Dissecting mechanisms of p97-assisted protein degradation by the proteasome in the human system.

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Efficient degradation of proteins by the proteasome is essential for protein homeostasis and cell signaling. Large cohorts of ubiquitylated proteasomal substrates require prior processing by the AAA+ ATPase p97 (also called VCP, or Cdc48 in yeast) suggesting that p97 unfolds proteins to facilitate subsequent degradation in the proteasome. p97 is being explored as a cancer drug target, while mutations in p97 cause neuromuscular degenerative disease, termed multisystem proteinopathy-1. In recent years, great progress has been made in using biochemical approaches to understand substrate targeting and threading to and through the central channel of p97 leading to substrate unfolding. However, how unfolded proteins are then handed over from the p97 exit pore to the 26S proteasome is not understood, particularly in the human system that involves additional factors. In this project, we will biochemically reconstitute the coupling of p97-mediated unfolding of ubiquitylated substrates with proteasomal degradation in the human system and combine these approaches with assays that monitor key aspects of the process in real time. Moreover, building on own structural and biochemical advances, we will analyze accessory proteins that bind close to the p97 exit pore and are believed to integrate substrate shuttling to the proteasome. We will validate our findings in cells and define affected substrate proteomes. Our results will provide direct mechanistic insight into the missing link in protein degradation, and allow us to cast light on the molecular basis of p97-associated and related disease.