Global characterization of ubiquitin information networks with mass spectrometry

利用质谱法对泛素信息网络进行全局表征

• 批准号: 8500915
• 负责人: STEVEN P GYGI
• 金额: $ 45.73万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-15 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8500915
• 关键词: Address; Affect; Affinity Chromatography; Alleles; Alzheimer' s Disease; Antibodies; Area; Atlases; Biological; Biology; Canavanine; Carbon; Cataloging; Catalogs; Cell Cycle; Cell Cycle Progression; Cell physiology; Cells; Collection; Communities; Complex; Data; Data Set; Defect; Detection; Diabetes Mellitus; Digestion; Disease; Elements; Environment; Enzymes; Essential Genes; Ethanol; Etiology; Event; F-Box Proteins; Fungal Genome; Galactose; Genes; Genomics; Growth; Health; Heat-Shock Response; Human; Hydrogen Peroxide; Individual; Information Networks; Intervention; Label; Learning; Libraries; Ligase; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measurement; Measures; Metabolic Pathway; Metabolism; Methods; Molecular Profiling; N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase; Neurodegenerative Disorders; Nocodazole; Normal Cell; Pathway interactions; Phase; Physiology; Play; Positioning Attribute; Proteasome Inhibition; Protein Analysis; Protein-Protein Interaction Map; Proteins; Proteome; Proteomics; Regulation; Regulator Genes; Research; Resources; Role; Solutions; Source; Stress; System; Techniques; Technology; Therapeutic Intervention; Time; Trypsin; Ubiquitin; Variant; Work; Yeasts; base; design; enzyme substrate; protein expression; protein folding; protein protein interaction; public health relevance; research study; response; ubiquitin ligase; ubiquitin-protein ligase

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.

描述（由申请人提供）：泛素（UB）途径中的缺陷通常是导致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系统是一个强烈的研究领域。但是，UB生物学非常复杂，例如在人类中，它得到了500多个UB连接酶和95个去泛素酶（DUB）的支持。目前，这种复杂性以及我们对玩家及其互动的有限理解是针对许多疾病的有针对性干预的严重障碍。质谱法（MS）技术的最新进展为以公正和全球的方式表征整个UB信息网络的令人兴奋的可能性。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，从而允许相对容易的全局蛋白质组学测量和蛋白质表达操作。我们将收集围绕与UB依赖性酶（所有E3连接酶，F-box蛋白，DUB）的约120个基因中的每个基因的全局测量结果。我们将其称为“泛素调节”基因的UR120集。数百个全球数据集的收集需要多重以维持吞吐量。具体而言，在AIM 1中，我们将在各个UR120缺失菌株中介绍UB共轭物，包括十个条件的面板，其中泛素化在细胞反应和调节中起着特定的作用，从而鉴定出以条件依赖性方式发生的泛素化事件。在AIM 2中，在与AIM 1相同的十个条件下，UR120基因的缺失菌株将在蛋白质表达下进行全局分类，以检测蛋白质表达的稳定和动态变化。在AIM 3中，我们将开发一条管道，以大规模分析UB途径的蛋白质相互作用。在内源表达水平下，将使用带有HA标记的蛋白质创建UR120基因的库及其通过亲和力纯化（AP）-MS鉴定的相互作用。将检查相同的十个条件，以了解UR120相互作用网络如何应对细胞环境的变化。我们还将对连接酶和去偶联酶的非活性变体进行AP-MS分析，以稳定酶 - 基底配对。这项工作的最终结果将是一个三管齐下的信息网络，其中包括共轭分析，表达分析和物理相互作用研究。由此，我们将准备一份首先的草稿，o全面的地图，详细介绍了有关连接酶和配音的所有潜在酶 - 基底关系。通过检查与UB生物学直接相关的多种条件下的UR120，我们可以开始了解不同元素在正常细胞周期中如何合作以及响应扰动。这些数据将对蛋白质泛素化谱进行全面评估，并可用于发现酶 - 基底关系。完成后，我们的下一步将是为人类基因创建互补的信息网络。