Mechanism of protein folding intermediate disaggregation by molecular chaperones

分子伴侣蛋白质折叠中间解聚机制

• 批准号: 8269836
• 负责人: HAYS S RYE
• 金额: $ 29.72万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8269836
• 关键词: Affect; Alzheimer' s Disease; Amino Acid Sequence; Amyloid Neuropathies; Amyotrophic Lateral Sclerosis; Anabolism; Binding; Biological Assay; Cells; Clathrin; Color; Complex; Coupled; Coupling; Cryoelectron Microscopy; Cystic Fibrosis; Deposition; Detection; Disease; Disease Progression; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Functional disorder; Goals; GroEL Protein; Growth; Huntington Disease; Kinetics; Life; Link; Macromolecular Complexes; Measurement; Mediating; Methods; Modeling; Modification; Molecular; Molecular Chaperones; Molecular Conformation; Nanosphere; Nature; Neuropathy; Parkinson Disease; Pathology; Play; Population; Population Distributions; Process; Protein Binding; Protein C Inhibitor; Proteins; Quality Control; Reaction; Role; Sampling; Set protein; Solutions; Spectrum Analysis; Stress; Structure; System; Techniques; Testing; Thalassemia; Time; Toxic effect; Translations; Work; analytical tool; base; design; flexibility; human disease; in vitro Model; monomer; particle; protein aggregate; protein aggregation; protein folding; protein misfolding; public health relevance; research study; single-molecule FRET

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.

描述（由申请人提供）：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线性氨基酸序列中，但将这些信息从一级序列翻译为天然结构通常会出错，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。例子包括囊性纤维化、地中海贫血、α 1-抗胰蛋白酶缺乏和几种神经病如阿尔茨海默氏病、亨廷顿病和帕金森氏病。这些疾病的进展通常与蛋白质原纤维的形成相关。然而，结构化原纤的生长和沉积通常在无定形和部分结构化的预原纤状态的形成之前。越来越多的证据表明，前原纤维，低阶聚集体在许多疾病的病理学中发挥着重要的作用。重要的是，蛋白质聚集受到细胞蛋白质质量控制机制的严重影响，涉及分子伴侣网络。确切地说，不同的分子伴侣系统如何合作，以拆除和重新激活聚集的蛋白质，以及分子伴侣作用如何影响疾病的进展，还没有很好地理解。一个显着的障碍，以更好地理解蛋白质聚集体的分子伴侣解体是固有的复杂性和异质性的聚集蛋白质样品。聚集蛋白通常形成各种各样的构象状态和组装。这种复杂而广泛的状态分布通常很难用当前的检测技术捕获。该建议的主要目标是克服这种分析限制，以开发一个详细的机制的理解，如何一个必要的分子伴侣网络，ClpB，DnaKJ-GrpE和GroEL-ES，提取和重折叠蛋白质的聚集体组成。为了实现这一目标，我们将：（1）开发一种基于单粒子荧光猝发检测的新分析工具，其能够快速定量聚集蛋白质群体内状态的特定摩尔分布，以及该分布如何随时间变化，（2）采用该方法来检查DnaKJ-GrpE和ClpB双分子伴侣系统对蛋白质聚集体分解的机制，和（3）采用荧光光谱和低温电子显微镜的组合来确定在从聚集体中提取后，DnaK与非天然蛋白质的结合如何影响随后GroEL对蛋白质的折叠。 公共卫生关系：多种人类疾病，如肌萎缩侧索硬化症、囊性纤维化、地中海贫血、α 1-抗胰蛋白酶缺乏症和多种淀粉样神经病，如阿尔茨海默氏病、亨廷顿病和帕金森氏病，都与细胞蛋白质的不受控制和病理性自缔合有共同的联系。这个过程被称为蛋白质聚集，如何导致疾病尚不清楚。活细胞内蛋白质聚集的进程直接受到一组称为分子伴侣的专门蛋白质的影响。我们的目标是发展一个清晰的理解蛋白质聚集体是如何识别和拆除的分子伴侣网络。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。