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.

项目总结/摘要 确定与Cdc 48的辅因子和底物相互作用的结构基础 Cdc 48（p97的酵母同源物）是参与蛋白质提取的必需AAA+ ATP酶 在整个细胞中从广泛的位置，包括蛋白质复合物，核糖体，染色质， 线粒体和内质网本研究将回答Cdc 48如何处理底物以及结构变化 它必须经历这样做，以及Cdc 48的衔接蛋白如何调节其活性并帮助靶向 cdc 48对特定底物的作用。为了研究Cdc 48的作用机制，我们将重点关注Cdc 48的辅因子 命名为Shp 1。我们将对底物结合的Cdc 48进行纯化， 使用ATP类似物ADP β-BeFx捕获底物。我们将使用冷冻电镜观察 底物与Cdc 48的结合以及Cdc 48所经历的构象变化。这将使我们能够 关于底物如何与Cdc 48结合并被Cdc 48加工的结论。 使用辅因子Shp 1来了解更多关于Cdc 48的信息将阐明Cdc 48-Shp 1复合物如何 靶和处理衬底。通过关注来自我们的冷冻EM显微照片的颗粒， 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Shp 1的相互作用 关于Cdc 48这超出了Shp 1和Cdc 48的直接相互作用，而是进入了Shp 1如何相互作用的机制 与底物相互作用，使其靶向Cdc 48的中心孔。然而，Shp 1不是唯一的辅因子。 Cdc48.其他辅因子如何与Cdc 48同步工作以靶向和加工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，我们已经表明，当纯化Car 2的辅因子时， Cdc48.从我们的数据来看，这种蛋白质似乎不是Cdc 48的底物，但似乎与Cdc 48有关。 纯化过程中Cdc 48的存在。Car 2不是Cdc 48的既定辅因子。但因为 Cdc 48和Car 2丰度之间的联系，我们怀疑Car 2可能与Cdc 48相互作用， 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 以及这些潜在相互作用背后的功能。