Global characterization of ubiquitin information networks with mass spectrometry

描述（由申请人提供）：泛素（Ub）通路的缺陷通常是癌症和破坏性神经退行性疾病的原因。由于其在生物回路中的核心作用和治疗干预的潜力，Ub系统是一个激烈的研究领域。然而，Ub生物学是高度复杂的，例如在人类中，它由超过500个Ub连接酶和95个去泛素酶（DUBs）支持。目前，这种复杂性和我们对参与者及其相互作用的有限理解严重阻碍了对许多疾病进行有针对性的干预。基于质谱（MS）技术的最新进展为以公正和全球的方式表征整个Ub信息网络开辟了令人兴奋的可能性。虽然从长远来看，我们的研究将在人类细胞中进行，但我们将首先在酵母中表征该网络，这使得相对容易的全球蛋白质组学测量和蛋白质表达操作。我们将收集约120个与ub依赖性酶相关的基因（所有E3连接酶，F-box蛋白，dub）的全球测量数据。我们称之为UR120组“泛素调节”基因。数百个全局数据集的收集需要多路复用以维持吞吐量。具体来说，在Aim 1中，我们将分析单个UR120缺失菌株中的Ub偶联物，包括十组泛素化在细胞反应和调节中起特定作用的条件，从而确定泛素化事件以条件依赖的方式发生。在Aim 2中，UR120基因缺失菌株将在与Aim 1相同的10个条件下进行全局蛋白表达谱分析，以检测蛋白表达的稳定和动态变化。在Aim 3中，我们将开发针对Ub途径的蛋白质相互作用的大规模分析管道。用ha标记的内源性表达水平的蛋白建立UR120基因文库，并通过亲和纯化(AP)-MS鉴定其相互作用。同样的十个条件将被检查，以了解UR120相互作用网络如何响应细胞环境的变化。我们还将对连接酶和脱醌酶的无活性变体进行AP-MS分析，以稳定酶-底物配对。这项工作的最终结果将是一个由共轭谱、表达谱和物理相互作用研究组成的三管齐下的信息网络。在此基础上，我们将为连接酶和dub的所有潜在酶-底物关系的综合图谱准备第一份草稿。通过检查与Ub生物学直接相关的多种条件下的UR120集，我们可以开始了解不同元素如何在整个正常细胞周期中合作并响应扰动。这些数据将提供蛋白质泛素化谱的全面评估，并可用于揭示酶-底物关系。一旦完成，我们的下一步将是创建人类基因的互补信息网络。

DESCRIPTION (provided by applicant): Defects in ubiquitin (Ub) pathways are often responsible for cancer and devastating neurodegenerative diseases. Because of its central role in biological circuits and the potential for therapeutic intervention, the Ub system is an intense research area. However, Ub biology is highly complex, e.g. in humans it is supported by over 500 Ub ligases and 95 deubiquitinases (DUBs). Currently, this complexity and our limited understanding of players and their interactions are a severe hindrance for targeted intervention for many diseases. Recent advances in mass spectrometry (MS)-based technologies open up the exciting possibility to characterize the entire Ub information network in an unbiased and global manner. While in the long term our studies will be performed in human cells, we will first characterize this network in yeast, which allows comparatively easy global proteomic measurements and protein expression manipulations. We will collect global measurements surrounding each of ~120 genes implicated as Ub-dependent enzymes (all E3 ligases, F-box proteins, DUBs). We term this the UR120 set for "ubiquitin-regulatory" genes. The collection of hundreds of global datasets requires multiplexing to sustain throughput. Specifically, in Aim 1, we will profile Ub conjugates across individual UR120 deletion strains including a panel of ten conditions where ubiquitylation plays specific roles in cellular response and regulation, thus identifying ubiquitylation events occurring in a condition-dependent manner. In Aim 2, deletions strains for the UR120 genes will be globally profiled for protein expression under the same ten conditions as in Aim 1 to detect stable and dynamic changes in protein expression. In Aim 3, we will develop a pipeline for the large-scale analysis of protein interactions specific to the Ub pathway. A library of the UR120 genes will be created with HA-tagged proteins under endogenous expression levels and their interactions identified by affinity purification (AP)-MS. The same ten conditions will be examined to learn how the UR120 interaction network responds to changes in cellular environment. We will also perform AP-MS analyses for inactive variants of ligases and dequbiquitinases to stabilize enzyme-substrate pairing. The final result of this work will be a three-pronged information network comprised of conjugate profiling, expression profiling, and physical interaction studies. From this, we will prepare a first-of-its-kind draft o a comprehensive map of all potential enzyme-substrate relationships for ligases and DUBs. By examining the UR120 set under multiple conditions directly relevant to Ub biology, we can begin to understand how different elements cooperate throughout a normal cell-cycle and in response to perturbations. These data will provide a comprehensive assessment of the protein ubiquitylation profiles and can be used to uncover enzyme-substrate relationships. Upon completion, our next step will be to create the complementary information networks for human genes.