Augmenter of Liver Regeneration: Substrate Specificity of a Flavin-dependent Oxidoreductase from the Mitochondrial Intermembrane Space

Hepatic regenerative stimulator substance (1), now termed hepatopoietin (2) or augmenter of liver regeneration (ALR1 (3)), was first identified as a protein that stimulated hepatocyte proliferation and liver regeneration (1–4). ALR is believed to activate the MAP kinase pathway (5, 6), inhibits natural killer cells (7) and influence the regeneration of Drosophila imaginal discs (8). The sequence of this growth factor showed homology with two yeast proteins, Erv1p and Erv2p (3, 9, 10). All three proteins were found to be FAD-linked sulfhydryl oxidases (11–14) and the subsequent crystal structures of Erv2p (15) and ALR (16) identified a new flavin-binding fold with the isoalloxazine ring inserted into the mouth of a 4-helix bundle. A depiction of dimeric ALR is shown in Figure 1A. Mammalian ALR is found in two main alternatively spliced forms (2, 6, 12, 17–20). The short form (sfALR, 15 kDa) is an extracellular cytokine and also participates in intracellular redox-dependent signaling pathways (1–4, 6, 18). The long form of the oxidase (lfALR, 23 kDa) exists predominantly in the mitochondrial intermembrane space (IMS) and contains an 80-amino acid N-terminal extension housing a second CxxC motif. The presence of additional redox-active disulfides in N- or C-terminal extensions from the core flavin binding domain is a common feature of the Erv/ALR family (21–23) where they may mediate the transfer of reducing equivalents between thiol substrate and flavin prosthetic group (see later). Fig 1B shows that these distal disulfides are placed within an N-terminal extension of lfALR and its yeast ortholog Erv1p. These regions show little sequence conservation and, in particular, the CxxC motif of lfALR is some 30 residues closer to the Erv/ALR flavin binding domain than the corresponding distal disulfide in Erv1p. FIGURE 1 The short form of the human ALR dimer and a comparison of the sequence of full length human ALR and of yeast Erv1p. Panel A: The homology model of the homodimer is based on the structure of the short form of rat ALR (16, 36). In this stereoview, the sulfur ... lfALR and yeast Erv1p play key roles in the generation of disulfide bonds in the mitochondrial intermembrane space (IMS) as depicted for Erv1p in Figure 2. Studies using yeast have suggested that oxidized Mia40 exchanges disulfides with a variety of proteins undergoing oxidative folding in the IMS: for example proteins with twin Cx3C motifs (the Tim 8, 9, 10, 12, and 13 proteins), twin Cx9C motifs (Cox17, Cox19, Mic14, Mic17, and Mdm35), the copper chaperone Ccs1, and Erv1p, itself (24–35). Reduced Mia40, so generated, is then the immediate substrate of Erv1p (Figure 2). FIGURE 2 Schematic representation of the pathway for generation of disulfide bonds in the yeast mitochondrial IMS. An unfolded protein transits the outer membrane through the TOM complex and is trapped by disulfide bond generation mediated by Mia40. Regeneration ... In contrast to the yeast Erv1p/Mia40 system, rather little is known concerning possible interaction between human ALR and potential thiol substrates in the IMS. Indeed, only sfALR has been subject to a detailed enzymological investigation (12, 36). This small dimeric flavoprotein (Figure 1A) is a very weak stand-alone sulfhydryl oxidase for reduced lysozyme (12). Dithiothreitol was later used as a convenient model substrate and showed kcat values of 50–75/min with a range of constructs of sfALR (36). In terms of the physiological oxidant of ALR, Farrell and Thorpe found that cytochrome c was a much better oxidant than molecular oxygen in vitro (36). They suggested that channeling reducing equivalents via the respiratory chain would avoid the generation of hydrogen peroxide associated with disulfide bond generation in the IMS (36). Support for this suggestion has subsequently come from in vitro and in vivo studies with yeast Erv1p (35, 37). Recently, Koehler and colleagues have suggested an alternate pathway involving cytochrome c peroxidase-mediated oxidation of cytochrome c driven by the hydrogen peroxide generated by Erv1p (38). The present work presents the first enzymological comparison of the long and short forms of human ALR. Reductive titrations and mutagenesis are used to explore the mechanistic consequences of the inclusion of an additional distal CxxC motif within the 80-residue N-terminal extension. While the properties of the full-length Erv1p (13, 39) are one benchmark for our continuing studies on ALR, their distal CxxC motifs occupy different locales in divergent N-terminal regions (see earlier, Figure 1B). We further extend our studies to include the presumed physiological substrate of human lfALR, Mia40. This work suggests unanticipated new roles for Mia40 and reinforces how much more we need to know concerning oxidative protein folding and metal trafficking systems in the mitochondrial IMS.