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.

项目摘要摘要 26S蛋白质组会进行最调节的蛋白质降解，并消除体内有毒蛋白质。这 蛋白酶是一种异常大而复杂的ATP依赖性蛋白酶，完成了近70个个体 多肽亚基。尽管传统的想法是蛋白酶体是通过这些组装而来的 一个单一的，僵化的逐步序列中的亚基，我们小组的最新证据和其他人出乎意料地表明 在体内可能存在更广泛的组装路线“景观”。尽管这种可能性尚未测试，但 这样的集会景观将确保这种基本的生物过程可以在 面部障碍的面孔，将提供一种有力的手段来调整蛋白酶体的速度或体积 生物发生响应细胞环境。对利用蛋白酶体的兴趣越来越多 生物发生，以治疗像癌症和神经退行性疾病的潜水员。理解 是否存在这样的集会景观，如果是这样，则如何利用它来确保快速和忠实 蛋白酶体的生物发生对于指导这种靶向装配靶向疗法的开发至关重要。 该多PI应用的目的是检验以下假设。 体内，并且通过该景观内可能路线的相对通量主要由动力学因素支配 响应细胞内环境的变化。通过结合PI的相对专业知识 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 用于蛋白酶体生物发生的范例。下面描述的拟议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完善但独立的目标。在AIM 1中，我们将使用新的 建立了尖端的单分子和整体荧光测定的收集，以表征 特定蛋白酶体组装步骤的动力学。该目标下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在动力学控制之下，但可以通过 外源性因素，例如配体或蛋白酶体相互作用的辅助蛋白。 AIM 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路线评估相对通量 在体内，并了解对环境刺激的响应的变化如何变化。在此下进行实验 AIM还将在活细胞中测试我们从我们在体外动力学模型中得出的预测。 目标1。这些研究的结果将导致对蛋白酶体生物学和对 一般而言，大分子组件也有望照亮癌症的新治疗途径 神经变性和其他疾病。