Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation

Autores
Campolo, Nicolás; Mastrogiovanni, Mauricio; Mariotti, Michele; Issoglio, Federico Matías; Estrin, Dario Ariel; Hägglund, Per; Grune, Tilman; Davies, Michael J.; Bartesaghi, Silvina; Radi, Rafael
Año de publicación
2023
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO−) in vitro. We found that ONOO− exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO− induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.
Fil: Campolo, Nicolás. Universidad de la Republica; Uruguay
Fil: Mastrogiovanni, Mauricio. Universidad de la Republica; Uruguay
Fil: Mariotti, Michele. Universidad de Copenhagen; Dinamarca
Fil: Issoglio, Federico Matías. Universidade Nova de Lisboa; Portugal. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina
Fil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina
Fil: Hägglund, Per. Universidad de Copenhagen; Dinamarca
Fil: Grune, Tilman. Universidad de Viena; Austria. German Center for Cardiovascular Research; Alemania. German Institute of Human Nutrition; Alemania
Fil: Davies, Michael J.. Universidad de Copenhagen; Dinamarca
Fil: Bartesaghi, Silvina. Universidad de la Republica; Uruguay
Fil: Radi, Rafael. Universidad de la Republica; Uruguay
Materia
AGGREGATION
DITYROSINE
FREE RADICALS
GLUTAMINE SYNTHETASE
HYDROGEN PEROXIDE
NITROTYROSINE
OXIDANTS
PEROXYNITRITE
THIOL OXIDATION
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/218973
Acceder
id CONICETDig_489f17a315beffc3db688055d2f8a36e
oai_identifier_str oai:ri.conicet.gov.ar:11336/218973
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregationCampolo, NicolásMastrogiovanni, MauricioMariotti, MicheleIssoglio, Federico MatíasEstrin, Dario ArielHägglund, PerGrune, TilmanDavies, Michael J.Bartesaghi, SilvinaRadi, RafaelAGGREGATIONDITYROSINEFREE RADICALSGLUTAMINE SYNTHETASEHYDROGEN PEROXIDENITROTYROSINEOXIDANTSPEROXYNITRITETHIOL OXIDATIONhttps://purl.org/becyt/ford/1.4https://purl.org/becyt/ford/1Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO−) in vitro. We found that ONOO− exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO− induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.Fil: Campolo, Nicolás. Universidad de la Republica; UruguayFil: Mastrogiovanni, Mauricio. Universidad de la Republica; UruguayFil: Mariotti, Michele. Universidad de Copenhagen; DinamarcaFil: Issoglio, Federico Matías. Universidade Nova de Lisboa; Portugal. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; ArgentinaFil: Hägglund, Per. Universidad de Copenhagen; DinamarcaFil: Grune, Tilman. Universidad de Viena; Austria. German Center for Cardiovascular Research; Alemania. German Institute of Human Nutrition; AlemaniaFil: Davies, Michael J.. Universidad de Copenhagen; DinamarcaFil: Bartesaghi, Silvina. Universidad de la Republica; UruguayFil: Radi, Rafael. Universidad de la Republica; UruguayAmerican Society for Biochemistry and Molecular Biology2023-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/218973Campolo, Nicolás; Mastrogiovanni, Mauricio; Mariotti, Michele; Issoglio, Federico Matías; Estrin, Dario Ariel; et al.; Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 299; 3; 3-2023; 1-220021-9258CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S002192582300073Xinfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.jbc.2023.102941info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:46:02Zoai:ri.conicet.gov.ar:11336/218973instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 09:46:03.224CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation
title Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation
spellingShingle Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation
Campolo, Nicolás
AGGREGATION
DITYROSINE
FREE RADICALS
GLUTAMINE SYNTHETASE
HYDROGEN PEROXIDE
NITROTYROSINE
OXIDANTS
PEROXYNITRITE
THIOL OXIDATION
title_short Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation
title_full Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation
title_fullStr Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation
title_full_unstemmed Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation
title_sort Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation
dc.creator.none.fl_str_mv Campolo, Nicolás
Mastrogiovanni, Mauricio
Mariotti, Michele
Issoglio, Federico Matías
Estrin, Dario Ariel
Hägglund, Per
Grune, Tilman
Davies, Michael J.
Bartesaghi, Silvina
Radi, Rafael
author Campolo, Nicolás
author_facet Campolo, Nicolás
Mastrogiovanni, Mauricio
Mariotti, Michele
Issoglio, Federico Matías
Estrin, Dario Ariel
Hägglund, Per
Grune, Tilman
Davies, Michael J.
Bartesaghi, Silvina
Radi, Rafael
author_role author
author2 Mastrogiovanni, Mauricio
Mariotti, Michele
Issoglio, Federico Matías
Estrin, Dario Ariel
Hägglund, Per
Grune, Tilman
Davies, Michael J.
Bartesaghi, Silvina
Radi, Rafael
author2_role author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv AGGREGATION
DITYROSINE
FREE RADICALS
GLUTAMINE SYNTHETASE
HYDROGEN PEROXIDE
NITROTYROSINE
OXIDANTS
PEROXYNITRITE
THIOL OXIDATION
topic AGGREGATION
DITYROSINE
FREE RADICALS
GLUTAMINE SYNTHETASE
HYDROGEN PEROXIDE
NITROTYROSINE
OXIDANTS
PEROXYNITRITE
THIOL OXIDATION
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.4
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO−) in vitro. We found that ONOO− exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO− induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.
Fil: Campolo, Nicolás. Universidad de la Republica; Uruguay
Fil: Mastrogiovanni, Mauricio. Universidad de la Republica; Uruguay
Fil: Mariotti, Michele. Universidad de Copenhagen; Dinamarca
Fil: Issoglio, Federico Matías. Universidade Nova de Lisboa; Portugal. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina
Fil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina
Fil: Hägglund, Per. Universidad de Copenhagen; Dinamarca
Fil: Grune, Tilman. Universidad de Viena; Austria. German Center for Cardiovascular Research; Alemania. German Institute of Human Nutrition; Alemania
Fil: Davies, Michael J.. Universidad de Copenhagen; Dinamarca
Fil: Bartesaghi, Silvina. Universidad de la Republica; Uruguay
Fil: Radi, Rafael. Universidad de la Republica; Uruguay
description Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO−) in vitro. We found that ONOO− exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO− induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.
publishDate 2023
dc.date.none.fl_str_mv 2023-03
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/218973
Campolo, Nicolás; Mastrogiovanni, Mauricio; Mariotti, Michele; Issoglio, Federico Matías; Estrin, Dario Ariel; et al.; Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 299; 3; 3-2023; 1-22
0021-9258
CONICET Digital
CONICET
url http://hdl.handle.net/11336/218973
identifier_str_mv Campolo, Nicolás; Mastrogiovanni, Mauricio; Mariotti, Michele; Issoglio, Federico Matías; Estrin, Dario Ariel; et al.; Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 299; 3; 3-2023; 1-22
0021-9258
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S002192582300073X
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jbc.2023.102941
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv American Society for Biochemistry and Molecular Biology
publisher.none.fl_str_mv American Society for Biochemistry and Molecular Biology
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.070432

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