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: 10201455
• 负责人: IAN COONEY
• 金额: $ 3.71万
• 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/10201455
• 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.

项目概要/摘要 确定辅因子和底物与 Cdc48 相互作用的结构基础 Cdc48（p97 的酵母同源物）是一种重要的 AAA+ ATP 酶，参与蛋白质提取 从广泛的位置遍及细胞，包括蛋白质复合物、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 它必须经历这样做，以及 Cdc48 的接头蛋白如何调节其活性并帮助靶向 Cdc48 针对特定底物。为了研究Cdc48的机制，我们将重点关注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 进行纯化 使用 ADP⋅BeFx（一种 ATP 类似物）捕获底物。我们将使用冷冻电镜来可视化结合 Cdc48 的底物以及 Cdc48 经历的构象变化。这将使我们能够 关于底物如何与 Cdc48 结合并被 Cdc48 处理的结论。 使用辅因子 Shp1 了解有关 Cdc48 的更多信息将阐明 Cdc48-Shp1 复合物如何 目标并处理基材。通过使用假定的 Shp1 来关注我们的冷冻电镜显微照片中的颗粒 密度，并使用交联来富集这些颗粒，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，并深入探讨了 Shp1 如何发挥作用的机制 与底物相互作用，将其靶向 Cdc48s 中心孔。然而，Shp1 并不是唯一的辅助因子 疾病预防控制中心48。其他辅助因子如何与 Cdc48 同步工作以靶向和处理底物仍然是一个开放的问题 研究领域。 根据我们自己未发表的数据，我们在纯化辅因子时显示了 Car2 的上调 疾病预防控制中心48。根据我们的数据，这种蛋白质似乎不是 Cdc48 的底物，但似乎与 纯化中存在 Cdc48。 Car2 不是 Cdc48 的既定辅助因子。然而，由于 Cdc48 和 Car2 丰度之间存在联系，我们怀疑 Car2 可能与 Cdc48 相互作用，并且可能充当 辅因子。我们将使用质谱和冷冻电镜来研究这些蛋白质可能发生的任何相互作用 以及这些潜在相互作用背后的功能。