Phospho-Binding Ligands and Substrates of BRCA1

描述（由申请人提供）：在越来越工业化的环境中，我们越来越多地接触到各种各样的外源化合物，其中许多是DNA损伤剂，导致癌症发病率增加。提高我们对细胞如何响应和修复DNA损伤的理解将使我们能够更好地评估暴露于环境因子时的遗传毒性应激，并开发新的分子策略来改善细胞对DNA损伤的反应。在本研究中，我们将重点研究乳腺癌1易感基因产物（BRCA1）对乳腺和卵巢特异性肿瘤抑制的机制，BRCA1是参与DNA损伤信号传导的关键分子。我们之前确定了BRCA1的BRCT结构域为磷丝氨酸/苏氨酸结合结构域，并且这种磷结合功能最近被证明对BRCA1的肿瘤抑制功能至关重要。我们将进行全面的蛋白质组筛选，以确定BRCA1直接依赖BRCT结构域的结合伙伴，以及雌激素应答组织中BRCA1与其结合伙伴BARD1复合物中泛素连接酶活性的下游靶点。提出的研究符合我们实验室的长期目标，即在分子细节上理解细胞信号通路的翻译后修饰，以及模块化结合域，如何调节细胞对DNA损伤和细胞周期进展的多个方面的反应。筛查结果应提供(1)全面、公正地鉴定雌激素反应性乳腺细胞中所有brca1相互作用蛋白，并确定它们是否为BRCA1-BARD1泛素连接酶活性的底物；(2)鉴定DNA损伤诱导的致癌风险增加的潜在靶点和生物标志物；(3)创建一种新的筛选方法，以全基因组方式鉴定磷酸化特异性蛋白-蛋白相互作用和E3泛素连接酶底物。由于下游靶点可能在缺乏正常BRCA1功能的细胞中被错误调节，我们的筛选有望揭示药物开发的新靶点和评估细胞对基因毒性应激反应的新生物标志物。

DESCRIPTION (provided by applicant): In an ever more industrialized environment, we are increasingly exposed to a wide range of xenobiotic compounds, many of which are DNA damaging agents that have contributed to an increasing incidence of cancer. Improving our understanding of how cells respond to and repair DNA damage will allow us to better assess genotoxic stress upon exposure to environmental agents and to develop novel molecular strategies to improve cellular responses to DNA damage. In the present proposal we focus on the mechanism responsible for breast and ovarian-specific tumor suppression by the Breast Cancer-1 susceptibility gene product (BRCA1), a key molecule involved in DNA damage signaling. We previously identified the BRCT domains of BRCA1 as phosphoserine/threonine-binding domains, and this phospho-binding function has recently been shown to be critical for BRCA1's tumor suppressor function. We will conduct a comprehensive proteome-wide screen to identify direct BRCT domain-dependent binding partners of BRCA1 and downstream targets for the ubiquitin ligase activity of BRCA1 in complex with its binding partner BARD1 in estrogen-responsive tissues. The proposed studies fit well within the long term goal of our laboratory to understand, in molecular detail, how post-translational modifications by cell signaling pathways, together with modular binding domains, regulate multiple aspects of the cellular response to DNA damage and cell cycle progression. The results of the screen should provide (1) a comprehensive unbiased identification of all BRCA1-interacting proteins in estrogen-responsive breast cells, along with determination of whether they are or are not substrates for BRCA1-BARD1 ubiquitin ligase activity; (2) the identification of potential targets and biomarkers of increased risk for DNA damage-induced carcinogenesis and (3) the creation of a novel screening approach to identify phospho-specific protein-protein interactions and E3 ubiquitin ligase substrates in a genome-wide manner. Since downstream targets are likely to be misregulated in cells lacking normal BRCA1 function, our screen is expected to reveal novel targets for drug development and novel biomarkers for assessing cellular responses to genotoxic stress. PUBLIC HEALTH RELEVANCE: BRCA1 is a tumor suppressor protein that plays a critically important role in the DNA damage response, but the mechanism by which BRCA1 prevents tumor formation is largely unknown. Mutations in BRCA1 causes dramatically increased risk for cancer in estrogen-responsive tissues, particularly the breast and ovary. In this proposal we develop a novel proteome-wide method for comprehensively identifying the binding partners and substrates of BRCA1 in order to better understand its anti-cancer function at the molecular level.