Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango

Autores
Mansilla, Santiago Nahuel; Tórtora, Verónica; Pignataro, María Florencia; Sastre, Santiago; Castro, Ignacio; Chiribao, María Laura; Robello, Carlos; Zeida Camacho, Ari Fernando; Santos, Javier; Castro, Laura
Año de publicación
2023
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Mitochondrial aconitase (ACO2) has been postulated as a redox sensor in the tricarboxylic acid cycle. Its high sensitivity towards reactive oxygen and nitrogen species is due to its particularly labile [4Fe–4S]2+ prosthetic group which yields an inactive [3Fe–4S]+ cluster upon oxidation. Moreover, ACO2 was found as a main oxidant target during aging and in pathologies where mitochondrial dysfunction is implied. Herein, we report the expression and characterization of recombinant human ACO2 and its interaction with frataxin (FXN), a protein that participates in the de novo biosynthesis of Fe–S clusters. A high yield of pure ACO2 (≥99%, 22 ± 2 U/mg) was obtained and kinetic parameters for citrate, isocitrate, and cis-aconitate were determined. Superoxide, carbonate radical, peroxynitrite, and hydrogen peroxide reacted with ACO2 with second-order rate constants of 108, 108, 105, and 102 M−1 s−1, respectively. Temperature-induced unfolding assessed by tryptophan fluorescence of ACO2 resulted in apparent melting temperatures of 51.1 ± 0.5 and 43.6 ± 0.2 °C for [4Fe–4S]2+ and [3Fe–4S]+ states of ACO2, sustaining lower thermal stability upon cluster oxidation. Differences in protein dynamics produced by the Fe–S cluster redox state were addressed by molecular dynamics simulations. Reactivation of [3Fe–4S]+-ACO2 by FXN was verified by activation assays and direct iron-dependent interaction was confirmed by protein-protein interaction ELISA and fluorescence spectroscopic assays. Multimer modeling and protein-protein docking predicted an ACO2-FXN complex where the metal ion binding region of FXN approaches the [3Fe–4S]+ cluster, supporting that FXN is a partner for reactivation of ACO2 upon oxidative cluster inactivation.
Fil: Mansilla, Santiago Nahuel. Universidad de la República; Uruguay
Fil: Tórtora, Verónica. Universidad de la República; Uruguay
Fil: Pignataro, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; Argentina
Fil: Sastre, Santiago. Universidad de la República; Uruguay
Fil: Castro, Ignacio. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; Argentina
Fil: Chiribao, María Laura. Universidad de la República; Uruguay
Fil: Robello, Carlos. Universidad de la República; Uruguay
Fil: Zeida Camacho, Ari Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; Argentina. Universidad de la República; Uruguay
Fil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de la República; Uruguay
Fil: Castro, Laura. Universidad de la República; Uruguay
Materia
ACONITASE
FRATAXIN
MITOCHONDRIA
INTERACTION
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/225425
Acceder
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repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tangoMansilla, Santiago NahuelTórtora, VerónicaPignataro, María FlorenciaSastre, SantiagoCastro, IgnacioChiribao, María LauraRobello, CarlosZeida Camacho, Ari FernandoSantos, JavierCastro, LauraACONITASEFRATAXINMITOCHONDRIAINTERACTIONhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Mitochondrial aconitase (ACO2) has been postulated as a redox sensor in the tricarboxylic acid cycle. Its high sensitivity towards reactive oxygen and nitrogen species is due to its particularly labile [4Fe–4S]2+ prosthetic group which yields an inactive [3Fe–4S]+ cluster upon oxidation. Moreover, ACO2 was found as a main oxidant target during aging and in pathologies where mitochondrial dysfunction is implied. Herein, we report the expression and characterization of recombinant human ACO2 and its interaction with frataxin (FXN), a protein that participates in the de novo biosynthesis of Fe–S clusters. A high yield of pure ACO2 (≥99%, 22 ± 2 U/mg) was obtained and kinetic parameters for citrate, isocitrate, and cis-aconitate were determined. Superoxide, carbonate radical, peroxynitrite, and hydrogen peroxide reacted with ACO2 with second-order rate constants of 108, 108, 105, and 102 M−1 s−1, respectively. Temperature-induced unfolding assessed by tryptophan fluorescence of ACO2 resulted in apparent melting temperatures of 51.1 ± 0.5 and 43.6 ± 0.2 °C for [4Fe–4S]2+ and [3Fe–4S]+ states of ACO2, sustaining lower thermal stability upon cluster oxidation. Differences in protein dynamics produced by the Fe–S cluster redox state were addressed by molecular dynamics simulations. Reactivation of [3Fe–4S]+-ACO2 by FXN was verified by activation assays and direct iron-dependent interaction was confirmed by protein-protein interaction ELISA and fluorescence spectroscopic assays. Multimer modeling and protein-protein docking predicted an ACO2-FXN complex where the metal ion binding region of FXN approaches the [3Fe–4S]+ cluster, supporting that FXN is a partner for reactivation of ACO2 upon oxidative cluster inactivation.Fil: Mansilla, Santiago Nahuel. Universidad de la República; UruguayFil: Tórtora, Verónica. Universidad de la República; UruguayFil: Pignataro, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; ArgentinaFil: Sastre, Santiago. Universidad de la República; UruguayFil: Castro, Ignacio. