Proteomics identification of ubiquitin enzyme substrates

描述（由申请人提供）：本项目的目标是建立一种通用方法，即连接酶底物（BULLS）的生物素和泛素（Ub）标记，用于定义体内单个泛素连接酶的特异性底物。在底物-E3相互作用期间，特异性底物将同时被生物素和泛素修饰，从而能够进行后续的亲和纯化和蛋白质组学鉴定。泛素是真核生物中一种重要的小分子蛋白质，调节几乎所有的细胞事件，其在神经发育和神经发病机制中的作用受到广泛重视。这种可逆的翻译后修饰是通过E1/E2/E3酶的酶促反应级联启动的，并被去泛素化酶（DUBs）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起着关键作用，并且Ub失调有助于神经系统疾病。然而，由于缺乏敏感的工具来识别这些Ub连接酶的相应底物，阻碍了对潜在分子机制的研究。我们已经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），重点是Ub信号在神经发育中的作用。在这里，我们建议发明一种双标记的方法来识别特定的基板修改个别E3酶的BULLS。我们将使用在神经元形态发生中起作用的E3连接酶思维炸弹1（Mib 1）来探索这一想法。两个具体目标是：（i）建立一种有效的体内生物素标记和体外纯化经连接酶修饰的特异性底物的方法;（ii）通过定量蛋白质组学系统地鉴定神经元中Mib 1底物。一旦开发，我们预计，这一战略将成为一个简单而敏感的方法来确定E3基板连接。 公共卫生相关性：泛素调节几乎所有的细胞事件，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的，在体内的标记方法，以确定特定的蛋白质底物修饰的泛素连接酶。该方法为构建泛素连接酶与底物的连接图谱提供了一种通用的方法，促进了泛素信号转导在神经科学中的研究。

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.