Exploring the immunoproteasome's substrate preferences for improved hydrolysis and selectivity

Abstract

The proteasome is an integral macromolecular machine responsible for regulated protein degradation, and its barrel-like core particle (CP) hydrolyzes protein substrates into peptide fragments. A proteasome isoform that is expressed under conditions of inflammation is known as the immunoproteasome (iCP), which incorporates different catalytic subunits of altered cleavage specificities from the standard proteasome (sCP). Probes and inhibitors have been generated to study iCP activity and for therapeutics, respectively; recently, the iCP has been harnessed as a prodrug enzyme to release bioactive compounds selectively into iCP-expressing cells. iCP-targeting probes, prodrugs, and inhibitors are based on peptide recognition sequences and their favorable interactions within the iCP's substrate channel. To better understand what unnatural substrates the iCP can recognize, we synthesized peptide-conjugated substrates and applied them to a liquid chromatography-mass spectrometry (LC-MS) method after incubation with purified human iCP. Structure–activity relationships of unnatural peptide-conjugated substrates revealed modifications that improved substrate selectively for the iCP by more than 3-fold compared to the original scaffold. As such, this report will be helpful to guide future iCP-targeting probes, prodrugs, and inhibitor design.

Graphical abstract: Exploring the immunoproteasome's substrate preferences for improved hydrolysis and selectivity

Supplementary files

Article information

Article type
Paper
Submitted
06 May 2025
Accepted
01 Jul 2025
First published
07 Jul 2025
This article is Open Access
Creative Commons BY-NC license

RSC Chem. Biol., 2025, Advance Article

Exploring the immunoproteasome's substrate preferences for improved hydrolysis and selectivity

C. S. Muli, C. A. Loy and D. J. Trader, RSC Chem. Biol., 2025, Advance Article , DOI: 10.1039/D5CB00114E

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