Quantitation of Ubiquitin Dynamics and Homeostasis

泛素动力学和稳态的定量

• 批准号: 9315902
• 负责人: Robert Cohen
• 金额: $ 44.88万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9315902
• 关键词: Acute; Affinity; Alpha Cell; Biochemical; Biochemical Pathway; Cell Cycle Regulation; Cell Extracts; Cells; Cellular Stress; Chromatin Structure; DNA Damage; Databases; Defect; Deubiquitinating Enzyme; Development; Equipment and supply inventories; Eukaryota; Fluorescent Dyes; Gene Expression; Genetic Transcription; Goals; Growth; Hela Cells; Histone H2B; Histones; Homeostasis; Human; Individual; Inflammation; Investigation; Label; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Measurement; Measures; Mediating; Methods; Mitochondria; Modeling; Monitor; Movement; Mutation; Natural Immunity; Neurodegenerative Disorders; Neurons; Pathway interactions; Peptides; Physiologic pulse; Polyubiquitin; Post-Translational Protein Processing; Process; Proteasome Inhibition; Protein Family; Proteins; RNA Polymerase II; Reagent; Regulation; Reporting; Resource Development; Role; Saccharomycetales; Signal Pathway; Signal Transduction; Site; Stress; Synapses; Testing; Time; Ubiquitin; Ubiquitination; Water; Yeasts; base; cell growth; experimental study; human disease; in vitro Assay; mathematical model; method development; multicatalytic endopeptidase complex; protein degradation; protein transport; proteotoxicity; public health relevance; response; sensor; tat Protein; thioester; tool

 DESCRIPTION (provided by applicant): In all eukaryotes, numerous pathways and signaling networks depend upon the protein ubiquitin (Ub) to regulate the amounts, localization, interactions, or activities of thousands of proteins. Ub is the archetype of a family of proteins tat regulate other proteins by covalent attachment. Like other post-translational modifications, Ub conjugation is reversible and tightly controlled. Ub and polyUb signals are used in diverse processes that include intracellular protein degradation, cell cycle control, inflammation and innate immunity, protein trafficking, chromatin structure and gene expression, and DNA damage response pathways. Considerable progress has been made to define the structural and biochemical bases for Ub's role in these and many other processes, yet our picture of how Ub-mediated pathways are controlled and interact is woefully incomplete. The fundamental premise of this proposal is that knowing the levels of free and conjugated ubiquitin under conditions of cell growth or stress, and how the flux of Ub through those pools is regulated, are necessary if we are to understand intracellular Ub-dependent signaling. Our Aims combine method and resource development with focused investigations of Ub homeostasis, polyUb stability and editing, and the regulation of histone ubiquitination. A specific long-term goal is to test the ide that changes in Ub flux through specific protein conjugates can identify regulatory nodes of signaling pathways, even in cases where steady-state levels of those conjugates do not change. We propose to develop and apply new tools that, for the first time, will enable quantitative measurements of (1) the distribution of free or conjugated Ub in live cells, and (2) the movement of Ub through specific protein conjugates. These measurements will be made in cultured yeast or mammalian cells to evaluate Ub dynamics during growth and in response to various stress conditions, to monitor the absolute flux of Ub through different forms of polyUb and Ub-histone conjugates, and to determine globally the relative fluxes (i.e., stabilities) of Ub among the cell' inventory of Ub-protein conjugates. A mathematical model developed to describe Ub's movement through its different biochemical forms will be refined based on measurements of intracellular Ub concentrations and flux. The ability of the model to predict effects of mutations and other perturbations of Ub pathways will be tested to evaluate its accuracy and utility.

 描述（由申请人提供）：在所有真核生物中，许多途径和信号网络依赖于蛋白质泛素（Ub）来调节数千种蛋白质的数量、定位、相互作用或活性，Ub 是蛋白质家族的原型。与其他翻译后修饰一样，Ub 缀合是可逆的并且受到严格控制，可用于包括细胞内蛋白质在内的多种过程。降解、细胞周期控制、炎症和先天免疫、蛋白质运输、染色质结构和基因表达以及 DNA 损伤反应途径在确定 Ub 在这些过程和许多其他过程中的作用的结构和生化基础方面已经取得了相当大的进展，但我们的研究还没有取得进展。不幸的是，关于 Ub 介导的途径如何被控制和相互作用的图景是不完整的，该提议的基本前提是了解细胞生长或应激条件下游离和结合的泛素的水平，以及 Ub 如何通过这些条件。如果我们要了解细胞内 Ub 依赖性信号传导，我们的目标是将方法和资源开发与 Ub 稳态、多聚 Ub 稳定性和编辑以及组蛋白泛素化调节的重点研究结合起来。为了测试通过特定蛋白质缀合物的 Ub 通量的变化可以识别信号通路的调节节点，即使在这些缀合物的稳态水平不改变的情况下，我们建议开发和应用新工具。这将首次能够测量（1）活细胞中游离或结合的 Ub 的分布，以及（2）Ub 通过特定蛋白质结合物的运动。这些测量将在培养的酵母或哺乳动物细胞中进行。评估生长过程中和响应各种应激条件的 Ub 动态，通过不同形式的多聚 Ub 和 Ub-组蛋白缀合物监测 Ub 的绝对通量，并总体确定 Ub 的相对通量（即稳定性）为描述 Ub 通过其不同生化形式的运动而开发的数学模型将根据细胞内 Ub 浓度和通量的测量进行完善，该模型预测突变和其他扰动的影响的能力。将测试 Ub 通路以评估其准确性和实用性。