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

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• dc.contributor.author de Cubas, Laura
• dc.contributor.author Mallor, Jorge
• dc.contributor.author Herrera-Fernández, Víctor
• dc.contributor.author Ayté del Olmo, José
• dc.contributor.author Vicente García, Rubén, 1978-
• dc.contributor.author Hidalgo Hernando, Elena
• dc.date.accessioned 2023-06-02T06:02:10Z
• dc.date.available 2023-06-02T06:02:10Z
• dc.date.issued 2023
• dc.description.abstract 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.
• dc.description.sponsorship 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).
• dc.format.mimetype application/pdf
• dc.identifier.citation de Cubas L, Mallor J, Herrera-Fernández V, Ayté J, Vicente R, Hidalgo E. 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. Antioxidants. 2023 Mar 13;12(3):706. DOI: 10.3390/antiox12030706
• dc.identifier.doi http://dx.doi.org/10.3390/antiox12030706
• dc.identifier.issn 2076-3921
• dc.identifier.uri http://hdl.handle.net/10230/57021
• dc.language.iso eng
• dc.publisher MDPI
• dc.relation.ispartof Antioxidants. 2023 Mar 13;12(3):706
• dc.relation.projectID info:eu-repo/grantAgreement/ES/3PE/PID2021-122837NB-I00
• dc.relation.projectID info:eu-repo/grantAgreement/ES/2PE/PID2019-106755RB-I00
• dc.rights © 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/).
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.rights.uri http://creativecommons.org/licenses/by/4.0/
• dc.subject.keyword H2O2 concentrations
• dc.subject.keyword H2O2 sensor
• dc.subject.keyword Jurkat
• dc.subject.keyword Fission yeast
• dc.subject.keyword roGFP-Tpx1.C169S
• dc.subject.keyword Zinc
• dc.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
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion