Mechanism of protein aggregate recognition and disassembly by molecular chaperones

描述：细胞必需蛋白质的错误折叠和聚集是所有人的根本问题。 活着的有机体。即使是非必需蛋白质的聚集也会导致像II型这样的衰弱疾病 糖尿病、阿尔茨海默氏症、亨廷顿氏症和帕金森氏症。重要的是，蛋白质的折叠和聚集 受到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。不同的伴侣系统究竟是如何协作拆除和重新激活聚合的 蛋白质，以及分子伴侣作用如何影响疾病进展，还不是很清楚。这 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特性会影响它们的拆分方式吗？第三，如何 临界小热休克(SHSP)类分子伴侣促进蛋白质聚集体分解？ 以详细和定量的方式解决这些问题是非常困难的，使用标准 方法，因为蛋白质聚集体的复杂性和异质性可能会掩盖关键 中间体和过渡体。单粒子分析，特别是一种称为猝发的荧光技术 分析光谱学(BAS)是克服这一问题的理想选择。BAS可以量化复杂程度 纳米颗粒在自由溶液中的分布，允许检测动态填充的中间体和 亚种群。本项目将使用BAS通过两个模型来研究蛋白质聚集体的分解 解聚酶系统，一个来自细菌，一个来自酵母，其详细程度是其他系统无法达到的 接近了。总体目标是发展对不同分子的机械理解 伴侣网络识别和分解具有不同物理特性的蛋白质聚集体 属性。为了实现这一目标，该项目将扩展BAS的功能，将多色和 Förster共振能量转移测量。该项目还将开发一套新的方法， 是对BAS的补充，并允许更详细地分析水动力和结构特性 利用(1)微流体流中水平光片激发和粒子跟踪技术制备纳米粒子 和(2)尖端增强拉曼光谱(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途径。此外，由于 本工作中检查的伴侣网络的核心组件是保守的，进一步预计 这些研究中的发现将有助于从根本上更好地理解分子 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。

DESCRIPTION: 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.