Biophysical characterisation of the recombinant human frataxin precursor

Autores
Castro, Ignacio Hugo; Ferrari, Alejandro; Herrera, Maria Georgina; Noguera, Martín Ezequiel; Maso, Lorenzo; Benini, Monica; Rufini, Alessandra; Testi, Roberto; Costantini, Paola; Santos, Javier
Año de publicación
2018
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Friedreich's ataxia is a disease caused by a decrease in the levels of expression or loss of functionality of the mitochondrial protein frataxin (FXN). The development of an active and stable recombinant variant of FXN is important for protein replacement therapy. Although valuable data about the mature form FXN81‐210 has been collected, not enough information is available about the conformation of the frataxin precursor (FXN1‐210). We investigated the conformation, stability and function of a recombinant precursor variant (His6‐TAT‐FXN1‐210), which includes a TAT peptide in the N‐terminal region to assist with transport across cell membranes. His6‐TAT‐FXN1‐210 was expressed in Escherichia coli and conditions were found for purifying folded protein free of aggregation, oxidation or degradation, even after freezing and thawing. The protein was found to be stable and monomeric, with the N‐terminal stretch (residues 1–89) mostly unstructured and the C‐terminal domain properly folded. The experimental data suggest a complex picture for the folding process of full‐length frataxin in vitro: the presence of the N‐terminal region increased the tendency of FXN to aggregate at high temperatures but this could be avoided by the addition of low concentrations of GdmCl. The purified precursor was translocated through cell membranes. In addition, immune response against His6‐TAT‐FXN1‐210 was measured, suggesting that the C‐terminal fragment was not immunogenic at the assayed protein concentrations. Finally, the recognition of recombinant FXN by cellular proteins was studied to evaluate its functionality. In this regard, cysteine desulfurase NFS1/ISD11/ISCU was activated in vitro by His6‐TAT‐FXN1‐210. Moreover, the results showed that His6‐TAT‐FXN1‐210 can be ubiquitinated in vitro by the recently identified frataxin E3 ligase RNF126, in a similar way as the FXN1‐210, suggesting that the His6‐TAT extension does not interfere with the ubiquitination machinery.
Fil: Castro, Ignacio Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Ferrari, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Herrera, Maria Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Noguera, Martín Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Maso, Lorenzo. Universita Di Padova. Dipartimento Di Biología; Italia
Fil: Benini, Monica. Fratagene Therapeutics Srl; Italia. University Of Rome Tor Vergata; Italia
Fil: Rufini, Alessandra. University Of Rome Tor Vergata; Italia. Fratagene Therapeutics Srl; Italia
Fil: Testi, Roberto. University Of Rome Tor Vergata; Italia. Fratagene Therapeutics Srl; Italia
Fil: Costantini, Paola. Universita Di Padova. Dipartimento Di Biología; Italia
Fil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Materia
FRIEDREICH'S ATAXIA
AGGREGATION
CONFORMATION
PRECURSOR
STABILITY
UNFOLDING
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/93982
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/93982
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Biophysical characterisation of the recombinant human frataxin precursorCastro, Ignacio HugoFerrari, AlejandroHerrera, Maria GeorginaNoguera, Martín EzequielMaso, LorenzoBenini, MonicaRufini, AlessandraTesti, RobertoCostantini, PaolaSantos, JavierFRIEDREICH'S ATAXIAAGGREGATIONCONFORMATIONPRECURSORSTABILITYUNFOLDINGhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1https://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Friedreich's ataxia is a disease caused by a decrease in the levels of expression or loss of functionality of the mitochondrial protein frataxin (FXN). The development of an active and stable recombinant variant of FXN is important for protein replacement therapy. Although valuable data about the mature form FXN81‐210 has been collected, not enough information is available about the conformation of the frataxin precursor (FXN1‐210). We investigated the conformation, stability and function of a recombinant precursor variant (His6‐TAT‐FXN1‐210), which includes a TAT peptide in the N‐terminal region to assist with transport across cell membranes. His6‐TAT‐FXN1‐210 was expressed in Escherichia coli and conditions were found for purifying folded protein free of aggregation, oxidation or degradation, even after freezing and thawing. The protein was found to be stable and monomeric, with the N‐terminal stretch (residues 1–89) mostly unstructured and the C‐terminal domain properly folded. The experimental data suggest a complex picture for the folding process of full‐length frataxin in vitro: the presence of the N‐terminal region increased the tendency of FXN to aggregate at high temperatures but this could be avoided by the addition of low concentrations of GdmCl. The purified precursor was translocated through cell membranes. In addition, immune response against His6‐TAT‐FXN1‐210 was measured, suggesting that the C‐terminal fragment was not immunogenic at the assayed protein concentrations. Finally, the recognition of recombinant FXN by cellular proteins was studied to evaluate its functionality. In this regard, cysteine desulfurase NFS1/ISD11/ISCU was activated in vitro by