Resolving the mechanism of cofactor and substrate interactions with Cdc48

解决辅因子和底物与 Cdc48 相互作用的机制

• 批准号: 10434738
• 负责人: IAN COONEY
• 金额: $ 3.78万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10434738
• 关键词: ATP phosphohydrolase; Adaptor Signaling Protein; Address; Area; Arginine; Binding; Biochemical; Biochemistry; CDC48 protein; Cells; Chromatin; Classification; Communication; Complex; Cryoelectron Microscopy; Data; Degenerative Disorder; Degradation Pathway; Disease; Enzymes; Hand; Heritability; Human; In Vitro; Learning; Link; Location; Malignant Neoplasms; Mass Spectrum Analysis; Metabolic; Mitochondria; Mitochondrial Proteins; Molecular Conformation; Molecular Machines; Mutation; N Domain; Names; Neurodegenerative Disorders; Nucleotides; Orthologous Gene; Outcome; Pathway interactions; Play; Preparation; Process; Proteins; Publishing; Research; Resolution; Ribosomes; Role; Saccharomyces cerevisiae; Sampling; Side; Source; Structure; Substrate Interaction; Up-Regulation; Work; Yeasts; analog; bone; cofactor; crosslink; density; feeding; improved; insight; multisystem proteinopathy; muscle degeneration; novel therapeutics; particle; protein activation; protein complex; reconstitution; recruit; targeted cancer therapy; yeast genetics

Project Summary/Abstract Determine the structural basis of cofactor and substrate interactions with Cdc48 Cdc48 (the yeast otholog of p97) is an essential AAA+ ATPase that is involved in extracting proteins throughout the cell from a vast range of locations, including protein complexes, ribosomes, chromatin, mitochondria and the ER. This study will answer how Cdc48 processes substrates and the structural changes it must undergo to do so as well as how adaptor proteins of Cdc48 modulate its activity and help to target Cdc48 to specific substrates. To investigate the mechanism of Cdc48, we will focus on a cofactor of Cdc48 named Shp1 in Saccharomyces cerevisiae. We will perform purifications of substrate bound Cdc48 using ADP⋅BeFx, an ATP analog, to trap substrates. We will use cryo-EM to visualize the binding of the substrate(s) to Cdc48 and the conformational changes that Cdc48 undergoes. This will allow us to make conclusions about how substrate binds to and is processed by Cdc48. Using the cofactor Shp1 to learn more about Cdc48 will illuminate how the Cdc48-Shp1 complex targets and processes substrates. By focusing on particles from our cryo-EM micrographs with putative Shp1 density, and using crosslinking to enrich for these particles, we aim to better visualize the interaction of Shp1 with Cdc48. This goes beyond the direct interaction of Shp1 and Cdc48 and into the mechanism of how Shp1 interacts with substrates to target them to Cdc48s central pore. However, Shp1 is not the only cofactor of Cdc48. How other cofactors work in synchrony with Cdc48 to target and process substrates remains an open area of study. From our own unpublished data, we have shown upregulation of Car2 when purifying cofactors of Cdc48. From our data, this protein does not seem to be a substrate of Cdc48 but does seem to be linked to the presence of Cdc48 in purifications. Car2 is not an established cofactor of Cdc48. However, because of the link between Cdc48 and Car2 abundance, we suspect that Car2 may interact with Cdc48 and perhaps act as a cofactor. We will use mass spectrometry and cryo-EM to investigate any interactions that these proteins may have and the function behind these potential interactions.

项目摘要/摘要 确定辅因子和底物与CDC48相互作用的结构基础 CDC48(p97的酵母同源基因)是一种参与蛋白质提取的必需的aaa+ATPase 包括蛋白质复合体、核糖体、染色质、 线粒体和内质网。这项研究将回答CDC48如何处理底物和结构变化 它必须经历这样的过程，以及CDC48的接头蛋白如何调节其活性和帮助靶向 CDC48结合到特定底物上。为了研究CDC48的作用机制，我们将重点研究CDC48的一个辅因子 在酿酒酵母中命名为SHP1。我们将对底物结合的CDC48进行提纯 使用三磷酸腺苷类似物adp⋅befx捕获底物。我们将使用冷冻-EM来可视化 底物(S)对CDC48的作用以及CDC48所经历的构象变化。这将使我们能够 关于底物如何与CDC48结合并被CDC48处理的结论。 使用辅因子SHP1了解更多关于CDC48的信息将阐明CDC48-SHP1复合体是如何 目标和加工衬底。通过聚焦于我们的冷冻-EM显微照片中的粒子，推测为SHP1 密度，并使用交联来丰富这些粒子，我们的目标是更好地可视化SHP1的相互作用 使用CDC48。这超越了SHP1和CDC48的直接相互作用，而进入了SHP1如何 与底物相互作用，使其靶向于cdc48s中心孔。然而，SHP1并不是唯一的辅助因子 CDC48。其他辅因子如何与CDC48同步作用以靶向和处理底物仍是一个未知数 研究领域。 从我们自己未发表的数据中，我们已经显示出在纯化辅因子时Car2的上调。 CDC48。从我们的数据来看，这种蛋白质似乎不是CDC48的底物，但似乎确实与 纯化过程中CDC48的存在。Car2不是CDC48的既定辅因子。然而，由于 CDC48和Car2丰度之间的联系，我们怀疑Car2可能与CDC48相互作用，并可能作为一种 辅因。我们将使用质谱仪和低温电子显微镜来研究这些蛋白质可能存在的任何相互作用 以及这些潜在相互作用背后的功能。

项目成果

Active conformation of the p97-p47 unfoldase complex.

• DOI: 10.1038/s41467-022-30318-3
• 发表时间: 2022-05-12
• 期刊: Nature communications
• 影响因子: 16.6