The capture proteasome assay: A method to measure proteasome activity in vitro.
Because of its crucial role in various cellular processes, the proteasome is the focus of intensive research for the development of proteasome inhibitors to treat cancer and autoimmune diseases. Here, we describe a new and easy assay to measure the different proteasome activities in vitro (chymotrypsin-like, caspase-like, and trypsin-like) based on proteasome capture on antibody-coated plates, namely the capture proteasome assay (CAPA). Applying the CAPA to lysates from cells expressing standard proteasome, immunoproteasome, or intermediate proteasomes β5i or β1i-β5i, we can monitor the activity of the four proteasome subtypes. The CAPA provided similar results as the standard whole-cell proteasome-Glo assay without the problem of contaminating proteases requiring inhibitors. However, the profile of trypsin-like activity differed between the two assays. This could be partly explained by the presence of MgSO4 in the proteasome-Glo buffer, which inhibits the trypsin-like activity of the proteasome. The CAPA does not need MgSO4 and, therefore, provides a more precise measurement of the trypsin-like activity. The CAPA provides a quick and accurate method to measure proteasome activity in vitro in a very specific manner and should be useful for the development of proteasome inhibitors.
Ludwig Institute for Cancer Research, B-1200 Brussels, Belgium; Walloon Excellence in Life Sciences and Biotechnology (WELBIO), B-1200 Brussels, Belgium; de Duve Institute, Université catholique de Louvain, B-1200 Brussels, Belgium. Electronic address: nathalie.vigneron@bru.licr.org.
Nathalie
N
Vigneron
Ludwig Institute for Cancer Research, B-1200 Brussels, Belgium; Walloon Excellence in Life Sciences and Biotechnology (WELBIO), B-1200 Brussels, Belgium; de Duve Institute, Université catholique de Louvain, B-1200 Brussels, Belgium.
Joanna
J
Abi Habib
Ludwig Institute for Cancer Research, B-1200 Brussels, Belgium; Walloon Excellence in Life Sciences and Biotechnology (WELBIO), B-1200 Brussels, Belgium; de Duve Institute, Université catholique de Louvain, B-1200 Brussels, Belgium.
Benoît J
BJ
Van den Eynde
D055910:Antibodies, Immobilized / Q000737:chemistry; D002460:Cell Line; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D049454:Firefly Luciferin / Q000031:analogs & derivatives / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000302:isolation & purification / Q000378:metabolism*; D061988:Proteasome Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D055910:Antibodies, Immobilized; D005456:Fluorescent Dyes; D010455:Peptides; D061988:Proteasome Inhibitors; D049454:Firefly Luciferin; D046988:Proteasome Endopeptidase Complex
Activity assay; Fluorogenic peptides; Proteasome; Proteasome–Glo