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

利用2D-DIGE系统鉴定新的乳腺癌相关基因及其功能分析

基本信息

批准号：
18591445
负责人：
FUKUDA Mamoru
金额：
$ 2.57万
依托单位：
St.Marianna University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591445/
关键词：
BRCA1 BARD1 familial breast cancer ubiquitin ligase HERC2 2D-DIGE proteomics

项目摘要

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.

分析乳腺癌抑制基因BRCA1的蛋白稳定性对于了解乳腺癌的发生机制具有重要意义。该项目的目的如下：1)筛选BRCA1泛素化的底物或调节BRCA1的因子。2)HERC2介导的BRCA1调控的功能分析。3)HERC2转基因小鼠的表型分析。结果：1)制备的兔抗BRCA1多克隆抗体可免疫沉淀大量BRCA1复合体。然而，通过筛选，我找不到任何调控BRCA1或受BRCA1调控的新因素。2)免疫沉淀和免疫印迹证实内源性HERC2与BRCA1之间存在相互作用。在细胞周期的S期，HERC2-BRCA1的相互作用达到最大，而BRCA1的表达最低。片段分析表明，含有Hect结构域的HERC2的C-末端与BRCA1的N-末端降解结构域相互作用。内源性HERC2主要定位于细胞质，并被CRM1抑制剂瘦素B输入细胞核，提示HERC2是一种胞质和核穿梭蛋白。这些数据表明，HERC2可能是BRCA1的E3连接酶，其靶向是BRCA1在S期的降解。3)获得13个转基因小鼠克隆，筛选出在MMTV诱导下乳腺组织表达HERC2 C末端基因的小鼠。然而，没有观察到明显的表型。