Arabidopsis thaliana Enolase 2 is modified by S-glutathionylation

Citation

Dumont S, Bykova NV, Rojas LM, Bélanger M, Rivoal J (2018) Arabidopsis thaliana Enolase 2 is modified by S-glutathionylation. Plant Biology 2018. A joint meeting of the American Society of Plant Biologists, Canadian Society of Plant Biologists/Societe Canadienne de Biologie Vegetale, and the International Society of Photosynthesis Research, Montreal, Québec, Canada, July 14-18, 2018. Poster

Plain language summary

Plants produce reactive oxygen species (ROS) as part of their aerobic metabolism. Several abiotic and biotic stresses such as detection of pathogen or herbivore attack can trigger an oxidative burst via a rapid release of ROS. Higher levels of ROS can adversely affect all cellular macromolecules, including lipids, carbohydrates, nucleic acids, and proteins. ROS can also act as signaling molecules involved in plant defense responses to different stress conditions. This role can be mediated via the promotion of redox posttranslational modifications (PTMs) on protein cysteine thiol groups. Enolase is a glycolytic enzyme that catalyses the conversion of 2-phosphoglycerate to phosphoenolpyruvate (PEP). PEP can be converted to pyruvate by pyruvate kinase but can also be used to fuel secondary metabolism through the shikimate pathway and phenylpropanoids synthesis.
In this project we studied the effect of redoxdependent protein PTMs on Enolase 2 (ENO2) from Arabidopsis thaliana. Recombinant ENO2 was heterologously expressed and purified to homogeneity. We found that the activity of the recombinant enzyme was affected differently upon incubation with redox reagents such as hydrogen peroxide, glutathione and diamide. In particular, S-glutathionylation (the formation of a disulfide bond between ENO2 and the small peptide glutathione) could be observed in vitro using a biotinylated analog of glutathione and western blot detection. ENO2 S-glutathionylation was reversible in vitro upon incubation with recombinant A. thaliana glutaredoxins. Using nanoLC-MS/MS analysis of the recombinant enzyme, we were able to identify two Cys residues modified by S-glutathionylation. Further characterization of the importance of these residues in ENO2 is currently underway. The possible implications of this redox modification of plant enolase in the defense against environmental stresses will be discussed.

Abstract

Plants produce reactive oxygen species (ROS) as part of their aerobic metabolism. Several abiotic and biotic stresses such as detection of pathogen or herbivore attack can trigger an oxidative burst via a rapid release of ROS. Higher levels of ROS can adversely affect all cellular macromolecules, including lipids, carbohydrates, nucleic acids, and proteins. ROS can also act as signaling molecules involved in plant defense responses to different stress conditions. This role can be mediated via the promotion of redox posttranslational modifications (PTMs) on protein cysteine thiol groups. Enolase is a glycolytic enzyme that catalyses the conversion of 2-phosphoglycerate to phosphoenolpyruvate (PEP). PEP can be converted to pyruvate by pyruvate kinase but can also be used to fuel secondary metabolism through the shikimate pathway and phenylpropanoids synthesis.
In this project we studied the effect of redoxdependent protein PTMs on Enolase 2 (ENO2) from Arabidopsis thaliana. Recombinant ENO2 was heterologously expressed and purified to homogeneity. We found that the activity of the recombinant enzyme was affected differently upon incubation with redox reagents such as hydrogen peroxide, glutathione and diamide. In particular, S-glutathionylation (the formation of a disulfide bond between ENO2 and the small peptide glutathione) could be observed in vitro using a biotinylated analog of glutathione and western blot detection. ENO2 S-glutathionylation was reversible in vitro upon incubation with recombinant A. thaliana glutaredoxins. Using nanoLC-MS/MS analysis of the recombinant enzyme, we were able to identify two Cys residues modified by S-glutathionylation. Further characterization of the importance of these residues in ENO2 is currently underway. The possible implications of this redox modification of plant enolase in the defense against environmental stresses will be discussed.

Publication date

2018-07-14

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