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KMID : 0923620220220030028
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Immune Network
2022 Volume.22 No. 3 p.28 ~ p.28
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Evaluation of Immunoproteasome-Specific Proteolytic Activity Using Fluorogenic Peptide Substrates
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Kim Su-Min
Park Seo-Hyeong
Choi Won-Hoon
Lee Min-Jae
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Abstract
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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.
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KEYWORD
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Proteasome, Immunoproteasome, Fluorogenic substrates, Proteasome inhibitors
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