Mechanism of Substrate Unfolding by the AAA+ ATPase p97 and Binding Partners

AAA ATPase p97 和结合伙伴的底物解折叠机制

• 批准号: 10678124
• 负责人: Deirdre Mack
• 金额: $ 4.77万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678124
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affect; Autophagocytosis; Binding; Biochemical; Biochemistry; Biological Assay; Cell physiology; Cells; Cellular Membrane; Chromatin; Co-Immunoprecipitations; Communication; Complex; Cryoelectron Microscopy; Degenerative Disorder; Deubiquitination; Development; Disease; Enzymes; Equilibrium; Failure; Fluorescence; Future; Golgi Apparatus; Grant; Health; Image; In Vitro; Knowledge; Maintenance; Mediating; Membrane; Mentors; Mentorship; Mitochondria; Modeling; Molecular Machines; Mutation; Negative Staining; Organ; Organelles; Pathway interactions; Play; Polyubiquitin; Process; Protein phosphatase; Proteins; Quality Control; Recombinants; Regulation; Reporting; Research; Resolution; Resources; Ribosomes; Role; Sampling; Science; Scientist; Structure; System; Training; Ubiquitin; Universities; Utah; Western Blotting; Work; analog; career; improved; multicatalytic endopeptidase complex; mutant; open data; p97 ATPase; postmitotic; protein degradation; proteostasis; reconstitution; recruit; skills; structural biology; therapeutic development; ubiquitin isopeptidase; unfoldase

Abstract Cells must maintain a balance between generating, folding, transporting, and degrading proteins in order to maintain proper protein homeostasis, or proteostasis. A central player in the maintenance of mammalian proteostasis is p97, a AAA+ ATPase (ATPase associated with diverse cellular activities) that leverages the power of ATP hydrolysis to pull ubiquitinated substrates from a variety of organelles and unfold them before proteasomal degradation. Mutations in p97 can lead to diseases associated with dysregulation of proteostasis; thus, while it is known that p97 is critical to cellular health, much about its mechanism remains unknown. In general, p97 must bind, translocate, and release the unfolded substrate. Each of these steps is dependent on p97’s interactions with multiple binding partners, yet how these interactions are coordinated has not been fully characterized. Studies have shown that the p97 binding partner, Otu1, trims ubiquitin moieties from p97 substrates to allow their efficient unfolding and release. Yet how this deubiquitination occurs remains an open question. Solving the p97-Otu1 structure will elucidate how polyubiquitinated substrates are deubiquitinated and will construct a more complete understanding of how p97 processes its substrates. Recent work has explored unfolding initiation in the context of the heterodimeric Ufd1/Npl4 (UN) binding partner. However, it is not known if this initiation mechanism extends to other polyubiquitin substrate recruiting binding partners or if they utilize a unique mechanism. One of the most important p97 binding partners is p47, which is involved in Golgi membrane remodeling. Historically, p47 was reported to only interact with specific non-ubiquitinated or monoubiquitinated proteins; however, recent evidence has also demonstrated that p47 also interacts with polyubiquitinated substrates. Exploring the structural and biochemical basis of this action would broaden understanding of how p97 complexes unfold polyubiquitinated substrates. To elucidate how p97 is regulated by binding partners and how those binding partners interact with polyubiquinated substrate, I will determine high-resolution structures of the p97-Otu1 complex and the p97-p47 complex in complex with polyubiquitinated substrates via cryo-EM. Under the support of this grant, I plan to gain expertise in biochemistry and structural biology, improve my mentoring skills, refine my science communication, and become a leader with sensitivity for those that also come from underrepresented backgrounds. Under the mentorship of Dr. Peter Shen, and with the cutting-edge resources provided by the University of Utah, I am confident that the training plan will maximize my development towards my future career as an independent scientist.

抽象的 细胞必须在蛋白质的生成、折叠、运输和降解之间保持平衡 为了维持适当的蛋白质稳态，或蛋白质稳态的维持的核心角色。 哺乳动物蛋白质稳态是 p97，一种 AAA+ ATP 酶（与多种细胞活动相关的 ATP 酶）， 利用 ATP 水解的力量从各种细胞器中提取泛素化底物并展开 p97 突变可能导致与 p97 失调相关的疾病。 因此，虽然 p97 对细胞健康至关重要，但其机制仍存在很多疑问。 一般来说，p97 必须结合、移位和释放未折叠的底物。 依赖于 p97 与多个结合伙伴的相互作用，但这些相互作用是如何协调的 尚未得到充分表征。 研究表明，p97 结合伴侣 Otu1 将 p97 底物中的泛素部分修剪为 然而，这种去泛素化如何发生仍然是一个悬而未决的问题。 解析 p97-Otu1 结构将阐明多泛素化底物是如何去泛素化的，并将 更全面地了解 p97 如何处理其底物。 最近的工作探索了异二聚 Ufd1/Npl4 (UN) 结合背景下的展开起始 然而，尚不清楚这种启动机制是否扩展到其他多聚泛素底物招募。 结合伙伴，或者如果它们利用独特的机制，最重要的 p97 结合伙伴之一是 p47， 据报道，p47 参与高尔基膜重塑。 然而，最近的证据也表明 p47 还与多泛素化底物相互作用，探索这种作用的结构和生化基础。 将拓宽对 p97 复合物如何展开多泛素化底物的理解，以阐明如何进行。 p97 受结合配偶体以及这些结合配偶体如何与多聚泛素化底物相互作用的调节，I 将确定 p97-Otu1 复合物和 p97-p47 复合物的高分辨率结构 通过冷冻电镜进行多泛素化底物。 在这笔资助的支持下，我计划获得生物化学和结构生物学方面的专业知识，提高 我的指导技能，完善我的科学沟通，并成为对那些同样具有敏感性的人的领导者 来自于未被充分代表的背景，在Peter Shen博士的指导下，并拥有最前沿的知识。 犹他大学提供的资源，我相信该培训计划将最大限度地提高我的能力 我未来作为一名独立科学家的职业发展。