Thiol Redox Regulation of Plant beta-Carbonic Anhydrase.

Dreyer A, Schackmann A, Kriznik A, et al. Thiol Redox Regulation of Plant beta-Carbonic Anhydrase. Biomolecules. 2020;10: 1125.beta-carbonic anhydrases (betaCA) accelerate the equilibrium formation between CO2 and carbonate. Two plant betaCA isoforms are targeted to the chloroplast and represent abundant proteins in the range of >1% of chloroplast protein. While their function in gas exchange and photosynthesis is well-characterized in carbon concentrating mechanisms of cyanobacteria and plants with C4-photosynthesis, their function in plants with C3-photosynthesis is less clear. The presence of conserved and surface-exposed cysteinyl residues in the betaCA-structure urged to the question whether betaCA is subject to redox regulation. Activity measurements revealed reductive activation of betaCA1, whereas oxidized betaCA1 was inactive. Mutation of cysteinyl residues decreased betaCA1 activity, in particular C280S, C167S, C230S, and C257S. High concentrations of dithiothreitol or low amounts of reduced thioredoxins (TRXs) activated oxidized betaCA1. TRX-y1 and TRX-y2 most efficiently activated betaCA1, followed by TRX-f1 and f2 and NADPH-dependent TRX reductase C (NTRC). High light irradiation did not enhance betaCA activity in wildtype Arabidopsis, but surprisingly in betaca1 knockout plants, indicating light-dependent regulation. The results assign a role of betaCA within the thiol redox regulatory network of the chloroplast

Enzymologie du mécanisme de régulation des Peroxyrédoxines par suroxydation au cours de la signalisation cellulaire redox

Peroxiredoxins from the Prx1 subfamily (Prx) are moonlighting peroxidases that operate in peroxide signaling and are regulated by sulfinylation. Prxs offer a major model of protein−thiol oxidative modification. They react with H2O2 to form a sulfenic acid intermediate that either engages into a disulfide bond, committing the enzyme into its peroxidase cycle, or again reacts with peroxide to produce a sulfinic acid that inactivates the enzyme. Sensitivity to sulfinylation depends on the kinetics of these two competing reactions and is critically influenced by a structural transition from a fully folded (FF) to locally unfolded (LU) conformation. Analysis of the reaction of the Tsa1 Saccharomyces cerevisiae Prx with H2O2 by Trp fluorescence-based rapid kinetics revealed a process linked to the FF/LU transition that is kinetically distinct from disulfide formation and suggested that sulfenate formation facilitates local unfolding. Use of mutants of distinctive sensitivities and of different peroxide substrates showed that sulfinylation sensitivity is not coupled to the resolving step kinetics but depends only on the sulfenic acid oxidation and FF-to-LU transition rate constants. From these two parameters, the relative sensitivities of Prxs toward hyperoxidation with different substrates can be predicted, as confirmed by in vitro and in vivo patterns.La suroxydation des peroxyrédoxines (Prx) est un mécanisme post-traductionnel essentiel impliqué dans la régulation et la signalisation cellulaire redox. Les Prx sont des peroxydases à thiol, qui réagissent avec les peroxydes pour former un intermédiaire acide sulfénique. Leur sensibilité à la suroxydation dépend de la compétition entre la réaction de sulfinylation et la formation d’un pont disulfure au cours du cycle peroxydase. Cette compétition est contrôlée par une transition conformationnelle entre deux conformations FF : structurée et LU : localement déstructurée. Dans ce projet, nous abordons le mécanisme de suroxydation de Tsa1, la principale Prx1 cytosolique de S. cerevisiae, par des analyses cinétiques à l'état pré-stationnaire en cinétique rapide, à l'état stationnaire et in vivo. Une phase cinétique correspondant à un changement de conformation associé à la transition FF/LU a été identifiée, montrant que la formation de l’acide sulfénique facilite cette transition. L’utilisation de mutants de sensibilité à la suroxydation altérée et de différents substrats peroxydes a permis de montrer que la sensibilité est découplée de l'étape de formation du pont disulfure et ne dépend que des constantes de vitesse de sulfinylation et de transition conformationnelle FF/LU. A partir de ces deux paramètres, nous pouvons prédire l'indice de sensibilité à la suroxydation CHyp1%, une prédiction démontrée in vitro et in vivo

Enzymology of the regulatory mechanism of Peroxiredoxins by hyperoxidation during redox cell signalling

