Activity-based probes trap early active intermediates during metacaspase activation

Vida Štrancar; Katarina P van Midden; Daniel Krahn; Kyoko Morimoto; Marko Novinec; Christiane Funk; Simon Stael; Christopher J Schofield; Marina Klemenčič; Renier A L van der Hoorn

doi:10.1016/j.isci.2022.105247

Activity-based probes trap early active intermediates during metacaspase activation

iScience. 2022 Sep 29;25(11):105247. doi: 10.1016/j.isci.2022.105247. eCollection 2022 Nov 18.

Authors

Affiliations

¹ Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia.
² The Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, South Park Road, Oxford OX1 3RB, UK.
³ Chemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute or Antimicrobial Research, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK.
⁴ Department of Chemistry, Umeå University, Umeå, Sweden.
⁵ Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.
⁶ VIB-UGent Center for Plant Systems Biology, Ghent, Belgium.

Abstract

Metacaspases 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 precursors in 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.

Keywords: Cell biology; Functional aspects of cell biology; Methodology in biological sciences.