Biophysical Characterization of the Recombinant Human Frataxin Precursor

HERRERA MARIA GEORGINA; BENINI MONICA; CONSTANTINI PAOLA; FERRARI ALEJANDRO; MASO LORENZO; TESTI ROBERTO; CASTRO IGNACIO HUGO; NOGUERA MARTÍN EZEQUIEL; RUFINI ALESSANDRA; SANTOS JAVIER

Pisa

Conferencia; The 2nd International Ataxia Research Conference; 2017

Introduction: One outstanding strategy to increase the concentration of active frataxin (FXN) inside the mitochondria is the production of recombinant variants of FXN with the capability to cross cell membranes using a TAT-derived peptide fused to FXN precursor [1-4]. For an eventual TAT-FXN-based therapy, the integrity of protein conformation is essential. Not enough information is available about the conformation of precursor FXN1-210. Here, we investigated the conformation and stability of a recombinant precursor (TAT-His6-FXN1-210), which includes a TAT peptide in the N-terminal region plus a histidine tag.Methods: His6-TAT-FXN1-210 was expressed in Escherichia coli codon plus ROSETTA2pLysDE3 cells and purified to ≥ 95% (SDS-PAGE). Mass and aggregation state were evaluated by ESI-MS and MALS/DLS. Circular dichroism and fluorescence measurements were performed. GdmCl and temperature-induced unfolding experiments were carried out. Cysteine desulfurase NFS1/ISD11 activation by FXN was investigated (methylene blue method). Protein transduction was studied using FITC-labeling TAT-His6-FXN1-210. Immune response against TAT-His6-FXN1-187-210 (0.05 or 0.25 nmol) was measured using C57 mice. Specific antibodies to FXN variants were tested by ELISA.Results: We optimized expression and purification conditions that maintain the protein soluble, even after freezing and thawing, free of aggregation, oxidation or degradation. His6-TAT-FXN1-210 is monomeric, with the N-terminal stretch (residues 1-89) mostly unstructured, and the C-terminal domain folded. GdmCl-induced unfolding of the precursor is reversible, cooperative and the protein is stable. Temperature-induced unfolding/aggregation was not reversible but the addition of low concentrations of GdmCl makes it reversible. His6-TAT-FXN1-210 activates desulfurase in vitro. Precursor was translocated across cell membranes and our results support the notion that the C- terminal fragment is not immunogenic at these concentrations.Conclusions: His6-TAT-FXN1-210 exhibits an enhanced propensity to aggregate. Conditions were set that keep the protein stable and soluble after freeze/thaw, with minimal autoproteolysis. Low GdmCl concentration prevents N- terminal-mediated aggregation.1. Marcus, D., et al., Heterologous mitochondrial targeting sequences can deliver functional proteins into mitochondria Therapeutic approaches for the treatment of Friedreich´s ataxia. Int J Biochem Cell Biol, 2016. 81(Pt A): p. 48-56.2. Vyas, P.M., et al., A TAT-frataxin fusion protein increases lifespan and cardiac function in a conditional Friedreich´s ataxia mouse model. Hum Mol Genet, 2012. 21(6): p. 1230-47.3. Kim, M.J., et al., Tat-Frataxin protects dopaminergic neuronal cells against MPTPinducedtoxicity in a mouse model of Parkinson´s disease. Biochimie, 2012. 94(11): p. 2448-56.4. Strawser, C.J., K.A. Schadt, and D.R. Lynch,Therapeutic approaches for the treatment of Friedreich´s ataxia. Expert Rev Neurother, 2014. 14(8): p. 949-57