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Evaluation of Immunoproteasome-Specific Proteolytic Activity Using Fluorogenic Peptide Substrates

Authors :
Sumin Kim
Seo Hyeong Park
Won Hoon Choi
Min Jae Lee
Source :
Immune network. 22(3)
Publication Year :
2022

Abstract

The 26S proteasome irreversibly hydrolyzes polyubiquitylated substrates to maintain protein homeostasis; it also regulates immune responses by generating antigenic peptides. An alternative form of the 26S proteasome is the immunoproteasome, which contains substituted catalytic subunits (β1i/PSMB9, β2i/PSMB10, and β5i/PSMB8) instead of constitutively expressed counterparts (β1/PSMB6, β2/PSMB7, and β5/PSMB5). The immunoproteasome expands the peptide repertoire presented on MHC class I molecules. However, how its activity changes in this context is largely elusive, possibly due to the lack of a standardized methodology to evaluate its specific activity. Here, we describe an assay protocol that measures the immunoproteasome activity of whole-cell lysates using commercially available fluorogenic peptide substrates. Our results showed that the most accurate assessment of immunoproteasome activity could be achieved by combining β5i-targeting substrate Ac-ANW-AMC and immunoproteasome inhibitor ONX-0914. This simple and reliable protocol may contribute to future studies of immunoproteasomes and their pathophysiological roles during viral infection, inflammation, and tumorigenesis.

Details

ISSN :
15982629
Volume :
22
Issue :
3
Database :
OpenAIRE
Journal :
Immune network
Accession number :
edsair.doi.dedup.....c6cf0b739f821fd8b082ab9c83a5619c