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。维护中的核心角色 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 它们在蛋白酶体降解之前。 p97 突变可导致与基因失调相关的疾病 蛋白质稳态；因此，虽然众所周知 p97 对细胞健康至关重要，但关于其机制的许多内容仍然存在 未知。一般来说，p97 必须结合、移位并释放未折叠的底物。这些步骤中的每一步都是 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 让它们有效地展开和释放。然而，这种去泛素化是如何发生的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 伙伴。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 具有约束力的合作伙伴或他们使用独特的机制。 p47 是最重要的 p97 结合伴侣之一， 参与高尔基膜重塑。据报道，历史上 p47 仅与特定的相互作用 非泛素化或单泛素化蛋白质；然而，最近的证据也表明 p47 还与多泛素化底物相互作用。探索该作用的结构和生化基础 将加深对 p97 复合物如何展开多泛素化底物的理解。阐明如何 p97 受结合伴侣的调节以及这些结合伴侣如何与多聚泛素化底物相互作用，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 来自代表性不足的背景。在Peter Shen博士的指导下，并与前沿的 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。