New in vivo approach to broaden the thioredoxin family interactome in chloroplasts
Fecha
2022Autor
Versión
Acceso abierto / Sarbide irekia
Tipo
Artículo / Artikulua
Versión
Versión publicada / Argitaratu den bertsioa
Impacto
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10.3390/antiox11101979
Resumen
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 trans ...
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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. [--]
Materias
Chloroplast,
Nicotiana,
NTRC,
Proteomics,
Redox regulation,
Target proteins,
Thioredoxin
Editor
MDPI
Publicado en
Antioxidants 2022, 11(10), 1979
Departamento
Universidad Pública de Navarra. Departamento de Ciencias de la Salud /
Nafarroako Unibertsitate Publikoa. Osasun Zientziak Saila /
Universidad Pública de Navarra/Nafarroako Unibertsitate Publikoa. Institute for Multidisciplinary Research in Applied Biology - IMAB
Versión del editor
Entidades Financiadoras
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”.