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.

摘要 细胞必须在产生、折叠、运输和降解蛋白质之间保持平衡， 以维持适当的蛋白质稳态或蛋白质稳态。一个维持 哺乳动物蛋白质稳态是p97，一种AAA + ATP酶（与多种细胞活性相关的ATP酶）， 利用ATP水解的力量从各种细胞器中提取泛素化底物， 在蛋白酶体降解之前。p97基因突变可导致与细胞凋亡调节异常相关的疾病。 因此，虽然已知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、移位和释放未折叠的底物。这些步骤中的每一个都 依赖于p97与多个结合伴侣的相互作用，然而这些相互作用如何协调， 还没有完全定性。 研究表明，p97结合配偶体Otu 1从p97底物中修剪泛素部分， 允许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。 解决p97-Otu 1结构将阐明多泛素化底物如何去泛素化， 构建一个更完整的理解如何p97处理其底物。 最近的工作已经探索了在异二聚体Ufd 1/Npl4（UN）结合的背景下的解折叠起始。 搭档然而，目前尚不清楚这种启动机制是否延伸到其他多聚泛素底物招募 结合伴侣，或者它们是否利用独特的机制。最重要的p97结合伴侣之一是p47， 参与高尔基体膜的重塑。历史上，据报道p47仅与特定的 非泛素化或单泛素化蛋白质;然而，最近的证据也表明，p47 也与多聚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。为了阐明 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， 将确定p97-Otu 1复合物和p97-p47复合物的高分辨率结构， 通过cryo-EM的多泛素化底物。 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， 我的指导技能，完善我的科学沟通，并成为一个领导者与那些也敏感 来自代表性不足的背景在沈彼得博士的指导下， 通过犹他州大学提供的资源，我相信培训计划将最大限度地提高我的 我未来的职业生涯是作为一名独立的科学家。