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Thiol Peroxidases as Major Regulators of Intracellular Levels of Peroxynitrite in Live Saccharomyces cerevisiae Cells
Thiol peroxidases (TP) are ubiquitous and abundant antioxidant proteins of the peroxiredoxin and glutathione peroxidase families that can catalytically and rapidly reduce biologically relevant peroxides, such as hydrogen peroxide and peroxynitrite. However, the TP catalytic cycle is complex, dependi...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278867/ https://www.ncbi.nlm.nih.gov/pubmed/32429358 http://dx.doi.org/10.3390/antiox9050434 |
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author | Condeles, André Luís Gomes, Fernando de Oliveira, Marcos Antonio Soares Netto, Luís Eduardo Toledo Junior, José Carlos |
author_facet | Condeles, André Luís Gomes, Fernando de Oliveira, Marcos Antonio Soares Netto, Luís Eduardo Toledo Junior, José Carlos |
author_sort | Condeles, André Luís |
collection | PubMed |
description | Thiol peroxidases (TP) are ubiquitous and abundant antioxidant proteins of the peroxiredoxin and glutathione peroxidase families that can catalytically and rapidly reduce biologically relevant peroxides, such as hydrogen peroxide and peroxynitrite. However, the TP catalytic cycle is complex, depending on multiple redox reactions and partners, and is subjected to branching and competition points that may limit their peroxide reductase activity in vivo. The goals of the present study were to demonstrate peroxynitrite reductase activity of TP members in live cells in real time and to evaluate its catalytic characteristics. To these ends, we developed a simple fluorescence assay using coumarin boronic acid (CBA), exploiting that fact that TP and CBA compete for peroxynitrite, with the expectation that higher TP peroxynitrite reductase activity will lower the CBA oxidation. TP peroxynitrite reductase activity was evaluated by comparing CBA oxidation in live wild type and genetically modified Δ8 (TP-deficient strain) and Δ8+TSA1 (Δ8 strain that expresses only one TP member, the TSA1 gene) Saccharomyces cerevisiae strains. The results showed that CBA oxidation decreased with cell density and increased with increasing peroxynitrite availability. Additionally, the rate of CBA oxidation decreased in the order Δ8 > Δ8+TSA1 > WT strains both in control and glycerol-adapted (expressing higher TP levels) cells, showing that the CBA competition assay could reliably detect peroxynitrite in real time in live cells, comparing CBA oxidation in strains with reduced and increased TP expression. Finally, there were no signs of compromised TP peroxynitrite reductase activity during experimental runs, even at the highest peroxynitrite levels tested. Altogether, the results show that TP is a major component in the defense of yeast against peroxynitrite insults under basal and increasing stressful conditions. |
format | Online Article Text |
id | pubmed-7278867 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-72788672020-06-12 Thiol Peroxidases as Major Regulators of Intracellular Levels of Peroxynitrite in Live Saccharomyces cerevisiae Cells Condeles, André Luís Gomes, Fernando de Oliveira, Marcos Antonio Soares Netto, Luís Eduardo Toledo Junior, José Carlos Antioxidants (Basel) Article Thiol peroxidases (TP) are ubiquitous and abundant antioxidant proteins of the peroxiredoxin and glutathione peroxidase families that can catalytically and rapidly reduce biologically relevant peroxides, such as hydrogen peroxide and peroxynitrite. However, the TP catalytic cycle is complex, depending on multiple redox reactions and partners, and is subjected to branching and competition points that may limit their peroxide reductase activity in vivo. The goals of the present study were to demonstrate peroxynitrite reductase activity of TP members in live cells in real time and to evaluate its catalytic characteristics. To these ends, we developed a simple fluorescence assay using coumarin boronic acid (CBA), exploiting that fact that TP and CBA compete for peroxynitrite, with the expectation that higher TP peroxynitrite reductase activity will lower the CBA oxidation. TP peroxynitrite reductase activity was evaluated by comparing CBA oxidation in live wild type and genetically modified Δ8 (TP-deficient strain) and Δ8+TSA1 (Δ8 strain that expresses only one TP member, the TSA1 gene) Saccharomyces cerevisiae strains. The results showed that CBA oxidation decreased with cell density and increased with increasing peroxynitrite availability. Additionally, the rate of CBA oxidation decreased in the order Δ8 > Δ8+TSA1 > WT strains both in control and glycerol-adapted (expressing higher TP levels) cells, showing that the CBA competition assay could reliably detect peroxynitrite in real time in live cells, comparing CBA oxidation in strains with reduced and increased TP expression. Finally, there were no signs of compromised TP peroxynitrite reductase activity during experimental runs, even at the highest peroxynitrite levels tested. Altogether, the results show that TP is a major component in the defense of yeast against peroxynitrite insults under basal and increasing stressful conditions. MDPI 2020-05-16 /pmc/articles/PMC7278867/ /pubmed/32429358 http://dx.doi.org/10.3390/antiox9050434 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Condeles, André Luís Gomes, Fernando de Oliveira, Marcos Antonio Soares Netto, Luís Eduardo Toledo Junior, José Carlos Thiol Peroxidases as Major Regulators of Intracellular Levels of Peroxynitrite in Live Saccharomyces cerevisiae Cells |
title | Thiol Peroxidases as Major Regulators of Intracellular Levels of Peroxynitrite in Live Saccharomyces cerevisiae Cells |
title_full | Thiol Peroxidases as Major Regulators of Intracellular Levels of Peroxynitrite in Live Saccharomyces cerevisiae Cells |
title_fullStr | Thiol Peroxidases as Major Regulators of Intracellular Levels of Peroxynitrite in Live Saccharomyces cerevisiae Cells |
title_full_unstemmed | Thiol Peroxidases as Major Regulators of Intracellular Levels of Peroxynitrite in Live Saccharomyces cerevisiae Cells |
title_short | Thiol Peroxidases as Major Regulators of Intracellular Levels of Peroxynitrite in Live Saccharomyces cerevisiae Cells |
title_sort | thiol peroxidases as major regulators of intracellular levels of peroxynitrite in live saccharomyces cerevisiae cells |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278867/ https://www.ncbi.nlm.nih.gov/pubmed/32429358 http://dx.doi.org/10.3390/antiox9050434 |
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