Štrancar Vida, van Midden Katarina P, Krahn Daniel, Morimoto Kyoko, Novinec Marko, Funk Christiane, Stael Simon, Schofield Christopher J, Klemenčič Marina, van der Hoorn Renier A L
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia.
The Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, South Park Road, Oxford OX1 3RB, UK.
iScience. 2022 Sep 29;25(11):105247. doi: 10.1016/j.isci.2022.105247. eCollection 2022 Nov 18.
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 . 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.
类半胱天冬酶是存在于植物、真菌和原生生物中的必需半胱氨酸蛋白酶，其通过尚未明确的机制受钙结合和蛋白水解成熟的调控。在此，我们开发并验证了针对三种主要类半胱天冬酶类型的基于活性的探针，并使用它们来研究钙介导的类半胱天冬酶从前体的激活过程。通过将含有酰氧基甲基酮（AOMK）反应基团的受底物启发的四肽探针与I型、II型和III型类半胱天冬酶的纯化代表相结合，我们能够证明成熟类半胱天冬酶的标记严格依赖于钙。对所有类半胱天冬酶具有最高亲和力的探针也仅在钙存在的情况下标记所有三种类半胱天冬酶的更高分子量蛋白形式，显示出活性的、未加工的类半胱天冬酶中间体。我们的数据表明，类半胱天冬酶的激活通过钙结合后在前体加工之前形成的先前未知的活性中间体进行。
