摘要：SummaryMetacaspases are essential cysteine proteases present in plants, fungi, and protists that are regulated by calcium binding and proteolytic maturation through mechanisms not yet understood. Here, we developed and validated activity-based probes for the three main metacaspase types, and used them to study calcium-mediated activation of metacaspases from their precursorsin vitro. By combining substrate-inspired tetrapeptide probes containing an acyloxymethylketone (AOMK) reactive group, with purified representatives of type-I, type-II, and type-III metacaspases, we were able to demonstrate that labeling of mature metacaspases is strictly dependent on calcium. The probe with the highest affinity for all metacaspases also labels higher molecular weight proteoforms of all three metacaspases only in the presence of calcium, displaying the active, unprocessed metacaspase intermediates. Our data suggest that metacaspase activation proceeds through previously unknown active intermediates that are formed upon calcium binding, before precursor processing.Graphical abstractDisplay OmittedHighlights•Four tetrapeptide acyloxymethylketone designed to label metacaspases•All four probes label purified metacaspases representing classes I, II, and III•Labeling and autocatalytic processing requires calcium•Highest affinity IRSK probe traps early intermediates during activationCell biology; Functional aspects of cell biology; Methodology in biological sciences
