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，我们越来越多地暴露于各种异生素化合物，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后的遗传毒性压力，并开发新的分子策略来改善细胞对DNA损伤的反应。在本提案中，我们专注于乳腺癌-1易感基因产物（BRCA 1），一种参与DNA损伤信号传导的关键分子，对乳腺和卵巢特异性肿瘤抑制的机制。我们以前确定BRCA 1的BRCT结构域为磷酸丝氨酸/苏氨酸结合结构域，这种磷酸结合功能最近被证明对BRCA 1的肿瘤抑制功能至关重要。我们将进行一个全面的蛋白质组范围内的筛选，以确定直接BRCA 1的BRCT结构域依赖性结合伙伴和下游目标的泛素连接酶活性的BRCA 1复合物与其结合伙伴BARD 1在雌激素反应组织。拟议的研究符合我们实验室的长期目标，即在分子细节上了解细胞信号传导途径的翻译后修饰如何与模块化结合结构域一起调节细胞对DNA损伤和细胞周期进程的反应的多个方面。筛选结果应提供（1）雌激素应答乳腺细胞中所有BRCA 1相互作用蛋白的全面无偏鉴定，沿着确定它们是否是BRCA 1-BARD 1遍在蛋白连接酶活性的底物;（2）鉴定DNA损伤诱导的致癌作用风险增加的潜在靶点和生物标志物，以及（3）创建一种新的筛选方法，以全基因组方式鉴定磷酸特异性蛋白质-蛋白质相互作用和E3泛素连接酶底物。由于下游靶点可能在缺乏正常BRCA 1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遗传毒性应激反应的新生物标志物。 公共卫生相关性：BRCA 1是一种肿瘤抑制蛋白，在DNA损伤反应中起着至关重要的作用，但BRCA 1防止肿瘤形成的机制在很大程度上是未知的。BRCA 1突变导致雌激素敏感组织（特别是乳腺和卵巢）癌症风险显著增加。在这项提案中，我们开发了一种新的蛋白质组范围的方法，用于全面识别BRCA 1的结合伴侣和底物，以便更好地了解其在分子水平上的抗癌功能。

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.