Journal Article

Nanobodies as tools for studying human frataxin biology

Garay-Alvarez, Alba, Parreño, Viviana, Bok, Marina, Noguera, Martín, Molina, Rafael, Hermoso, Juan A., Santos, Javier, Pavan, María Florencia, Ibañez, Lorena Itatí, Fernández, Natalia Brenda, Aran, Martín, Rodríguez, Naira Antonia, Gentili, Hernán G., Pignataro, María Florencia, Muñoz, Inés G., Vila, Antonella, García, Augusto E., Grossi, Julián

2026

Overview

Iron-sulfur clusters are essential cofactors for the accurate cellular function of many proteins. In eukaryotic cells, the biogenesis of most iron-sulfur clusters occurs in the mitochondria and involves the action of the Cys desulfurase supercomplex, which is activated by the protein frataxin (FXN). The decrease of FXN expression and/or function results in Friedreich's ataxia (FRDA).In this work, several nanobodies specific to human FXN were selected via phage display, demonstrating a wide range of effects on Cys desulfurase activity and a strong interaction with FXN. Nanobody interaction stabilized wild-type and FRDA-related FXN variants in vitro. FXN-nanobody complexes were characterized by NMR, SAXS, and X-ray crystallography. Additionally, Nanobody expression was studied in human cells. The subcellular localization, direct interaction with FXN by in situ proximity ligation assay, effect on cell viability, Fe-S-dependent enzymatic activities, and oxygen consumption rates were analyzed. Significantly, nanobody expression did not alter these key metabolic variables, suggesting that the interaction with FXN did not disrupt the pathway.As a whole, our results suggest that nanobodies can serve as binding partners for mitochondrial FXN. However, the specific effect of the nanobodies on the conformational stability of FRDA-related FXN variants in cells should be investigated.