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。由于它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种Ub连接酶和95种去泛素化酶（DUB）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了解严重阻碍了对许多疾病的靶向干预。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信息网络提供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这个网络，这使得相对容易的全局蛋白质组学测量和蛋白质表达操纵成为可能。我们将收集全球测量约120个基因涉及的Ub依赖性酶（所有E3连接酶，F-box蛋白，DUB）。我们称之为“泛素调节”基因的UR 120集。数百个全球数据集的收集需要多路复用来维持吞吐量。具体而言，在目标1中，我们将分析单个UR 120缺失菌株中的Ub缀合物，包括一组10种条件，其中泛素化在细胞应答和调节中起特定作用，从而鉴定以条件依赖性方式发生的泛素化事件。在目标2中，将在与目标1相同的10种条件下对UR 120基因缺失菌株的蛋白质表达进行全局分析，以检测蛋白质表达的稳定和动态变化。在目标3中，我们将开发一个管道，用于大规模分析特定于Ub途径的蛋白质相互作用。将用HA标记的蛋白质在内源表达水平下创建UR 120基因的文库，并通过亲和纯化（AP）-MS鉴定它们的相互作用。将检查相同的十种条件以了解UR 120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dequbiquitinases的失活变体进行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三管齐下的信息网络，包括共轭分析，表达分析和物理相互作用的研究。由此，我们将准备一个第一个的草案，一个全面的地图的所有潜在的酶底物关系的连接酶和DUB。通过在与Ub生物学直接相关的多种条件下检查UR 120集，我们可以开始理解不同元素如何在正常细胞周期中合作以及对扰动的反应。这些数据将提供一个全面的评估的蛋白质泛素化概况，并可用于揭示酶-底物的关系。完成后，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.