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 细胞必须在生成、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平衡，或蛋白质平衡。在维护中扮演核心角色 哺乳动物的蛋白平衡是p97，一种AAA+ATPase(与多种细胞活动相关的ATPase)，它 利用ATP水解的力量，从各种细胞器中拉出泛素化的底物并展开 在蛋白酶体降解之前。P97基因突变可导致与基因调控失调相关的疾病 蛋白质平衡；因此，虽然我们知道p97对细胞健康是至关重要的，但关于它的许多机制仍然存在。 未知。一般来说，p97必须结合、移位和释放未折叠的底物。其中的每一步都是 依赖于p97‘S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 没有被充分描述的。 研究表明，p97结合伙伴Otu1将泛素部分从p97底物修剪成 让它们高效地展开和释放。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-Otu1结构将阐明多泛素化底物是如何去泛素化的，并将 构建对p97如何处理其底物的更完整的理解。 最近的工作探索了在异二聚体Ufd1/Npl4(UN)结合的背景下展开的启动 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 有约束力的伙伴，或者如果他们利用一个独特的机制。最重要的p97结合伙伴之一是p47， 参与高尔基体膜重塑。从历史上看，P47被报道只与特定的 非泛素化或单泛素化的蛋白质；然而，最近的证据也表明P47 也与多泛素化的底物相互作用。探索这一作用的结构和生化基础 将扩大对p97复合体如何展开多泛素化底物的理解。为了阐明如何 P97受结合伙伴以及这些结合伙伴如何与多泛素底物相互作用的调节，即 将确定p97-Otu1复合体和p97-P47复合体的高分辨结构 通过冷冻-EM制备多泛素化底物。 在这笔赠款的支持下，我计划获得生物化学和结构生物学方面的专业知识，改进 我的指导技能，完善我的科学交流，并成为一名对那些也 来自不被充分代表的背景。在沈彼得博士的指导下，与最先进的 由犹他大学提供的资源，我相信培训计划将最大限度地提高我的 对我未来作为一名独立科学家的职业发展。