Structural Biology of the Ubiquitin Conjugation System

 描述（由申请人提供）：泛素（Ub）对蛋白质的可逆翻译后修饰是一种调控机制，几乎控制真核生物细胞功能的所有方面。UB缀合改变靶蛋白的性质，例如其稳定性、亚细胞定位、分子间相互作用和构象/活性。正是通过这些作用的组合，Ub与关键靶蛋白的缀合调节细胞周期控制、信号转导、免疫和分化等过程。Ub途径与人类健康的相关性通过以下事实来强调：其失调与诸如癌症、神经系统疾病、心血管疾病和免疫疾病等病理学有关，并且其是FDA批准的用于延长多发性骨髓瘤患者生命的药物的癌症治疗干预的经验证的靶标。通过E1、E2和E3三种酶的顺序相互作用和活性，Ub与靶蛋白的缀合进行。E1执行两个主要功能：（1）在两步过程中激活Ub，导致在Ub和E1催化半胱氨酸之间形成硫酯键，以及（2）募集E2酶，然后将Ub转移到E2催化半胱氨酸（硫酯转移）。在E1-E2硫酯转移后，所得E2-Ub中间体与Ub E3连接酶的三个不同家族的成员相互作用，所述E3连接酶通过不同的机制催化Ub与靶蛋白的缀合。在通过环间环（RBR）E3家族成员催化Ub缀合之前，Ub必须以与E1-E2硫酯转移类似的机制从E2催化半胱氨酸转移到RBR E3催化半胱氨酸，但其结构基础未知。在人类中，有两种Ub E1（Uba 1和Uba 6）对30多种不同的Ub E2表现出重叠但不同的特异性，并且有~12个RBR E3家族成员与E2-Ub中间体的重叠但不同的子集一起发挥作用。特异性是必不可少的Ub信号的完整性，然而，在预测的接触位点E1，E2和RBR E3的保守性差，表明在E1-E2和E2-RBR接口，排除了我们的能力，建立规则的基础上序列分析的特异性。通过使用生物化学，生物物理学和结构方法，本提案中的研究旨在建立E1-E2（Aim 1）和E2-RBR E3（Aim 2）相互作用中的分子识别规则，并确定Ub在最终与靶蛋白缀合之前从E1转移到E2再到RBR E3的结构基础。

 DESCRIPTION (provided by applicant): Reversible post-translational modification of proteins by ubiquitin (Ub) is a regulatory mechanism that controls nearly all aspects of cellular function i eukaryotes. Ub conjugation alters properties of the target protein such as its stability, subcellulr localization, intermolecular interactions, and conformation/activity. It is through a combination o these effects that Ub conjugation to key target proteins regulates processes such as cell cycle control, signal transduction, immunity, and differentiation. The relevance of the Ub pathway to human health is underscored by the fact that its dysregulation is implicated in pathologies such as cancers, 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 conjugation to target proteins proceeds through the sequential interactions and activities of three enzymes, E1, E2, and E3. E1 performs two main functions: (1) activation of Ub in a two-step process that results in the formation of a thioester bond between Ub and the E1 catalytic cysteine, and (2) recruitment of E2 enzymes followed by transfer of Ub to an E2 catalytic cysteine (thioester transfer). After E1-E2 thioester transfer, the resulting E2-Ub intermediate interacts with members of three different families of Ub E3 ligases that catalyze Ub conjugation to target proteins by distinct mechanisms. Prior to catalysis of Ub conjugation by RING-between-RING (RBR) E3 family members, Ub must be transferred from the E2 catalytic cysteine to an RBR E3 catalytic cysteine in a process that is mechanistically analogous to E1-E2 thioester transfer, but the structural basis for which i unknown. In humans, there are two Ub E1s (Uba1 and Uba6) that exhibit overlapping but distinct specificities for more than 30 different Ub E2s and there are ~12 RBR E3 family members that function with overlapping but distinct subsets of E2-Ub intermediates. Specificity is essential to the integrity of Ub signaling, however, poor conservation across E1s, E2s, and RBR E3s at predicted contact sites suggests structural plasticity at E1-E2 and E2-RBR interfaces that precludes our ability to establish the rules governing specificity based on sequence analysis. Through use of biochemical, biophysical, and structural approaches, the research in this proposal aims establish the rules governing molecular recognition in E1-E2 (Aim 1) and E2-RBR E3 (Aim 2) interactions and to determine the structural basis by which Ub is transferred from E1 to E2 to RBR E3 prior to finally being conjugated to the target protein.