Structural and Functional Studies of Hsp70 Molecular Chaperones

Hsp70 分子伴侣的结构和功能研究

• 批准号: 9067389
• 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/9067389
• 关键词: ATP phosphohydrolase; Accounting; Allosteric Regulation; Alzheimer' s Disease; Binding; Binding Sites; Biochemical; Biochemistry; Color; Coupling; Disease; Escherichia coli; Genetic; Goals; Health; Heart; Homeostasis; Human; Kinetics; Length; Life; Link; Malignant Neoplasms; Manuscripts; Membrane; Modeling; Molecular; Molecular Chaperones; 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disease; innovation; insight; interdisciplinary approach; novel; novel therapeutics; polypeptide; protein folding; unpublished works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（由申请人提供）：Hsp70 是普遍存在且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，在维持细胞蛋白质稳态方面发挥多种重要作用。 维持蛋白质稳态的根本重要性不可避免地将 Hsp70 与许多破坏性人类疾病联系起来，尤其是癌症和神经退行性疾病，如帕金森病和阿尔茨海默病。因此，阐明 Hsp70 的结构和生化特性不仅将增进我们对 Hsp70 辅助蛋白质折叠的基本分子机制的理解，而且还为如何靶向 Hsp70 进行癌症和神经退行性疾病的治疗干预提供重要见解。 Hsp70 具有三种关键的生化活性，它们是伴侣活性的核心：ATP 酶、肽底物结合和 ATP 诱导的变构偶联。 尽管付出了广泛的努力，但由于缺乏对两个关键生化活性的深入了解，Hsp70 辅助蛋白质折叠的基本机制仍然不明确：1) ATP 诱导的变构偶联是 Hsp70 伴侣活性的核心~然而，之前的所有研究都未能揭示其分子机制。 2) Hsp70 上已确立的单肽结合位点使得解释有效的伴侣活性变得困难。 Hsp70 上是否存在额外的肽结合位点来实现高效率？ 因此，本提案的总体目标是分析这两个关键的生化活性，以剖析 Hsp70 伴侣功能的基本机制。 最近，我们解决了变构活性状态下完整Hsp70的第一个晶体结构，并发现了一种新的晶体结构。 Hsp70 上的肽底物结合位点。基于这些原始发现，我们提出以下两个具体目标：1）阐明 Hsp70 中 ATP 驱动的变构偶联的分子机制，2）表征 Hsp70 上的新肽结合位点并研究其在蛋白质折叠中的作用。 为了实现我们的目标，我们采用结合 X 射线晶体学、生物化学、NMR、EPR、计算化学以及酵母和大肠杆菌遗传学的多学科方法。我们期望该提案的成功完成将有助于我们实现我们的长期目标，即建立对 Hsp70 分子伴侣活性在蛋白质折叠中的基本机制的透彻理解。