Investigation of the proteasome assembly landscape

项目摘要摘要 26S蛋白酶体进行最受调控的蛋白质降解，并在体内消除有毒蛋白质。这个 蛋白酶体是一种异常庞大和复杂的依赖于ATP的蛋白酶，由近70个个体组成 多肽亚基。尽管传统的观点认为蛋白酶体是由这些蛋白质组装而成 来自我们小组和其他人的最新证据出人意料地表明，亚基是单一的、严格的逐步序列 在活体中，组装路线可能会有更广阔的“景观”。虽然这种可能性还没有得到检验， 这样的集合格局将确保这一基本的生物过程能够在 面对组装障碍，并将提供一种强大的手段来调整蛋白酶体的速度或体积 对细胞环境作出反应的生物发生。对利用蛋白酶体的兴趣与日俱增 帮助治疗癌症和神经退行性疾病等各种疾病的生物发生。理解 这样的集会环境是否存在，如果存在，如何利用它来确保快速和忠实 蛋白酶体生物发生，将是关键，以指导这种组装靶向治疗的发展。 这个多PI应用程序的目标是测试蛋白酶体组装环境存在于 在这一景观中，可能的路线的相对流量在很大程度上受动力学因素的影响 这种变化是对细胞内环境的反应。通过将PI各自的专业知识结合在一起 蛋白酶体生物学以及在酶动力学和单分子生物物理学中，我们希望验证这一新的 蛋白酶体生物发生的范式。拟议的研究将增加一个重要的新方面，如下所述- 时间--让我们了解蛋白酶体在体内的组装。 我们的实验方法包含两个互补但独立的目标。在目标1中，我们将使用一个新的 建立了尖端的单分子和集合荧光分析集合，以表征 特定蛋白酶体组装步骤的动力学。在这一目标下的实验旨在检验这一假设。 通过两条可能的装配路线的相对通量主要受动力学控制，但可以通过 外源因素，如配体或与蛋白酶体相互作用的辅助蛋白。AIM 2将雇佣一套 新开发的化学遗传学方法，通过两种可能的组装路线评估相对通量 在体内，并了解通量如何变化对环境刺激的反应。在此基础上的实验 AIM还将在活细胞中测试我们建立的体外组装动力学模型的预测 目的1.这些研究的结果将有助于更深入地理解蛋白酶体生物学和 大分子组装，也有望照亮癌症的新治疗途径， 神经退行性变等疾病。

Project Summary Abstract The 26S proteasome conducts most regulated protein degradation and eliminates toxic proteins in vivo. The proteasome is an unusually large and complex ATP-dependent protease comprising nearly 70 individual polypeptide subunits. Although the conventional thinking has been that the proteasome is assembled from these subunits in a single, rigid stepwise sequence, recent evidence from our group and others unexpectedly suggests a broader “landscape” of assembly routes may exist in vivo. Although this possibility has not yet been tested, such an assembly landscape would ensure that this essential biological process can continue effectively in the face of assembly roadblocks, and would provide a powerful means to adjust the speed or volume of proteasome biogenesis in response to the cellular environment. There is an increasing interest in harnessing proteasome biogenesis to help treat conditions as diverse as cancer and neurodegenerative disorders. Understanding whether such an assembly landscape exists, and if so, how it is harnessed to ensure rapid and faithful proteasome biogenesis, will be critical to guide development of such assembly-targeted therapies. The goal of this multi-PI application is to test the hypothesis that a proteasome assembly landscape exists in vivo, and that the relative flux through possible routes within this landscape is governed largely by kinetic factors that change in response to the intracellular environment. By combining the PIs’ respective expertise in proteasome biology and in enzyme kinetics and single molecular biophysics, we hope to validate this new paradigm for proteasome biogenesis. The proposed studies, described below, will add a critical new dimension— time—to our understanding of proteasome assembly in vivo. Our experimental approach contains two complementary but independent Aims. In Aim 1, we will utilize a newly established collection of cutting-edge single-molecule and ensemble fluorescence assays to characterize the kinetics of specific proteasome assembly steps. Experiments under this aim are designed to test the hypothesis that the relative flux through two possible assembly routes is primarily under kinetic control, but can be tuned by exogenous factors such as ligands or proteasome-interacting accessory proteins. Aim 2 will employ a suite of newly developed chemical-genetic approaches to assess the relative flux through two possible assembly routes in vivo, and to understand how the flux changes in response to environmental stimuli. Experiments under this Aim will also test in living cells the predictions derived from our in vitro kinetic model of assembly established in Aim 1. The outcomes of these studies will lead to a deeper understanding of proteasome biology and of macromolecular assembly in general, and also promise to illuminate new therapeutic avenues for cancer, neurodegeneration, and other diseases.