Activity-based probes trap early active intermediates during metacaspase activation
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...
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| Format: | Article |
| Language: | English |
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Elsevier
2022-11-01
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| Series: | iScience |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S258900422201519X |
| _version_ | 1811325882091962368 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Vida Štrancar |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T14:40:21Z |
| format | Article |
| id | doaj.art-24c8f0d0a12d4df987ff05d968906b07 |
| institution | Directory Open Access Journal |
| issn | 2589-0042 |
| language | English |
| last_indexed | 2024-04-13T14:40:21Z |
| publishDate | 2022-11-01 |
| publisher | Elsevier |
| record_format | Article |
| series | iScience |
| spelling | doaj.art-24c8f0d0a12d4df987ff05d968906b072022-12-22T02:42:54ZengElsevieriScience2589-00422022-11-012511105247Activity-based probes trap early active intermediates during metacaspase activationVida Štrancar0Katarina P. van Midden1Daniel Krahn2Kyoko Morimoto3Marko Novinec4Christiane Funk5Simon Stael6Christopher J. Schofield7Marina Klemenčič8Renier A.L. van der Hoorn9Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, SloveniaDepartment of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, SloveniaThe 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, UKThe Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, South Park Road, Oxford OX1 3RB, UKDepartment of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, SloveniaDepartment of Chemistry, Umeå University, Umeå, SwedenDepartment of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium; VIB-UGent Center for Plant Systems Biology, Ghent, BelgiumChemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute or Antimicrobial Research, University of Oxford, Mansfield Road, Oxford OX1 3TA, UKDepartment of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia; Department of Chemistry, Umeå University, Umeå, SwedenThe Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, South Park Road, Oxford OX1 3RB, UK; Corresponding authorSummary: 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.http://www.sciencedirect.com/science/article/pii/S258900422201519XCell biologyFunctional aspects of cell biologyMethodology in biological sciences |
| spellingShingle | 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 Activity-based probes trap early active intermediates during metacaspase activation iScience Cell biology Functional aspects of cell biology Methodology in biological sciences |
| title | Activity-based probes trap early active intermediates during metacaspase activation |
| title_full | Activity-based probes trap early active intermediates during metacaspase activation |
| title_fullStr | Activity-based probes trap early active intermediates during metacaspase activation |
| title_full_unstemmed | Activity-based probes trap early active intermediates during metacaspase activation |
| title_short | Activity-based probes trap early active intermediates during metacaspase activation |
| title_sort | activity based probes trap early active intermediates during metacaspase activation |
| topic | Cell biology Functional aspects of cell biology Methodology in biological sciences |
| url | http://www.sciencedirect.com/science/article/pii/S258900422201519X |
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