和FAT10对肿瘤的发生和发展都很重要，了解这种相互作用的机制可以为寻找新的战略靶点进行药物筛选和分子分型提供线索，并可能为开发新的有效的肝细胞癌治疗策略提供线索。
BackgroundFAT10 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.MethodsThe 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.ResultsIn 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.ConclusionsWe 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.