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; ArgentinaFil: Chiribao, María Laura. Universidad de la República; UruguayFil: Robello, Carlos. Universidad de la República; UruguayFil: Zeida Camacho, Ari Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; Argentina. Universidad de la República; UruguayFil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de la República; UruguayFil: Castro, Laura. Universidad de la República; UruguayElsevier Science Inc.2023-02info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/225425Mansilla, Santiago Nahuel; Tórtora, Verónica; Pignataro, María Florencia; Sastre, Santiago; Castro, Ignacio; et al.; Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango; Elsevier Science Inc.; Free Radical Biology and Medicine; 197; 2-2023; 71-840891-5849CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0891584923000497info:eu-repo/semantics/altIdentifier/doi/10.1016/j.freeradbiomed.2023.01.028info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:31:56Zoai:ri.conicet.gov.ar:11336/225425instacron: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 10:31:56.975CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango
title Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango
spellingShingle Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango
Mansilla, Santiago Nahuel
ACONITASE
FRATAXIN
MITOCHONDRIA
INTERACTION
title_short Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango
title_full Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango
title_fullStr Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango
title_full_unstemmed Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango
title_sort Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango
dc.creator.none.fl_str_mv Mansilla, Santiago Nahuel
Tórtora, Verónica
Pignataro, María Florencia
Sastre, Santiago
Castro, Ignacio
Chiribao, María Laura
Robello, Carlos
Zeida Camacho, Ari Fernando
Santos, Javier
Castro, Laura
author Mansilla, Santiago Nahuel
author_facet Mansilla, Santiago Nahuel
Tórtora, Verónica
Pignataro, María Florencia
Sastre, Santiago
Castro, Ignacio
Chiribao, María Laura
Robello, Carlos
Zeida Camacho, Ari Fernando
Santos, Javier
Castro, Laura
author_role author
author2 Tórtora, Verónica
Pignataro, María Florencia
Sastre, Santiago
Castro, Ignacio
Chiribao, María Laura
Robello, Carlos
Zeida Camacho, Ari Fernando
Santos, Javier
Castro, Laura
author2_role author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv ACONITASE
FRATAXIN
MITOCHONDRIA
INTERACTION
topic ACONITASE
FRATAXIN
MITOCHONDRIA
INTERACTION
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Mitochondrial aconitase (ACO2) has been postulated as a redox sensor in the tricarboxylic acid cycle. Its high sensitivity towards reactive oxygen and nitrogen species is due to its particularly labile [4Fe–4S]2+ prosthetic group which yields an inactive [3Fe–4S]+ cluster upon oxidation. Moreover, ACO2 was found as a main oxidant target during aging and in pathologies where mitochondrial dysfunction is implied. Herein, we report the expression and characterization of recombinant human ACO2 and its interaction with frataxin (FXN), a protein that participates in the de novo biosynthesis of Fe–S clusters. A high yield of pure ACO2 (≥99%, 22 ± 2 U/mg) was obtained and kinetic parameters for citrate, isocitrate, and cis-aconitate were determined. Superoxide, carbonate radical, peroxynitrite, and hydrogen peroxide reacted with ACO2 with second-order rate constants of 108, 108, 105, and 102 M−1 s−1, respectively. Temperature-induced unfolding assessed by tryptophan fluorescence of ACO2 resulted in apparent melting temperatures of 51.1 ± 0.5 and 43.6 ± 0.2 °C for [4Fe–4S]2+ and [3Fe–4S]+ states of ACO2, sustaining lower thermal stability upon cluster oxidation. Differences in protein dynamics produced by the Fe–S cluster redox state were addressed by molecular dynamics simulations. Reactivation of [3Fe–4S]+-ACO2 by FXN was verified by activation assays and direct iron-dependent interaction was confirmed by protein-protein interaction ELISA and fluorescence spectroscopic assays. Multimer modeling and protein-protein docking predicted an ACO2-FXN complex where the metal ion binding region of FXN approaches the [3Fe–4S]+ cluster, supporting that FXN is a partner for reactivation of ACO2 upon oxidative cluster inactivation.