La suroxydation des peroxyrédoxines (Prx) est un mécanisme post-traductionnel essentiel impliqué dans la régulation et la signalisation cellulaire redox. Les Prx sont des peroxydases à thiol, qui réagissent avec les peroxydes pour former un intermédiaire acide sulfénique. Leur sensibilité à la suroxydation dépend de la compétition entre la réaction de sulfinylation et la formation d’un pont disulfure au cours du cycle peroxydase. Cette compétition est contrôlée par une transition conformationnelle entre deux conformations FF : structurée et LU : localement déstructurée. Dans ce projet, nous abordons le mécanisme de suroxydation de Tsa1, la principale Prx1 cytosolique de S. cerevisiae, par des analyses cinétiques à l'état pré-stationnaire en cinétique rapide, à l'état stationnaire et in vivo. Une phase cinétique correspondant à un changement de conformation associé à la transition FF/LU a été identifiée, montrant que la formation de l’acide sulfénique facilite cette transition. L’utilisation de mutants de sensibilité à la suroxydation altérée et de différents substrats peroxydes a permis de montrer que la sensibilité est découplée de l'étape de formation du pont disulfure et ne dépend que des constantes de vitesse de sulfinylation et de transition conformationnelle FF/LU. A partir de ces deux paramètres, nous pouvons prédire l'indice de sensibilité à la suroxydation CHyp1%, une prédiction démontrée in vitro et in vivo.Peroxiredoxins from the Prx1 subfamily (Prx) are moonlighting peroxidases that operate in peroxide signaling and are regulated by sulfinylation. Prxs offer a major model of protein−thiol oxidative modification. They react with H2O2 to form a sulfenic acid intermediate that either engages into a disulfide bond, committing the enzyme into its peroxidase cycle, or again reacts with peroxide to produce a sulfinic acid that inactivates the enzyme. Sensitivity to sulfinylation depends on the kinetics of these two competing reactions and is critically influenced by a structural transition from a fully folded (FF) to locally unfolded (LU) conformation. Analysis of the reaction of the Tsa1 Saccharomyces cerevisiae Prx with H2O2 by Trp fluorescence-based rapid kinetics revealed a process linked to the FF/LU transition that is kinetically distinct from disulfide formation and suggested that sulfenate formation facilitates local unfolding. Use of mutants of distinctive sensitivities and of different peroxide substrates showed that sulfinylation sensitivity is not coupled to the resolving step kinetics but depends only on the sulfenic acid oxidation and FF-to-LU transition rate constants. From these two parameters, the relative sensitivities of Prxs toward hyperoxidation with different substrates can be predicted, as confirmed by in vitro and in vivo patterns

Effect of the Processing Temperature on the Degradation of Food Flavonoids: Kinetic and Calorimetric Studies on Model Solutions

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Redox signaling : insights into the interaction mechanism of sulfiredoxin with peroxiredoxin and ATP

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Structural Analysis of the Plasmodial Proteins ZNHIT3 and NUFIP1 Provides Insights into the Selectivity of a Conserved Interaction

International audienceMalaria is a widespread and lethal disease caused by the Plasmodium parasites that can infect human beings through Anopheles mosquitoes. For that reason, the biology of Plasmodium needs to be studied to develop antimalarial treatments. By determining the three-dimensional structures of macromolecules, structural biology helps to understand the function of proteins and can reveal how interactions occur between biological partners. Here, we studied the ZNHIT3 and NUFIP1 proteins from Plasmodium falciparum, two proteins tightly linked to the ribosome biology. Due to their important functions in post-translational modifications of ribosomal RNAs and in ribophagy, these proteins participate in the survival of cells. In this study, we solved the three-dimensional structure of a thermally stable and species-dependent complex between fragments of these proteins. Our results were compared to the AlphaFold predictions, which motivated the study of the free ZNHIT3 fragment that binds NUFIP1. We showed that the latter fragment multimerized in vitro but also had the inner ability to change its conformation to escape the solvent exposition of key hydrophobic residues involved in the interaction with NUFIP1. Our data could open the gate to selective drug discovery processes involving these two proteins

Dynamics of a determinant conformational transition in peroxiredoxin sulfinylation mechanism

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Interaction between dietary bioactive peptides of short length and bile salts in submicellar or micellar state

International audienc

Dynamics of a key conformational transition in the mechanism of peroxiredoxin sulfinylation

International audiencePeroxiredoxins from the Prx1 subfamily (Prx) are moonlighting peroxidases that operate in peroxide signaling and are regulated by sulfinylation. Prxs offer a major model of protein−thiol oxidative modification. They react with H 2 O 2 to form a sulfenic acid intermediate that either engages into a disulfide bond, committing the enzyme into its peroxidase cycle, or again reacts with peroxide to produce a sulfinic acid that inactivates the enzyme. Sensitivity to sulfinylation depends on the kinetics of these two competing reactions and is critically influenced by a structural transition from a fully folded (FF) to locally unfolded (LU) conformation. Analysis of the reaction of the Tsa1 Saccharomyces cerevisiae Prx with H 2 O 2 by Trp fluorescencebased rapid kinetics revealed a process linked to the FF/LU transition that is kinetically distinct from disulfide formation and suggested that sulfenate formation facilitates local unfolding. Use of mutants of distinctive sensitivities and of different peroxide substrates showed that sulfinylation sensitivity is not coupled to the resolving step kinetics but depends only on the sulfenic acid oxidation and FF-to-LU transition rate constants. In addition, stabilization of the active site FF conformation, the determinant of sulfinylation kinetics, is only moderately influenced by the Prx C-terminal tail dynamics that determine the FF → LU kinetics. From these two parameters, the relative sensitivities of Prxs toward hyperoxidation with different substrates can be predicted, as confirmed by in vitro and in vivo patterns of sulfinylation