THE DOUBLE LIFE OF THE APOPTOSIS INDUCING FACTOR (AIF): THE PRO-VITAL ROLE OF A PRO-DEATH PROTEIN

Beside its apoptotic function, the Apoptosis Inducing Factor (AIF), a highly conserved mitochondrial flavoprotein, plays a pro-vital role in eukaryotic cells through its interaction with CHCHD4, a mitochondrial protein that contributes to oxidative folding of respiratory complexes’ subunits. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer complex (CT complex) upon reaction with NAD(H), which leads to protein dimerization modulating the affinity for its ligands. To date, nine point mutations of the human AIF gene were found to cause rare and severe neurodegenerative mitochondriopathies. To define the molecular bases of the pathogenicity of AIF variants, we selected a set of AIF mutations (G337E, D236G, G261S and F133L) and investigated their effects on both AIF molecular properties and its interaction with CHCHD4. To this aim, a combination of biophysical techniques, Microscale Thermophoresis (MST) and structural biology was used. AIF variants CT complex stability was evaluated studying its reoxidation by O2. Interestingly, CT complex of G337E and G261S forms displayed a faster oxidation compared to wild type AIF, indicating a lower stability. Moreover, the 3D structures of the CT complex of AIF-D236G and of AIF-F133L both in oxidized and CT complex state were obtained, showing, however, no significant structural rearrangements with respect to the wild type protein. Hence, we investigated possible alterations of the interaction with CHCHD4 induced by AIF pathogenic mutations. Through a MST approach, for the first time we quantitatively studied redox-dependent effects of AIF amino acid replacements on its affinity for CHCHD4, revealing a possible involvement of the G337 residue in protein-protein complex formation. We characterized the AIF-CHCHD4 complex also from a structural point of view using the Small-Angle X-ray Scattering (SAXS) technique, through which we identified a putative interaction region between the two proteins. In addition, all SAXS models obtained revealed that the solvent-exposed G337 residue is localized in the neighborhood of the identified region, in agreement with a possible role of this residue in complex formation. Our results, not only shed new light on AIF-CHCHD4 relationship, but also provide a possible explanation of the pathogenicity of the AIF G337E allelic variant.