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
Affiliations
Vida Štrancar
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia
Katarina P. van Midden
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia
Daniel Krahn
The Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, South Park Road, Oxford OX1 3RB, UK; Chemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute or Antimicrobial Research, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK
Kyoko Morimoto
The Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, South Park Road, Oxford OX1 3RB, UK
Marko Novinec
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia
Christiane Funk
Department of Chemistry, Umeå University, Umeå, Sweden
Simon Stael
Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium; VIB-UGent Center for Plant Systems Biology, Ghent, Belgium
Christopher J. Schofield
Chemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute or Antimicrobial Research, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK
Marina Klemenčič
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia; Department of Chemistry, Umeå University, Umeå, Sweden
Renier A.L. van der Hoorn
The Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, South Park Road, Oxford OX1 3RB, UK; Corresponding author
Summary: 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.
