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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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Autores principales: Ancín, María, Fernandez-Irigoyen, Joaquin, Santamaria, Enrique, Larraya, Luis, Fernández-San Millán, Alicia, Veramendi, Jon, Farran, Inmaculada
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9598788/
https://www.ncbi.nlm.nih.gov/pubmed/36290702
http://dx.doi.org/10.3390/antiox11101979
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author Ancín, María
Fernandez-Irigoyen, Joaquin
Santamaria, Enrique
Larraya, Luis
Fernández-San Millán, Alicia
Veramendi, Jon
Farran, Inmaculada
author_facet Ancín, María
Fernandez-Irigoyen, Joaquin
Santamaria, Enrique
Larraya, Luis
Fernández-San Millán, Alicia
Veramendi, Jon
Farran, Inmaculada
author_sort Ancín, María
collection PubMed
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.
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spelling pubmed-95987882022-10-27 New In Vivo Approach to Broaden the Thioredoxin Family Interactome in Chloroplasts Ancín, María Fernandez-Irigoyen, Joaquin Santamaria, Enrique Larraya, Luis Fernández-San Millán, Alicia Veramendi, Jon Farran, Inmaculada Antioxidants (Basel) Article 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. MDPI 2022-10-04 /pmc/articles/PMC9598788/ /pubmed/36290702 http://dx.doi.org/10.3390/antiox11101979 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/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/).
spellingShingle Article
Ancín, María
Fernandez-Irigoyen, Joaquin
Santamaria, Enrique
Larraya, Luis
Fernández-San Millán, Alicia
Veramendi, Jon
Farran, Inmaculada
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
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_short 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
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9598788/
https://www.ncbi.nlm.nih.gov/pubmed/36290702
http://dx.doi.org/10.3390/antiox11101979
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