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

体内交联促进全蛋白质组蛋白质鉴定

• 批准号: 7750238
• 负责人: Jinhwan Eugene Lee
• 金额: $ 5.17万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7750238
• 关键词: Address; Affinity Chromatography; BRCA1 Mutation; BRCA1 Protein; BRCA1 gene; Cancer Patient; Cell membrane; Cell physiology; Cells; Chemicals; Complex; Culture Media; Detection; Discipline; Etiology; Formaldehyde; Functional disorder; Genetic; Knowledge; Malignant - descriptor; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Methodology; Mission; Modification; Molecular; Mutation; Nature; Organism; Physiological; Proteins; Proteome; Proteomics; Public Health; Research; Role; Shewanella; Techniques; Technology; Tumor Suppressor Proteins; Ubiquitin; Ubiquitin-Conjugating Enzymes; United States National Institutes of Health; Woman; Work; Yeasts; base; biological systems; crosslink; human disease; in vivo; insight; lifetime risk; malignant breast neoplasm; novel; protein complex; protein function; protein protein interaction; tool; ubiquitin ligase

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.

描述(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密集研究的主题。然而，对生物系统内蛋白质相互作用的全球检测对当前的技术构成了重大挑战。例如，结合串联亲和纯化的质谱学是在全球范围内研究蛋白质相互作用的有用工具，但这种技术无法恢复瞬时/弱蛋白质相互作用-这些相互作用在串联亲和纯化步骤中丢失。在这项建议中，我们建议通过引入一种化学交联策略来克服这个问题，目的是在体内保存瞬时蛋白质复合体。利用这一策略，我们将试图了解BRCA1的分子基础，BRCA1是乳腺癌病因学中的关键肿瘤抑制因子。BRCA1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BRCA1的细胞功能将大大提高我们对乳腺癌的认识，但我们对BRCA1功能的了解仍然不完整。为了确定BRCA1的细胞功能，有必要确定BRCA1泛素化的底物及其泛素修饰的细胞后果。以前通过传统的质谱学来解决这个问题的尝试产生了好坏参半的结果，可能是由于涉及BRCA1的蛋白质相互作用的瞬变性质。-特异性AIMS-1.在哺乳动物细胞的活体交联和质谱学中，为酵母蛋白质组的质谱学分析而开发的在体交联技术将被优化用于哺乳动物细胞。2.BRCA1抑瘤作用的机制BRCA1的细胞底物和相互作用蛋白将用化学交联法确定。-相关性-BRCA1突变使患乳腺癌的终身风险达到60%-85%，乳腺癌是美国女性中最常见的癌症。BRCA1在零星病例中也经常下调，这表明它在抑制癌症方面发挥了核心作用。因此，我们的提案旨在了解BRCA1是如何抑制乳腺癌的，这与公共卫生和NIH的任务有关。