Mitochondrial disruption in Drosophila apoptosis

Mitochondrial disruption is a conserved aspect of apoptosis, seen in many species from mammals to nematodes. Despite significant conservation of other elements of the apoptotic pathway in Drosophila, a broad role for mitochondrial changes in apoptosis in flies remains unconfirmed. Here, we show that Drosophila mitochondria become permeable in response to the expression of Reaper and Hid, endogenous regulators of developmental apoptosis. Caspase activation in the absence of Reaper and Hid is not sufficient to permeabilize mitochondria, but caspases play a role in Reaper- and Hid-induced mitochondrial changes. Reaper and Hid rapidly localize to mitochondria, resulting in changes in mitochondrial ultrastructure. The dynamin-related protein, Drp1, is important for Reaper- and DNA-damage-induced mitochondrial disruption. Significantly, we show that inhibition of Reaper or Hid mitochondrial localization or inhibition of Drp1 significantly inhibits apoptosis, indicating a role for mitochondrial disruption in fly apoptosis.     

Reaper-induced dissociation of a Scythe-sequestered cytochrome c-releasing activity.

Reaper is a potent apoptotic inducer critical for programmed cell death in the fly Drosophila melanogaster. While Reaper homologs from other species have not yet been reported, ectopic expression of Reaper in cells of vertebrate origin can also trigger apoptosis, suggesting that Reaper-responsive pathways are likely to be conserved. We recently reported that Reaper-induced mitochondrial cytochrome c release and caspase activation in a cell-free extract of Xenopus eggs requires the presence of a 150 kDa Reaper-binding protein, Scythe. We now show that Reaper binding to Scythe causes Scythe to release a sequestered apoptotic inducer. Upon release, the Scythe-sequestered factor(s) is sufficient to induce cytochrome c release from purified mitochondria. Moreover, addition of excess Scythe to egg extracts impedes Reaper-induced apoptosis, most likely through rebinding of the released factors. In addition to Reaper, Scythe binds two other Drosophila apoptotic regulators: Grim and Hid. Surprisingly, however, the region of Reaper which is detectably homologous to Grim and Hid is dispensable for Scythe binding.     

The mitochondrial ARTS protein promotes apoptosis through targeting XIAP

ARTS is an unusual septin-like mitochondrial protein that was originally shown to mediate TGF-beta-induced apoptosis. Recently, we found that ARTS is also important for cell killing by other pro-apoptotic factors, such as arabinoside, etoposide, staurosporine and Fas. In Drosophila, the IAP antagonists Reaper, Hid and Grim are essential for the induction of virtually all apoptotic cell death. We found that mutations in peanut, which encodes a Drosophila homologue of ARTS, can dominantly suppress cell killing by Reaper, Hid and Grim, indicating that peanut acts downstream or in parallel to these. In mammalian cells, ARTS is released from mitochondria upon pro-apoptotic stimuli and then binds to XIAP. Binding of ARTS to XIAP is direct, as recombinant ARTS and XIAP proteins can bind to each other in vitro. ARTS binding to XIAP is specific and related to its pro-apoptotic function, as mutant forms of ARTS (or related septins) that fail to bind XIAP failed to induce apoptosis. ARTS leads to decreased XIAP protein levels and caspase activation. Our data suggest that ARTS induces apoptosis by antagonizing IAPs.     

Role of mitochondrial remodeling in programmed cell death in Drosophila melanogaster

The role of mitochondria in Drosophila programmed cell death remains unclear, although certain gene products that regulate cell death seem to be evolutionarily conserved. We find that developmental programmed cell death stimuli in vivo and multiple apoptotic stimuli ex vivo induce dramatic mitochondrial fragmentation upstream of effector caspase activation, phosphatidylserine exposure, and nuclear condensation in Drosophila cells. Unlike genotoxic stress, a lipid cell death mediator induced an increase in mitochondrial contiguity prior to fragmentation of the mitochondria. Using genetic mutants and RNAi-mediated knockdown of drp-1, we find that Drp-1 not only regulates mitochondrial fission in normal cells, but mediates mitochondrial fragmentation during programmed cell death. Mitochondria in drp-1 mutants fail to fragment, resulting in hyperplasia of tissues in vivo and protection of cells from multiple apoptotic stimuli ex vivo. Thus, mitochondrial remodeling is capable of modifying the propensity of cells to undergo death in Drosophila.     

