Drosophila Omi, a mitochondrial-localized IAP antagonist and proapoptotic serine protease.
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.
Project description:Mitochondrial AAA (ATPases Associated with diverse cellular Activities) proteases i-AAA (intermembrane space-AAA) and m-AAA (matrix-AAA) are closely related and have major roles in inner membrane protein homeostasis. Mutations of m-AAA proteases are associated with neuromuscular disorders in humans. However, the role of i-AAA in metazoans is poorly understood. We generated a deletion affecting Drosophila i-AAA, dYME1L (dYME1L(del)). Mutant flies exhibited premature aging, progressive locomotor deficiency and neurodegeneration that resemble some key features of m-AAA diseases. dYME1L(del) flies displayed elevated mitochondrial unfolded protein stress and irregular cristae. Aged dYME1L(del) flies had reduced complex I (NADH/ubiquinone oxidoreductase) activity, increased level of reactive oxygen species (ROS), severely disorganized mitochondrial membranes and increased apoptosis. Furthermore, inhibiting apoptosis by targeting dOmi (Drosophila Htra2/Omi) or DIAP1, or reducing ROS accumulation suppressed retinal degeneration. Our results suggest that i-AAA is essential for removing unfolded proteins and maintaining mitochondrial membrane architecture. Loss of i-AAA leads to the accumulation of oxidative damage and progressive deterioration of membrane integrity, which might contribute to apoptosis upon the release of proapoptotic molecules such as dOmi. Containing ROS level could be a potential strategy to manage mitochondrial AAA protease deficiency.
Project description:Mitochondrial proteins such as cytochrome c, Smac/DIABLO and Omi/HtrA2 play important roles in the cell death pathways of mammalian cells. In Drosophila, the role of mitochondria in cell death is less clear. Here, we report the identification and characterization of the Drosophila ortholog of human Omi/HtrA2. We show that Drosophila Omi/HtrA2 is imported into the mitochondria where it undergoes proteolytic maturation to yield two isoforms, dOmi-L and dOmi-S. dOmi-L contains a canonical N-terminal IAP-binding motif (AVVS), whereas dOmi-S contains a distinct N-terminal motif (SKMT). DIAP1 was able to bind to both isoforms via its BIR1 and BIR2 domains. This resulted in cleavage of the linker region of DIAP1 between the BIR1 and BIR2 domains and further degradation of the BIR1 domain by the proteolytic activity of dOmi. The binding of DIAP1 to dOmi also resulted in DIAP1-mediated polyubiquitination of dOmi, suggesting that DIAP1 could target dOmi for proteasomal degradation. Consistent with this, expression of DIAP1 in Drosophila eye discs protected them from dOmi-induced eye ablation, indicating that DIAP1 plays an important role in protecting cells from the potentially lethal effects of dOmi. The ability of IAPs to bind to and ubiquitinate mitochondrial proteins such as dOmi may be a key conserved function to counterbalance the lethal effects of these proteins if accidentally released into the cytosol.
Project description:Drosophila inhibitor of apoptosis (IAP) 1 (DIAP1) is an E3 ubiquitin ligase that regulates apoptosis in flies, in large part through direct inhibition and/or ubiquitinylation of caspases. IAP antagonists, such as Reaper, Hid, and Grim, are thought to induce cell death by displacing active caspases from baculovirus IAP repeat domains in DIAP1, but can themselves become targets of DIAP1-mediated ubiquitinylation. Herein, we demonstrate that Grim self-associates in cells and is ubiquitinylated by DIAP1 at Lys136 in an UbcD1-dependent manner, resulting in its rapid turnover. K48-linked ubiquitin chains are added almost exclusively to BIR2-bound Grim as a result of its structural proximity to DIAP1's RING domain. However, active caspases can simultaneously cleave Grim at Asp132, removing the lysine necessary for ubiquitinylation as well as any existing ubiquitin conjugates. Cleavage therefore enhances the stability of Grim and initiates a feed-forward caspase amplification loop, resulting in greater cell death. In summary, Grim is a caspase substrate whose cleavage promotes apoptosis by limiting, in a target-specific fashion, its ubiquitinylation and turnover by the proteasome.
Project description:Ubiquitin-mediated inactivation of caspases has long been postulated to contribute to the regulation of apoptosis. However, detailed mechanisms and functional consequences of caspase ubiquitylation have not been demonstrated. Here we show that the Drosophila Inhibitor of Apoptosis 1, DIAP1, blocks effector caspases by targeting them for polyubiquitylation and nonproteasomal inactivation. We demonstrate that the conjugation of ubiquitin to drICE suppresses its catalytic potential in cleaving caspase substrates. Our data suggest that ubiquitin conjugation sterically interferes with substrate entry and reduces the caspase's proteolytic velocity. Disruption of drICE ubiquitylation, either by mutation of DIAP1's E3 activity or drICE's ubiquitin-acceptor lysines, abrogates DIAP1's ability to neutralize drICE and suppress apoptosis in vivo. We also show that DIAP1 rests in an "inactive" conformation that requires caspase-mediated cleavage to subsequently ubiquitylate caspases. Taken together, our findings demonstrate that effector caspases regulate their own inhibition through a negative feedback mechanism involving DIAP1 "activation" and nondegradative polyubiquitylation.
