Steroidogenic acute regulatory protein-binding protein cloned by a yeast two-hybrid system
Steroidogenic acute regulatory protein-binding protein cloned by a yeast two-hybrid system
复制标题
DOI：
10.1074/jbc.m302291200
复制
发表时间：
2003-10-24
影响因子：
4.8
通讯作者：
Fujimoto, S
中科院分区：
文献类型：
--
作者：
Sugawara, T;Shimizu, H;Fujimoto, S
类固醇生成急性调节蛋白（星星）在胆固醇从线粒体外膜向内膜转运中起着关键作用。据报道，缺少前62个氨基酸的星星突变蛋白（N-62星星蛋白）与野生型星星蛋白一样有效。在本研究中，我们研究了星星蛋白刺激类固醇生成的机制。利用基于Gal 4的酵母双杂交系统鉴定与N-62星星蛋白相互作用的蛋白质。从筛选1 × 106个克隆中获得9个阳性克隆。pull-down分析和哺乳动物双杂交分析的结果证实了N-62星星蛋白和克隆4翻译产物之间的相互作用。克隆4的翻译产物被命名为StAR结合蛋白（SBP）。我们通过将SBP cDNA插入pTarget载体制备表达质粒（pSBP）。在与人细胞色素P450 SCC系统、星星表达载体和pSBP共转染后，COS-1细胞产生的双烯醇酮的量增加。类固醇激素产生的细胞进行小干扰RNA处理的类固醇激素的量小于由控制细胞产生的。总之，SBP结合细胞中的星星蛋白并增强星星蛋白促进类固醇激素合成的能力。
Steroidogenic acute regulatory ( StAR) protein plays a key role in the transport of cholesterol from the outer mitochondrial membrane to the inner membrane. A StAR mutant protein lacking the first 62 amino acids (N-62 StAR protein) has been reported to be as effective as wild-type StAR protein. In the present study, we examined the mechanism by which StAR protein stimulates steroidogenesis. A Gal4-based yeast two-hybrid system was used to identify proteins interacting with N-62 StAR protein. Nine positive clones were obtained from screening 1 x 10(6) clones. The results of pull-down assays and mammalian two-hybrid assays confirmed interaction between N-62 StAR protein and the clone 4 translated product. The clone 4 translated product was named StAR-binding protein (SBP). We prepared an expression plasmid (pSBP) by inserting SBP cDNA into the pTarget vector. After cotransfection with the human cytochrome P450scc system, StAR expression vector, and pSBP, the amount of pregnenolone produced by COS-1 cells was increased. The amount of steroid hormones produced by steroidogenic cells subjected to small interfering RNA treatment was less than that produced by control cells. In conclusion, SBP binds StAR protein in cells and enhances the ability of StAR protein to promote syntheses of steroid hormones.