Viral modulators of cell death provide new links to old pathways

By observing how viruses facilitate their parasitic relationships with host cells, we gain insights into key regulatory pathways of the cell. Not only are mitochondria key players in the regulation of programmed cell death, but many viral regulators of cell death also alter mitochondrial functions either directly or indirectly. Although cytomegalovirus vMIA and Epstein-Barr virus BHRF1 seem to have opposite effects on mitochondrial morphology, they both inhibit cell death. Drosophila Reaper, a regulator of developmental cell death, acts on IAP (inhibitor of apoptosis) proteins to activate caspases, but can regulate mitochondrial permeability in vitro. Despite its pivotal role in Drosophila, homologues of Reaper in other species were not previously known. Recently, amino acid sequence similarity was recognized between Drosophila Reaper and a protein known to be important for the replication and virulence of mosquito-borne bunyaviruses that cause human encephalitis. Thus, viral mechanisms for regulating apoptosis are diverse and not fully elucidated but promise to provide new insights.     

The RHG motifs of Drosophila Reaper and Grim are important for their distinct cell death-inducing abilities

Reaper, Hid, and Grim are three Drosophila cell death activators that each contain a conserved NH(2)-terminal Reaper, Hid, Grim (RHG) motif. We have analyzed the importance of the RHG motifs in Reaper and Grim for their different abilities to activate cell death during development. Analysis of chimeric R/Grim and G/Reaper proteins indicated that the Reaper and Grim RHG motifs are functionally distinct and help to determine specific cell death activation properties. A truncated GrimC protein lacking the RHG motif retained an ability to induce cell death, and unlike Grim, R/Grim, or G/Reaper, its actions were not efficiently blocked by the cell death inhibitors, Diap1, Diap2, p35, or a dominant/negative Dronc caspase. Finally, we identified a second region of sequence similarity in Reaper, Hid, and Grim, that may be important for shared RHG motif-independent activities.     

The DIAP1 RING finger mediates ubiquitination of Dronc and is indispensable for regulating apoptosis

Members of the Inhibitor of Apoptosis Protein (IAP) family block activation of the intrinsic cell death machinery by binding to and neutralizing the activity of pro-apoptotic caspases. In Drosophila melanogaster, the pro-apoptotic proteins Reaper (Rpr), Grim and Hid (head involution defective) all induce cell death by antagonizing the anti-apoptotic activity of Drosophila IAP1 (DIAP1), thereby liberating caspases. Here, we show that in vivo, the RING finger of DIAP1 is essential for the regulation of apoptosis induced by Rpr, Hid and Dronc. Furthermore, we show that the RING finger of DIAP1 promotes the ubiquitination of both itself and of Dronc. Disruption of the DIAP1 RING finger does not inhibit its binding to Rpr, Hid or Dronc, but completely abrogates ubiquitination of Dronc. Our data suggest that IAPs suppress apoptosis by binding to and targeting caspases for ubiquitination.     

sickle, a novel Drosophila death gene in the reaper/hid/grim region, encodes an IAP-inhibitory protein

Inhibitors of apoptosis proteins (IAPs) interact with caspases and inhibit their protease activity, whereas the IAP-inhibitory proteins Smac/DIABLO in mammals and Reaper, Hid, and Grim in flies relieve IAP-mediated inhibition to induce cell death. Here we describe the functional characterization of the novel Drosophila cell death protein Sickle (Skl), which binds to IAPs and neutralizes their apoptotic inhibitory activity. Skl exhibits no sequence homology to Reaper, Hid, Grim, or Smac/DIABLO, except within the 4 residue N-terminal IAP binding motif. Skl interacts with Drosophila and mammalian IAPs and can promote caspase activation in the presence of IAPs. Consistent with these findings, expression of Skl in Drosophila and mammalian cell lines or in Drosophila embryos induces apoptosis. Skl can also synergize with Grim to induce cell death in the Drosophila eye imaginal disc. Based on biochemical and structural data, the N terminus of Skl, like that of the mammalian Smac/DIABLO, is absolutely required for its apoptotic and caspase-promoting activities and its ability to interact with IAPs. These findings point to conservation in the structure and function of the IAP-inhibitory proteins across species and suggest the existence of other family members.     

