Modulation of the activity of the iron-sulfur cluster assembly mitochondrial supercomplex through affitin interaction

HERRERA, MARÍA GEORGINA; ARAN, MARTÍN; CASTRO, IGNACIO HUGO; NOGUERA MARTÍN EZEQUIEL; PIGNATARO, MARÍA FLORENCIA; SEWELL, KARL ELLIOTH; SANTOS, JAVIER

Congreso; Primeras Jornadas Virtuales SAB 2020; 2020

The development of molecules that can specifically bind to protein targets plays a majorrole not only in therapeutics and diagnostics but also in basic and applied research. Inthis work, we selected human frataxin (FXN) high affinity binders. We employed Sac7dvariants (affitins) and Ribosome Display technology for the in-vitro selection process. Adecrease in the expression of FXN, the kinetic activator of (NFS1/ACP-ISD11/ISCU/FXN)2Fe-S cluster supercomplex in mitochondria, leads to Friedreich?s ataxia (FRDA), a rareinherited disease that causes progressive cardiac and nervous system damage. After 4-5rounds of selection, we obtained a set of six different binders (affitins 10, 12, 37, 60, 186and 224). Affitins were expressed in E. coli, purified and characterized by UV-Vis and CDSpectroscopy. Also, some of them (37, 186 and 224) showed a marked tendency to formdimers (as judged by SEC-FPLC). To further test affitin-FXN interaction, we performedELISA experiments with different FXN plate orientation and also soluble FXN competitionexperiments. As a whole, affitin 37 and 224 were the most promising ones in terms ofthe binding affinity constant obtained by SPR (Surface Plasmon Resonance) experiments.Additionally, by the analysis of chemical shift perturbations of 15N FXN we were able tomap the binding site of Affitin 224 on the region of the acidic ridge of FXN. Furthermore,we investigated the effect of affitin addition on the desulfurase activity in the human(NFS1-ACP-ISD11-ISCU-FXN)2 supercomplex. In this sense, to prove affitin interaction inthe context of the supercomplex, we labeled FXNS202C variant and also affitin 224 withmaleimide Texas Red to perform anisotropy experiments. Importantly, to determine ifthese affitins will interact with frataxin in the mitochondria environment, we propose twodifferent strategies: a) to produce a Trojan variant of Affitin 224 to deliver the affitin tothe mitochondrial matrix and b) to transfect the affitin 224 gene (with a mitochondrialsignal) to HEK 293T cells or FRDA fibroblasts cell lines.