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（由适用提供）：在所有真核生物中，许多途径和信号网络都取决于蛋白质泛素（UB）来调节数千种蛋白质的量，定位，相互作用或活性。 Ub是通过共价附着来调节其他蛋白质家族的原型。像其他翻译后修改一样，UB共轭是可逆的，并且受到严格控制。 UB和Polyub信号用于包括细胞内蛋白质降解，细胞周期控制，注射和先天免疫，蛋白质运输，蛋白质结构和基因表达以及DNA损伤反应途径的不同过程。已经取得了相当大的进步来定义UB在这些和许多其他过程中的作用的结构和生化基础，但是我们关于如何控制UB介导的途径和相互作用的图片是严重的。该提案的基本前提是，如果我们要理解和细胞内UB依赖性信号传导，则必须了解细胞生长或压力的自由和偶联泛素的水平，以及如何通过这些池的通量来调节UB的通量。我们的目标将方法和资源开发与UB体内稳态，polyub稳定性和编辑以及组蛋白泛素化的调节相结合。一个特定的长期目标是测试IDE，即使在这些结合物的稳态水平不变的情况下，也可以通过特定的蛋白质结合物在特定的蛋白质结合物中变化可以识别信号传导途径的调节节点。我们建议开发和应用新工具，这首先将实现（1）活细胞中游离或共轭UB的分布的定量测量，以及（2）UB通过特定蛋白质结合物的运动。 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., conditions) of Ub among the cell' inventory of Ub-protein conjugates.开发出一种数学模型来描述UB通过其不同的生化形式的运动，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UB途径扰动的影响的能力将进行测试以评估其准确性和效用。