Abstract
We recently developed a new assay to measure proteasome activity in vitro (CAPA for capture proteasome assay) [1], based on proteasome capture on an antibody-coated plate. When used with lysates originating from cells expressing either standard proteasome, immunoproteasome or intermediate proteasomes β5i or β1i-β5i, this assay allows the individual monitoring of the chymotrypsin-like, trypsin-like and caspase-like activities of the corresponding proteasome subtypes. The efficiency and specificity of four proteasome inhibitors were studied using the CAPA assay, demonstrating the potential of this assay for the development of subtype-specific proteasome inhibitors.
Highlights
We recently developed a new assay to measure proteasome activity in vitro (CAPA for capture proteasome assay) [1], based on proteasome capture on an antibody-coated plate
When used with lysates originating from cells expressing either standard proteasome, immunoproteasome or intermediate proteasomes β5i or β1i-β5i, this assay allows the individual monitoring of the chymotrypsin-like, trypsin-like and caspase-like activities of the corresponding proteasome subtypes
To facilitate the in vitro study of proteasome activity, we have designed a new type of assay (CAPA for capture proteasome assay) based on the specific capture of proteasomes on 96-well plates [1] (Fig. 1)
Summary
To facilitate the in vitro study of proteasome activity, we have designed a new type of assay (CAPA for capture proteasome assay) based on the specific capture of proteasomes on 96-well plates [1] (Fig. 1) Using this assay, proteasomes contained in any type of human cell lysate are captured on a black Maxisorp plate pre-coated with the anti-proteasome α2 subunit antibody MCP21 and tested for their ability to degrade fluorogenic peptides suc-LLVY-AMC, Z-LLE-AMC or Boc-LRR-AMC. As a proof-of-concept, we have used the CAPA assay to test the effect of four different proteasome inhibitors (bortezomib, lactacystin, epoxomicin and PR-957) on the chymotrypsin-like, caspase-like and trypsin-like activities of the four proteasome subtypes (see Figure 7 and 8 in Ref [1]). PR-957 was not effective on the caspase-like and trypsinlike activities of the proteasomes Overall these results show the potential of the CAPA assay for the study and identification of proteasome subtype-specific inhibitors
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