2. Modulation of the Cys Desulfurase Supercomplex. Function by Nanobody-Frataxin Interaction. simposio

JAVIER SANTOS

Córdoba

Conferencia; SAB; 2023

SAB

Modulation of the Cys Desulfurase Supercomplex Function by Nanobody-Frataxin InteractionJavier SantosInstituto de Biociencias, Biotecnología y Biología Traslacional (iB3). DFBMC, DQB, Facultad de Ciencias Exactas y Naturales (FCEN), Universidad de Buenos Aires, Buenos Aires, Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Godoy Cruz 2290 (C1425FQB) CABA – República Argentina.ABSTRACTIron-sulfur (Fe-S) clusters are essential cellular cofactors. Hundreds of proteins require such cofactors to work. In eukaryotic cells, the biogenesis of most Fe-S clusters occurs in the mitochondria. The process involves the Cys desulfurase supercomplex which is activated by frataxin. We propose to modulate the frataxin stability and the supercomplex function by means of nanobody-frataxin interaction. We selected several frataxin-specific nanobodies by phage display. Nanobodies were expressed in E. coli and purified. The interaction with frataxin was characterized by size exclusion chromatography, interferometry, and NMR. We found a strong interaction with slow dissociation equilibrium and high affinity (KD= 1-30nM). Furthermore, nanobody interaction yielded in vitro stabilization of the FRDA frataxin variant G130V, characterized by an increase in Tm of 15 C compared to G130V variant alone, a value even higher than that of the wild type frataxin. Moreover, the 3D structure of the nanobody-frataxin complexes was resolved by X-ray diffraction. This allowed us to map the nanobody-frataxin on the structure of the desulfurase supercomplex. The modulation of the frataxin function was study in vitro by an Cys desulfurase activation assay. Interestingly, the presence of nanobodies produced a broad range of effects on enzymatic activity. To evaluate the biological effects of nanobody interactions in the cellular environment, we first expressed the nanobodies in human cells (HEK-293T, HeLa Kyoto). Nanobody expression, subcellular localization, cell viability, Fe-S cluster dependent enzymatic activities, and oxygen consumption rates were analyzed. Nanobody expression did not significantly alter these metabolic variables in HEK-293T cells, suggesting that the interaction with frataxin did not perturb the interaction of the latter with the Cys desulfurase supercomplex in the mitochondria. Currently, nanobody expression, function modulation, and function dissection are being investigated in FRDA cellular models.