Fil: Mansilla, Santiago Nahuel. Universidad de la República; Uruguay
Fil: Tórtora, Verónica. Universidad de la República; Uruguay
Fil: Pignataro, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; Argentina
Fil: Sastre, Santiago. Universidad de la República; Uruguay
Fil: Castro, Ignacio. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; Argentina
Fil: Chiribao, María Laura. Universidad de la República; Uruguay
Fil: Robello, Carlos. Universidad de la República; Uruguay
Fil: Zeida Camacho, Ari Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; Argentina. Universidad de la República; Uruguay
Fil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de la República; Uruguay
Fil: Castro, Laura. Universidad de la República; Uruguay
description Mitochondrial aconitase (ACO2) has been postulated as a redox sensor in the tricarboxylic acid cycle. Its high sensitivity towards reactive oxygen and nitrogen species is due to its particularly labile [4Fe–4S]2+ prosthetic group which yields an inactive [3Fe–4S]+ cluster upon oxidation. Moreover, ACO2 was found as a main oxidant target during aging and in pathologies where mitochondrial dysfunction is implied. Herein, we report the expression and characterization of recombinant human ACO2 and its interaction with frataxin (FXN), a protein that participates in the de novo biosynthesis of Fe–S clusters. A high yield of pure ACO2 (≥99%, 22 ± 2 U/mg) was obtained and kinetic parameters for citrate, isocitrate, and cis-aconitate were determined. Superoxide, carbonate radical, peroxynitrite, and hydrogen peroxide reacted with ACO2 with second-order rate constants of 108, 108, 105, and 102 M−1 s−1, respectively. Temperature-induced unfolding assessed by tryptophan fluorescence of ACO2 resulted in apparent melting temperatures of 51.1 ± 0.5 and 43.6 ± 0.2 °C for [4Fe–4S]2+ and [3Fe–4S]+ states of ACO2, sustaining lower thermal stability upon cluster oxidation. Differences in protein dynamics produced by the Fe–S cluster redox state were addressed by molecular dynamics simulations. Reactivation of [3Fe–4S]+-ACO2 by FXN was verified by activation assays and direct iron-dependent interaction was confirmed by protein-protein interaction ELISA and fluorescence spectroscopic assays. Multimer modeling and protein-protein docking predicted an ACO2-FXN complex where the metal ion binding region of FXN approaches the [3Fe–4S]+ cluster, supporting that FXN is a partner for reactivation of ACO2 upon oxidative cluster inactivation.
publishDate 2023
dc.date.none.fl_str_mv 2023-02
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/11336/225425
Mansilla, Santiago Nahuel; Tórtora, Verónica; Pignataro, María Florencia; Sastre, Santiago; Castro, Ignacio; et al.; Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango; Elsevier Science Inc.; Free Radical Biology and Medicine; 197; 2-2023; 71-84
0891-5849
CONICET Digital
CONICET
url http://hdl.handle.net/11336/225425
identifier_str_mv Mansilla, Santiago Nahuel; Tórtora, Verónica; Pignataro, María Florencia; Sastre, Santiago; Castro, Ignacio; et al.; Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango; Elsevier Science Inc.; Free Radical Biology and Medicine; 197; 2-2023; 71-84
0891-5849
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/S0891584923000497
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.freeradbiomed.2023.01.028
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier Science Inc.
publisher.none.fl_str_mv Elsevier Science Inc.
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 13.069144

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