Visualizing the Mechanisms of Protein Quality Control

项目总结/摘要 最佳的细胞功能需要平衡的网络，以维持蛋白质的合成，折叠，运输， 重塑和降解。Cdc 48/p97/VCP AAA ATP酶是一种必需的、丰富的分子生物学活性物质， 这台机器有助于维持真核生物的这种平衡，是许多细胞的关键控制点。 途径。Cdc 48的最佳特征在于其在蛋白酶体靶向泛素化蛋白中的作用。 这种酶不仅可以降解，而且还在广泛的其他重要途径中发挥作用，包括细胞周期 进展、自噬、膜融合和基因表达。人类Cdc 48基因突变导致 临床表现为包涵体肌病、佩吉特氏病 骨病、额颞叶痴呆（统称为IBMPFD）和家族性肌萎缩侧索硬化症 硬化症（fALS）。此外，Cdc 48表达在几种肿瘤中升高，其抑制剂正在出现。 用于癌症治疗的一类疗法。尽管有这些关键作用，令人惊讶的是， 分子机制，使Cdc 48执行其无数的细胞功能。超过30个适配器， 已知辅因子与Cdc 48相互作用，但细胞如何将这些结合伴侣组织成功能性的 复合物并不被很好地理解。我的研究计划旨在确定Cdc 48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 计算处理方法来可视化和表征Cdc 48组装体在其天然的 组成和构象状态。在项目期间，我们建议实现两个主要目标： 首先，我们的目标是确定天然Cdc 48组装体的结构及其分子决定簇， 在功能上分离跨多个细胞通路的酶;第二，我们试图解决如何Cdc 48 将ATP水解产生的能量转化为拉力，重塑并展开其蛋白质底物。 解决这些问题对于理解Cdc 48如何驱动广泛的细胞过程至关重要。 并提供其功能障碍和失调如何导致退行性疾病和癌症的见解。 我们为实现这些目标而开发的工具可能会广泛适用于定义结构化 其他具有挑战性的分子机器的景观。

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 a critical control point for many cellular pathways. 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 cause 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 not well understood. 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 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 structures of native Cdc48 assemblies and their molecular determinants that functionally separate the enzyme 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 provide insights into 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.