Structure of functionally important dynamic states of the proteasome

蛋白酶体功能重要动态的结构

• 批准号: 8696127
• 负责人: Philip Coffino
• 金额: $ 44.88万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8696127
• 关键词: 26S proteasome; ATP Hydrolysis; ATP phosphohydrolase; Address; Alpha Particles; Binding; Binding Sites; Biochemical; Biochemistry; Biological; Biomechanics; Cells; Communication; Complex; Coupled; Cryoelectron Microscopy; Data; Disease; Elements; Event; Excision; Failure; Genetic Transcription; Goals; Huntington Disease; Hybrids; Hydrolysis; Invaded; Investigation; Knowledge; Laboratories; Link; Mechanics; Mediating; Methods; Modeling; Molecular; Molecular Conformation; Movement; Mutate; Mutation; Nature; Neurodegenerative Disorders; Nucleosome Core Particle; Nucleotides; Outcome; Positioning Attribute; Process; Proteins; Proteolysis; Regulation; Research; Resolution; Rest; Site; Structure; Testing; Therapeutic Intervention; Thinking; Work; base; constriction; grasp; insight; multicatalytic endopeptidase complex; mutant; novel; particle; polypeptide; protein degradation; protein misfolding; public health relevance; tool

DESCRIPTION (provided by applicant): Proteasomes are giant molecular complexes that degrade intracellular proteins in a regulated manner. They are composed of two subassemblies- a 19S regulatory particle (RP) and a proteolytic 20S catalytic particle (CP). The objective of this application is to understand functionally relevant proteasome structural dynamics. Recent findings from several labs provide subnanometer resolution structures of the proteasome. These beautiful models, however, are incomplete. First, substrate is absent. In the first Specific Aim it will be determined how protein substrates engage the proteasome. This Aim will investigate the mode of interaction between substrate and three key proteasome sites of action: substrate binding, extended polypeptide undergoing active translocation and the constriction against which a folded domain unravels. Second, the present models are static. In the second Specific Aim various biochemical approaches will be used to trap substrate engagement with proteasome in a set of specific conformational states associated with distinct functional states. A widely accepted yet untested paradigm is that protein unfolding and translocation is coupled with large-scale conformational changes of the proteasome. This view will be tested by determining structures of proteasomes with substrate processing in specific states. The third Specific Aim will investigate the structural basis of recent biochemical observations demonstrating homotropic allostery within the proteasome CP. Communication within the CP may be important in biochemical regulation and in determining how the cell allocates the CP pool among various forms of classic and hybrid proteasomes. The rationale for structuring this application with two PIs is that accomplishing the scientific goals of this project requires their continuing interaction to exploit and further develop methods that originated in the labs of each of the applicants: Novel cryoEM methods, which enable rapid high-resolution structure determination, and novel proteasome substrates, which enable formation of stable and tunable proteasome-substrate complexes for structural studies. The proposed studies will visualize functionally relevant proteasome complexes at atomic- level resolution. Proteasomes are fueled by ATP binding and hydrolysis and use the energy so derived to move and unfold their substrates. However, little information is available that relates proteasome function to structural dynamics. The long term goal of these investigations is to understand the biomechanics of proteasome action. The proposed research is significant because it is a first step in transforming our understanding of proteasomes from static objects to dynamic players.

描述（由申请人提供）：蛋白酶体是以调节方式降解细胞内蛋白质的巨型分子复合物。它们由两个亚摘要组成 - A 19S调节颗粒（RP）和蛋白水解20S催化粒子（CP）。这个目的 应用是了解与功能相关的蛋白酶体结构动力学。来自几个实验室的最新发现提供了蛋白酶体的亚纳米分辨率结构。但是，这些美丽的模型是不完整的。首先，底物不存在。在第一个特定目标中 将确定蛋白质底物如何参与蛋白酶体。该目标将研究底物与三个关键蛋白酶体作用位点之间的相互作用方式：底物结合，扩展的多肽经历了活跃的易位，以及折叠域分散的收缩。其次，目前的模型是静态的。在第二个特定目的中，各种生化方法将用于将与蛋白酶体的蛋白酶体捕获在与不同功能状态相关的一组特定构象状态下。一个 广泛接受但未经测试的范例是蛋白质展开和转运与蛋白酶体的大规模构象变化相结合。该观点将通过确定特定状态下底物处理的蛋白酶体的结构来测试。第三个具体目的将研究最近的生化观察的结构基础，这些观察结果表明蛋白酶体CP中的同型变构。 CP内的通信在生化调节中可能很重要，并且在确定细胞如何在各种形式的经典和杂种蛋白酶体之间分配CP池。用两个PI构建此应用的理由是，实现该项目的科学目标需要持续相互作用来利用和进一步开发源自每个申请人实验室的方法：新型的冷冻方法，从而实现快速的高分辨率结构确定，并确定新颖的蛋白酶体底物，以实现稳定的蛋白酶体和调节性的蛋白质复杂的构成稳定的蛋白质复合体。拟议的研究将在原子水平分辨率下可视化与功能相关的蛋白酶体复合物。蛋白酶体是通过ATP结合和水解助力的，并利用如此衍生的能量移动和展开其底物。但是，很少有将蛋白酶体功能与结构性功能相关的信息 动力学。这些研究的长期目标是了解蛋白酶体作用的生物力学。拟议的研究很重要，因为它是将我们对蛋白酶体从静态对象转变为动态玩家的第一步。