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Activity-based probes trap early active intermediates during metacaspase activation.

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.

SUBMITTER: Strancar V

PROVIDER: S-EPMC9626678 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Activity-based probes trap early active intermediates during metacaspase activation.

Štrancar Vida V van Midden Katarina P KP Krahn Daniel D Morimoto Kyoko K Novinec Marko M Funk Christiane C Stael Simon S Schofield Christopher J CJ Klemenčič Marina M van der Hoorn Renier A L RAL

iScience 20220929 11

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 <i>in vitro</i>. By combining substrate-inspired tetrapeptide probes containing an acyloxymethylketone (AOMK) reactive group, wit ...[more]

PMID: 36339259

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Activity-based probes trap early active intermediates during metacaspase activation.

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Activity-based probes trap early active intermediates during metacaspase activation.

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