Project description:Omi/HtrA2 is a mitochondrial serine protease that is released into the cytosol during apoptosis to antagonize inhibitors of apoptosis (IAPs) and contribute to caspase-independent cell death. Here, we demonstrate that Omi/HtrA2 directly cleaves various IAPs in vitro, and the cleavage efficiency is determined by its IAP-binding motif, AVPS. Cleavage of IAPs such as c-IAP1 substantially reduces its ability to inhibit and ubiquitylate caspases. In contrast to the stoichiometric anti-IAP activity by Smac/DIABLO, Omi/HtrA2 cleavage of c-IAP1 is catalytic and irreversible, thereby more efficiently inactivating IAPs and promoting caspase activity. Elimination of endogenous Omi by RNA interference abolishes c-IAP1 cleavage and desensitizes cells to apoptosis induced by TRAIL. In addition, overexpression of cleavage-site mutant c-IAP1 makes cells more resistant to TRAIL-induced caspase activation. This IAP cleavage by Omi is independent of caspase. Taken together, these results indicate that unlike Smac/DIABLO, Omi/HtrA2's catalytic cleavage of IAPs is a key mechanism for it to irreversibly inactivate IAPs and promote apoptosis.
Project description:While apoptosis regulation has been studied extensively in Drosophila melanogaster, similar studies in other insects, including disease vectors, lag far behind. In D. melanogaster, the inhibitor of apoptosis (IAP) protein DIAP1 is the major negative regulator of caspases, while IAP antagonists induce apoptosis, in part, by binding to DIAP1 and inhibiting its ability to regulate caspases. In this study, we characterized the roles of two IAP antagonists, Michelob_x (Mx) and IMP, in apoptosis in the yellow fever mosquito Aedes aegypti. Overexpression of Mx or IMP caused apoptosis in A. aegypti Aag2 cells, while silencing expression of mx or imp attenuated apoptosis. Addition of recombinant Mx or IMP, but not cytochrome c, to Aag2 cytosolic extract caused caspase activation. Consistent with this finding, AeIAP1 bound and inhibited both initiator and effector caspases from A. aegypti, and Mx and IMP competed with caspases for binding to AeIAP1. However, a difference was observed in the BIR domains responsible for Dronc binding by AeIAP1 versus DIAP1. These findings demonstrate that the mechanisms by which IAP antagonists regulate apoptosis are largely conserved between A. aegypti and D. melanogaster, although subtle differences exist.
Project description:The proapoptotic factors Reaper, Hid, Grim, and Sickle regulate apoptosis in Drosophila by inhibiting the antiapoptotic factor DIAP1 (Drosophila inhibitor of apoptosis 1). Heat, UV light, x-rays, and developmental signals can all increase the proapoptotic factors, but the control of transcription of the diap1 gene is unclear. We show that in imaginal discs the single Drosophila STAT protein (STAT92E) when activated can directly increase DIAP1 through binding to STAT DNA-binding sites in the diap1 promoter. The STAT92E contribution to DIAP1 production is required for cell survival after x-irradiation but not under unstressed conditions. Because DIAP1 prevents apoptosis after a variety of stresses, STAT92E may have a role in regulating stress responses in general.
Project description:Apart from their roles as chaperones, heat shock proteins are involved in other vital activities including apoptosis with mammalian Hsp60 being ascribed proapoptotic as well as antiapoptotic roles. Using conditional RNAi or overexpression of Hsp60D, a member of the Hsp60 family in Drosophila melanogaster, we show that the downregulation of this protein blocks caspase-dependent induced apoptosis. GMR-Gal4-driven RNAi for Hsp60D in developing eyes dominantly suppressed cell death caused by expression of Reaper, Hid, or Grim (RHG), the key activators of canonical cell death pathway. Likewise, Hsp60D-RNAi rescued cell death induced by GMR-Gal4-directed expression of full-length and activated DRONC. Overexpression of Hsp60D enhanced cell death induced either by directed expression of RHG or DRONC. However, the downregulation of Hsp60D failed to suppress apoptosis caused by unguarded caspases in DIAP1-RNAi flies. Furthermore, in DIAP1-RNAi background, Hsp60D-RNAi also failed to inhibit apoptosis induced by RHG expression. The Hsp60 and DIAP1 show diffuse and distinct granular overlapping distributions in the photoreceptor cells with the bulk of both proteins being outside the mitochondria. Depletion of either of these proteins disrupts the granular distribution of the other. We suggest that in the absence of Hsp60D, DIAP1 is unable to dissociate from effecter and executioner caspases, which thus remain inactive.
Project description:The release of mitochondrial intermembrane space proteins to the cytosol is a key event during apoptosis. We used in situ fluorescent labeling of proteins tagged with a short tetracysteine-containing sequence to follow the release of Smac, Omi, adenylate kinase-2, cytochrome c, and apoptosis-inducing factor (AIF) during apoptosis and compared the release with that of cytochrome c tagged with GFP in individual cells observed over time. We observed a caspase-independent, simultaneous release of cytochrome c, Smac, Omi, and adenylate kinase-2. Although AIF release also was caspase-independent and commenced with that of the other proteins, it proceeded much more slowly and incompletely from mitochondria, perhaps because of a requirement for a secondary event. These results suggest that these proteins are released through the same mitochondrial pore and that apoptosis may not be regulated through a selective release of individual mitochondrial proteins. The timing and extent of AIF release makes it unlikely that it is involved in the induction of apoptosis, either upstream or downstream of mitochondrial outer membrane permeabilization.
Project description:Proapoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define proapoptotic BCL-2 protein composition after stress and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for proapoptotic BCL-2 family function. Cells with hyperfragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAX?9·membrane interactions. This work identifies a mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis.