Biophysical characterisation of the recombinant human frataxin precursor
| Autor: | Lorenzo Maso, Monica Benini, Alessandra Rufini, Paola Costantini, Roberto Testi, Maria Georgina Herrera, Martín Ezequiel Noguera, Javier Santos, Ignacio Hugo Castro, Alejandro Ferrari |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
conformation
0301 basic medicine precursor Friedreich's ataxia aggregation stability unfolding medicine.disease_cause General Biochemistry Genetics and Molecular Biology law.invention Ciencias Biológicas purl.org/becyt/ford/1 [https] FRIEDREICH'S ATAXIA 03 medical and health sciences Protein replacement therapy Ubiquitin law medicine purl.org/becyt/ford/1.6 [https] PRECURSOR Escherichia coli Research Articles Settore MED/04 - Patologia Generale STABILITY 030102 biochemistry & molecular biology biology Cysteine desulfurase Chemistry UNFOLDING Bioquímica y Biología Molecular AGGREGATION CONFORMATION Ubiquitin ligase Cell biology 030104 developmental biology biology.protein Frataxin Recombinant DNA ISCU CIENCIAS NATURALES Y EXACTAS Research Article |
| Zdroj: | CONICET Digital (CONICET) Consejo Nacional de Investigaciones Científicas y Técnicas instacron:CONICET FEBS Open Bio |
| ISSN: | 2211-5463 |
| DOI: | 10.1002/2211-5463.12376 |
| Popis: | 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 |
| Databáze: | OpenAIRE |
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