促凋亡因子Smac/DIABLO与核受体NR4A1相互作用的筛选和鉴定

目的 利用酵母双杂交技术筛选线粒体促凋亡因子野生型Smac/DIABLO的相互作用蛋白.方法 ①设计促凋亡因子野生型Smac/DIABLO引物,利用PCR在成人肝cDNA文库中扩增野生型Smac/DIABLO基因片段,构建酵母真核表达载体pDBLeu-Smac/DIABLO.②pDBLeu-Smac/DIABLO诱饵质粒转化酵母MaV203,Western blot检测融合蛋白在酵母中的正确表达,自激活作用的检测并确定抑制His基础表达的3AT浓度.③对成人肝cDNA文库的筛选.选择能同时激活His、Ura、LacZ 3个报告基因的克隆为阳性,利用回转实验和GST-pull down对阳性克隆进行鉴定.结果 成功构建pDBLeu-Smac/DIABLO载体,转化酵母细胞MaV203,无细胞毒性,无自激活作用,3AT浓度为25 mmol/L.经回转实验和GST-pull down证实了线粒体促凋亡因子Smac/DIABLO与核受体NR4A1之间具有相互作用.结论 Smac/DIABLO可能通过与核受体NR4A1的相互作用来影响核受体NR4A1对细胞生存或者凋亡的调控.

Isolation and identification of NR4A1 interacting with Smac/DIABLO

Objective To screen proteins in human adult liver cDNA library interacting with Smac/DIABLO with yeast two-hybrid technique. Methods The wild Smac/DIABLO was amplified with PCR in human adult liver cDNA library. The recombinant vector pDBLeu-Smac/DIABLO was constructed and transformed into yeast MaV203, and the expression of GAL4 BD-Smac/DIABLO fusion protein was dectected by Western blotting. The self-activation was examined, and 3-Amino-1,2,4-Triazole (3AT) concentration required to titrate basal HIS expression levels was determined. The human adult liver cDNA library was screened sequentially. The clones that grew on SC-Leu-Trp-His-Ura plates was employed to test if they could activate His, Ura and LacZ report genes. The plasmids from those clones that could activate the 3 report genes were isolated, then cotransformed with pDBLeu-Smac/DIABLO, and verified with GST-pull down after sequencing. Results The bait vector pDBLeu-Smac/DIABLO was successfully constructed and expressed correctly in MaV203. One clone nuclear receptor subfamily 4, group A, member 1 (NR4A1) was obtained and could interact with wild Smac/DIABLO via cotransformation and GST-pull down. Conclusion Smac/DIABLO may interact with NR4A1 and affect its function on cell growth and apoptosis.