Apoptosis-Inducing Factor 1, Mitochondrial
Mitochondria play a central role in both cellular redox metabolism and programmed cell death (PCD) induction, since they contain certain agents relevant in these two key cellular functions. Among them is the apoptosis-inducing factor (AIF), initially described as the first caspase-independent apoptotic inducer provoking PCD after liberation from mitochondria (Susin et al. 1999). In healthy cells, AIF is found in the mitochondrial intermembrane space (IMS), where it also has a vital role in the redox metabolism of this organelle that is not well described yet. AIF is released from the IMS to the cytosol in response to apoptotic stimuli and then translocated into the nucleus, where it acts as a proapoptotic factor.
AIFM1 Processing and Localization in the Cell
AIFM1 NADH-Dependent Redox Activity
Main AIFM1 Structural Characteristics
The second NAD(H) molecule, NAD(H)B, binds at the si-face of the flavin, with the side chain of W483 stacking at one side with its nicotinamide ring and at the other with the flavin ring (Fig. 3c) (Ferreira et al. 2014). F582 and particularly W196 are displaced to accommodate the adenine of NAD(H)B through stacking interactions, also contributing to stabilize its pyrophosphate, ribose, and nicotinamide moieties. Comparison of the oxidized and reduced structures shows that binding of NAD(H)B induces conformational changes in the 190–202 β-hairpin and in the 509–560 segment (Fig. 3d), as well as in the molecular dimerization interface (439–453 segment). Noticeably, the 509–560 insertion in mammalian enzymes is a part of the apoptotic C-terminal domain that in the oxidized enzyme shows its 517–533 moiety folded into two short helices which decrease the solvent accessibility to the flavin ring active site. In the complex of the reduced protein with the coenzyme these helices become disordered, and their positions are occupied by NAD(H)B.
To date, six pathological hAIF mutations have been associated with neurological disorders. Five of them (ΔR201, V243L, G262S, G308E, and G338E) cause severe mitochondriopathies associated to reduced expression of respiratory chain complexes and OXPHOS failure, while the sixth mutation (E493V) results in increased cell death via apoptosis causing the Cowchock syndrome but without affecting the OXPHOS activity (Rinaldi et al. 2012; Ardissone et al. 2015; Kettwig et al. 2015; Ghezzi et al. 2010). Mutations E493V and ΔR201 locate in the NAD(H)B site. The E493V mutation probably produces a negative impact in the allocation of NAD(H)B. The variant ΔR201 is structurally unstable and prone to FAD release, facts probably related with R201 stabilizing the two short helices in the oxidized structure (Ghezzi et al. 2010). These two mutations support the functional importance of the folding conservation in the NAD(H)B binding site in both redox states of the protein. The rest of mutations are distributed all over the hAIF structure, but in general contribute to structural elements involved in the allocation of the adenine moiety portions of either NAD(H)A or FAD. Thus, mutations at residues V243 and G262 led to a decreased level of protein expression probably related to a defective folding (Ardissone et al. 2015; Kettwig et al. 2015) or FAD incorporation, while those at G308 and G338 were critical for NAD(H)A stabilization. Structural alterations in ligand binding sites in the pathogenic mutations highlight the importance of the proper folding of the protein as well as the correct cofactor and coenzyme binding for its suitable functioning.
AIFM1 has been hypothesized to act as a sensor of the NADH/NAD+ intracellular levels, with coenzyme binding and flavin reduction modulating its monomer-dimer equilibrium and the interaction with other molecules that could be determining its biological activity. So far, it has been described in the interaction of AIF with a variety of proteins in the different cellular compartments where it can be located. Nevertheless, other partners might still be unknown, and the exact role of AIF in mitochondria remains a conundrum. In mitochondria the interaction of the AIFred−NAD+ complex with CHCHD4, a protein that participates in the import of several mitochondrial proteins and their oxidative folding by disulfide bond formation, has linked AIF to the biogenesis of respiratory chain complexes (Hangen et al. 2015; Lui and Kong 2007). The interaction of AIFΔ1–101 with Hsp70 in the cytosol induces cellular survival (Lui and Kong 2007), while its interaction with cyclophilin A (CypA) in the same compartment promotes the optimal nuclear translocation of both proteins to induce PCD (Cande et al. 2004). Once in the nucleus, AIF forms a degradosome with CypA and the phosphorylated histone H2AX that provokes chromatin condensation and DNA degradation (Artus et al. 2010; Cande et al. 2004). In addition, the cytosolic and nuclear interactions of AIF with reduced thioredoxin 1 (Trx1) are proposed to provide a mechanism to attenuate the AIF lethal action (Shelar et al. 2015). Under proapoptotic oxidative stress conditions, Trx1 becomes oxidized in the cytosol and the Trx1-AIF complex dissociates, which likely promote nuclear translocation of AIF. In the nucleus, the complexation of AIF with reduced Trx1 would hinder its interaction with DNA and, as a consequence, apoptosis (Shelar et al. 2015).
AIFM1 is a ubiquitously distributed protein in mammals that regulates caspase-independent programmed cell death, but it is also a key factor in the biogenesis of the respiratory complexes through its NADH-oxidoreductase activity. In each particular cell the main AIFM1 function is determined by its subcellular localization, with the protein in the mitochondrial intermembrane space in healthy cells being translocated to the nucleus upon apoptotic stimuli. In addition, current research has provided compelling evidences supporting the critical role of the AIFM1 redox state and monomer-dimer equilibrium in both its apoptotic and respiratory chain maintenance functions. At the molecular level AIFM1 folds in three domains, two of them related with its oxidoreductase activity and the C-terminal with its apoptotic function. Upon reduction by NADH it forms a FADH−-NAD+ CTC highly stable versus oxygen reoxidation and dimerizes, being in addition able to allocate two coenzyme molecules, one in the oxidoreductase portion and the other one in the apoptotic domain, whose nicotinamide portions get placed in the flavin ring environment. In addition, AIFM1 is able to interact with several protein partners, being such interactions modulated by the enzyme subcellular localization, redox state, and oligomeric state. Further elucidations of the mechanisms of processes involving AIFM1 are at this point required, since they will surely provide a better understanding of the molecular mechanisms underlying the diseases in which it is involved and will help to develop novel pharmacological therapies.
We thank Luis Martinez Lostao (University of Zaragoza) for helping us with the graphical illustration in Figure 1.
- Sorrentino L, Calogero AM, Pandini V, Vanoni MA, Sevrioukova IF, Aliverti A. Key role of the adenylate moiety and integrity of the adenylate-binding site for the NAD(+)/H binding to mitochondrial apoptosis-inducing factor. Biochemistry. 2015;54:6996–7009. doi: 10.1021/acs.biochem.5b00898.CrossRefPubMedGoogle Scholar