His6‐TAT‐FXN1‐210. Moreover, the results showed that His6‐TAT‐FXN1‐210 can be ubiquitinated in vitro by the recently identified frataxin E3 ligase RNF126, in a similar way as the FXN1‐210, suggesting that the His6‐TAT extension does not interfere with the ubiquitination machinery.Fil: Castro, Ignacio Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Ferrari, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Herrera, Maria Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Noguera, Martín Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Maso, Lorenzo. Universita Di Padova. Dipartimento Di Biología; ItaliaFil: Benini, Monica. Fratagene Therapeutics Srl; Italia. University Of Rome Tor Vergata; ItaliaFil: Rufini, Alessandra. University Of Rome Tor Vergata; Italia. Fratagene Therapeutics Srl; ItaliaFil: Testi, Roberto. University Of Rome Tor Vergata; Italia. Fratagene Therapeutics Srl; ItaliaFil: Costantini, Paola. Universita Di Padova. Dipartimento Di Biología; ItaliaFil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaWiley Blackwell Publishing, Inc2018-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/93982Castro, Ignacio Hugo; Ferrari, Alejandro; Herrera, Maria Georgina; Noguera, Martín Ezequiel; Maso, Lorenzo; et al.; Biophysical characterisation of the recombinant human frataxin precursor; Wiley Blackwell Publishing, Inc; FEBS Open Bio; 8; 3; 3-2018; 390-4052211-5463CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://febs.onlinelibrary.wiley.com/doi/full/10.1002/2211-5463.12376info:eu-repo/semantics/altIdentifier/doi/10.1002/2211-5463.12376info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-10T13:22:37Zoai:ri.conicet.gov.ar:11336/93982instacron: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:34982025-09-10 13:22:37.388CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Biophysical characterisation of the recombinant human frataxin precursor
title Biophysical characterisation of the recombinant human frataxin precursor
spellingShingle Biophysical characterisation of the recombinant human frataxin precursor
Castro, Ignacio Hugo
FRIEDREICH'S ATAXIA
AGGREGATION
CONFORMATION
PRECURSOR
STABILITY
UNFOLDING
title_short Biophysical characterisation of the recombinant human frataxin precursor
title_full Biophysical characterisation of the recombinant human frataxin precursor
title_fullStr Biophysical characterisation of the recombinant human frataxin precursor
title_full_unstemmed Biophysical characterisation of the recombinant human frataxin precursor
title_sort Biophysical characterisation of the recombinant human frataxin precursor
dc.creator.none.fl_str_mv Castro, Ignacio Hugo
Ferrari, Alejandro
Herrera, Maria Georgina
Noguera, Martín Ezequiel
Maso, Lorenzo
Benini, Monica
Rufini, Alessandra
Testi, Roberto
Costantini, Paola
Santos, Javier
author Castro, Ignacio Hugo
author_facet Castro, Ignacio Hugo
Ferrari, Alejandro
Herrera, Maria Georgina
Noguera, Martín Ezequiel
Maso, Lorenzo
Benini, Monica
Rufini, Alessandra
Testi, Roberto
Costantini, Paola
Santos, Javier
author_role author
author2 Ferrari, Alejandro
Herrera, Maria Georgina
Noguera, Martín Ezequiel
Maso, Lorenzo
Benini, Monica
Rufini, Alessandra
Testi, Roberto
Costantini, Paola
Santos, Javier
author2_role author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv FRIEDREICH'S ATAXIA
AGGREGATION
CONFORMATION
PRECURSOR
STABILITY
UNFOLDING
topic FRIEDREICH'S ATAXIA
AGGREGATION
CONFORMATION
PRECURSOR
STABILITY
UNFOLDING
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Friedreich's ataxia is a disease caused by a decrease in the levels of expression or loss of functionality of the mitochondrial protein frataxin (FXN). The development of an active and stable recombinant variant of FXN is important for protein replacement therapy. Although valuable data about the mature form FXN81‐210 has been collected, not enough information is available about the conformation of the frataxin precursor (FXN1‐210). We investigated the conformation, stability and function of a recombinant precursor variant (His6‐TAT‐FXN1‐210), which includes a TAT peptide in the N‐terminal region to assist with transport across cell membranes. His6‐TAT‐FXN1‐210 was expressed in Escherichia coli and conditions were found for purifying folded protein free of aggregation, oxidation or degradation, even after freezing and thawing. The protein was found to be stable and monomeric, with the N‐terminal stretch (residues 1–89) mostly unstructured and the C‐terminal domain properly folded. The experimental data suggest a complex picture for the folding process of full‐length frataxin in vitro: the presence of the N‐terminal region increased the tendency of FXN to aggregate at high temperatures but this could be avoided by the addition of low concentrations of GdmCl. The purified precursor was translocated through cell membranes. In addition, immune response against His6‐TAT‐FXN1‐210 was measured, suggesting that the C‐terminal fragment was not immunogenic at the assayed protein concentrations. Finally, the recognition of recombinant FXN by cellular proteins was studied to evaluate its functionality. In this regard, cysteine desulfurase NFS1/ISD11/ISCU was activated in vitro by His6‐TAT‐FXN1‐210. Moreover, the results showed that His6‐TAT‐FXN1‐210 can be ubiquitinated in vitro by the recently identified frataxin E3 ligase RNF126, in a similar way as the FXN1‐210, suggesting that the His6‐TAT extension does not interfere with the ubiquitination machinery.
