Plant Metacaspase Activation and Activity
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Metacaspases are essential for cell death regulation in plants. Further understanding of biochemistry of metacaspases and their molecular function in plant biology requires a set of robust methods for detection of metacaspase activation and quantitative analysis of corresponding proteolytic activity. Here we describe methods for purification of recombinant metacaspases, measurement of enzymatic activity of recombinant and endogenous metacaspases in vitro and in cell lysates, respectively, and finally detection of metacaspase activation in vivo. Additionally, an in vitro metacaspase protein substrate cleavage assay based on the cell-free production of substrate protein followed by proteolysis with recombinant metacaspase is presented. These methods have been originally developed for type II metacaspases from Arabidopsis and Norway spruce (Picea abies), but they can be used as templates for type I metacaspases, as well as for type II metacaspases from other species.
Key wordsCell lysate Metacaspase Natural substrate Programmed cell death Proteolytic activity Synthetic substrate
S.S. is indebted to the Special Research Fund of Ghent University for a postdoctoral fellowship. F.V.B acknowledges support from grants of the Ghent University Multidisciplinary Research Partnership “Ghent BioEconomy” 27 (project 01MRB510W), the Belgian Science Policy Office (project IAP7/29), and the Research Foundation Flanders (FWO-Vlaanderen; project G.0038.09). E.A.M. and P.V.B. acknowledge support from grants of the Swedish Research Council (VR), Knut and Alice Wallenberg Foundation, the Swedish Foundation for Strategic Research (SSF), Pehrssons Fund and Olle Engkvist Byggmästare Foundation.