新基因HSPCSET的蛋白结构分析和功能初探

Preliminary structural and functional study on a novel gene HSPCSET

目的研究人脐血CD34＋造血干／祖细胞来源的新基因HSPCSET的结构和生物学功能。方法采用生物信息学技术对HSPCSET蛋白进行结构分析和同源性比较。酵母双杂交实验筛选与HSPCSET蛋白相互作用的蛋白分子，测序后筛选出功能较明确的阳性克隆进一步回复性杂交，采用G—gal实验和GST拉下试验验证。免疫荧光共定位方法分析HSPCSET和相关基因细胞内定位情况。结果蛋白结构分析和同源性比对揭示HSPCSET是古老的SET基因家族的成员，可能具有非常保守而重要的生物学功能。应用酵母双杂交技术筛选能与之相互作用的蛋白，获得13个阳性克隆，分别涉及信号传导、转录调控、细胞凋亡、肿瘤发生、发育等途径，与细胞增殖、分化、凋亡及肿瘤发生密切相关。采用GST拉下试验进一步在体外水平验证了其中4个基因：GADD34（凋亡相关分子）、SIVA（凋亡相关分子）、DNAJ（分子伴侣）和PHF1（转录相关因子）与HSPCSET蛋白确实存在相互作用。免疫荧光共定位方法显示HSPCSET和GADD34共转染细胞株后共同定位于细胞核内，两者之间存在强烈的相互作用。结论新基因HSPCSET可能参与凋亡途径、具有非常保守而重要的生物学功能。本研究结果为进一步研究HSPCSET在造血调控中的作用及研究肿瘤发生奠定基础。

Objective To characterize the structural and the functional feature of a novel gene HSPCSET isolated from human CD34 ＋ hematopoietic stem/progenitor cells （HS/PCs）. Methods Bioinformatic technology was used to identify the structural features of the HSPCSET protein and perform the multiple sequence alignment. Yeast-two-hybrid system was used to identify the proteins interacting with the HSPCSET protein. After sequencing,we selected out the positive clones which had clear functions, and carried out β- gal experiment and GST pull clown assay to confirm the results. The cellular location of the HSPCSET was checked by immunofluorescence assay. Results The HSPCSET protein belongs to a SET domain family, which is evolutionarily conserved across species. It implied that HSPCSET may have biologically important function. Using yeast-two-hybrid system, we showed that the protein sequence with SET domain might bind to 13 proteins,which involved in signaling transduction, transcriptional regulation, apoptosis, tumorigenesis, development, etc. And 4 proteins （GADD34, SIVA, DNAJ and PHF1） were confirmed by one-on-one back of the hybrid experiment, β-gal test and GST pull down assay. When GADD34 and HSPCSET were co-transfected, they co-localized in the nucleus, suggesting a strong interaction. Conclusion The novel gene HSPCSET is likely to have biologically important function. This study provides the basis for further studies of its function in hematopoiesis and tumorigenesis.