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 细胞必须保持生成，折叠，运输和降解蛋白质之间的平衡 为了维持适当的蛋白质稳态或蛋白质的稳态。维护的中心参与者 哺乳动物蛋白质抑制性是p97，一种AAA+ ATPase（与多种细胞活性相关的ATPase） 利用ATP水解的力量从各种细胞器中拉出泛素化底物并展开 它们在蛋白酶体降解之前。 P97中的突变会导致与失调相关的疾病 蛋白毒酸；因此，虽然众所周知，p97对细胞健康至关重要，但关于其机制的很多仍然存在 未知。通常，p97必须结合，转移和释放展开的底物。这些步骤中的每一个都是 取决于p97与多个绑定伙伴的互动，但是这些交互如何协调的 没有充分表征。 研究表明，p97结合伴侣OTU1，将泛素部分从p97底物缩小到泛素部分 允许其有效的展开和释放。然而，这种去泛素化的发生仍然是一个悬而未决的问题。 求解P97-OTU1结构将阐明多泛素化的底物是去泛素化的方式，并且将会 对P97如何处理其底物进行更完整的了解。 最近的工作探索了在异二聚体UFD1/NPL4（UN）绑定的背景下的发展启动 伙伴。但是，尚不清楚该倡议机制是否扩展到其他多泛素底物募集 约束伙伴或使用独特的机制。最重要的P97绑定伙伴之一是P47， 这与高尔基膜重塑有关。从历史上看，据报道P47仅与特定相互作用 非泛素化或单泛素化蛋白；但是，最近的证据也证明了P47 还与多泛素化的底物相互作用。探索此行动的结构和生化基础 将扩大对P97复合物如何展开多泛素化底物的理解。阐明如何 p97受结合伙伴的调节，以及那些结合伙伴如何与多偶联的底物相互作用，I 将确定p97-OTU1复合物的高分辨率结构和与 通过冷冻EM的多泛素化底物。 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，改善 我的心理技能，完善我的科学沟通，并成为那些也是如此的领导者 来自代表性不足的背景。在彼得·沉（Peter Shen）博士的精神统治下，并具有尖端 犹他大学提供的资源，我相信培训计划将使我的 发展成为我作为独立科学家的未来职业。