Immune Netw.  2022 Jun;22(3):e28. 10.4110/in.2022.22.e28.

Evaluation of ImmunoproteasomeSpecific Proteolytic Activity Using Fluorogenic Peptide Substrates

• Kim S
• Park SH
• Choi WH
• Lee MJ

• KMID: 2536255
• DOI: http://doi.org/10.4110/in.2022.22.e28

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 β5itargeting 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.

Keyword

Proteasome; Immunoproteasome; Fluorogenic substrates; Proteasome inhibitors