SYSTEMATIC ANALYSIS OF THE DUB PROTEOME

描述（由申请人提供）：泛素系统控制许多细胞途径。蛋白质通过E1-E2-E3级联用泛素标记，这经常导致通过蛋白酶体降解。去泛素化酶（Dubs）的作用是从蛋白质中去除泛素，导致泛素化事件产生的信号输入逆转。少数Dubs与转录控制、肿瘤发生、肿瘤抑制和神经退行性变相关的重要细胞通路有关，但大多数Dubs的生物学功能、相互作用伴侣和底物尚不清楚。我们最近开发了一种新的信息和实验平台，用于蛋白质复合物的半高通量蛋白质组学分析，并将该技术应用于人类基因组编码的95个Dubs中的75个，使用293 T细胞作为初始细胞系统。这些研究表明，相当一部分的Dubs与以前鉴定的和新的蛋白质复合物稳定相关。在许多情况下，相关蛋白质的身份提供了生物功能或途径的第一个迹象，其中未表征的Dubs可能会运作。已经在基于shRNA的检查点筛选中鉴定了几种Dubs和/或其相关蛋白，为许多Dubs及其新鉴定的复合物提供了候选生物学途径。出现了几个主题：1）Dubs经常是大分子机器的组成部分，2）Dubs经常与E3相关，3）含有WD 40重复序列的蛋白质（包括最近被证明与泛素结合的WD 40蛋白质）经常与Dubs相关，鉴于最近发现WDR 48激活Usp 1，这是有趣的。我们的方法鉴定了许多紧密相关的蛋白质，但未鉴定出少量充分研究的Dubs的已知底物，这表明需要替代方法来鉴定Dubs的更弱结合的底物。该建议旨在进一步阐明Dubs控制的功能和途径。在目标1中，我们将完成我们的Dub蛋白质组的系统分析，并将检查“底物捕获”Dub突变体的蛋白质组，以确定弱结合底物的目标。还建议进一步加强和整合我们的信息平台和数据库。目的2的广泛目标是阐明Dubs功能和调节中的新兴主题，包括：1）WD 40蛋白作为Dubs的激活剂和底物受体亚基的作用，以及2）Dubs，E3和E3靶点的交叉调节。这些主题将使用我们通过蛋白质组学分析确定的2个特定Dub网络来解决。总之，这些实验将提供一个强大的资源领域致力于阐明的功能和目标的配音，并将开始发现重要的监管网络，似乎是普遍采用的调节配音功能。公共卫生相关性：泛素对蛋白质的修饰构成了细胞中蛋白质调节的主要模式，并构成了多种信号传导途径的基础。泛素的去除是许多途径中的关键步骤，并且可以导致通过信号传导途径的通量的改变。负责去除泛素的酶被称为去泛素化酶（Dubs）。一些Dubs与关键信号系统有关，包括p53调节，DNA损伤反应，DNA修复和神经退行性疾病。我们采用蛋白质组学来阐明与人类基因组中95个Dubs中的75个Dubs相关的蛋白质和蛋白质复合物。在许多情况下，我们提供了特定Dub功能的途径的第一个迹象。在这个提议中，我们试图通过在Dub靶标的系统分析中采用“底物捕获”突变体来继续阐明Dub途径，并且我们提议阐明迄今为止从我们的蛋白质组学分析中出现的2个主要调控主题：1）由WD 40蛋白激活Dub和由Dub相互调控E3。这项工作将继续加强我们对Dubs功能和监管的系统性理解。

DESCRIPTION (provided by applicant): The ubiquitin system controls many cellular pathways. Proteins are tagged with ubiquitin via an E1-E2-E3 cascade, and this frequently leads to degradation via the proteasome. Deubiquitinating enzymes (Dubs) act to remove ubiquitin from proteins, leading to reversal of the signaling input generated by the ubiquitination event. A small number of Dubs have been linked to important cellular pathways linked to transcriptional control, oncogenesis, tumor suppression, and neurodegeneration, yet the biological functions, interaction partners, and substrates of the majority of Dubs are unknown. We have recently developed a new informatic and experimental platform for semi high-throughput proteomic analysis of protein complexes and have applied this technology to 75 of the 95 Dubs encoded by the human genome, using 293T cells as an initial cell system. These studies reveal that a substantial fraction of Dubs are stably associated with previously identified and novel protein complexes. In many cases, the identity of associated proteins provides the first indications of the biological functions or pathways in which uncharacterized Dubs may operate. Several Dubs and/or their associated proteins have been identified in shRNA-based checkpoint screens, providing candidate biological pathways for many Dubs and their newly identified complexes. Several themes have emerged: 1) Dubs are frequently components of large molecular machines, 2) Dubs frequently associate with E3s, and 3) WD40- repeat containing proteins (including WD40 proteins that have recently been shown to bind ubiquitin) are frequently associated with Dubs, which is interesting in light of the recent finding that WDR48 activates Usp1. Our approach identified many tightly associated proteins but known substrates of a small number of well- studied Dubs were not identified, suggesting that alternative approaches are required to identify more weakly bound substrates of Dubs. This proposal seeks to further elucidate the functions and pathways controlled by Dubs. In aim 1,we will complete our systematic analysis of the Dub proteome, and will examine the proteome of "substrate-trapping" Dub mutants with the goal of identifying weakly bound substrates. Further enhancement and integration of our informatics platform and database is also proposed. Aim 2 has the broad goal of elucidating emerging themes in the function and regulation of Dubs, including: 1) the role of WD40 proteins as activator and substrate receptor subunits of Dubs, and 2) the cross-regulation of Dubs, E3s, and targets of E3s. These themes will be addressed using 2 specific Dub networks we have identified via our proteomics analysis. Together, these experiments will provide a powerful resource for the field devoted to elucidating the functions and targets of Dubs and will begin to uncover important regulatory networks that appear to be commonly employed to regulate Dub function. PUBLIC HEALTH RELEVANCE: Modification of proteins by ubiquitin constitutes a primary mode of protein regulation in cells and underlies diverse signaling pathways. Removal of ubiquitin is a key step in many pathways and can lead to alterations in the flux through signaling pathways. The enzymes responsible for ubiquitin removal are referred to as deubiquitinating enzymes (Dubs). Several Dubs have been implicated in key signaling systems, including p53 regulation, the DNA damage response, DNA repair, and neurodegenerative diseases. We have employed proteomics to elucidate the proteins and protein complexes associated with 75 of the 95 Dubs present in the human genome. In many cases, we provide the first indications of the pathways within which a particular Dub functions. In this proposal, we seek to continue to elucidate Dub pathways by employing "substrate trapping" mutants in a systematic analysis of Dub targets, and we propose to elucidate 2 major regulatory themes that have emerged from our proteomic analysis thus far: 1) activation of Dubs by WD40 proteins and reciprocal regulation of E3s by Dubs. This work will continue to strengthen our understanding of the functions and regulation of Dubs in a systematic manner.