Apoptosis-inducing factor

Although the seminal paper by Kerr, Wyllie and Currie (Kerr et al., 1972), who coined the word ‘apoptosis’, stated that only nuclei but not cytoplasmic organelles would undergo major modifications in the dying cell, recent evidence suggests that no cellular compartment is spared (Ferri and Kroemer, 2001). Mitochondria are particularly affected early during the apoptotic process, and they are now thought to act as central regulators of cell death. Indeed, several pro-apoptotic signal transduction and damage pathways converge on mitochondria to induce mitochondrial membrane permeabilization (MMP), and this phenomenon is under the control of Bcl-2-related proteins (Kroemer and Reed, 2000). MMP can occur as a result of a primary caspase activation but it often results from caspase-independent pathways (Green and Kroemer, 1998). Moreover, MMP leads to the activation of caspases and caspase-independent death effectors, mainly through the release of soluble intermembrane proteins that are normally secured behind the outer mitochondrial membrane. One caspase-activating intermembrane protein is cytochrome c (Liu et al., 1996), which triggers the proteolytic maturation of caspases within the apoptosome, the caspase activation complex including Apaf-1, caspase-9 and caspase-3 (Budijardjo et al., 1999). Other caspase activators include Hsp10 (which stimulates the apoptosome), Smac/DIABLO and HtrA2 (which both inhibit caspase-inhibitory IAP proteins). Caspase-independent effects can be attributed to ‘apoptosisinducing factor’ (AIF) (Susin et al., 1999), endonuclease G, as well as HtrA2, which also posesses a serine protease activity (Ravagnan et al., 2002). Here, we summarize recent progress on the structure and function of AIF. AIF: a phylogenetically old flavoprotein with a glutathione reductase-like fold The mammalian AIF precursor contains an N-terminal mitochondrial localization sequence (MLS, residues 1-100) and a large C-terminal part (121-610) that shares similarity with bacterial oxidoreductases (Susin et al., 1999). AIF homologs are also found in invertebrates, including insects, nematodes, fungi, and plants, meaning that the AIF gene has been conserved throughout the eukaryotic kingdom (Fig. 1). In humans, one AIF homolog (AMID/PRG3), which is distantly related to AIF (Fig. 1), has been recently described to exert a pro-apoptotic function (Ohiro et al., 2002; Wu et al., 2002). According to conflicting reports, AMID/PRG3 may either be associated with the outer mitochondrial membrane (Wu et al., 2002) or localized in the non-mitochondrial cytoplasma (Ohiro et al., 2002). The mature form of AIF (57 kDa) is generated by cleaving of the MLS, after import into the mitochondrial intermembrane space. Because it can stably bind FAD, AIF falls in the category of flavoproteins. AIF displays NAD(P)H oxidase as well as monodehydroascorbate reductase activities (Miramar et al., 2001). The overall crystal structure of mature mouse AIF has been recently resolved at 2.0 Å resolution. AIF displays a glutathione-reductase-like fold, with an FAD-binding domain (aa 122-262 and 400-477), an NADH-binding domain (263399), and a C-terminal domain (478-610) that bears a small AIF-specific insertion (509-559) not found in glutathione reductase (Fig. 2a). The amino acids interacting with FAD and NADH have been mapped precisely, and the mutants E313A and K176A have been shown to reduce FAD binding (Mate et al., 2002). Human mature AIF (which is 92% identical to 4727