Visualizing the Mechanisms of Protein Quality Control

项目摘要/摘要 最佳的细胞功能需要维持蛋白质合成、折叠、运输、 重塑和退化。CDC48/p97/VCP AAA ATPase是一种必需和丰富的分子 帮助在真核生物生命中维持这种平衡的机器，因此是 蛋白质平衡。CDC48最好的特征是它在蛋白酶体的泛素化蛋白靶向方面的作用 降解，但该酶也在广泛的其他基本途径中发挥作用，包括细胞周期 进展、自噬、膜融合和基因表达。人类CDC48基因突变是致病因素 一种临床表现为包涵体肌病的多系统蛋白病，Paget‘s 骨病、额颞部痴呆(统称为IBMPFD)和家族性肌萎缩侧索硬化 硬化症(FALS)。此外，CDC48在几种肿瘤中表达升高，其抑制物是一种新兴的 癌症治疗的治疗学类。尽管扮演着这些关键角色，但令人惊讶的是，人们对 CDC48发挥多种细胞功能的分子机制。三十多个适配器和 已知辅因子与CDC48相互作用，但细胞如何将这些结合伙伴组织成功能性的 复合体也是未知的。我的研究计划旨在定义CDC48功能的潜在机制 通过使用内源性纯化、蛋白质组学、冷冻-EM成像和计算的综合方法 加工方法。我们的方法将用于可视化和表征CDC48程序集 其天然的组成和构象状态。在项目期间，我们提出了两个主要的目标 目标：首先，我们的目标是确定在多个细胞中从功能上分离CDC48的分子线索 第二，我们试图解决CDC48如何将ATP水解产生的能量转化为 重塑并打开它的蛋白质底物。解决这些问题对于理解CDC48如何 推动广泛的细胞过程，以及其功能障碍和调控不当如何导致退行性变 疾病和癌症。我们为实现这些目标而开发的工具可能会广泛应用于 定义了其他具有挑战性的分子机器的结构图景。

Project Summary / Abstract Optimal cellular function requires balanced networks that maintain protein synthesis, folding, trafficking, remodeling and degradation. The Cdc48/p97/VCP AAA ATPase is an essential and abundant molecular machine that helps maintain this balance across eukaryotic life and is therefore a critical control point for proteostasis. Cdc48 is best characterized for its role in targeting ubiquitylated proteins for proteasomal degradation, but the enzyme also functions in a wide range of other essential pathways, including cell cycle progression, autophagy, membrane fusion, and gene expression. Mutations in human Cdc48 are causative of a multisystem proteopathy that clinically manifests as a combination of Inclusion Body Myopathy, Paget's Disease of Bone, Frontotemporal Dementia (collectively known as IBMPFD), and familial Amyotrophic Lateral Sclerosis (fALS). Moreover, Cdc48 expression is elevated in several tumors and its inhibitors are an emerging class of therapeutics for cancer treatment. Despite these critical roles, surprisingly little is known about the molecular mechanisms that allow Cdc48 to perform its myriad cellular functions. More than thirty adaptors and cofactors are known to interact with Cdc48, but how cells organize these binding partners into functional complexes is also unknown. My research program aims to define the mechanisms underlying Cdc48 functions by using an integrative approach of endogenous purification, proteomics, cryo-EM imaging, and computational processing methods. Our approach will be used to visualize and characterize Cdc48 assemblies in an array of its native compositional and conformational states. During the project period, we propose to achieve two major goals: first, we aim to determine the molecular cues that functionally separate Cdc48 across multiple cellular pathways; second, we seek to resolve how Cdc48 converts energy from ATP hydrolysis into a pulling force that remodels and unfolds its protein substrates. Addressing these questions is essential to understand how Cdc48 drives a wide range of cellular processes and how its dysfunction and misregulation contribute to degenerative disease and cancer. The tools we develop to accomplish these goals will likely be broadly applicable in defining the structural landscapes of other challenging molecular machines.