Quantitation of Ubiquitin Dynamics and Homeostasis

 描述(申请人提供)：在所有真核生物中，无数的通路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是一个蛋白质家族的原型，TAT通过共价结合来调节其他蛋白质。与其他翻译后修饰一样，Ub共轭是可逆的，并且受到严格控制。Ub和PolyUb信号被用于不同的过程，包括细胞内蛋白质降解、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损伤反应通路。在确定Ub在这些和许多其他过程中作用的结构和生化基础方面已经取得了相当大的进展，但我们对Ub介导的途径是如何控制和相互作用的图景还很不完整。这一建议的基本前提是，如果我们要了解细胞内Ub依赖的信号转导，就必须知道细胞生长或应激条件下游离和结合泛素的水平，以及Ub通过这些池的流量是如何调节的。我们的目标是将方法和资源开发与Ub动态平衡、PolyUb稳定性和编辑以及组蛋白泛素化调控的重点研究结合起来。一个特定的长期目标是测试IDE，即通过特定蛋白质结合物改变Ub通量可以识别信号通路的调节节点，即使在这些结合物的稳定水平没有改变的情况下也是如此。我们建议开发和应用新的工具，这将首次使定量测量(1)活细胞中游离或结合的Ub的分布，以及(2)通过特定的蛋白质结合物移动Ub。这些测量将在培养的酵母或哺乳动物细胞中进行，以评估Ub在生长过程中和对各种应激条件的反应动态，监测通过不同形式的PolyUb和Ub-组蛋白结合物的Ub的绝对通量，并确定Ub在细胞的Ub-蛋白质结合物库存中的全局相对通量(即稳定性)。为描述Ub通过其不同的生化形式移动而开发的数学模型将基于对细胞内Ub浓度和通量的测量进行改进。将测试该模型预测Ub途径突变和其他扰动的影响的能力，以评估其准确性和实用性。

 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.