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的结构基础 cdc48（p97的酵母菌）是必不可少的AAA+ ATPase，参与提取蛋白质 整个细胞中的各种位置，包括蛋白质复合物，核糖体，染色质， 线粒体和急诊室。这项研究将回答CDC48如何处理底物和结构变化 它必须这样做，以及如何调整Cdc48的适配器蛋白调节其活性并有助于靶向 cdc48到特定的底物。为了研究CDC48的机制，我们将重点关注CDC48的辅助因子 在酿酒酵母中命名为SHP1。我们将执行固定cdc48的纯化 使用adp·befx，一个ATP类似物来捕获基材。我们将使用Cryo-Em可视化的结合 底物至Cdc48和Cdc48经历的构象变化。这将使我们能够 关于底物如何与Cdc48结合并处理的结论。 使用Cofactor SHP1了解有关CDC48的更多信息，将阐明CDC48-SHP1复合 目标和过程底物。通过专注于带有推定SHP1的Cryo-Em显微照片的粒子 密度，并使用交联以丰富这些颗粒，我们的目标是更好地可视化SHP1的相互作用 与CDC48。这超出了SHP1和CDC48的直接相互作用以及SHP1的机制 与底物相互作用将其靶向Cdc48s中心孔。但是，SHP1不是唯一的辅助因子 CDC48。其他辅助因子如何与CDC48同步以靶向和过程底物为开放 研究领域。 从我们自己的未发表的数据中，我们在净化CAR2的上调净化辅助器时 CDC48。从我们的数据中，该蛋白质似乎不是Cdc48的底物，但似乎确实与 纯化中Cdc48的存在。 CAR2不是CDC48的既定辅助因子。但是，由于 CDC48和CAR2抽象之间的联系，我们怀疑CAR2可能与Cdc48相互作用，也许可以充当 辅因子。我们将使用质谱法和冷冻EM来研究这些蛋白可能的相互作用 具有这些潜在相互作用的功能。

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