Thiol Peroxidases as Major Regulators of Intracellular Levels of Peroxynitrite in Live Saccharomyces cerevisiae Cells
Autor(a) principal: | |
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Data de Publicação: | 2020 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.3390/antiox9050434 http://hdl.handle.net/11449/196973 |
Resumo: | 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 Delta 8 (TP-deficient strain) and Delta 8+TSA1 (Delta 8 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 Delta 8 > Delta 8+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. |
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Thiol Peroxidases as Major Regulators of Intracellular Levels of Peroxynitrite in Live Saccharomyces cerevisiae Cellsthiol peroxidasesperoxiredoxinsthioredoxinsperoxynitriteboronatenitric oxideSaccharomyces cerevisiaeThiol 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 Delta 8 (TP-deficient strain) and Delta 8+TSA1 (Delta 8 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 Delta 8 > Delta 8+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.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ Sao Paulo, Dept Quim, Fac Filosofia Ciencias & Letras Ribeirao Preto, BR-14040901 Ribeirao Preto, BrazilUniv Sao Paulo, Inst Biociencias, Dept Genet & Biol Evolut, BR-05508090 Sao Paulo, SP, BrazilUniv Estadual Paulista, Inst Biociencias, Campus Litoral Paulista, BR-11330900 Sao Paulo, SP, BrazilUniv Estadual Paulista, Inst Biociencias, Campus Litoral Paulista, BR-11330900 Sao Paulo, SP, BrazilFAPESP: 2013/07937-8CAPES: 001FAPESP: 2017/09443-3MdpiUniversidade de São Paulo (USP)Universidade Estadual Paulista (Unesp)Condeles, Andre LuisGomes, FernandoOliveira, Marcos Antonio de [UNESP]Soares Netto, Luis EduardoToledo Junior, Jose Carlos2020-12-10T20:02:14Z2020-12-10T20:02:14Z2020-05-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article14http://dx.doi.org/10.3390/antiox9050434Antioxidants. Basel: Mdpi, v. 9, n. 5, 14 p., 2020.http://hdl.handle.net/11449/19697310.3390/antiox9050434WOS:000539284200077Web of Sciencereponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengAntioxidantsinfo:eu-repo/semantics/openAccess2021-10-23T09:20:04Zoai:repositorio.unesp.br:11449/196973Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T15:16:31.909130Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
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 |
spellingShingle |
Thiol Peroxidases as Major Regulators of Intracellular Levels of Peroxynitrite in Live Saccharomyces cerevisiae Cells Condeles, Andre Luis thiol peroxidases peroxiredoxins thioredoxins peroxynitrite boronate nitric oxide Saccharomyces cerevisiae |
title_short |
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_sort |
Thiol Peroxidases as Major Regulators of Intracellular Levels of Peroxynitrite in Live Saccharomyces cerevisiae Cells |
author |
Condeles, Andre Luis |
author_facet |
Condeles, Andre Luis Gomes, Fernando Oliveira, Marcos Antonio de [UNESP] Soares Netto, Luis Eduardo Toledo Junior, Jose Carlos |
author_role |
author |
author2 |
Gomes, Fernando Oliveira, Marcos Antonio de [UNESP] Soares Netto, Luis Eduardo Toledo Junior, Jose Carlos |
author2_role |
author author author author |
dc.contributor.none.fl_str_mv |
Universidade de São Paulo (USP) Universidade Estadual Paulista (Unesp) |
dc.contributor.author.fl_str_mv |
Condeles, Andre Luis Gomes, Fernando Oliveira, Marcos Antonio de [UNESP] Soares Netto, Luis Eduardo Toledo Junior, Jose Carlos |
dc.subject.por.fl_str_mv |
thiol peroxidases peroxiredoxins thioredoxins peroxynitrite boronate nitric oxide Saccharomyces cerevisiae |
topic |
thiol peroxidases peroxiredoxins thioredoxins peroxynitrite boronate nitric oxide Saccharomyces cerevisiae |
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 Delta 8 (TP-deficient strain) and Delta 8+TSA1 (Delta 8 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 Delta 8 > Delta 8+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. |
publishDate |
2020 |
dc.date.none.fl_str_mv |
2020-12-10T20:02:14Z 2020-12-10T20:02:14Z 2020-05-01 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.3390/antiox9050434 Antioxidants. Basel: Mdpi, v. 9, n. 5, 14 p., 2020. http://hdl.handle.net/11449/196973 10.3390/antiox9050434 WOS:000539284200077 |
url |
http://dx.doi.org/10.3390/antiox9050434 http://hdl.handle.net/11449/196973 |
identifier_str_mv |
Antioxidants. Basel: Mdpi, v. 9, n. 5, 14 p., 2020. 10.3390/antiox9050434 WOS:000539284200077 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Antioxidants |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
14 |
dc.publisher.none.fl_str_mv |
Mdpi |
publisher.none.fl_str_mv |
Mdpi |
dc.source.none.fl_str_mv |
Web of Science reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
_version_ |
1808128492678676480 |