MOLECULAR BASIS OF NEURODEGENERATIVE DISEASES: THE PATHOGENIC G308E VARIANT OF THE APOPTOSIS INDUCING FACTOR (AIF)

The apoptosis inducing factor (AIF) is a highly conserved mitochondrial flavoprotein known to play two opposite roles in eukaryotic cells: in mitochondria it is required for efficient oxidative phosphorylation (OXPHOS), while, when released into the cytoplasm, it triggers caspase-independent apoptosis. The mechanism of AIF-induced cell death was extensively investigated, whereas its mitochondrial role is poorly understood. There are many evidences of the AIF importance for mitochondrial morphology and function. Recently, the discovery of its direct interaction with CHCHD4, a key regulator of the import and assembly of respiratory complex subunits in mitochondria, was reported. A unique feature of AIF, probably pivotal for its vital functions, is the ability to form a tight, air-stable charge-transfer (CT) complex with NAD(H) and to undergo dimerization. Although some aspects of AIF interaction with NAD(H) have been analyzed in the recent years, its precise mechanism is not fully understood. Moreover, over the last five years, six point mutations of the AIFM1 gene, located on the X chromosome, have been associated with severe mitochondriopathies displaying neurodegeneration as a common feature. Most of the resulting mutant AIF variants have been biochemically characterized, except for the G308E replacement, discovered in 2011 and associated with a severe encephalopathy, characterized by OXPHOS defect. Aim of the present PhD project was therefore the thorough biochemical characterization of the apoptogenic form of murine AIF, carrying the equivalent amino acyl substitution (G307E) of the human one to understand how it could alter the AIF properties at the molecular level. To do so, we analysed how the wild type and G307E forms of murine AIF interact with NAD(H) and nicotinamide mononucleotides, NMN(H), finding that the pathogenic replacement resulted in a dramatic and specific decrease of the rate for CT complex formation and consequent protein dimerization only in the case of the physiological ligand. Our results demonstrate that the adenylate moiety of NAD(H) is crucial for the ligand binding process and that the G307E replacement causes an alteration of the adenylate-binding site of AIF that drastically decreases the affinity for and the association rate of the ligand. In addition, we shed new light on the mechanism of the dimerization process, demonstrating that FAD reduction, rather than NAD(H) binding, initiates the conformational rearrangement of AIF that leads to the quaternary structure transition. Taken together, our results contribute to define how AIF works at the molecular level in binding NAD(H) and in undergoing dimerization, and also point out that the pathogenic G308E replacement in human AIF, responsible of a rare neurodegenerative disease, has the selective effect of slowing down the formation of the dimeric CT complex between the flavoprotein and the nicotinamide dinucleotide.