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

基本信息

批准号：
10439743
负责人：
Daniel Ryland Southworth
金额：
$ 40.25万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
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/10439743
关键词：
ATP Hydrolysis Address Alzheimer&apos 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 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

项目摘要

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.

项目概要/摘要蛋白质解聚和周转对于蛋白质稳态（蛋白质稳态）和细胞活力至关重要。细胞应激和衰老过程中会发生功能障碍，加速有害蛋白质聚集和淀粉样蛋白提高对机制的理解对于确定蛋白质稳态途径如何失效以及如何形成至关重要。预防神经退行性疾病和其他蛋白质错误折叠疾病的治疗目标。热休克蛋白 (Hsp) 保守 AAA+ 家族的 100 个成员作为蛋白质在所有生命中发挥着关键功能它们形成六聚体、ATP驱动的机器，催化易位然后，未折叠的蛋白质通过 Hsp 分子重新折叠。分子伴侣或被相关蛋白酶降解，例如在蛋白酶体的情况下。实现功能状态结构的挑战导致了跨领域的相互冲突的机械模型 AAA+ 超家族，重点关注保守的 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化结构，克服了这些挑战我们确定了 AAA+ 解聚酶的第一个底物结合结构。（Hsp104）处于不同的易位状态，并发现这些机器通过旋转机构运行，涉及自我们上次提交以来，精确的底物抓取和释放状态以及两个氨基酸易位步骤。在本申请中，我们确定了 ClpAP AAA+ 蛋白酶的多种结构，这些结构经历了活性底物展开和蛋白水解我们的发现共同揭示了 AAA+ 的新范例机械展开基质的下一个要解决的主要问题是：易位机制如何。（现在被认为在 AAA+ 中高度保守）与特定的细胞功能相结合？目标是确定如何针对不同的蛋白质稳态和细胞应激精确调整易位和展开该应用的目的是确定耦合的关键变构控制机制。 ATP 驱动的易位至底物识别、解折叠和降解： (SA1) 确定。 ClpAP“细菌蛋白酶体”复合物的蛋白质解折叠和蛋白水解机制（SA2）确定； Hsp104 如何与天然底物和淀粉样蛋白相互作用并分解；以及 (SA3) 确定如何 Hsp70 伴侣与 Hsp104 合作促进底物加载。完成这项工作后，我们将。识别构象网络和蛋白质：定义核心易位循环的蛋白质相互作用变构连接以指定这些 AAA+ 机器的独特细胞功能。