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Evaluation of Immunoproteasome-Specific Proteolytic Activity Using Fluorogenic Peptide Substrates
Cited 2 time in
Web of Science
Cited 2 time in Scopus
- Authors
- Issue Date
- 2022-06
- Publisher
- 대한면역학회
- Citation
- Immune Network, Vol.22 No.3, p. 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 (beta 1i/PSMB9, beta 2i/PSMB10, and beta 5i/PSMB8) instead of constitutively expressed counterparts (beta 1/PSMB6, beta 2/PSMB7, and beta 5/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 beta 5i-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.
- ISSN
- 1598-2629
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