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Activity-based probes trap early active intermediates during metacaspase activation.
Vida Štrancar1, Katarina P van Midden1, Daniel Krahn2,3
1Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia.
Iscience
|November 7, 2022
Summary
Calcium binding activates essential metacaspases (cysteine proteases) in plants and fungi. New probes reveal active, unprocessed metacaspase intermediates form upon calcium binding before precursor processing.
Area of Science:
- Biochemistry
- Molecular Biology
- Protease research
Background:
- Metacaspases are crucial cysteine proteases in plants, fungi, and protists.
- Their regulation by calcium and proteolytic maturation mechanisms remain largely unknown.
Purpose of the Study:
- To develop and validate activity-based probes for metacaspase types.
- To investigate calcium-mediated activation of metacaspases from precursors in vitro.
Main Methods:
- Designed substrate-inspired tetrapeptide probes with an acyloxymethylketone (AOMK) reactive group.
- Utilized purified type-I, type-II, and type-III metacaspases for in vitro studies.
- Analyzed calcium-dependent labeling of metacaspases and their proteoforms.
Main Results:
- Metacaspase labeling by probes was strictly dependent on calcium presence.
- A high-affinity probe labeled mature and higher molecular weight metacaspase proteoforms.
- Labeling of unprocessed metacaspase intermediates occurred only with calcium.
Conclusions:
- Metacaspase activation is calcium-dependent.
- Calcium binding triggers the formation of previously unidentified active metacaspase intermediates.
- These intermediates precede the final precursor processing in metacaspase activation.
