Discrimination of Differentially Inhibited Cysteine Proteases by Activity-Based Profiling Using Cystatin Variants with Tailored Specificities

Abstract

Recent research has shown the possibility of tailoring the inhibitory specificity of plant cystatins toward cysteine (Cys) proteases by single mutations at positively selected amino acid sites. Here we devised a cystatin activity-based profiling approach to assess the impact of such mutations at the proteome scale using single variants of tomato cystatin SlCYS8 and digestive Cys proteases of the herbivorous insect, Colorado potato beetle, as a model. Biotinylated forms of SlCYS8 and SlCYS8 variants were used to capture susceptible Cys proteases in insect midgut protein extracts by biotin immobilization on avidin-embedded beads. A quantitative LC-MS/MS analysis of the captured proteins was performed to compare the inhibitory profile of different SlCYS8 variants. The approach confirmed the relevance of phylogenetic inferences categorizing the insect digestive Cys proteases into six functionally distinct families. It also revealed significant variation in protease family profiles captured with N-terminal variants of SlCYS8, in line with in silico structural models for Cys protease-SlCYS8 interactions suggesting a functional role for the N-terminal region. Our data confirm overall the usefulness of cystatin activity-based protease profiling for the monitoring of Cys protease-inhibitor interactions in complex biological systems. They also illustrate the potential of biotinylated cystatins to identify recombinant cystatin candidates for the inactivation of specific Cys protease targets.

An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves

“…J05176) were flanked with the N-terminal signal peptide of a protein disulphide isomerase (PDI) from alfalfa [37], to give the expression vectors pEAQ- Sl CYS8 and pEAQ-α 1 -ACT, respectively (Fig 1). A pEAQ vector for Q47P, an inactive form of Sl CYS8 produced by site-directed substitution of residue Gln-47 for a proline in the Sl CYS8 donor clone, was used as a functional negative control for Sl CYS8 in planta [38]. All gene constructs were verified by DNA sequencing before plant transfection assays.…”

Phytocystatins: Defense Proteins against Phytophagous Insects and Acari

“…For the same purpose, the discrimination of differentially inhibited L. decemlineata CysProt was assessed by biotinylated forms of SlCYS8 and SlCYS8 variants [57]. These tailored cystatins were used to capture susceptible CysProt in protein extracts of the insect midgut by biotin immobilization on avidin-embedded beads.…”

“…These tailored cystatins were used to capture susceptible CysProt in protein extracts of the insect midgut by biotin immobilization on avidin-embedded beads. A quantitative LC-MS/MS analysis of the captured proteins was performed to compare the inhibitory profiles within SlCYS8 variants and to identify candidates for the inactivation of specific CysProt targets in the pest [57]. However, rebalancing of midgut proteases by the pest in response to the cystatins may occur.…”

Review: The future of cystatin engineering

“…Despite a similar mechanism of action and structural homology among them, cystatins naturally exhibit distinct differences in enzyme affinities [20,40]. Unfortunately, the majority of sequence data currently available is still either from a model plant, such as Arabidopsis, or from plants where commercial interest has strongly driven the genomic and transcriptomic exploration in these plants.…”

“…The general potential of such multi-proteins has been demonstrated in several studies with various expression strategies. These include stable fusion protein production, cleavable poly-proteins or polycistronic DNA constructs with the co-translational release of proteins [40]. More simple two component multi-cystatins are cystatin combinations to obtain simultaneous resistance against an insect and a phyto-pathogen [48].…”