Proteome-wide protein Identification Facilitated by in Vivo Cross-linking

描述（由申请人提供）：异常的蛋白质-蛋白质相互作用与许多人类疾病有关，了解蛋白质相互作用网络已成为不同学科深入研究的主题。然而，生物系统内蛋白质相互作用的全球检测对当前技术提出了重大挑战。例如，质谱结合串联亲和纯化是一个有用的工具，在全球范围内研究蛋白质的相互作用，但这种技术无法恢复瞬时/弱蛋白质的相互作用，这些相互作用在串联亲和纯化步骤中丢失。在这个建议中，我们建议通过引入一种化学交联策略来克服这个问题，该策略旨在保留体内的瞬时蛋白质复合物。使用这种策略，我们将试图了解BRCA 1的分子基础，BRCA 1是乳腺癌病因学中的一个关键肿瘤抑制因子。BRCA 1编码一种泛素连接酶，其酶活性被乳腺癌患者中发现的突变所消除。阐明BRCA 1的细胞功能将大大提高我们对乳腺癌的认识，但我们对BRCA 1功能的理解仍然是不完整的。为了确定BRCA 1的细胞功能，有必要确定BRCA 1泛素化的底物及其泛素修饰的细胞后果。以前试图解决这个问题的常规质谱产生了混合的结果，可能是由于涉及BRCA 1的蛋白质相互作用的瞬时性质。- 具体目标- 1.哺乳动物细胞中的体内交联和质谱分析已经开发用于酵母蛋白质组的质谱分析的体内交联技术将针对哺乳动物细胞进行优化。2. BRCA 1的肿瘤抑制功能的机制将使用化学交联策略来确定BRCA 1的细胞底物和相互作用蛋白。BRCA 1突变赋予60-85%的终身患乳腺癌的风险，这是美国女性中最常见的癌症。因此，我们旨在了解BRCA 1如何抑制乳腺癌的建议与公共卫生和NIH的使命有关。

DESCRIPTION (provided by applicant): Aberrant protein-protein interactions are implicated in a number of human diseases, and understanding protein interaction networks has become the subject of intense research in diverse disciplines. Yet, global detection of protein interactions within biological systems poses a significant challenge for current technology. For instance, mass spectrometry combined with tandem affinity purification is a useful tool for investigating protein interactions on a global scale, but this technique fails to recover transient/weak protein interactions-these interactions are lost during the tandem affinity purification steps. In this proposal, we propose to overcome this problem by introducing a chemical cross-linking strategy aimed at preserving transient protein complexes in vivo. Using this strategy, we will attempt to understand the molecular basis for BRCA1, a critical tumor suppressor in the etiology of breast cancer. BRCA1 encodes a ubiquitin ligase, whose enzymatic activity is abolished by mutations found in breast cancer patients. Elucidation of the cellular function of BRCA1 will greatly enhance our knowledge on breast cancer, but our understanding on BRCA1 function is still incomplete. It is necessary to identify the substrates ubiquitinated by BRCA1 and cellular consequences of their ubiquitin modification in order to define the cellular function of BRCA1. Previous attempts to address this issue by conventional mass spectrometry have produced mixed results probably due to the transient nature of the protein interactions involving BRCA1. -Specific Aims- 1. In Vivo Cross-linking & Mass spectrometry in Mammalian Cells An in vivo cross-linking technique that has been developed for the mass spectrometric analyses of the yeast proteome will be optimized for the mammalian cells. 2. Mechanism of the Tumor Suppressor Function of BRCA1 The cellular substrates and interacting proteins for BRCA1 will be determined using a chemical cross-linking strategy. -Relevance- BRCA1 mutations confer 60-85% lifetime risk of developing breast cancer, which is the most common cancer among women in the U.S. BRCA1 is also frequently down-regulated in sporadic cases, indicating its central role in suppressing the cancer. Thus, our proposal aimed at comprehending how BRCA1 inhibits breast cancer is relevant to public health and NIH mission.