Expression of the H2O2 biosensor roGFP-Tpx1.C160S in fission and budding yeasts and jurkat cells to compare intracellular H2O2 levels, transmembrane gradients, and response to metals

Intracellular hydrogen peroxide (H2O2) levels can oscillate from low, physiological concentrations, to intermediate, signaling ones, and can participate in toxic reactions when overcoming certain thresholds. Fluorescent protein-based reporters to measure intracellular H2O2 have been developed in rec...

ver descrição completa

Detalhes bibliográficos
Autores: de Cubas, Laura, Mallor, Jorge, Herrera-Fernández, Víctor, Ayté del Olmo, José, Vicente García, Rubén, 1978-, Hidalgo Hernando, Elena
Formato: artículo
Estado:Versión publicada
Fecha de publicación:2023
País:España
Recursos:Universitat Pompeu Fabra
Repositorio:Repositorio Digital de la UPF
OAI Identifier:oai:repositori.upf.edu:10230/57021
Acesso em linha:http://hdl.handle.net/10230/57021
http://dx.doi.org/10.3390/antiox12030706
Access Level:acceso abierto
Palavra-chave:H2O2 concentrations
H2O2 sensor
Jurkat
Fission yeast
roGFP-Tpx1.C169S
Zinc
id ES_bb3c70db47e31e6597dedbf5cc0fd739
oai_identifier_str oai:repositori.upf.edu:10230/57021
network_acronym_str ES
network_name_str España
repository_id_str
spelling Expression of the H2O2 biosensor roGFP-Tpx1.C160S in fission and budding yeasts and jurkat cells to compare intracellular H2O2 levels, transmembrane gradients, and response to metalsde Cubas, LauraMallor, JorgeHerrera-Fernández, VíctorAyté del Olmo, JoséVicente García, Rubén, 1978-Hidalgo Hernando, ElenaH2O2 concentrationsH2O2 sensorJurkatFission yeastroGFP-Tpx1.C169SZincIntracellular hydrogen peroxide (H2O2) levels can oscillate from low, physiological concentrations, to intermediate, signaling ones, and can participate in toxic reactions when overcoming certain thresholds. Fluorescent protein-based reporters to measure intracellular H2O2 have been developed in recent decades. In particular, the redox-sensitive green fluorescent protein (roGFP)-based proteins fused to peroxiredoxins are among the most sensitive H2O2 biosensors. Using fission yeast as a model system, we recently demonstrated that the gradient of extracellular-to-intracellular peroxides through the plasma membrane is around 300:1, and that the concentration of physiological H2O2 is in the low nanomolar range. Here, we have expressed the very sensitive probe roGFP2-Tpx1.C169S in two other model systems, budding yeast and human Jurkat cells. As in fission yeast, the biosensor is ~40-50% oxidized in these cell types, suggesting similar peroxide steady-state levels. Furthermore, probe oxidation upon the addition of extracellular peroxides is also quantitatively similar, suggesting comparable plasma membrane H2O2 gradients. Finally, as a proof of concept, we have applied different concentrations of zinc to all three model systems and have detected probe oxidation, demonstrating that an excess of this metal can cause fluctuations of peroxides, which are moderate in yeasts and severe in mammalian cells. We conclude that the principles governing H2O2 fluxes are very similar in different model organisms.This work is supported by grants PID2021-122837NB-I00/MICIN/AEI/10.13039/501100011033/FEDER, UE to E.H. and PID2019-106755RB-I00/AEI/10.13039/501100011033 to R.V., funded by Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación, and Fondo Europeo de Desarrollo Regional, and by a Redox Biology and Medicine Research Network grant (Red2018-102576-T) to E.H. The Oxidative Stress and Cell Cycle group is also supported by the Generalitat de Catalunya (Spain) (2021 SGR 00007). E.H., J.A., and R.V. are also funded by the Unidad de Excelencia María de Maeztu, funded by the AEI (CEX2018-000792-M) (Spain). E.H. is the recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain). L.C. and J.M. are recipients of María de Maeztu predoctoral fellowships (FPI) from the Ministerio de Economía y Competitividad (Spain).MDPI202320232023info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfapplication/pdfhttp://hdl.handle.net/10230/57021http://dx.doi.org/10.3390/antiox12030706reponame:Repositorio Digital de la UPFinstname:Universitat Pompeu FabraInglésAntioxidants. 2023 Mar 13;12(3):706info:eu-repo/grantAgreement/ES/3PE/PID2021-122837NB-I00info:eu-repo/grantAgreement/ES/2PE/PID2019-106755RB-I00© 2023 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 (https://creativecommons.org/licenses/by/4.0/).http://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:repositori.upf.edu:10230/570212026-06-12T07:21:37Z
dc.title.none.fl_str_mv Expression of the H2O2 biosensor roGFP-Tpx1.C160S in fission and budding yeasts and jurkat cells to compare intracellular H2O2 levels, transmembrane gradients, and response to metals
