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Discrimination of differentially inhibited cysteine proteases by activity-based profiling using cystatin variants with tailored specificities.
J Proteome Res. 2012 Dec 07; 11(12):5983-93.JP

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.

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Publisher Full Text (DOI)

Authors+Show Affiliations

Sainsbury F
Département de Phytologie, Université Laval, Québec, Canada.
Rhéaume AJ
No affiliation info available
Goulet MC
No affiliation info available
Vorster J
No affiliation info available
Michaud D
No affiliation info available

MeSH

AnimalsColeopteraCystatinsCysteine ProteasesCysteine Proteinase InhibitorsEnzyme AssaysInsect ProteinsLarvaModels, BiologicalMolecular Docking SimulationMultiprotein ComplexesMutationPhylogenyPlant ProteinsProtein BindingProtein Interaction MapsProteomeProteomicsStructure-Activity RelationshipSubstrate Specificity

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

23082957

Citation

Sainsbury, Frank, et al. "Discrimination of Differentially Inhibited Cysteine Proteases By Activity-based Profiling Using Cystatin Variants With Tailored Specificities." Journal of Proteome Research, vol. 11, no. 12, 2012, pp. 5983-93.
Sainsbury F, Rhéaume AJ, Goulet MC, et al. Discrimination of differentially inhibited cysteine proteases by activity-based profiling using cystatin variants with tailored specificities. J Proteome Res. 2012;11(12):5983-93.
Sainsbury, F., Rhéaume, A. J., Goulet, M. C., Vorster, J., & Michaud, D. (2012). Discrimination of differentially inhibited cysteine proteases by activity-based profiling using cystatin variants with tailored specificities. Journal of Proteome Research, 11(12), 5983-93. https://doi.org/10.1021/pr300699n
Sainsbury F, et al. Discrimination of Differentially Inhibited Cysteine Proteases By Activity-based Profiling Using Cystatin Variants With Tailored Specificities. J Proteome Res. 2012 Dec 7;11(12):5983-93. PubMed PMID: 23082957.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Discrimination of differentially inhibited cysteine proteases by activity-based profiling using cystatin variants with tailored specificities. AU - Sainsbury,Frank, AU - Rhéaume,Ann-Julie, AU - Goulet,Marie-Claire, AU - Vorster,Juan, AU - Michaud,Dominique, Y1 - 2012/11/06/ PY - 2012/10/23/entrez PY - 2012/10/23/pubmed PY - 2013/5/25/medline SP - 5983 EP - 93 JF - Journal of proteome research JO - J Proteome Res VL - 11 IS - 12 N2 - 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. SN - 1535-3907 UR - https://www.unboundmedicine.com/medline/citation/23082957/Discrimination_of_differentially_inhibited_cysteine_proteases_by_activity_based_profiling_using_cystatin_variants_with_tailored_specificities_ L2 - https://doi.org/10.1021/pr300699n DB - PRIME DP - Unbound Medicine ER -