Proteome-wide analysis of E3 ubiquitin ligase-substrate relationships

E3 泛素连接酶-底物关系的全蛋白质组分析

• 批准号: 7333968
• 负责人: Lea Starita
• 金额: $ 4.68万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-16 至 2009-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7333968
• 关键词: Affinity; Affinity Chromatography; Biology; Biotin; Chimeric Proteins; Chromatography; Development; Disease; Engineering; Etiology; Goals; Human Genome; Human Ubiquitin; In Vitro; Knowledge; Learning; Ligase; Light; Mass Spectrum Analysis; Molecular Biology; Mutate; Parkinson Disease; Peptides; Play; Proteins; Proteome; Role; Saccharomyces cerevisiae; Speed; Streptavidin; Substrate Interaction; System; Techniques; Technology; Testing; Time; Tumor Suppressor Proteins; Ubiquitin; Ubiquitin-Protein Ligase Complexes; Yeasts; base; biotin 2; early onset; enzyme substrate; human disease; malignant breast neoplasm; man; multicatalytic endopeptidase complex; new technology; parkin gene/protein; parkin protein; tissue/cell culture; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): The ubiquitin proteasome system plays a key role in many human diseases, with proteins such as the breast cancer-specific tumor suppressor BRCA1 (breast cancer-1) and the protein mutated in early onset Parkinson's disease Parkin acting as ubiquitin ligases. However, the elucidation of enzyme-substrate relationships between ubiquitin protein ligases and their targets poses a significant challenge. The goal of this proposal is to develop a new technology that enables rapid and simple identification of the substrates for ubiquitin ligases. My approach is to tag the ubiquitin moieties used by a specific ubiquitin ligase with biotin, which then becomes associated with substrate proteins and allows their facile identification. The tagging is carried out by the bacterial biotin ligase, BirA, which attaches biotin to proteins that contain a biotin-acceptor peptide. Two fusion proteins are constructed: one of a ubiquitin ligase to BirA, and the other of ubiquitin to the biotin-acceptor peptide. In this arrangement, when the biotin-acceptor ubiquitin is brought to the ubiquitin ligase, the attached BirA will biotinylate the ubiquitin, marking it as having been acted on by that specific ubiquitin ligase. Substrate proteins to which the biotinylated ubiquitin is subsequently attached can thus be purified using streptavidin chromatography and identified by mass spectrometry. As a first test of the system, I have constructed three BirA-ubiquitin ligase fusions and the biotin-acceptor ubiquitin in the yeast Saccharomyces cerevisiae, and shown that: 1) all proteins are expressed; and 2) the biotin-acceptor ubiquitin is functional, used by ubiquitin ligases in yeast, and biotinylated in vitro by BirA. Once this technology is established, I plan to expand it to carry out the determination of substrates for all the known ubiquitin ligases in yeast, providing a critically needed proteome-wide view of ubiquitin ligases and their substrates. Ultimately, I would adapt the system for the study of ubiquitin ligases in mammalian tissue culture cells. The determination of substrates for human ubiquitin ligases like BRCA1 and Parkin is currently a daunting task, and this system should speed their discovery and shed light on the etiology of several human diseases.

描述（申请人提供）：泛素蛋白酶体系统在许多人类疾病中发挥着关键作用，诸如乳腺癌特异性肿瘤抑制因子BRCA1（乳腺癌-1）和早发性帕金森病突变蛋白Parkin等蛋白质发挥着关键作用。泛素连接酶。然而，阐明泛素蛋白连接酶及其靶标之间的酶-底物关系提出了重大挑战。该提案的目标是开发一种新技术，能够快速、简单地识别泛素连接酶的底物。我的方法是用生物素标记特定泛素连接酶使用的泛素部分，然后生物素与底物蛋白相关联，并可以轻松识别它们。标记是由细菌生物素连接酶 BirA 进行的，它将生物素连接到含有生物素受体肽的蛋白质上。构建了两种融合蛋白：一种是泛素连接酶 BirA，另一种是泛素连接酶生物素受体肽。在这种安排中，当生物素受体泛素被带到泛素连接酶时，附着的 BirA 将使泛素生物素化，将其标记为已被该特定泛素连接酶作用。因此，可以使用链霉亲和素层析来纯化随后附着有生物素化泛素的底物蛋白，并通过质谱法进行鉴定。作为该系统的第一次测试，我在酿酒酵母中构建了三个 BirA-泛素连接酶融合体和生物素受体泛素，并表明：1）所有蛋白质均表达； 2) 生物素受体泛素具有功能，被酵母中的泛素连接酶使用，并在体外被 BirA 生物素化。一旦这项技术建立起来，我计划将其扩展到对酵母中所有已知的泛素连接酶的底物进行测定，从而提供急需的泛素连接酶及其底物的整个蛋白质组视图。最终，我将调整该系统以研究哺乳动物组织培养细胞中的泛素连接酶。目前，确定 BRCA1 和 Parkin 等人类泛素连接酶的底物是一项艰巨的任务，该系统应能加快这些底物的发现速度，并揭示多种人类疾病的病因学。