Identification of novel breast cancer-related genes using 2D-DIGE system and their functional analyses

分析乳腺肿瘤抑制因子BRCA1蛋白稳定性调控对了解乳腺癌发生机制具有重要意义。该项目的目的如下：1)筛选被BRCA1泛素化的底物或BRCA1调控因子。2) herc2介导的BRCA1调控的功能分析。3) HERC2转基因小鼠表型分析。结果：1)制备了兔BRCA1多克隆抗体，免疫沉淀大量BRCA1复合物。然而，我无法通过筛选确定任何调节或受BRCA1调节的新因素。2)内源性HERC2与BRCA1的相互作用通过免疫沉淀和免疫印迹证实。细胞中HERC2-BRCA1的相互作用在细胞周期的S期最大，此时BRCA1的表达量最小。片段分析表明，HERC2中含有HECT结构域的c端与BRCA1的n端degron结构域相互作用。内源性HERC2主要定位于细胞质，并通过Crm1抑制剂leptomycin B导入细胞核，提示HERC2是一种细胞质和细胞核穿梭蛋白。这些数据表明HERC2可能是BRCA1的E3连接酶，靶向BRCA1在S期降解。3)我们获得了13个转基因小鼠克隆，并选择了MMTV诱导下乳腺组织中表达HERC2 c端mRNA的小鼠。然而，没有观察到显著的表型。

Analyses for regulation of protein stability of breast tumor suppressor BRCA1 is important to understand the mechanisms underlying breast carcinogenesis. The purposes of the project were as following: 1) Screen of substrates ubiquitinated by BRCA1 or of factors that regulates BRCA1. 2) Functional analyses of HERC2-mediated regulation of BRCA1. 3) Phenotypic analyses of HERC2 transgenic mice. Results: 1) Rabbit polyclonal antibodies to BRCA1 were generated to immunoprecipitate large amounts of BRCA1 complexes. However, I could not identify any novel factors that regulates or were regulated by BRCA1 using the screens. 2) The interaction between endogenous HERC2 and BRCA1 was confirmed by immunoprecipitation followed by immunoblotting. HERC2-BRCA1 interaction in cells maximized in the S phase of the cell cycle, when the BRCA1 expression minimized. Fragment analyses revealed that C-terminus of HERC2 that contains HECT domain interacts with N-terminal degron domain of BRCA1. Endogenous HERC2 mainly localized in the cytoplasm and was imported to the nuclear by the Crm1 inhibitor leptomycin B, suggesting that HERC2 was a cytoplasmic and nuclear shuttle protein. These data suggest that HERC2 is a possible E3 ligase for BRCA1 that targets BRCA1 for degradation in S phase. 3) We obtained 13 clones of transgenic mice and selected mice that expressed mRNA of HERC2 C-terminus in the breast tissue under MMTV induction. However, no significant phenotype was observed.