Mechanisms of Protein Disaggregation and Turnover by AAA+ Chaperones

AAA 分子伴侣的蛋白质解聚和周转机制

• 批准号: 10219751
• 负责人: Daniel Ryland Southworth
• 金额: $ 39.27万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10219751
• 关键词: ATP Hydrolysis; Address; Alzheimer' s Disease; Amino Acids; Amyloid; Architecture; Biochemical; Biological Assay; Cell Aging; Cell Survival; Cell physiology; Cells; Cellular Stress; Communication; Complex; Conflict (Psychology); Coupled; Coupling; Cryoelectron Microscopy; Crystallization; Disease; Event; Family; Filament; Fluorescence Resonance Energy Transfer; Future; Goals; Heat shock proteins; Hydrolysis; Life; Mass Spectrum Analysis; Mechanics; Mediating; Methods; Mitochondria; Modeling; Molecular Chaperones; Molecular Conformation; Motor; Nature; Neurodegenerative Disorders; Parkinson Disease; Pathway interactions; Peptide Hydrolases; Proteins; Proteolysis; Public Health; Resolution; Rotation; Science; Site; Stress; Structural Models; Structure; Substrate Interaction; Surface; System; To specify; Variant; Work; Yeasts; alpha synuclein; amyloid formation; base; biological adaptation to stress; biophysical techniques; crosslink; genetic regulatory protein; human disease; improved; insight; member; multicatalytic endopeptidase complex; mutant; novel strategies; polypeptide; prevent; protein aggregation; protein misfolding; protein protein interaction; proteostasis; single-molecule FRET; sup35; therapeutic target; unfoldase; yeast prion

PROJECT SUMMARY/ABSTRACT Protein disaggregation and turnover are essential for protein homeostasis (proteostasis) and cell viability. Malfunction occurs during cell stress and aging, accelerating deleterious protein aggregation and amyloid formation. Improved mechanistic understanding is critical for determining how proteostasis pathways fail and for identifying therapeutic targets in preventing neurodegenerative disease and other protein mis-folding diseases. Heat shock protein (Hsp) 100 members of the conserved AAA+ family serve critical functions in all life as protein unfoldases and disaggregases. They form hexameric, ATP-driven machines that catalyze the translocation of polypeptide substrates through a central channel. The unfolded proteins are then refolded by Hsp molecular chaperones or degraded by an associated protease, such as in the case of the proteasome. Challenges in achieving structures of functional states have led to conflicting mechanistic models across the AAA+ superfamily. Focusing on conserved Hsp100 members, yeast Hsp104 and the bacterial Clp proteins, we have overcome these challenges by using cryo-electron microscopy to determine structures of biochemically defined, functional complexes. We determined the first substrate-bound structures of a AAA+ disaggregase (Hsp104) in distinct translocation states and discovered these machines operate by a rotary mechanism involving precise substrate gripping and release states and a two amino acid translocation step. Since our last submission of this application, we have determined multiple structures of the ClpAP AAA+ protease undergoing active substrate unfolding and proteolysis Together our discoveries reveal a new paradigm for how AAA+s mechanically unfold substrates. The next major question to address is: How is the translocation mechanism (which is now considered highly conserved among AAA+s) coupled to specific cellular functions? Our long-term goal is to determine how translocation and unfolding are precisely tuned for different proteostasis and cell stress response functions. The objective for this application is to identify key allosteric control mechanisms that couple ATP-driven translocation to substrate recognition, unfolding and degradation. Here we will: (SA1) Determine mechanisms of protein unfolding and proteolysis by the ClpAP “bacterial proteasome” complex; (SA2) Determine how Hsp104 interacts with and disaggregates native substrates and amyloids; and (SA3) Determine how the Hsp70 chaperone collaborates with Hsp104 to promote substrate loading. At the completion of this work we will identify conformational networks and protein:protein interactions that define how the core translocation cycle connects allosterically to specify distinct cellular functions of these AAA+ machines.

项目摘要/摘要 蛋白质分解和周转对于蛋白质稳态（蛋白抑制剂）和细胞活力至关重要。 故障发生在细胞应激和衰老期间，加速有害蛋白质聚集和淀粉样蛋白 形成。改善的机理理解对于确定蛋白质的途径如何失败至关重要 鉴定治疗靶标在预防神经退行性疾病和其他蛋白质错误折叠疾病方面。 热休克蛋白（HSP）配置的AAA+家族的100名成员在所有生命中都具有关键功能 展开和分裂。它们形成了六聚体ATP驱动的机器，催化了易位 多肽底物通过中央通道。然后通过HSP分子重折叠展开的蛋白 伴侣或由相关蛋白酶降解，例如蛋白酶体。 实现功能状态结构的挑战导致了跨越的机械模型 AAA+超家族。专注于配置的HSP100成员，酵母HSP104和细菌CLP蛋白， 我们通过使用冷冻电子显微镜来确定生物化学结构来克服这些挑战 定义的功能复合物。我们确定了AAA+分解酶的第一个基材结合结构 （HSP104）在不同的易位状态下，发现这些机器通过涉及的旋转机制运行 精确的底物握住和释放状态以及两个氨基酸易位步骤。自上次提交以来 在此应用程序中，我们确定了经历活跃的CLPAP AAA+蛋白酶的多个结构 底物展开和蛋白水解一起我们的发现揭示了一个新的范式，以实现AAA+s 机械展开底物。要解决的下一个主要问题是：易位机制如何 （现在认为这是在AAA+s之间高度构成的）与特定的细胞函数耦合？我们的长期 目标是确定针对不同蛋白质和细胞应力的易位和展开的精确调整 响应功能。该应用的目的是确定夫妇的关键变构控制机制 ATP驱动的易位转化为底物识别，展开和退化。我们将在这里：（sa1）确定 Clpap“细菌蛋白酶体”复合物的蛋白质的机制； （SA2）确定 HSP104如何与天然底物和淀粉样蛋白相互作用并分解； （sa3）确定 HSP70伴侣与HSP104合作以促进底物加载。完成这项工作时，我们将 识别会议网络和蛋白质：定义核心转运周期的蛋白质相互作用 定构连接以指定这些AAA+机器的不同蜂窝函数。