Fil: Castro, Ignacio Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Ferrari, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Herrera, Maria Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Noguera, Martín Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Maso, Lorenzo. Universita Di Padova. Dipartimento Di Biología; Italia
Fil: Benini, Monica. Fratagene Therapeutics Srl; Italia. University Of Rome Tor Vergata; Italia
Fil: Rufini, Alessandra. University Of Rome Tor Vergata; Italia. Fratagene Therapeutics Srl; Italia
Fil: Testi, Roberto. University Of Rome Tor Vergata; Italia. Fratagene Therapeutics Srl; Italia
Fil: Costantini, Paola. Universita Di Padova. Dipartimento Di Biología; Italia
Fil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
description Friedreich's ataxia is a disease caused by a decrease in the levels of expression or loss of functionality of the mitochondrial protein frataxin (FXN). The development of an active and stable recombinant variant of FXN is important for protein replacement therapy. Although valuable data about the mature form FXN81‐210 has been collected, not enough information is available about the conformation of the frataxin precursor (FXN1‐210). We investigated the conformation, stability and function of a recombinant precursor variant (His6‐TAT‐FXN1‐210), which includes a TAT peptide in the N‐terminal region to assist with transport across cell membranes. His6‐TAT‐FXN1‐210 was expressed in Escherichia coli and conditions were found for purifying folded protein free of aggregation, oxidation or degradation, even after freezing and thawing. The protein was found to be stable and monomeric, with the N‐terminal stretch (residues 1–89) mostly unstructured and the C‐terminal domain properly folded. The experimental data suggest a complex picture for the folding process of full‐length frataxin in vitro: the presence of the N‐terminal region increased the tendency of FXN to aggregate at high temperatures but this could be avoided by the addition of low concentrations of GdmCl. The purified precursor was translocated through cell membranes. In addition, immune response against His6‐TAT‐FXN1‐210 was measured, suggesting that the C‐terminal fragment was not immunogenic at the assayed protein concentrations. Finally, the recognition of recombinant FXN by cellular proteins was studied to evaluate its functionality. In this regard, cysteine desulfurase NFS1/ISD11/ISCU was activated in vitro by His6‐TAT‐FXN1‐210. Moreover, the results showed that His6‐TAT‐FXN1‐210 can be ubiquitinated in vitro by the recently identified frataxin E3 ligase RNF126, in a similar way as the FXN1‐210, suggesting that the His6‐TAT extension does not interfere with the ubiquitination machinery.
publishDate 2018
dc.date.none.fl_str_mv 2018-03
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/11336/93982
Castro, Ignacio Hugo; Ferrari, Alejandro; Herrera, Maria Georgina; Noguera, Martín Ezequiel; Maso, Lorenzo; et al.; Biophysical characterisation of the recombinant human frataxin precursor; Wiley Blackwell Publishing, Inc; FEBS Open Bio; 8; 3; 3-2018; 390-405
2211-5463
CONICET Digital
CONICET
url http://hdl.handle.net/11336/93982
identifier_str_mv Castro, Ignacio Hugo; Ferrari, Alejandro; Herrera, Maria Georgina; Noguera, Martín Ezequiel; Maso, Lorenzo; et al.; Biophysical characterisation of the recombinant human frataxin precursor; Wiley Blackwell Publishing, Inc; FEBS Open Bio; 8; 3; 3-2018; 390-405
2211-5463
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://febs.onlinelibrary.wiley.com/doi/full/10.1002/2211-5463.12376
info:eu-repo/semantics/altIdentifier/doi/10.1002/2211-5463.12376
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Wiley Blackwell Publishing, Inc
publisher.none.fl_str_mv Wiley Blackwell Publishing, 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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