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为研究真核翻译起始因子eIF4E的调控,利用酵母双杂交系统从人骨髓cDNA文库筛选eIF4E相互作用蛋白质,核苷酸序列分析及同源性检索表明,其中一个约为0.4 kb的克隆与HUWE1(HECT,UBA和WWE domain containing 1,又称ARF-BP1,HECTH9,HUWE1)高度同源。细胞免疫共沉淀实验结果显示,在哺乳动物细胞水平能够检测到eIF4E蛋白与全长HUWE1的特异相互作用。利用构建好的含有HECT结构域的突变体进行相互作用位点研究,结果表明HUWE1的HECT结构域可以与eIF4E蛋白相互作用。
In order to investigate the regulation of eIF4E, a eukaryotic translation initiation factor (eIF4E), the yeast two-hybrid system was used to screen eIF4E-interacting proteins from human bone marrow cDNA library. Nucleotide sequence analysis and homology search revealed that a 0.4 kb clone was associated with HUWE1 (HECT, UBA and WWE domain containing 1, also known as ARF-BP1, HECTH9, HUWE1). Cellular coimmunoprecipitation assay results showed that specific interaction of eIF4E protein with full-length HUWE1 was detected at mammalian cell level. Mutations of constructed HECT domain-containing mutants were used to study the interaction sites. The results showed that the HECT domain of HUWE1 could interact with eIF4E protein.