Protein folding in the cell: Challenges and coping mechanisms

细胞中的蛋白质折叠：挑战和应对机制

• 批准号: 10410352
• 负责人: LILA M GIERASCH
• 金额: $ 54.43万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10410352
• 关键词: Affect; Affinity; Alzheimer' s Disease; Amino Acid Substitution; Binding; Binding Sites; Biochemical; Biological Assay; C-terminal; Cell physiology; Cells; Charge; Computer Models; Cystic Fibrosis; Disease; Dissociation; Docking; Fluorescence; Guanine Nucleotide Exchange Factors; Health; Huntington Disease; Hydrophobicity; Lead; Learning; Light; Literature; Mass Spectrum Analysis; Mediating; Methods; Modeling; Molecular Chaperones; Molecular Conformation; N-terminal; Neurons; Nuclear Magnetic Resonance; Nucleotides; Parkinson Disease; Pathologic; Peptides; Post-Translational Protein Processing; Property; Proteins; Proteome; Quality Control; Research; Role; S-nitro-N-acetylpenicillamine; Synaptic Vesicles; Thinness; Translating; Work; coping mechanism; loss of function; misfolded protein; polypeptide; protein folding; protein function; proteostasis; therapeutic target

Project Summary Hsp70 molecular chaperones are central players in protein homeostasis and quality control. They mediate a diverse set of cellular functions using a deceptively simple allosteric mechanism. In their ATP-bound states, they bind substrates with rapid on/off rates and relatively low affinity, and in their ADP-bound states, substrates bind more tightly with slow association/dissociation. Hsp70s bind short sequences within unfolded regions of their substrates, preferring hydrophobic residues with flanking positively charged residues. Their transition between a high substrate affinity, ADP- bound state, and a low substrate affinity, ATP-bound state, involves major conformational rearrangement of both the N-terminal nucleotide-binding domain, and the C-terminal substrate- binding domain. While recent research has shed light on this allosteric conformational change, many questions remain and are the focus of the proposed research. What are the features of their substrate-binding sites that enable binding to many but not all sequences (they are “selectively promiscuous”)? Hsp70s work with partner co-chaperones, the J-proteins that help with cellular localization and delivery of specific substrates, and the nucleotide-exchange factors that facilitate replacement of ADP by ATP in the allosteric cycle. What are the structural origins of these partnerships, and how do they affect substrate binding and release? Preliminary results and literature observations suggest that Hsp70s can bind nearly isoenergetically to their extended polypeptide substrates in either an N- to C-orientation or a C- to N-orientation. This provocative bimodal substrate binding may have functional implications. The proposed work will examine the structural origins of this capability as well as the potential impact on the functions of the chaperone. Past work shows that the allosteric properties of Hsp70 are tunable by amino acid substitutions and by post-translational modifications. The proposed work will delve into the structural origins of this tuning, and the consequences of changes in the allosteric energy landscape for specific Hsp70 functions. Past work leaned heavily on peptide models to understand how Hsp70s bind their substrates. We will characterize the binding of Hsp70s to protein substrates to learn how the affinity for short sequence motifs translates into a hierarchy of site binding: are flanking sequences involved in selection of binding sites? How pivotal is accessibility? Lastly, the role of the Hsc70 chaperone in preparing SNAP-25 to participate in pre- synaptic vesicle docking and fusion in neurons will be studied. Methods that will be deployed in the proposed work include biochemical assays, nuclear magnetic resonance, fluorescence, mass spectrometry, and computational modeling.

项目摘要 热休克蛋白70分子伴侣是蛋白质稳态和质量控制的核心参与者。他们 使用看似简单的变构机制来调节多种细胞功能。在 它们的ATP结合状态，它们以快速的开/关速率和相对低的亲和力结合底物， 在ADP结合状态下，底物结合更紧密，缔合/解离更慢。热休克蛋白70 在其底物的未折叠区域内结合短序列，优选疏水残基 两侧带正电荷的残基。它们在高底物亲和力，ADP- 结合状态，和低底物亲和力，ATP结合状态，涉及主要构象 N-末端核苷酸结合结构域和C-末端底物的重排， 结合域虽然最近的研究揭示了这种变构构象变化， 许多问题仍然存在，是拟议研究的重点。它们的特点是什么？ 能够结合许多但不是所有序列的底物结合位点（它们是“选择性地 滥交”）？热休克蛋白70与伴侣辅助分子伴侣（辅助分子伴侣）一起工作，辅助分子伴侣是J蛋白， 特定底物的定位和递送，以及促进 在变构循环中ATP取代ADP。它们的结构起源是什么 伙伴关系，以及它们如何影响底物结合和释放？初步结果和 文献观察表明，Hsp 70可以几乎等能地结合到它们的延伸， 在一个实施方案中，所述多核苷酸可以以N-至C-取向或C-至N-取向与多肽底物结合。这一挑衅 双峰底物结合可能具有功能上的意义。拟议的工作将审查 这种能力的结构起源以及对联合国系统功能的潜在影响。 监护人过去的工作表明，Hsp 70的变构特性是可调的氨基酸， 取代和翻译后修饰。拟议的工作将深入研究 这种调整的结构起源，以及变构能量变化的后果 Hsp 70的具体功能。过去的工作严重依赖于肽模型， 了解热休克蛋白70如何结合其底物。我们将描述Hsp 70与 蛋白质底物，以了解对短序列基序的亲和力如何转化为 位点结合：侧翼序列是否参与结合位点的选择？关键在于 可访问性？最后，Hsc 70分子伴侣在准备SNAP-25参与预处理中的作用。 将研究神经元中的突触囊泡对接和融合。中部署的方法 建议的工作包括生物化学测定、核磁共振、荧光、质谱 光谱分析和计算机建模。