Mechanism of protein folding intermediate disaggregation by molecular chaperones

描述（由申请人提供）：虽然编码蛋白质的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，但将这些信息从一级序列翻译为天然结构经常出错，导致蛋白质错误折叠和聚集。在某些情况下，蛋白质聚集会引发严重的细胞功能障碍和疾病。例子包括囊性纤维化、地中海贫血、α - 1-抗胰蛋白酶缺乏症和一些神经疾病，如阿尔茨海默氏症、亨廷顿氏症和帕金森病。这些疾病的进展往往与蛋白原纤维的形成有关。然而，结构原纤维的生长和沉积通常是在无定形和部分结构的前原纤维状态形成之前。越来越多的证据表明，纤原前低阶聚集体在许多疾病的病理中起着核心和共同的作用。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。确切地说，不同的伴侣系统是如何合作拆除和重新激活聚集的蛋白质的，以及分子伴侣的作用是如何影响疾病进展的，目前还不清楚。一个显著的障碍，以更好地理解蛋白质聚集分解的分子伴侣是固有的复杂和异质性的性质，聚集的蛋白质样品。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，用目前的检测技术很难捕捉到这种复杂而广泛的状态分布。该提案的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折叠蛋白质进行详细的机制理解。为实现这一目标，我们将：(1)开发了一种新的基于单粒子荧光爆发检测的分析工具，该工具能够快速量化聚集蛋白群体中特定状态的摩尔分布，以及该分布如何随时间变化；(2)利用该方法研究了DnaKJ-GrpE和ClpB双伴侣蛋白系统对蛋白质聚集体分解的机制；(3)采用荧光光谱和低温电镜相结合的方法来确定从聚集体中提取非天然蛋白质后，DnaK与蛋白质的结合如何影响GroEL随后对蛋白质的折叠。

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.