Structural and Functional Studies of Hsp70 Molecular Chaperones

Hsp70 分子伴侣的结构和功能研究

• 批准号: 9279173
• 负责人: Qinglian Liu
• 金额: $ 28.98万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9279173
• 关键词: ATP phosphohydrolase; Allosteric Regulation; Alzheimer' s Disease; Binding; Binding Sites; Biochemical; Biochemistry; Coupling; Crystallization; Disease; Escherichia coli; Genetic; Goals; Heart; Human; Hydrophobicity; Kinetics; Length; Life; Link; Malignant Neoplasms; Manuscripts; Membrane; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Nature; Neurodegenerative Disorders; Nucleotides; Organism; P2 peptide; Parkinson Disease; Peptide Conformation; Peptides; Play; Property; Proteins; Research; Resolution; Role; Side; Site; Structure; Substrate Domain; Testing; Therapeutic Intervention; Transportation; X-Ray Crystallography; Yeasts; base; computational chemistry; design; human disease; innovation; insight; interdisciplinary approach; novel; novel therapeutics; polypeptide; protein folding; proteostasis; public health relevance; unpublished works

DESCRIPTION (provided by applicant): Hsp70s are ubiquitous and highly conserved molecular chaperones that play multiple essential roles in maintaining cellular protein homeostasis through assisting in protein folding, assembly, degradation, and transportation across membrane. The fundamental importance of maintaining protein homeostasis inevitably links Hsp70s with many destructive human diseases, most notably cancers and neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Thus, elucidating the structural and biochemical properties of Hsp70s will not only advance our understanding of the basic molecular mechanism of Hsp70-assited protein folding, but also provide crucial insights regarding how to target Hsp70s for therapeutic interventions in treating cancers and neurodegenerative disorders. Hsp70s have three key biochemical activities that are at the heart of the chaperone activity: ATPase, peptide substrate binding, and ATP-induced allosteric coupling. In spite of extensive efforts, the very basic mechanism of Hsp70-assisted protein folding is still ill-defined due to the lack of in-depth understanding o two key biochemical activities: 1) ATP-induced allosteric coupling is central to Hsp70s' chaperone activity~ however, all previous studies had failed to reveal the molecular mechanism. 2) The well-established single peptide binding site on Hsp70s has made it difficult to explain the efficient chaperone activity. Could there be additional peptide binding sites on Hsp70s that account for the high efficiency? Thus, the overall objective of this proposal is to analyze these two key biochemical activities in order to dissect the basic mechanisms of Hsp70 chaperone function. Recently, we solved the first crystal structure of an intact Hsp70 in the allosteric active state, and discovered a novel peptide substrate binding site on Hsp70s. Based on these original discoveries, we propose the following two Specific Aims: 1) elucidate the molecular mechanism of the ATP-driven allosteric coupling in Hsp70s, and 2) characterize a novel peptide binding site on Hsp70s and investigate its role in protein folding. To achieve our goal, we use a multidisciplinary approach combining X-ray crystallography, biochemistry, NMR, EPR, computational chemistry, and yeast and E.coli genetics. We expect that successful completion of this proposal will help us realize our long-term goal, which is to establish a thoroug mechanism understanding of the very basic mechanism of Hsp70 chaperone activity in protein folding.

描述(申请人提供)：Hsp70是普遍存在的高度保守的分子伴侣，通过协助蛋白质折叠、组装、降解和跨膜运输，在维持细胞蛋白质动态平衡方面发挥多种重要作用。保持蛋白质动态平衡的根本重要性不可避免地将Hsp70与许多破坏性的人类疾病联系在一起，最明显的是癌症和神经退行性疾病，如帕金森氏症和阿尔茨海默氏症。因此，阐明Hsp70的结构和生化特性不仅有助于我们理解Hsp70相关蛋白折叠的基本分子机制，而且还将为如何靶向Hsp70用于治疗癌症和神经退行性疾病提供重要的见解。Hsp70有三个关键的生化活性，它们是伴侣活性的核心：ATPase，肽底物结合，和ATP诱导的变构偶联。尽管进行了广泛的研究，但由于对两个关键的生化活动缺乏深入的了解，Hsp70辅助蛋白质折叠的基本机制仍然不清楚：1)ATP诱导的变构偶联是Hsp70伴侣活性的核心。然而，以前的研究都未能揭示其分子机制。2)Hsp70上已确定的单肽结合位点使得很难解释有效的伴侣活性。Hsp70上有没有额外的多肽结合位点可以解释这种高效率？因此，这项建议的总体目标是分析这两个关键的生化活动，以剖析Hsp70伴侣功能的基本机制。最近，我们解决了第一个完整的变构活化态Hsp70的晶体结构，并发现了一个新的 Hsp70上的多肽底物结合位点。在这些原始发现的基础上，我们提出了以下两个具体目标：1)阐明Hsp70中ATP驱动的变构偶联的分子机制；2)鉴定Hsp70上一个新的肽结合部位，并研究其在蛋白质折叠中的作用。为了实现我们的目标，我们使用了一种结合了X射线结晶学、生物化学、核磁共振、电子顺磁共振、计算化学以及酵母和大肠杆菌遗传学的多学科方法。我们期待这项计划的成功完成将帮助我们实现我们的长期目标，即建立一个彻底的机制，了解蛋白质折叠中Hsp70伴侣活性的最基本机制。