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.

项目摘要 26S蛋白酶体在体内进行大多数调节的蛋白质降解并消除有毒蛋白质。的 蛋白酶体是一种非常大和复杂的ATP依赖性蛋白酶，包含近70个单独的 多肽亚基。虽然传统的观点认为蛋白酶体是由这些蛋白质组装而成的， 亚基在单一的，严格的逐步序列，最近的证据，从我们的小组和其他人出乎意料地表明， 体内可能存在更广泛的组装途径"景观"。虽然这种可能性尚未得到验证， 这样一个装配景观将确保这一基本的生物过程能够在 面对组装的障碍，并将提供一个强大的手段来调节蛋白酶体的速度或体积 对细胞环境的反应。人们对利用蛋白酶体越来越感兴趣 生物发生，以帮助治疗癌症和神经退行性疾病等多种疾病。理解 是否存在这样一个装配景观，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，将是至关重要的，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景观存在于 体内，并通过可能的路线在这一景观的相对流量主要是由动力学因素 它们会随着细胞内环境的变化而变化通过结合PI各自的专业知识， 蛋白酶体生物学和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。下文所述的拟议研究将增加一个重要的新层面- 时间，我们了解蛋白酶体组装在体内。 我们的实验方法包含两个互补但独立的目标。在目标1中，我们将使用新的 建立了尖端的单分子和集合荧光测定的集合，以表征 特定蛋白酶体组装步骤的动力学。根据这一目标设计的实验是为了检验这一假设 通过两种可能的组装路线的相对通量主要是在动力学控制下，但可以通过以下方式调节： 外源因子如配体或蛋白酶体相互作用辅助蛋白。目标2将采用一套 新开发的化学遗传学方法，通过两种可能的组装途径评估相对通量 在体内，并了解如何流量变化，以响应环境刺激。实验在此 Aim还将在活细胞中测试我们在1999年建立的体外组装动力学模型的预测。 目标1.这些研究的结果将导致对蛋白酶体生物学的更深入理解， 大分子组装，也有望照亮癌症的新治疗途径， 神经变性和其他疾病。