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  • 제22권3호
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  • Pages.28.1-28.11
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  • 2022
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  • 1598-2629(pISSN)
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  • 2092-6685(eISSN)
대한면역학회 (The Korean Association of Immunobiologists)
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Evaluation of Immunoproteasome-Specific Proteolytic Activity Using Fluorogenic Peptide Substrates

  • Sumin Kim (Department of Biochemistry & Molecular Biology, Seoul National University College of Medicine) ;
  • Seo Hyeong Park (Department of Biochemistry & Molecular Biology, Seoul National University College of Medicine) ;
  • Won Hoon Choi (Department of Biochemistry & Molecular Biology, Seoul National University College of Medicine) ;
  • Min Jae Lee (Department of Biochemistry & Molecular Biology, Seoul National University College of Medicine)
  • 투고 : 2022.01.04
  • 심사 : 2022.04.05
  • 발행 : 2022.06.30
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초록

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.

키워드

과제정보

S.K., S.H.P, and W.H.C. were supported by the BK21 FOUR Biomedical Science Program funded by the Ministry of Education and National Research Foundation (NRF) of Korea. This work was also supported by other grants from the NRF (2020R1A5A1019023 and 2021R1A2C2008023 to M.J.L.), the Korea Health Industry Development Institute and Korea Dementia Research Center (HU21C0071 to M.J.L.), and the Creative-Pioneering Researchers Program through Seoul National University.

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