Mechanism of protein folding intermediate disaggregation by molecular chaperones

描述(申请人提供)：虽然编码蛋白质二级和三级结构所需的所有信息都包含在其氨基酸的线性序列中，但这种信息从一级序列到自然结构的翻译经常出错，导致蛋白质错误折叠和聚集。在某些情况下，蛋白质的聚集可引发严重的细胞功能障碍和疾病。例如囊性纤维化、地中海贫血、α1抗胰蛋白酶缺乏症，以及阿尔茨海默氏症、亨廷顿氏症和帕金森氏症等几种神经性疾病。这些疾病的进展通常与蛋白质原纤维的形成有关。然而，结构纤维的生长和沉积通常先于无定形和部分结构的前纤维状态的形成。越来越多的证据表明，前纤维状、低阶聚集体在许多疾病的病理中发挥着核心和常见的作用。重要的是，蛋白质聚集在很大程度上受到细胞蛋白质质量控制机制的影响，包括分子伴侣网络。不同的伴侣系统如何合作分解和重新激活聚集的蛋白质，以及分子伴侣的作用如何影响疾病的进展，目前还不清楚。更好地理解分子伴侣对蛋白质聚集体分解的一个重要障碍是聚集性蛋白质样品固有的复杂性和异质性。聚集的蛋白质通常形成各种各样的构象状态和组装。一般来说，用当前的检测技术很难捕捉到这种复杂而广泛的状态分布。这项提议的一个主要目标是克服这一分析限制，以便对由ClpB、DNAKJ-GRPE和GroEL-ES组成的基本分子伴侣网络如何从聚集体中提取和复性蛋白质产生详细的机制理解。为了实现这一目标，我们将：(1)开发一种新的基于单粒子荧光爆发检测的分析工具，它能够快速定量聚集体中状态的特定摩尔分布以及该分布随时间的变化；(2)利用该方法研究DnaKJ-GRPE和ClpB双伴侣系统对蛋白质聚集体的分解机制；(3)利用荧光光谱和低温电子显微镜相结合的方法，确定从聚集体中提取非本地蛋白质后，DNAK与蛋白质的结合如何影响GroEL随后的蛋白质折叠。 公共卫生相关性：各种人类疾病，如肌萎缩侧索硬化症、囊性纤维化、地中海贫血、α1抗胰蛋白酶缺乏症，以及各种淀粉样神经病，如阿尔茨海默病、亨廷顿病和帕金森病，都与细胞蛋白的不受控制和病理性的自我联系有关。这个被称为蛋白质聚集的过程是如何导致疾病的，目前还不清楚。活细胞内蛋白质聚集的进程直接受到一组被称为分子伴侣的特殊蛋白质的影响。我们的目标是清楚地了解蛋白质聚集体是如何被分子伴侣网络识别和分解的。更好地了解这一过程将直接影响我们对与蛋白质错误折叠和聚集有关的疾病大家族的理解。

DESCRIPTION (provided by applicant): While all the information necessary to encode the secondary and tertiary structure of a protein is contained in its linear sequence of amino acids, translation of this information from primary sequence to native structure often goes awry, resulting in protein mis-folding and aggregation. In some cases, aggregation of proteins can trigger severe cellular dysfunction and disease. Examples include cystic fibrosis, thalassemias, alpha1- antitrypsin deficiency, and several neuropathies such as Alzheimer's, Huntington's and Parkinson's diseases. The progression of these diseases is often correlated with the formation of protein fibrils. However, the growth and deposition of structured fibrils is generally preceded by the formation of amorphous and partially structured, pre-fibrillar states. Growing evidence suggests that pre-fibrillar, low-order aggregates play a central and common role in the pathology of many diseases. Importantly, protein aggregation is 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. A significant impediment to a better understanding of protein aggregate disassembly by molecular chaperones is the inherently complex and heterogeneous nature of an aggregating protein sample. Aggregating proteins typically form a wide variety of conformational states and assemblies. This complex and broad distribution of states is, in general, very difficult to capture with current detection techniques. A principle goal of this proposal is to overcome this analytical limitation in order to develop a detailed mechanistic understanding of how an essential molecular chaperone network, consisting of ClpB, DnaKJ-GrpE and GroEL-ES, extracts and refolds proteins from aggregates. To accomplish this goal, we will: (1) develop a new analytical tool based on single-particle fluorescence burst detection that is capable of rapidly quantifying the specific molar distribution of states within an aggregated protein population, as well as how that distribution changes with time, (2) employ this method to examine the mechanism of protein aggregate disassembly by the DnaKJ-GrpE and ClpB bi-chaperone system, and (3) employ a combination of fluorescence spectroscopy and cryo-electron microscopy to determine how the binding of a non-native protein by DnaK, following extraction from an aggregate, affects the subsequent folding of the protein by GroEL. PUBLIC HEALTH RELEVANCE: Human diseases as diverse as amyotrophic lateral sclerosis, cystic fibrosis, thalassemias, alpha1-antitrypsin deficiency, and a variety of amyloid neuropathies such as Alzheimer's, Huntington's and Parkinson's disease share in common a link to the uncontrolled and pathological self-association of cellular proteins. How this process, known as protein aggregation, leads to disease is not well understood. The progression of protein aggregation inside a living cell is directly influenced by a specialized set of proteins known as molecular chaperones. Our goal is to develop a clear understanding of how protein aggregates are recognized and dismantled by networks of molecular chaperones. A better picture of this process will have a direct impact on our understanding of the large family of diseases that have been linked to protein misfolding and aggregation.