New In Vivo Approach to Broaden the Thioredoxin Family Interactome in Chloroplasts

Post-translational redox modifications provide an important mechanism for the control of major cellular processes. Thioredoxins (Trxs), which are key actors in this regulatory mechanism, are ubiquitous proteins that catalyse thiol-disulfide exchange reactions. In chloroplasts, Trx f, Trx m and NADPH...

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Detalhes bibliográficos
Autores: Ancín, María, Fernández-Irigoyen, Joaquín, Santamaría, Enrique, Larraya, Luis, Fernández San Millán, Alicia, Veramendi, Jon, Farrán, Inmaculada
Formato: artículo
Estado:Versión publicada
Fecha de publicación:2022
País:España
Recursos:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:dnet:digitalcsic_::2d9ce67c19077378ee329fa399341cfa
Acesso em linha:http://hdl.handle.net/10261/296374
Access Level:acceso abierto
Palavra-chave:Chloroplast
Thioredoxin
NTRC
Nicotiana
Proteomics
Redox regulation
Target proteins
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spelling New In Vivo Approach to Broaden the Thioredoxin Family Interactome in ChloroplastsAncín, MaríaFernández-Irigoyen, JoaquínSantamaría, EnriqueLarraya, LuisFernández San Millán, AliciaVeramendi, JonFarrán, InmaculadaChloroplastThioredoxinNTRCNicotianaProteomicsRedox regulationTarget proteinsPost-translational redox modifications provide an important mechanism for the control of major cellular processes. Thioredoxins (Trxs), which are key actors in this regulatory mechanism, are ubiquitous proteins that catalyse thiol-disulfide exchange reactions. In chloroplasts, Trx f, Trx m and NADPH-dependent Trx reductase C (NTRC) have been identified as transmitters of the redox signal by transferring electrons to downstream target enzymes. The number of characterised Trx targets has greatly increased in the last few years, but most of them were determined using in vitro procedures lacking isoform specificity. With this background, we have developed a new in vivo approach based on the overexpression of His-tagged single-cysteine mutants of Trx f, Trx m or NTRC into Nicotiana benthamiana plants. The over-expressed mutated Trxs, capable of forming a stable mixed disulfide bond with target proteins in plants, were immobilised on affinity columns packed with Ni-NTA agarose, and the covalently linked targets were eluted with dithiothreitol and identified by mass spectrometry-based proteomics. The in vivo approach allowed identification of 6, 9 and 42 new potential targets for Trx f, Trx m and NTRC, respectively, and an apparent specificity between NTRC and Trxs was achieved. Functional analysis showed that these targets are involved in several cellular processes.M.A. was supported by the “Juan de la Cierva-formación” postdoctoral fellowship from the Spanish Ministry of Science and Innovation MCIN/AEI/ 10.13039/501100011033 and the European Union “Next Generation EU/PRTR”.Molecular Diversity Preservation InternationalMinisterio de Ciencia, Innovación y Universidades (España)Agencia Estatal de Investigación (España)European CommissionConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2023202320222023info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://hdl.handle.net/10261/296374reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)InglésThe underlying dataset has been published as supplementary material of the article in the publisher platform at 10.3390/antiox11101979http://dx.doi.org/10.3390/antiox11101979Síinfo:eu-repo/semantics/openAccessoai:dnet:digitalcsic_::2d9ce67c19077378ee329fa399341cfa2026-05-22T06:33:51Z
dc.title.none.fl_str_mv New In Vivo Approach to Broaden the Thioredoxin Family Interactome in Chloroplasts
title New In Vivo Approach to Broaden the Thioredoxin Family Interactome in Chloroplasts
spellingShingle New In Vivo Approach to Broaden the Thioredoxin Family Interactome in Chloroplasts
Ancín, María
Chloroplast
Thioredoxin
NTRC
Nicotiana
Proteomics
Redox regulation
Target proteins
title_short New In Vivo Approach to Broaden the Thioredoxin Family Interactome in Chloroplasts
title_full New In Vivo Approach to Broaden the Thioredoxin Family Interactome in Chloroplasts
title_fullStr New In Vivo Approach to Broaden the Thioredoxin Family Interactome in Chloroplasts
title_full_unstemmed New In Vivo Approach to Broaden the Thioredoxin Family Interactome in Chloroplasts
title_sort New In Vivo Approach to Broaden the Thioredoxin Family Interactome in Chloroplasts
dc.creator.none.fl_str_mv Ancín, María
Fernández-Irigoyen, Joaquín
Santamaría, Enrique
Larraya, Luis
Fernández San Millán, Alicia
Veramendi, Jon
Farrán, Inmaculada
author Ancín, María
author_facet Ancín, María
Fernández-Irigoyen, Joaquín
Santamaría, Enrique
Larraya, Luis
Fernández San Millán, Alicia
Veramendi, Jon
Farrán, Inmaculada
author_role author
author2 Fernández-Irigoyen, Joaquín
Santamaría, Enrique
Larraya, Luis
Fernández San Millán, Alicia
Veramendi, Jon
Farrán, Inmaculada
author2_role author
author
author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Ciencia, Innovación y Universidades (España)
Agencia Estatal de Investigación (España)
European Commission
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Chloroplast
Thioredoxin
NTRC
Nicotiana
Proteomics
Redox regulation
Target proteins
topic Chloroplast
Thioredoxin
NTRC
Nicotiana
Proteomics
Redox regulation
Target proteins
description Post-translational redox modifications provide an important mechanism for the control of major cellular processes. Thioredoxins (Trxs), which are key actors in this regulatory mechanism, are ubiquitous proteins that catalyse thiol-disulfide exchange reactions. In chloroplasts, Trx f, Trx m and NADPH-dependent Trx reductase C (NTRC) have been identified as transmitters of the redox signal by transferring electrons to downstream target enzymes. The number of characterised Trx targets has greatly increased in the last few years, but most of them were determined using in vitro procedures lacking isoform specificity. With this background, we have developed a new in vivo approach based on the overexpression of His-tagged single-cysteine mutants of Trx f, Trx m or NTRC into Nicotiana benthamiana plants. The over-expressed mutated Trxs, capable of forming a stable mixed disulfide bond with target proteins in plants, were immobilised on affinity columns packed with Ni-NTA agarose, and the covalently linked targets were eluted with dithiothreitol and identified by mass spectrometry-based proteomics. The in vivo approach allowed identification of 6, 9 and 42 new potential targets for Trx f, Trx m and NTRC, respectively, and an apparent specificity between NTRC and Trxs was achieved. Functional analysis showed that these targets are involved in several cellular processes.
publishDate 2022
dc.date.none.fl_str_mv 2022
2023
2023
2023
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/296374
url http://hdl.handle.net/10261/296374
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv The underlying dataset has been published as supplementary material of the article in the publisher platform at 10.3390/antiox11101979
http://dx.doi.org/10.3390/antiox11101979
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Molecular Diversity Preservation International
publisher.none.fl_str_mv Molecular Diversity Preservation International
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,228081

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