PER1与RACK1蛋白作用位点分析

目的 筛选人下丘脑视交叉上核(SCN)区域内与PERIOD1(PER1)相互作用的新蛋白,研究RACK1与PER1的作用特点,明确其结合的关键结构域.方法 采用酵母双杂交方法,筛选得到人SCN区域内与PER1-PAS结构域相互作用的新蛋白,并构建5种表达不同长度RACK1片段的酵母文库质粒与PER1诱饵质粒共转染酵母AH109进行杂交,通过营养缺陷筛选、报告基因检测获得阳性克隆,并采用体外转录、免疫共沉淀实验证实阳性克隆蛋白间的相互作用.结果 酵母双杂交筛选得到人SCN区域内表达RACK1蛋白的克隆,重组RACK1表达质粒与PER-PAS诱饵质粒进行酵母双杂交筛选后得到三个阳性克隆:RACK1(WD1-7)、RACK1(WD4-7)和RACK1 (WD5-7),β-半乳糖苷酶测试阳性证实报告基因表达,免疫共沉淀结果显示阳性克隆与PER1蛋白间存在相互作用.结论 RACK1与PER1存在直接相互作用,RACK1含有7个WD40结构域,本研究发现与PER1结合的最小区域位于Ⅴ、Ⅵ、Ⅶ三个WD40结构域,提示其碳端序列为其结合的关键部位.

Identification of RACK1-PER1 Protein Interaction Sites

OBJECTIVE: By screening the cDNA library of suprachiasmatic nucleus (SCN) region of human hypothalamus, we try to capture the novel proteins interacting with PER1 and investigate the interplaying characteristic of RACK1 and PER1. METHODS: By yeast two-hybrid system, the new protein in human SCN, which was associating with PER1-PAS domain was obtained. Then five truncated fragments of RACK1 cDNA was cloned into yeast expression vector to form recombinant library plasmid and was co-transformed into yeast strain AH109 with bait plasmid containing PER1-PAS domain. The transformants were selected via nutrition-deficient medium, and the positive clones were identified or obtained by checking the expressin of report gene. At last all the protein interactions were confirmed by co-immunoprecipitation tests. RESULTS: one of the positive clones in human SCN cDNA library were identified with part of the RACK1 protein sequence. Three positive clones, which contained respectively the fragment of RACK1 (WD1-7), RACK1 (WD4-7) or RACK1 (WD5-7) were obtained through yeast two-hybrid screen with various RACK1 fragments and PER1-PAS domain. The RACK1 and PER1 protein interaction was determined by beta-galactosidase assay and co-immunopreipitation. CONCLUSION: The direct interaction between RACK1 and PER1 has been approved. RACK1 is composed of seven WD40 repeats and the minimal interacting sites are limited in V-VII WD40 domains in the present study,which means C-terminal amino acid of RACK1 may be essential to the interacting.