Quantitation of Ubiquitin Dynamics and Homeostasis

 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是一类通过共价连接调节其他蛋白质达特蛋白质家族的原型。与其他翻译后修饰一样，Ub结合是可逆的，并且受到严格控制。Ub和polyUb信号用于多种过程，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损伤反应途径。在确定Ub在这些和许多其他过程中的作用的结构和生物化学基础方面已经取得了相当大的进展，但我们对Ub介导的途径如何控制和相互作用的了解却非常不完整。这个建议的基本前提是，如果我们要了解细胞内的泛素依赖性信号，了解细胞生长或应激条件下游离和结合的泛素水平，以及如何调节通过这些池的Ub流量是必要的。我们的目标是将联合收割机方法和资源开发与重点研究的Ub稳态，polyUb稳定性和编辑，以及组蛋白泛素化的调控相结合。一个具体的长期目标是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传导途径的调节节点的想法，即使在那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的工具，这将是第一次，将能够定量测量（1）在活细胞中的游离或结合的Ub的分布，和（2）通过特定的蛋白质结合物的Ub的运动。这些测量将在培养的酵母或哺乳动物细胞中进行，以评估生长期间和响应于各种应激条件的Ub动力学，以监测Ub通过不同形式的聚Ub和Ub-组蛋白缀合物的绝对通量，并全面确定相对通量（即，稳定性）。一个数学模型，以描述通过其不同的生化形式的Ub的运动将细化细胞内的Ub浓度和流量的测量的基础上。将测试该模型预测突变和Ub途径的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.