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：必需细胞蛋白的错误折叠和聚集是所有人的基本问题 活生物体。即使是非必需蛋白的聚集也会导致诸如II型这样的使人衰弱的疾病 糖尿病，阿尔茨海默氏症，亨廷顿和帕金森氏病。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 伴侣。精确地不同的伴侣系统如何协调拆卸和重新激活聚集 蛋白质以及分子伴侣作用如何影响疾病进展，尚不清楚。这 提案将解决影响此问题的三个基本问题：首先，最准确的是什么 分子伴侣分解蛋白质骨料的物理描述？第二，以什么方式采用 骨料纳米颗粒的结构特性会影响它们如何拆开？第三，如何 临界小热休克（SHSP）类分子伴侣会增强蛋白质骨料拆卸？ 使用标准，以详细和定量的方式解决这些问题非常困难 方法，因为蛋白质聚集体的复杂和异质性可能会掩盖钥匙 中间和过渡。单个粒子分析，特别是一种称为爆发的荧光技术 分析光谱（BAS）非常适合克服这个问题。 BAS可以量化复合物 自由溶液中的纳米颗粒分布，允许检测动态人群的中间体和 子人群。该项目将采用BAS来研究两个模型的蛋白质聚集体的分解 分类系统，一种来自细菌，一种来自酵母菌的系统，在其他细节上无法到达其他细节 方法。总体目标是对不同分子的机械理解发展 伴侣网络识别和拆除具有独特物理的蛋白质聚集体 特性。为了实现这一目标，该项目将扩展BAS的功能，以结合多色和 Förster共振能量传递测量。该项目还将开发一套新方法 是BAS的完整性，并允许对流体动力和结构特性进行更详细的分析 通过（1）微流动流中的水平光板兴奋和粒子跟踪，骨料纳米颗粒 （2）尖端增强的拉曼光谱法（TERS）。预计这些技术的结合 将提供独特的强大方法来理解蛋白质分解。另外，因为 这项工作中检查的伴侣网络的核心组成部分是保存的，进一步预期 这些研究中提出的发现将有助于对分子的方式有帮助 伴侣识别并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。