Structural and Functional Analysis of Proteasome Core Particle Biogenesis

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Project Summary/Abstract Protein misfolding is a key feature of many human diseases including most neurodegenerative diseases and many cancers. Destruction of misfolded proteins is largely mediated by the proteasome, a 2.5 MDa multisubunit complex which is the most sophisticated protease ever described. The proteasome's active sites are sequestered within a barrel-shaped cylindrical chamber, known as the core particle (CP). Access of substrates to the CP is mediated by the regulatory particle (RP), which recognizes proteasome substrates via their ubiquitin tags. The RP unfolds, deubiquitinates, and injects the substrate into the CP where it is rendered into small peptides. Pharmacologic inhibition of the proteasome is an established anti-cancer therapy, most notably in multiple myeloma. Conversely, the possibility of enhancing proteasome function has generated considerable interest in recent years. Such a strategy might ameliorate diseases caused by protein misfolding. A key step in the generation of active proteasomes is the assembly of the 700 kDa 28-subunit CP, which precedes assembly of the full proteasome and occurs by an ordered multistep pathway that requires the function of five dedicated chaperone proteins. Structural analysis of CP maturation has been hampered by challenges in isolating and characterizing assembly intermediates due to their low abundance and transitory nature. Here we hypothesized that defined CP mutants may be enriched for assembly intermediates. We have developed a productive work-flow for the affinity purification and structural analysis of these mutants, and have already generated eight high resolution structures. In Aim 1, we will carry out this structural analysis of CP mutants using Cryo-Electron Microscopy, coupled with detailed structure-function analyses. In Aim 2, we will characterize a long-known but poorly understood regulator of the CP known as PI31/Fub1. We will attempt to determine its structure in complex with the CP using Cryo-Electron Microscopy, and test a number of specific hypotheses regarding its function. In Aim 3, we will characterize a novel protein which is a previously unrecognized transcriptional target of the Rpn4-mediated proteasome biogenesis regulon, and which appears to be a new proteasome-interacting protein. This proposal is expected to provide significant insight into proteasome assembly and overall function, information which could lead to novel therapeutic strategies based on modulating proteasome activity to treat diseases characterized by protein misfolding.