Visualizing the Mechanisms of Protein Quality Control

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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.