title Expression of the H2O2 biosensor roGFP-Tpx1.C160S in fission and budding yeasts and jurkat cells to compare intracellular H2O2 levels, transmembrane gradients, and response to metals
spellingShingle Expression of the H2O2 biosensor roGFP-Tpx1.C160S in fission and budding yeasts and jurkat cells to compare intracellular H2O2 levels, transmembrane gradients, and response to metals
de Cubas, Laura
H2O2 concentrations
H2O2 sensor
Jurkat
Fission yeast
roGFP-Tpx1.C169S
Zinc
title_short Expression of the H2O2 biosensor roGFP-Tpx1.C160S in fission and budding yeasts and jurkat cells to compare intracellular H2O2 levels, transmembrane gradients, and response to metals
title_full Expression of the H2O2 biosensor roGFP-Tpx1.C160S in fission and budding yeasts and jurkat cells to compare intracellular H2O2 levels, transmembrane gradients, and response to metals
title_fullStr Expression of the H2O2 biosensor roGFP-Tpx1.C160S in fission and budding yeasts and jurkat cells to compare intracellular H2O2 levels, transmembrane gradients, and response to metals
title_full_unstemmed Expression of the H2O2 biosensor roGFP-Tpx1.C160S in fission and budding yeasts and jurkat cells to compare intracellular H2O2 levels, transmembrane gradients, and response to metals
title_sort Expression of the H2O2 biosensor roGFP-Tpx1.C160S in fission and budding yeasts and jurkat cells to compare intracellular H2O2 levels, transmembrane gradients, and response to metals
dc.creator.none.fl_str_mv de Cubas, Laura
Mallor, Jorge
Herrera-Fernández, Víctor
Ayté del Olmo, José
Vicente García, Rubén, 1978-
Hidalgo Hernando, Elena
author de Cubas, Laura
author_facet de Cubas, Laura
Mallor, Jorge
Herrera-Fernández, Víctor
Ayté del Olmo, José
Vicente García, Rubén, 1978-
Hidalgo Hernando, Elena
author_role author
author2 Mallor, Jorge
Herrera-Fernández, Víctor
Ayté del Olmo, José
Vicente García, Rubén, 1978-
Hidalgo Hernando, Elena
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv H2O2 concentrations
H2O2 sensor
Jurkat
Fission yeast
roGFP-Tpx1.C169S
Zinc
topic H2O2 concentrations
H2O2 sensor
Jurkat
Fission yeast
roGFP-Tpx1.C169S
Zinc
description Intracellular hydrogen peroxide (H2O2) levels can oscillate from low, physiological concentrations, to intermediate, signaling ones, and can participate in toxic reactions when overcoming certain thresholds. Fluorescent protein-based reporters to measure intracellular H2O2 have been developed in recent decades. In particular, the redox-sensitive green fluorescent protein (roGFP)-based proteins fused to peroxiredoxins are among the most sensitive H2O2 biosensors. Using fission yeast as a model system, we recently demonstrated that the gradient of extracellular-to-intracellular peroxides through the plasma membrane is around 300:1, and that the concentration of physiological H2O2 is in the low nanomolar range. Here, we have expressed the very sensitive probe roGFP2-Tpx1.C169S in two other model systems, budding yeast and human Jurkat cells. As in fission yeast, the biosensor is ~40-50% oxidized in these cell types, suggesting similar peroxide steady-state levels. Furthermore, probe oxidation upon the addition of extracellular peroxides is also quantitatively similar, suggesting comparable plasma membrane H2O2 gradients. Finally, as a proof of concept, we have applied different concentrations of zinc to all three model systems and have detected probe oxidation, demonstrating that an excess of this metal can cause fluctuations of peroxides, which are moderate in yeasts and severe in mammalian cells. We conclude that the principles governing H2O2 fluxes are very similar in different model organisms.
publishDate 2023
dc.date.none.fl_str_mv 2023
2023
2023
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10230/57021
http://dx.doi.org/10.3390/antiox12030706
url http://hdl.handle.net/10230/57021
http://dx.doi.org/10.3390/antiox12030706
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Antioxidants. 2023 Mar 13;12(3):706
info:eu-repo/grantAgreement/ES/3PE/PID2021-122837NB-I00
info:eu-repo/grantAgreement/ES/2PE/PID2019-106755RB-I00
dc.rights.none.fl_str_mv http://creativecommons.org/licenses/by/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv MDPI
publisher.none.fl_str_mv MDPI
dc.source.none.fl_str_mv reponame:Repositorio Digital de la UPF
instname:Universitat Pompeu Fabra
instname_str Universitat Pompeu Fabra
reponame_str Repositorio Digital de la UPF
collection Repositorio Digital de la UPF
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869418007724294144
score 15.811543

Registos relacionados