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 HSP70分子伴侣是蛋白质稳态和质量控制的中心参与者。他们 使用一种欺骗性的简单变构机制介导一组各种细胞功能。在 他们与ATP结合的状态，它们以快速开/关和相对较低的亲和力结合基板，以及 在其ADP结合状态下，底物与较慢的关联/解离更紧密地结合。 HSP70S 结合其底物展开区域内的短序列，更喜欢疏水残差 带有积极充电的侧面保留。它们之间的高基质亲和力adp-之间的过渡 约束状态和低基材亲和力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。尽管最近的研究阐明了这种变构会议的变化，但 仍然存在许多问题，并且是拟议研究的重点。他们的特征是什么 底物结合位点可以使许多序列结合到许多序列（它们是选择性的） 混杂”）？ 特定底物的定位和传递，以及促进核苷酸 - 交换因子 在变构周期中，ATP替换ADP。这些的结构起源是什么 伙伴关系，它们如何影响底物的约束和释放？初步结果 文献观察表明，HSP70几乎可以将其与它们的扩展绑定 N-至C-取向或c-至n取向的多肽底物。这个挑衅 双峰底物结合可能具有功能含义。拟议的工作将检查 该能力的结构起源以及对功能的潜在影响 伴侣。过去的工作表明，HSP70的变构特性可通过氨基酸调谐 替换和通过翻译后修改。拟议的工作将深入研究 这种调音的结构起源以及变构能量的变化的后果 特定HSP70功能的景观。过去的工作大力依靠胡椒模型 了解HSP70如何绑定其底物。我们将表征HSP70与 蛋白质底物了解短序列基序的亲和力如何转化为层次结构 位点结合：侧翼序列是否参与结合位点的选择？关键是多么关键 可访问性？最后，HSC70伴侣在准备SNAP-25方面的作用以参与Pre- 神经元中的突触囊泡对接和融合将进行研究。将部署在 拟议的工作包括生化测定，核磁共振，荧光，质量 光谱和计算建模。