Mechanism of protein aggregate recognition and disassembly by molecular chaperones

分子伴侣识别和拆卸蛋白质聚集体的机制

• 批准号: 10581972
• 负责人: HAYS S RYE
• 金额: $ 3.9万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10581972
• 关键词: Address; Affect; Alzheimer' s Disease; Amyloid Neuropathies; Amyotrophic Lateral Sclerosis; Bacteria; Cells; Color; Complex; Cystic Fibrosis; Detection; Disease; Disease Progression; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Heat-Shock Response; Human; Huntington Disease; Lead; Light; Link; Measurement; Microfluidics; Modeling; Molecular Chaperones; Nature; Non-Insulin-Dependent Diabetes Mellitus; Organism; Parkinson Disease; Pathologic; Process; Property; Proteins; Quality Control; Raman Spectrum Analysis; Services; Spectrum Analysis; System; Techniques; Thalassemia; Work; Yeasts; alpha 1-Antitrypsin Deficiency; human disease; nanoparticle; novel strategies; particle; physical property; protein aggregation; protein folding; protein misfolding

SUMMARY: The misfolding and aggregation of essential cellular proteins is a fundamental problem for all living organisms. Aggregation of even non-essential proteins can lead to debilitating diseases like type II diabetes, Alzheimer's, Huntington's and Parkinson's diseases. Importantly, protein folding and aggregation are heavily influenced by the cellular protein quality control machinery, involving networks of molecular chaperones. Precisely how different chaperone systems cooperate to dismantle and reactivate aggregated proteins, and how molecular chaperone action affects disease progression, is not well understood. This proposal will address three fundamental questions that impact this problem: First, what is the most accurate physical description of protein aggregate disassembly by molecular chaperones? Second, in what way do the structural properties of an aggregate nanoparticle impact how an they are taken apart? Third, how does the critical small heat shock (sHsp) class of molecular chaperones enhance protein aggregate disassembly? Addressing these questions in a detailed and quantitative manner is exceedingly difficult using standard approaches, because the complex and heterogeneous nature of protein aggregates can obscure key intermediates and transitions. Single particle analysis, in particular a fluorescence technique known as Burst Analysis Spectroscopy (BAS), is ideally suited to overcome this problem. BAS can quantify complex nanoparticle distributions in free solution, allowing for the detection of dynamically populated intermediates and sub-populations. This project will employ BAS to study the disassembly of protein aggregates by two model disaggregase systems, one from bacteria and one from yeast, at a level of detail unreachable by other approaches. The overall goal is to develop a mechanistic understanding of how different molecular chaperone networks recognize and dismantle protein aggregates that possess distinct physical properties. In service of this goal, this project will extend the capabilities of BAS to incorporate multi-color and Förster resonance energy transfer measurements. This project will also develop a set of new approaches that are complementary to BAS and permit more detailed analysis of the hydrodynamic and structural properties of aggregate nanoparticles by using (1) horizontal light sheet excitation and particle tracking in microfluidic flow and (2) Tip-Enhanced Raman spectroscopy (TERS). It is anticipated that the combination of these techniques will provide uniquely powerful approach to understanding protein disaggregation. Additionally, because the core components of the chaperone networks examined in this work are conserved, it is further expected that the discoveries made in these studies will contribute to a fundamentally better understanding of how molecular chaperones recognize and process protein aggregates in human cells impacted by protein misfolding diseases.

摘要：细胞中重要蛋白质的错误折叠和聚集是一个基本问题 活的有机体即使是非必需蛋白质的聚集也会导致像II型这样的衰弱性疾病 糖尿病、阿尔茨海默氏症、亨廷顿舞蹈症和帕金森氏症。重要的是，蛋白质折叠和聚集是 受细胞蛋白质质量控制机制的严重影响，涉及分子网络 监护人不同的伴侣系统如何合作， 蛋白质，以及分子伴侣作用如何影响疾病进展，目前还没有很好的了解。这 该提案将解决影响这一问题的三个基本问题：第一，什么是最准确的 分子伴侣对蛋白质聚集体分解的物理描述？第二，以什么方式 聚集体纳米颗粒的结构性质影响它们如何被分开？第三， 临界小热休克（sHsp）类分子伴侣增强蛋白质聚集体的拆卸？ 用标准的方法来详细和定量地解决这些问题是极其困难的。 方法，因为蛋白质聚集体的复杂性和异质性可以掩盖关键 中间体和过渡。单颗粒分析，特别是称为Burst的荧光技术 分析光谱（BAS）非常适合解决这个问题。BAS可以量化复杂的 纳米粒子在自由溶液中的分布，允许检测动态填充的中间体， 亚种群。本计画将利用BAS技术，以两种模式来研究蛋白质聚集体的拆解 解聚酶系统，一个来自细菌，一个来自酵母，在一个细节的水平无法达到其他 接近。总体目标是对不同的分子 分子伴侣网络识别并分解具有不同物理性质的蛋白质聚集体， 特性.为了实现这一目标，该项目将扩展BAS的功能， 福斯特共振能量转移测量。该项目还将开发一套新的方法， 是BAS的补充，并允许更详细地分析流体动力学和结构特性， 通过使用（1）微流体流中的水平光片激发和颗粒跟踪来聚集纳米颗粒 （2）针尖增强拉曼光谱（TERS）。预计这些技术的组合 将为理解蛋白质解聚提供独特而有力的方法。此外，由于 在这项工作中检查的伴侣蛋白网络的核心组分是保守的，进一步预期， 这些研究中的发现将有助于从根本上更好地理解分子 分子伴侣识别和处理受蛋白质错误折叠影响的人类细胞中的蛋白质聚集体 疾病