In vivo protein tyrosine nitration in Arabidopsis thaliana

Nitration of tyrosine (Y) residues of proteins is a low abundant post-translational modification that modulates protein function or fate in animal systems. However, very little is known about the in vivo prevalence of this modification and its corresponding targets in plants. Immunoprecipitation, ba...

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Autores: Lozano-Juste, Jorge|||0000-0001-7034-566X, León, José|||0000-0002-7332-1572, Colom Moreno, Rosa María
Tipo de recurso: artículo
Fecha de publicación:2011
País:España
Institución:Universitat Politècnica de València (UPV)
Repositorio:RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia
Idioma:inglés
OAI Identifier:oai:riunet.upv.es:10251/78549
Acceso en línea:https://riunet.upv.es/handle/10251/78549
Access Level:acceso abierto
Palabra clave:AminoY
Arabidopsis
Nitric oxide
Nitrotyrosine
NitroY
Post-translational modification
Protein nitration
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network_name_str España
repository_id_str
dc.title.none.fl_str_mv In vivo protein tyrosine nitration in Arabidopsis thaliana
title In vivo protein tyrosine nitration in Arabidopsis thaliana
spellingShingle In vivo protein tyrosine nitration in Arabidopsis thaliana
Lozano-Juste, Jorge|||0000-0001-7034-566X
AminoY
Arabidopsis
Nitric oxide
Nitrotyrosine
NitroY
Post-translational modification
Protein nitration
title_short In vivo protein tyrosine nitration in Arabidopsis thaliana
title_full In vivo protein tyrosine nitration in Arabidopsis thaliana
title_fullStr In vivo protein tyrosine nitration in Arabidopsis thaliana
title_full_unstemmed In vivo protein tyrosine nitration in Arabidopsis thaliana
title_sort In vivo protein tyrosine nitration in Arabidopsis thaliana
dc.creator.none.fl_str_mv Lozano-Juste, Jorge|||0000-0001-7034-566X
León, José|||0000-0002-7332-1572
Colom Moreno, Rosa María
author Lozano-Juste, Jorge|||0000-0001-7034-566X
author_facet Lozano-Juste, Jorge|||0000-0001-7034-566X
León, José|||0000-0002-7332-1572
Colom Moreno, Rosa María
author_role author
author2 León, José|||0000-0002-7332-1572
Colom Moreno, Rosa María
author2_role author
author
dc.contributor.none.fl_str_mv Instituto Universitario Mixto de Biología Molecular y Celular de Plantas
Ministerio de Educación y Ciencia
Ministerio de Ciencia e Innovación
Ministerio de Ciencia y Tecnología
Consejo Superior de Investigaciones Científicas
Fundación Bancaja
Repositorio Institucional de la Universitat Politècnica de València Riunet
dc.subject.none.fl_str_mv AminoY
Arabidopsis
Nitric oxide
Nitrotyrosine
NitroY
Post-translational modification
Protein nitration
topic AminoY
Arabidopsis
Nitric oxide
Nitrotyrosine
NitroY
Post-translational modification
Protein nitration
description Nitration of tyrosine (Y) residues of proteins is a low abundant post-translational modification that modulates protein function or fate in animal systems. However, very little is known about the in vivo prevalence of this modification and its corresponding targets in plants. Immunoprecipitation, based on an anti-3-nitroY antibody, was performed to pulldown potential in vivo targets of Y nitration in the Arabidopsis thaliana proteome. Further shotgun liquid chromatography–mass spectrometry (LC-MS/MS) proteomic analysis of the immunoprecipitated proteins allowed the identification of 127 proteins. Around 35% of them corresponded to homologues of proteins that have been previously reported to be Y nitrated in other non-plant organisms. Some of the putative in vivo Y-nitrated proteins were further confirmed by western blot with specific antibodies. Furthermore, MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) analysis of protein spots, separated by two-dimensional electrophoresis from immunoprecipitated proteins, led to the identification of seven nitrated peptides corresponding to six different proteins. However, in vivo nitration sites among putative targets could not be identified by MS/MS. Nevertheless, an MS/MS spectrum with 3-aminoY318 instead of the expected 3-nitroY was found for cytosolic glyceraldehyde-3- phosphate dehydrogenase. Reduction of nitroY to aminoY during MS-based proteomic analysis together with the in vivo low abundance of these modifications made the identification of nitration sites difficult. In turn, in vitro nitration of methionine synthase, which was also found in the shotgun proteomic screening, allowed unequivocal identification of a nitration site at Y287.
publishDate 2011
dc.date.none.fl_str_mv 2011
2011-06-01
dc.type.none.fl_str_mv journal article
http://purl.org/coar/resource_type/c_6501
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://riunet.upv.es/handle/10251/78549
url https://riunet.upv.es/handle/10251/78549
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.relation.none.fl_str_mv Ministerio de Ciencia y Tecnología https://doi.org/10.13039/501100006280 GEN2003-20477-C02-02
Ministerio de Educación y Ciencia https://doi.org/10.13039/501100008743 BIO2005-00222
Ministerio de Ciencia e Innovación http://dx.doi.org/10.13039/501100004837 BIO2008-00839 BIOSINTESIS Y FUNCION DEL OXIDO NITRICO EN ARABIDOPSIS. CONEXION CON LOS ACIDOS ABSCISICO, SALICILICO Y JASMONICO
Ministerio de Educación y Ciencia https://doi.org/10.13039/501100008743 CSD2007-00057 Función y potencial biotecnológico de los factores de transcripción de las plantas.
