Investigation of the proteasome assembly landscape

蛋白酶体组装景观的研究

• 批准号: 10344955
• 负责人: Zucai Suo
• 金额: $ 43.02万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10344955
• 关键词: 26S proteasome; ATP phosphohydrolase; ATP-Dependent Proteases; Affinity; Binding; Biogenesis; Biological Assay; Biological Process; Biology; Biophysics; Cells; Chemicals; Collection; Complex; Development; Dimensions; Disease; Dissociation; Docking; Ensure; Environment; Enzyme Kinetics; Exogenous Factors; Fluorescence; Genetic; Goals; Human; Impairment; In Vitro; Individual; Investigation; Kinetics; Knowledge; Ligands; Literature; Malignant Neoplasms; Measurement; Measures; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Monitor; Mutation; Nature; Nerve Degeneration; Neurodegenerative Disorders; Nucleosome Core Particle; Outcome Study; Pathologic; Pathway interactions; Physiological; Proteins; Recycling; Route; Site; Spectrum Analysis; Speed; Stimulus; Stress; Testing; Thinking; Time; Work; Yeasts; base; chemical genetics; design; experimental study; flexibility; genetic approach; human disease; in vivo; interest; kinetic model; macromolecular assembly; multicatalytic endopeptidase complex; mutant; novel therapeutic intervention; novel therapeutics; particle; polypeptide; protein degradation; response; single molecule; stoichiometry; targeted treatment; tool

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.

项目概要 摘要 26S 蛋白酶体进行最受调节的蛋白质降解并消除体内有毒蛋白质。这 蛋白酶体是一种异常大且复杂的 ATP 依赖性蛋白酶，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物质组装而成的 我们小组和其他人的最新证据出人意料地表明，亚基以单一的、严格的逐步顺序排列 体内可能存在更广泛的组装路线“景观”。虽然这种可能性还没有被测试过， 这样的组装环境将确保这一重要的生物过程能够在 面对装配障碍，并将提供一种强有力的手段来调节蛋白酶体的速度或体积 响应细胞环境的生物发生。人们对利用蛋白酶体越来越感兴趣 生物发生有助于治疗癌症和神经退行性疾病等多种疾病。理解 这样的装配环境是否存在，如果存在，如何利用它来确保快速和忠实 蛋白酶体生物发生对于指导此类组装靶向疗法的开发至关重要。 此多 PI 应用程序的目标是检验蛋白酶体组装景观存在于 体内，并且通过该景观中可能的路线的相对通量很大程度上受动力学因素控制 响应细胞内环境而变化。通过结合 PI 各自的专业知识 蛋白酶体生物学以及酶动力学和单分子生物物理学，我们希望验证这一新的 蛋白酶体生物发生的范例。如下所述，拟议的研究将增加一个关键的新维度—— 时间——我们对体内蛋白酶体组装的理解。 我们的实验方法包含两个互补但独立的目标。在目标 1 中，我们将利用新的 建立了一系列尖端的单分子和整体荧光测定来表征 特定蛋白酶体组装步骤的动力学。在此目标下进行的实验旨在检验假设 通过两个可能的组装路线的相对通量主要受动力学控制，但可以通过以下方式调整 外源因素，例如配体或蛋白酶体相互作用的辅助蛋白。目标 2 将采用一套 新开发的化学遗传方法可通过两种可能的组装路线评估相对通量 体内，并了解通量如何响应环境刺激而变化。在此下进行实验 Aim 还将在活细胞中测试根据我们建立的体外组装动力学模型得出的预测。 目标 1. 这些研究的结果将导致人们对蛋白酶体生物学和蛋白酶体生物学有更深入的了解。 一般而言，大分子组装，也有望阐明癌症的新治疗途径， 神经退行性疾病和其他疾病。