ABSTRACT
Although essential in mammals, in flies the importance of mitochondrial outer membrane permeabilization for apoptosis remains highly controversial. Herein, we demonstrate that Drosophila Omi (dOmi), a fly homologue of the serine protease Omi/HtrA2, is a developmentally regulated mitochondrial intermembrane space protein that undergoes processive cleavage, in situ, to generate two distinct inhibitor of apoptosis (IAP) binding motifs. Depending upon the proapoptotic stimulus, mature dOmi is then differentially released into the cytosol, where it binds selectively to the baculovirus IAP repeat 2 (BIR2) domain in Drosophila IAP1 (DIAP1) and displaces the initiator caspase DRONC. This interaction alone, however, is insufficient to promote apoptosis, as dOmi fails to displace the effector caspase DrICE from the BIR1 domain in DIAP1. Rather, dOmi alleviates DIAP1 inhibition of all caspases by proteolytically degrading DIAP1 and induces apoptosis both in cultured cells and in the developing fly eye. In summary, we demonstrate for the first time in flies that mitochondrial permeabilization not only occurs during apoptosis but also results in the release of a bona fide proapoptotic protein.
Subject(s)
Apoptosis , Drosophila Proteins/antagonists & inhibitors , Drosophila melanogaster/cytology , Drosophila melanogaster/enzymology , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Mitochondria/enzymology , Mitochondrial Proteins/metabolism , Serine Endopeptidases/metabolism , Amino Acid Sequence , Animals , Binding Sites , Caspase Inhibitors , Caspases/metabolism , Cell Line , Conserved Sequence , Crystallography, X-Ray , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Gene Expression Regulation, Developmental , High-Temperature Requirement A Serine Peptidase 2 , Humans , Inhibitor of Apoptosis Proteins/chemistry , Inhibitor of Apoptosis Proteins/genetics , Mitochondria/metabolism , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Models, Molecular , Molecular Sequence Data , Protein Structure, Quaternary , Protein Structure, Tertiary , Protein Transport , Sequence Alignment , Serine Endopeptidases/chemistry , Serine Endopeptidases/geneticsABSTRACT
Teleost fish show a remarkable capability of nerve regeneration in their CNS, while injuries to axon fibers in the CNS of mammals result in degeneration and loss of function. Understanding this difference has biomedical consequences to humans. Both extrinsic factors from the neuronal environment and intrinsic neuronal factors seem to play a role in successful nerve regeneration. Among the intrinsic factors, a number of proteins termed axonal growth associated proteins (GAPs) are strongly induced during axon regeneration. RICH proteins are axonal GAPs that show homology to mammalian myelin marker proteins termed CNPases. Sequence analysis distinguishes three domains in these proteins. In this report, mutant versions of zebrafish RICH proteins were generated to study the roles of the domains of the protein at biochemical and cellular levels. The central CNPase homology domain was sufficient for catalytic activity. The amino terminal acidic domain causes the anomalous electrophoretic migration observed for RICH proteins. The small C-terminal domain bears an isoprenylation motif and is necessary for the interaction of zRICH with cellular membranes. At the cellular level, expression of wild-type zRICH protein in PC12 cells did not induce neurite generation. Additionally, neither the expression of wild-type zRICH nor the expression of mutant versions of the protein interfered with the levels of differentiation of PC12 cells induced by nerve growth factor, suggesting that, at least in this model of neuronal differentiation, zRICH proteins do not participate in the process of generation of neurites.
Subject(s)
Nerve Growth Factors/genetics , Nerve Regeneration/physiology , Optic Nerve/physiology , Zebrafish Proteins/genetics , Animals , Blotting, Western , Catalysis , Cell Differentiation/genetics , Cells, Cultured , Cytosol/metabolism , Electrophoresis, Polyacrylamide Gel , Humans , Immunohistochemistry , Kinetics , Mutation/physiology , Nerve Growth Factors/physiology , PC12 Cells , Plasmids/genetics , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Transfection , Zebrafish , Zebrafish Proteins/physiologyABSTRACT
X-linked inhibitor of apoptosis (XIAP), known primarily for its caspase inhibitory properties, has recently been shown to interact with and regulate the levels of COMMD1, a protein associated with a form of canine copper toxicosis. Here, we describe a role for XIAP in copper metabolism. We find that XIAP levels are greatly reduced by intracellular copper accumulation in Wilson's disease and other copper toxicosis disorders and in cells cultured under high copper conditions. Elevated copper levels result in a profound, reversible conformational change in XIAP due to the direct binding of copper to XIAP, which accelerates its degradation and significantly decreases its ability to inhibit caspase-3. This results in a lowering of the apoptotic threshold, sensitizing the cell to apoptosis. These data provide an unsuspected link between copper homeostasis and the regulation of cell death through XIAP and may contribute to the pathophysiology of copper toxicosis disorders.
