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几乎所有 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 突出的事实是，它的调节失调与癌症、神经疾病等病理有关 疾病，心血管疾病和免疫紊乱，它是一个有效的治疗目标 用FDA批准的延长多发性骨髓瘤患者生命的药物干预癌症。 UB信号需要三种酶E1、E2和E3的顺序相互作用和活性，这三种酶在 串联将Ub与靶蛋白偶联。人类有两个Ub E1s，Uba1和Uba6，它们催化Ub 活化和硫酯转移到数十个E2的不同谱系。而Uba1完全致力于Ub 激活，Uba6是非常不寻常的，因为它也能够激活FAT10(一种参与 有丝分裂进程和免疫)，并随后将其转移到高度Uba6特异性的E2，UBE2Z。 维护Ub信令的完整性是必不可少的，但Uba6混杂Ub的机制 和FAT10，以及控制E1/E2相互作用中的特异性/混杂的分子规则仍然很差 明白了。在E_1-E_2硫酯转移后，E_2~Ub中间体与数百种不同的酶系相互作用 E3连接酶分为三个家族，它们作为单个分子催化目标蛋白的泛素化 通过Ub上特定的赖氨酸残基连接在一起的聚合链。因为它是一个主要的决定因素 泛素化的功能结果，控制组装在底物蛋白质上的多聚Ub链的类型是 对于E3S环催化的反应，PolyUb链的特异性在很大程度上由E2决定 它们的功能与之相关。尽管有这种根本的重要性，但支配的分子机制 大多数PolyUb连锁类型在催化中的特异性仍不清楚。 通过使用结构、生化/生物物理和基于细胞的方法，该提案旨在发现： 1)人Ub-E1酶底物识别和催化活性的结构基础2)催化 UbE_1/E_2硫酯转移机制及专一性/杂交性的分子调控 相互作用，以及3)特定类型的多聚Ub链被E2/环E3对催化的机制。 UB信号是癌症和其他人类病理以及更深层次疾病治疗干预的靶点 了解E1、E2和E3如何协同工作来控制基本的细胞过程，这将导致 这些研究为新的小分子疗法的发展提供了平台。

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.