Proteome-wide analysis of E3 ubiquitin ligase-substrate relationships

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关键作用，如乳腺癌特异性肿瘤抑制基因BRCA1(乳腺癌-1)和早发性帕金森病帕金森病中突变的蛋白质充当泛素连接酶。然而，阐明泛素蛋白连接酶及其靶标之间的酶-底物关系是一个巨大的挑战。这项提议的目标是开发一种新的技术，能够快速和简单地鉴定泛素连接酶的底物。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联系起来，使它们能够方便地识别。标记是由细菌生物素连接酶BIRA执行的，它将生物素附着到含有生物素受体多肽的蛋白质上。构建了两种融合蛋白：一种是泛素连接酶与Bira结合，另一种是泛素与生物素受体多肽结合。在这种安排中，当生物素受体泛素被带到泛素连接酶上时，附着的BIRA将使泛素生物素化，标志着它已经被特定的泛素连接酶作用。因此，生物素化泛素随后连接到的底物蛋白可以用链霉亲和素层析纯化，并通过质谱学鉴定。作为系统的第一次测试，我已经在酿酒酵母中构建了三个biA-泛素连接酶融合和生物素受体泛素，结果表明：1)所有蛋白质都得到了表达；2)生物素受体泛素具有功能，在酵母中被泛素连接酶利用，并在体外被biA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，提供急需的泛素连接酶及其底物的蛋白质组学全景。最终，我将使该系统适用于哺乳动物组织培养细胞中泛素连接酶的研究。像BRCA1和Parkin这样的人类泛素连接酶底物的确定目前是一项艰巨的任务，这个系统应该会加速它们的发现，并揭示几种人类疾病的病因。

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.