# Structural and Functional Analysis of Proteasome Core Particle Biogenesis

> **NIH NIH R01** · BRIGHAM AND WOMEN'S HOSPITAL · 2024 · $400,476

## Abstract

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.

## Key facts

- **NIH application ID:** 10802314
- **Project number:** 5R01GM144367-03
- **Recipient organization:** BRIGHAM AND WOMEN'S HOSPITAL
- **Principal Investigator:** John W Hanna
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $400,476
- **Award type:** 5
- **Project period:** 2022-04-15 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10802314

## Citation

> US National Institutes of Health, RePORTER application 10802314, Structural and Functional Analysis of Proteasome Core Particle Biogenesis (5R01GM144367-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10802314. Licensed CC0.

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