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（由申请人提供）：泛素蛋白酶体系统在许多人类疾病中起关键作用，其中蛋白质如乳腺癌特异性肿瘤抑制因子BRCA 1（乳腺癌-1）和早发性帕金森病帕金中突变的蛋白质充当泛素连接酶。然而，泛素蛋白连接酶和它们的目标之间的酶-底物关系的阐明提出了一个重大的挑战。该提案的目标是开发一种新的技术，能够快速和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，然后与底物蛋白质结合，并允许它们容易识别。标记通过细菌生物素连接酶BirA进行，BirA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：一种是泛素连接酶与BirA的融合蛋白，另一种是泛素与生物素受体肽的融合蛋白。在这种安排中，当生物素受体泛素被带到泛素连接酶时，连接的BirA将生物素化泛素，标记它已被该特定的泛素连接酶作用。因此，可以使用链霉亲和素色谱法纯化随后连接生物素化泛素的底物蛋白，并通过质谱法鉴定。作为该系统的第一个测试，我已经构建了三个BirA-泛素连接酶融合体和酵母酿酒酵母中的生物素受体泛素，并表明：1）所有蛋白质都表达; 2）生物素受体泛素是功能性的，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，我计划将其扩展到确定酵母中所有已知的泛素连接酶的底物，为泛素连接酶及其底物提供急需的蛋白质组范围的视图。最后，我将调整系统，用于研究哺乳动物组织培养细胞中的泛素连接酶。确定人类泛素连接酶（如BRCA 1和Parkin）的底物目前是一项艰巨的任务，该系统应加速其发现并阐明几种人类疾病的病因。