Protein folding in the cell: Challenges and coping mechanisms Administrative Supplement for Equipment Purchase

细胞内蛋白质折叠：挑战与应对机制设备采购行政补充

• 批准号: 10795171
• 负责人: LILA M GIERASCH
• 金额: $ 11.93万
• 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/10795171
• 关键词: Administrative Supplement; 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; Learning; Literature; Mass Spectrum Analysis; Mediating; Methods; Modality; 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; Synaptic Vesicles; Thinness; Translating; Work; coping mechanism; equipment acquisition; loss of function; misfolded protein; polypeptide; presynaptic; 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 HSP70分子伴侣在蛋白质动态平衡和质量控制中发挥着重要作用。他们 使用一种看似简单的变构机制来调节一系列不同的细胞功能。在……里面 它们的ATP结合状态，它们以快速的开/关速率和相对较低的亲和力结合底物，以及 在ADP结合态，底物结合更紧密，结合/解离较慢。Hsp70 在底物的未折叠区域结合短序列，偏好疏水残基 侧翼有带正电荷的残基。它们在高底物亲和力、ADP- 结合态和底物亲和力低的ATP结合态涉及主要构象 N端核苷酸结合域和C端底物的重排- 结合结构域。虽然最近的研究揭示了这种变构构象的变化， 许多问题仍然存在，也是拟议研究的重点。他们的特点是什么？ 底物结合位点，能够与许多但不是所有序列结合(它们是有选择性的 乱交“)？Hsp70与伴侣辅伴蛋白协同工作，这是一种帮助细胞 特定底物的定位和传递，以及促进 在变构循环中用三磷酸腺苷取代ADP。这些结构的起源是什么？ 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，Hsp70可以几乎等能量地结合到其延伸的 N-到C-取向或C-到N-取向的多肽底物。这是挑衅性的 双峰底物结合可能具有功能暗示。拟议的工作将审查 这一能力的结构根源以及对 监护人。过去的工作表明，Hsp70的变构性质是可以通过氨基酸调节的 替换和翻译后修饰。拟议的工作将深入研究 这种调谐的结构起源，以及变构能量变化的结果 适用于特定HSP70功能的景观。过去的工作严重依赖于多肽模型来 了解Hsp70是如何结合它们的底物的。我们将描述Hsp70与 蛋白质底物，以了解短序列基序的亲和力如何转化为 结合位点：侧翼序列是否参与结合位点的选择？关键是什么？ 可访问性？最后，Hsc70伴侣在准备SNAP-25参与Pre-Pre中的作用 将研究突触小泡在神经元中的对接和融合。将部署在 拟议的工作包括生化分析、核磁共振、荧光、质量 光谱分析和计算建模。