Activity Profiling of Papain-Like Cysteine Proteases in Plants

Abstract

Transcriptomic and proteomic technologies are generating a wealth of data that are frequently used by scientists to predict the function of proteins based on their expression or presence. However, activity of many proteins, such as transcription factors, kinases, and proteases, depends on posttranslational modifications that frequently are not detected by these technologies. Therefore, to monitor activity of proteases rather than their abundance, we introduce protease activity profiling in plants. This technology is based on the use of biotinylated, irreversible protease inhibitors that react with active proteases in a mechanismbased manner. Using a biotinylated derivative of the Cys protease inhibitor E-64, we display simultaneous activities of many papain-like Cys proteases in extracts from various tissues and from different plant species. Labeling is pH dependent, stimulated with reducing agents, and inhibited specifically by Cys protease inhibitors but not by inhibitors of other protease classes. Using one-step affinity capture of biotinylated proteases followed by sequencing mass spectrometry, we identified proteases that include xylem-specific XCP2, desiccation-induced RD21, and cathepsin B-and aleurain-like proteases. Together, these results demonstrate that this technology can identify differentially activated proteases and/or characterize the activity of a particular protease within complex mixtures.

Granulosain I, a Cysteine Protease Isolated from Ripe Fruits of Solanum granuloso -leprosum (Solanaceae)

“…Little is known about the physiological functions of these peptidase. In plants, for example, they have been associated with the mobilization of nutrients and defence against pathogens [3,28,47]. Many of these enzymes have important applications in medicine and biotechnology.…”

“…These enzymes are usually synthesized as inactive or less active precursors. Activation takes place by limited intra-or intermolecular proteolysis [3]. To date, the amino acid sequences of more than 50 papain-like and 15 legumain-like plant proteinases have been established [4].…”

An Effector-Targeted Protease Contributes to Defense against Phytophthora infestans and Is under Diversifying Selection in Natural Hosts

“…Pellets were dissolved in SDS gel loading buffer. Detection of biotinylated proteins was done as described previously using streptavidin-HRP polymer (Sigma-Aldrich; van der Hoorn et al, 2004).…”

Serpin1 and WSCP differentially regulate the activity of the cysteine protease RD21 during plant development in Arabidopsis thaliana

“…1 A, b). Protein sequencing and immunoprecipitation studies identified RD21, a PLCP previously identified by DCG-04 activity profiling (25), in protein extracts of 4.5-d-old etiolated seedlings ( Fig. 1 A, a, lane 3 vs. lane 2, product C1).…”