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
Reserva de todos los derechos
http://rightsstatements.org/vocab/InC/1.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
Reserva de todos los derechos
http://rightsstatements.org/vocab/InC/1.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Oxford University Press (OUP)
publisher.none.fl_str_mv Oxford University Press (OUP)
dc.source.none.fl_str_mv reponame:RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia
instname:Universitat Politècnica de València (UPV)
instname_str Universitat Politècnica de València (UPV)
reponame_str RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia
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spelling In vivo protein tyrosine nitration in Arabidopsis thalianaLozano-Juste, Jorge|||0000-0001-7034-566XLeón, José|||0000-0002-7332-1572Colom Moreno, Rosa MaríaAminoYArabidopsisNitric oxideNitrotyrosineNitroYPost-translational modificationProtein nitrationNitration of tyrosine (Y) residues of proteins is a low abundant post-translational modification that modulates protein function or fate in animal systems. However, very little is known about the in vivo prevalence of this modification and its corresponding targets in plants. Immunoprecipitation, based on an anti-3-nitroY antibody, was performed to pulldown potential in vivo targets of Y nitration in the Arabidopsis thaliana proteome. Further shotgun liquid chromatography–mass spectrometry (LC-MS/MS) proteomic analysis of the immunoprecipitated proteins allowed the identification of 127 proteins. Around 35% of them corresponded to homologues of proteins that have been previously reported to be Y nitrated in other non-plant organisms. Some of the putative in vivo Y-nitrated proteins were further confirmed by western blot with specific antibodies. Furthermore, MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) analysis of protein spots, separated by two-dimensional electrophoresis from immunoprecipitated proteins, led to the identification of seven nitrated peptides corresponding to six different proteins. However, in vivo nitration sites among putative targets could not be identified by MS/MS. Nevertheless, an MS/MS spectrum with 3-aminoY318 instead of the expected 3-nitroY was found for cytosolic glyceraldehyde-3- phosphate dehydrogenase. Reduction of nitroY to aminoY during MS-based proteomic analysis together with the in vivo low abundance of these modifications made the identification of nitration sites difficult. In turn, in vitro nitration of methionine synthase, which was also found in the shotgun proteomic screening, allowed unequivocal identification of a nitration site at Y287.We thank Rafael Ruiz-Partida (CIPF) for his valuable help with protein modelling. We also thank Renate Scheibe (Universitat Osnabruck, Germany), Dorothee Staiger (University of Bielefeld, Germany), Mariam Sahrawy (EEZ-CSIC, Granada, Spain), Joe Ogas (Purdue University, USA), and Dominique Rumeau (Universite de la Mediterranee, France) for their kind donation of antibodies against GAPDH, GRP7, fructose bisphosphatase, PICKEL, and carbonic anhydrase, respectively. The AtMS1 cDNA fused to the 6xHis tag was kindly donated by David Dixon (University of Durham, UK). MS-based protein identification was performed by the Proteomic Service of CIPF-PROTEORED (Valencia, Spain). This work was supported by Ministerio de Educacion y Ciencia from Spain and FEDER funds from EU grants GEN2003-20477-C02-02, BIO2005-00222, BIO2008-00839, and CONSOLIDER TRANSPLANTA CSD2007-00057 (to JL), a fellowship from the Bancaja-CSIC Programme (to JLJ), and a contract of the I3P Programme of CSIC (co-financed with FEDER funds of the EU, to RC-M).Oxford University Press (OUP)Instituto Universitario Mixto de Biología Molecular y Celular de PlantasMinisterio de Educación y CienciaMinisterio de Ciencia e InnovaciónMinisterio de Ciencia y TecnologíaConsejo Superior de Investigaciones CientíficasFundación BancajaRepositorio Institucional de la Universitat Politècnica de València Riunet20112011-06-01journal articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttps://riunet.upv.es/handle/10251/78549reponame:RiuNet. Repositorio Institucional de la Universitat Politécnica de Valénciainstname:Universitat Politècnica de València (UPV)InglésengMinisterio de Ciencia y Tecnología https://doi.org/10.13039/501100006280 GEN2003-20477-C02-02Ministerio de Educación y Ciencia https://doi.org/10.13039/501100008743 BIO2005-00222Ministerio de Ciencia e Innovación http://dx.doi.org/10.13039/501100004837 BIO2008-00839 BIOSINTESIS Y FUNCION DEL OXIDO NITRICO EN ARABIDOPSIS. CONEXION CON LOS ACIDOS ABSCISICO, SALICILICO Y JASMONICOMinisterio de Educación y Ciencia https://doi.org/10.13039/501100008743 CSD2007-00057 Función y potencial biotecnológico de los factores de transcripción de las plantas.open accesshttp://purl.org/coar/access_right/c_abf2Reserva de todos los derechoshttp://rightsstatements.org/vocab/InC/1.0/info:eu-repo/semantics/openAccessoai:riunet.upv.es:10251/785492026-06-13T07:49:27Z
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