Activity-based probes trap early active intermediates during metacaspase activation

• DOI: 10.1016/j.isci.2022.105247
• Publication Date: 2022-09-29T19:21:56Z
• Authors: Vida Štrancar; Katarina P. van M...; Daniel Krahn; Kyoko Morimoto; Marko Novinec; Christiane Funk; Simon Stael; Christopher J. Sc...; Marina Klemenčič; Renier A.L. van d...
• Keywords: Cell biology; 0303 health sciences; IDENTIFICATION; Science; Methodology in biological sciences; Q; Biology and Life Sciences; SLOW-BINDING; CALCIUM; Article; PROGRAMMED CELL-DEATH; 03 medical and health sciences; Functional aspects of cell biology; TRYPANOSOMA-BRUCEI; ARABIDOPSIS-THALIANA; MALT1 PARACASPASE; CRYSTAL-STRUCTURE; Plant Biotechnology; INHIBITORS; Växtbioteknologi

Metacaspases are essential cysteine proteases present in plants, fungi, and protists that regulated by calcium binding proteolytic maturation through mechanisms not yet understood. Here, we developed validated activity-based probes for the three main metacaspase types, used them to study calcium-mediated activation of metacaspases from their precursors vitro. By combining substrate-inspired tetrapeptide containing an acyloxymethylketone (AOMK) reactive group, with purified representatives type-I, type-II, type-III metacaspases, were able demonstrate labeling mature is strictly dependent on calcium. The probe highest affinity all also labels higher molecular weight proteoforms only presence calcium, displaying active, unprocessed intermediates. Our data suggest proceeds previously unknown active intermediates formed upon binding, before precursor processing.