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The In Vitro Interaction of 12-Oxophytodienoic Acid and Related Conjugated Carbonyl Compounds with Thiol Antioxidants.

ABSTRACT: α,β-unsaturated carbonyls interfere with numerous plant physiological processes. One mechanism of action is their reactivity toward thiols of metabolites like cysteine and glutathione (GSH). This work aimed at better understanding these interactions. Both 12-oxophytodienoic acid (12-OPDA) and abscisic acid (ABA) conjugated with cysteine. It was found that the reactivity of α,β-unsaturated carbonyls with GSH followed the sequence trans-2-hexenal < 12-OPDA ≈ 12-OPDA-ethylester < 2-cyclopentenone << methyl vinylketone (MVK). Interestingly, GSH, but not ascorbate (vitamin C), supplementation ameliorated the phytotoxic potential of MVK. In addition, 12-OPDA and 12-OPDA-related conjugated carbonyl compounds interacted with proteins, e.g., with members of the thioredoxin (TRX)-fold family. 12-OPDA modified two cysteinyl residues of chloroplast TRX-f1. The OPDAylated TRX-f1 lost its activity to activate the Calvin-Benson-cycle enzyme fructose-1,6-bisphosphatase (FBPase). Finally, we show that 12-OPDA interacts with cyclophilin 20-3 (Cyp20-3) non-covalently and affects its peptidyl-prolyl-cis/trans isomerase activity. The results demonstrate the high potential of 12-OPDA as a diverse interactor and cellular regulator and suggest that OPDAylation may occur in plant cells and should be investigated as novel regulatory mechanism.

SUBMITTER: Maynard D

PROVIDER: S-EPMC8003295 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nitroalkene fatty acids are formed in vivo and exert protective and anti-inflammatory effects via reversible Michael addition to thiol-containing proteins in key signaling pathways. Nitro-conjugated linoleic acid (NO2-CLA) is preferentially formed, constitutes the most abundant nitrated fatty acid in humans, and contains two carbons that could potentially react with thiols, modulating signaling actions and levels. In this work, we examined the reactions of NO2-CLA with low molecular weight thiols (glutathione, cysteine, homocysteine, cysteinylglycine, and ?-mercaptoethanol) and human serum albumin. Reactions followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2-CLA located on the ?- and ?-carbons with respect to the nitro group. The differential reactivity was confirmed by computational modeling of the electronic structure. The rates (kon and koff) and equilibrium constants for both reactions were determined for different thiols. LC-UV-Visible and LC-MS analyses showed that the fast reaction corresponds to ?-adduct formation (the kinetic product), while the slow reaction corresponds to the formation of the ?-adduct (the thermodynamic product). The pH dependence of the rate constants, the correlation between intrinsic reactivity and thiol pKa, and the absence of deuterium solvent kinetic isotope effects suggested stepwise mechanisms with thiolate attack on NO2-CLA as rate-controlling step. Computational modeling supported the mechanism and revealed additional features of the transition states, anionic intermediates, and final neutral products. Importantly, the detection of cysteine-?-adducts in human urine provided evidence for the biological relevance of this reaction. Finally, human serum albumin was found to bind NO2-CLA both non-covalently and to form covalent adducts at Cys-34, suggesting potential modes for systemic distribution. These results provide new insights into the chemical basis of NO2-CLA signaling actions.

Project description:BackgroundDue to high neutral lipids accumulation in the cytoplasm, in vitro-produced embryos from Bos primigenius indicus and their crosses are more sensitive to chilling and cryopreservation than those from Bos primigenius taurus. The objective of the present study was to evaluate the effects of trans-10, cis-12 conjugated linoleic acid (CLA) on the development and cryotolerance of crossbred Bos primigenius taurus x Bos primigenius indicus embryos produced in vitro, and cultured in the presence of fetal calf serum. Bovine zygotes (n = 1,692) were randomly assigned to one of the following treatment groups: 1) Control, zygotes cultured in Charles Rosenkrans 2 amino acid (CR2aa) medium (n = 815) or 2) CLA, zygotes cultured in CR2aa medium supplemented with 100 μmol/L of trans-10, cis-12 CLA (n = 877). Embryo development (cleavage and blastocyst rates evaluated at days 3 and 8 of culture, respectively), lipid content at morula stage (day 5) and blastocyst cryotolerance (re-expansion and hatching rates, evaluated 24 and 72 h post-thawing, respectively) were compared between groups. Additionally, selected mRNA transcripts were measured by Real-Time PCR in blastocyst stage.ResultsThe CLA treatment had no effect on cleavage and blastocyst rates, or on mRNA levels for genes related to cellular stress and apoptosis. On the other hand, abundance of mRNA for the 1-acylglycerol-3-phosphate 0-acyltransferase-encoding gene (AGPAT), which is involved in triglycerides synthesis, and consequently neutral lipid content, were reduced by CLA treatment. A significant increase was observed in the re-expansion rate of embryos cultured with trans-10, cis-12 CLA when compared to control (56.3 vs. 34.4%, respectively, P = 0.002). However, this difference was not observed in the hatching rate (16.5 vs. 14.0%, respectively, P = 0.62).ConclusionsThe supplementation with trans-10, cis-12 CLA isomer in culture medium reduced the lipid content of in vitro produced bovine embryos by reducing the gene expression of 1-acylglycerol 3-phosphate 0-acyltransferase (AGPAT) enzyme. However, a possible improvement in embryo cryotolerance in response to CLA, as suggested by increased blastocyst re-expansion rate, was not confirmed by hatching rates.

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The In Vitro Interaction of 12-Oxophytodienoic Acid and Related Conjugated Carbonyl Compounds with Thiol Antioxidants.

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