Structural Biology of the Ubiquitin Conjugation System

项目摘要 泛素（Ub）对蛋白质的翻译后修饰是一种调控机制，几乎控制了所有的蛋白质翻译后修饰。 真核细胞生物学方面。泛素化改变靶蛋白的性质，如稳定性、亚细胞 定位、分子间相互作用和活性，从而调节细胞周期等过程 控制、DNA修复、信号转导和免疫。Ub信号与人类健康的相关性是 其失调与病理学如癌症、神经系统疾病、 它是一种有效的治疗靶点， 用FDA批准的药物干预癌症，延长多发性骨髓瘤患者的生命。 Ub信号传导需要三种酶E1、E2和E3的连续相互作用和活性，这三种酶作用于 串联以将Ub缀合至靶蛋白。人类携带两个Ub E1，Uba 1和Uba 6，催化Ub 活化和硫酯转移到数十个E2的不同库。虽然Uba 1完全致力于Ub 虽然Uba 6能够激活FAT 10（一种参与细胞凋亡的Ub样蛋白），但它是非常不寻常的，因为它也能够激活FAT 10。 有丝分裂进展和免疫），随后将其转移到高度Uba 6特异性的E2，UBE 2 Z。 维持Ub信号传导的完整性是必不可少的，然而Ub的Uba 6混杂的潜在机制 以及E1/E2相互作用中特异性/混杂性的分子规则仍然很差 明白在E1-E2硫酯转移后，E2~Ub中间体与数百个不同的库相互作用。 E3连接酶分为三个家族，催化靶蛋白作为单一分子的泛素化， 作为通过Ub上的特定赖氨酸残基连接在一起的聚合链。因为它是一个主要决定因素 泛素化的功能结果，控制在底物蛋白上组装的polyUb链的类型， 对于RING E3催化的反应，polyUb链特异性主要由E2决定。 它们的功能。尽管有这种根本的重要性， 大多数polyUb连接类型催化特异性仍然未知。 通过使用结构，生物化学/生物物理和基于细胞的方法，该提案旨在发现： 1)人Ub E1酶的底物识别和催化活性的结构基础2）催化活性 Ub E1/E2中E1-E2硫酯转移的机制和控制特异性/混杂性的分子规则 E2/RING E3对催化特定类型polyUb链的机制。 UB信号传导是癌症和其他人类病理学治疗干预的靶点， 了解E1，E2和E3如何共同控制将导致的基本细胞过程 所提出的研究可以为开发新的小分子治疗剂提供平台。

Project Summary Posttranslational modification of proteins by ubiquitin (Ub) is a regulatory mechanism that controls nearly all aspects eukaryotic cell biology. Ubiquitination alters properties of target proteins such as stability, subcellular localization, intermolecular interactions, and activity and thereby regulates processes such as cell cycle control, DNA repair, signal transduction, and immunity. The relevance of Ub signaling to human health is underscored by the fact that its dysregulation is implicated in pathologies such as cancer, neurological disorders, cardiovascular disease, and immune disorders and that it is a validated target for therapeutic intervention in cancer with FDA-approved medications extending the lives of multiple myeloma patients. Ub signaling requires the sequential interactions and activities of three enzymes, E1, E2, and E3, which act in tandem to conjugate Ub to target proteins. Humans harbor two Ub E1s, Uba1 and Uba6, that catalyze Ub activation and thioester transfer to distinct repertoires of tens of E2s. While Uba1 is fully dedicated to Ub activation, Uba6 is highly unusual in that it is also capable of activating FAT10 (a Ub-like protein involved in mitotic progression and immunity), and subsequently transferring it to a highly Uba6-specific E2, UBE2Z. Maintenance of the integrity of Ub signaling is essential, yet mechanisms underlying Uba6 promiscuity for Ub and FAT10, as well as the molecular rules governing specificity/promiscuity in E1/E2 interactions remain poorly understood. After E1-E2 thioester transfer, E2~Ub intermediates interact with distinct repertoires of hundreds of E3 ligases grouped into three families that catalyze ubiquitination of target proteins as a single molecule or as polymeric chains linked together by specific lysine residues on Ub. Because it is a major determinant of the functional outcome of ubiquitination, control of the type of polyUb chains assembled on substrate proteins is essential and for reactions catalyzed by RING E3s, polyUb chain specificity is largely determined by the E2 with which they function. Despite this fundamental importance, the molecular mechanisms governing specificity in catalysis of most polyUb linkage types remain unknown. Through use of structural, biochemical/biophysical, and cell-based approaches, this proposal aims to discover: 1) the structural basis for substrate recognition and catalytic activities of human Ub E1 enzymes 2) the catalytic mechanism of E1-E2 thioester transfer and molecular rules governing specificity/promiscuity in Ub E1/E2 interactions, and 3) mechanisms by which specific types of polyUb chains are catalyzed by E2/RING E3 pairs. Ub signaling is a target for therapeutic intervention in cancer and other human pathologies and the deeper understanding of how E1, E2, and E3 work together to control essential cellular processes that will result from the proposed studies could provide a platform for the development of novel small molecule therapeutics.