Proteomics identification of ubiquitin enzyme substrates

描述（由申请人提供）：该项目的目标是建立一种通用方法，连接酶底物（BULLS）的生物素和泛素（Ub）标记，用于定义体内各个泛素连接酶的特定底物。在底物-E3相互作用过程中，特定底物将同时被生物素和泛素修饰，从而实现后续的亲和纯化和蛋白质组学鉴定。泛素是真核细胞中必需的小蛋白，调节几乎所有细胞事件，其在神经发育和神经发病机制中的作用得到广泛认可。这种可逆的翻译后修饰是由 E1/E2/E3 酶的一系列酶促反应引发的，并被去泛素化酶 (DUB) 去除。人类蛋白质组包含两种 Ub E1 酶、约 40 种不同的 E2、至少 600 种 E3 连接酶和约 95 种 DUB。 Ub 信号传导的特异性主要归因于不同 E3 连接酶对蛋白质底物的选择性修饰。遗传和生化研究表明，越来越多的 Ub 连接酶在神经发育中发挥着关键作用，而 Ub 失调会导致神经系统疾病。然而，由于缺乏识别这些 Ub 连接酶相应底物的敏感工具，对潜在分子机制的研究受到阻碍。我们开发了一系列基于质谱（MS）的技术来分析泛素化蛋白质组（ubiquitinome），重点关注Ub信号在神经发育中的作用。在这里，我们建议发明一种 BULLS 的双重标记方法，用于识别由单个 E3 酶修饰的特定底物。我们将使用在神经元形态发生中发挥作用的 E3 连接酶 Mind Bomb 1 (Mib1) 来探索这个想法。两个具体目标是：(i) 建立一种有效的方法，用于体内生物素标记和连接酶修饰的特定底物的体外纯化，(ii) 通过定量蛋白质组学系统地识别神经元中的 Mib1 底物。一旦开发出来，我们预计该策略将成为确定 E3 基板连接的简单而灵敏的方法。 公共健康相关性：泛素调节几乎所有细胞事件，包括神经发育、突触可塑性、学习和记忆。我们建议开发一种新颖的体内标记方法来识别泛素连接酶修饰的特定蛋白质底物。该方法将为建立泛素连接酶和底物之间的连接图提供通用方法，促进神经科学中泛素信号传导的研究。

DESCRIPTION (provided by applicant): The goal of this project is to establish a generic approach, biotin and ubiquitin (Ub) labeling of ligase substrates (BULLS), for defining specific substrates of individual ubiquitin ligases in vivo. Specific substrates will be modified by biotin and ubiquitin simultaneously during the substrate-E3 interaction, enabling subsequent affinity purification and proteomics identification. Ubiquitin, an essential small protein in eukaryotic cels, regulates nearly all cellular events, and its role in neurodevelopment and neurological pathogenesis is widely appreciated. This reversible posttranslational modification is initiated by a cascade of enzymatic reactions through E1/E2/E3 enzymes, and is removed by deubiquitinating enzymes (DUBs). The human proteome contains two Ub E1 enzymes, ~40 different E2s, at least 600 E3 ligases and ~95 DUBs. The specificity in Ub signaling is primarily attributed to selective modification of protein substrates by diverse E3 ligases. Genetic and biochemical studies reveal that a growing list of Ub ligases plays critical roles in neurodevelopment, and that Ub dysregulation contributes to neurological disorders. The investigation of underlying molecular mechanisms, however, is hampered by the lack of sensitive tools to identify corresponding substrates of these Ub ligases. We have developed a series of mass spectrometry (MS)-based technologies to analyze the ubiquitinated proteome (ubiquitinome), with a focus on the role of Ub signaling in neurodevelopment. Here we propose to invent a dual labeling approach of BULLS for identifying specific substrates modified by individual E3 enzymes. We will explore this idea using the E3 ligase mind bomb 1 (Mib1) that functions in neuronal morphogenesis. Two specific aims are: (i) to set up an efficient approach for in vivo biotin labeling and in vitro purification of specific substrates modified by a ligase, nd (ii) to systematically identify Mib1 substrates in neurons by quantitative proteomics. Once developed, we anticipate that this strategy will become a simple and sensitive approach for determining the E3-substrate connection. PUBLIC HEALTH RELEVANCE: Ubiquitin regulates nearly all cellular events including neurodevelopment, synaptic plasticity, learning and memory. We propose to develop a novel, in vivo labeling method to identify specific protein substrates modified by ubiquitin ligases. The method will provide a generic approach for establishing the connection map between ubiquitin ligases and substrates, promoting the investigation of ubiquitin signaling in neuroscience.