Modulation of the Mitochondrial Iron-Sulfur Cluster Assembly Function by Frataxin-Nanobody Interaction

PIGNATARO MF; FERNANDEZ NB; PAVÁN MF; GARAY A; GROSSI J; NOGUERA ME; VILA A; GENTILI H; RODRIGUEZ N; ARAN M; HERMOSO DOMINGUEZ JA; IBAÑEZ LI; SANTOS J

Congreso; LI Reunión Anual de la Sociedad Argentina de Biofísica; 2023

Friedreich Ataxia (FA) is a neurodegenerative disease caused by reduced levels of FXN (and/or unstable forms of this protein). The primary contributor to cellular dysfunction in FA is the impairment of Iron-sulfur (Fe-S) cluster production. Fe-S clusters are essential cofactors found in all known life forms, with hundreds of proteins reliant on these cofactors for their function. In eukaryotes, the majority of Fe-S cluster biogenesis occurs within the mitochondria and is facilitated by the Iron Sulfur Cluster assembly machinery (ISC). This machinery consists of the NFS1-ISD11-ACP complex, which includes the cysteine desulfurase NFS1—an enzyme responsible for desulfurizing L-Cys to produce LAla, along with a persulfur (R-S-SH), and frataxin (FXN), which is necessary for efficient Fe-S cluster biogenesis. In this work, in order to modulate FXN conformational stability and function, we propose the quaternary addition of humanized llama nanobodies (NB) targeting FXN. We obtained NB libraries and then, several nanobodies showing affinity for FXN were selected using phage display. The interaction was firstly evaluated by SEC-FLPC and NMR. The activation of Cys-desulfurase function by FXN was evaluated in presence ofNBs and a broad range of effects were found. Importantly, nanobodies were able to stabilize a pathological FXN variant (G130V) in vitro. We obtained the dissociation constants (Kd) for some FXN-NB complexes by Biolayer Interferometry (BLI) (Kds between 1-30 nM). Moreover, to better understand the FXN modulation, the structure of the selected NB-FXN complexes was resolved by X-ray diffraction, which allowed us to map the complex on the structure of the decameric supercomplex (NFS1/ACP-ISD11/ISCU/ FXN)2. Importantly, the most promising NB were expressed in HEK 293T cells showing high levels of expression and mitochondrial localization. NB expression did not significantly alter Fe-S cluster dependent enzymatic activities and mitochondria oxygenconsumption rates. We are presently examining NB expression and functional modulation in FRDA cellular models to address the possibility of rescuing FXN function. Additionally, these novel tools will enhance our comprehension of this complex catalytic process.