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dc.contributor.authorRasoolizadeh, Asieh
dc.contributor.authorGoulet, Marie-Claire
dc.contributor.authorSainsbury, Frank
dc.contributor.authorCloutier, Conrad
dc.contributor.authorMichaud, Dominique
dc.description.abstractA causal link has been reported between positively selected amino acids in plant cystatins and the inhibitory range of these proteins against insect digestive cysteine (Cys) proteases. Here we assessed the impact of single substitutions to closely related amino acids on the contribution of positive selection to cystatin diversification. Cystatin sequence alignments, while confirming hypervariability, indicated a preference for related amino acids at positively selected sites. For example, the non-polar residues leucine (Leu), isoleucine (Ile) and valine (Val) were shown to predominate at positively selected site 2 in the N-terminal region, unlike selected sites 6 and 10, where polar residues are preferred. The model cystatin SlCYS8 and single variants with Leu, Ile or Val at position 2 were compared with regard to their ability to bind digestive proteases of the coleopteran pest Leptinotarsa decemlineata and to induce compensatory responses in this insect. A functional proteomics procedure to capture target Cys proteases in midgut extracts allowed confirmation of distinct binding profiles for the cystatin variants. A shotgun proteomics procedure to monitor whole Cys protease complements revealed protease family specific compensatory responses in the insect, dependent on the variant ingested. Our data confirm the contribution of closely related amino acids to the functional diversity of positively selected plant cystatins in a broader structure/function context imposing physicochemical constraints to primary structure alterations. They also underline the complexity of protease/inhibitor interactions in plant-insect systems, and the challenges still to be met in order to harness the full potential of ectopically expressed protease inhibitors in crop protection.
dc.publisherJohn Wiley & Sons
dc.relation.ispartofjournalFEBS Journal
dc.subject.fieldofresearchMedicinal and biomolecular chemistry
dc.subject.fieldofresearchBiochemistry and cell biology
dc.subject.fieldofresearchMedical biochemistry and metabolomics
dc.subject.keywordsScience & Technology
dc.subject.keywordsLife Sciences & Biomedicine
dc.subject.keywordsBiochemistry & Molecular Biology
dc.subject.keywordsactivity-based functional proteomics
dc.subject.keywordsfunctional diversification
dc.titleSingle substitutions to closely related amino acids contribute to the functional diversification of an insect-inducible, positively selected plant cystatin
dc.typeJournal article
dc.type.descriptionC1 - Articles
dcterms.bibliographicCitationRasoolizadeh, A; Goulet, M-C; Sainsbury, F; Cloutier, C; Michaud, D, Single substitutions to closely related amino acids contribute to the functional diversification of an insect-inducible, positively selected plant cystatin, FEBS Journal, 2016, 283 (7), pp. 1323-1335
gro.hasfulltextNo Full Text
gro.griffith.authorSainsbury, Frank

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