Lack of involvement of mitochondrial factors in caspase activation in a Drosophila cell-free system

Although mitochondrial proteins play well-defined roles in caspase activation in mammalian cells, the role of mitochondrial factors in caspase activation in Drosophila is unclear. Using cell-free extracts, we demonstrate that mitochondrial factors play no apparent role in Drosophila caspase activation. Cytosolic extract from apoptotic S2 cells, in which caspases were inhibited, induced caspase activation in cytosolic extract from normal S2 cells. Mitochondrial extract did not activate caspases, nor did it influence caspase activation by cytosolic extract. Silencing of Hid, Reaper, or Grim reduced caspase activation by apoptotic cell extract. Furthermore, a peptide representing the amino terminus of Hid was sufficient to activate caspases in cytosolic extract, and this activity was not enhanced by addition of mitochondria or mitochondrial lysate. The Hid peptide also induced apoptosis when introduced into S2 cells. These results suggest that caspase activation in Drosophila is regulated solely by cytoplasmic factors and does not involve any mitochondrial factors.     

Mechanism of action of Drosophila Reaper in mammalian cells: Reaper globally inhibits protein synthesis and induces apoptosis independent of mitochondrial permeability

Drosophila Reaper can bind inhibitor of apoptosis proteins (IAP) and thereby rescue caspases from proteasomal degradation. In insect cells, this is sufficient to induce apoptosis. Reaper can also induce apoptosis in mammalian cells, in which caspases need to be activated, usually via the mitochondrial pathway. Nevertheless, we find that Reaper efficiently induces apoptosis in mammalian cells in the absence of mitochondrial permeabilisation and cytochrome c release. Moreover, this capacity was only marginally affected by deletion of Reaper's amino-terminal IAP-binding motif. Independent of this motif, Reaper could globally suppress protein synthesis. Deletion of 20 amino acids from the carboxy-terminus of Reaper fully abrogated its potential to inhibit protein synthesis and to induce apoptosis in the absence of IAP-binding. Our findings indicate that the newly identified capacity of Reaper to suppress protein translation can operate in mammalian cells and may be key to its pro-apoptotic activity.     

Mitochondrial localization of Reaper to promote inhibitors of apoptosis protein degradation conferred by GH3 domain-lipid interactions

Morphological hallmarks of apoptosis result from activation of the caspase family of cysteine proteases, which are opposed by a pro-survival family of inhibitors of apoptosis proteins (IAPs). In Drosophila, disruption of IAP function by Reaper, HID, and Grim (RHG) proteins is sufficient to induce cell death. RHG proteins have been reported to localize to mitochondria, which, in the case of both Reaper and Grim proteins, is mediated by an amphipathic helical domain known as the GH3. Through direct binding, Reaper can bring the Drosophila IAP (DIAP1) to mitochondria, concomitantly promoting IAP auto-ubiquitination and destruction. Whether this localization is sufficient to induce DIAP1 auto-ubiquitination has not been reported. In this study we characterize the interaction between Reaper and the mitochondria using both Xenopus and Drosophila systems. We find that Reaper concentrates on the outer surface of mitochondria in a nonperipheral manner largely mediated by GH3-lipid interactions. Importantly, we show that mitochondrial targeting of DIAP1 alone is not sufficient for degradation and requires Reaper binding. Conversely, Reaper able to bind IAPs, but lacking a mitochondrial targeting GH3 domain (DeltaGH3 Reaper), can induce DIAP1 turnover only if DIAP1 is otherwise targeted to membranes. Surprisingly, targeting DIAP1 to the endoplasmic reticulum instead of mitochondria is partially effective in allowing DeltaGH3 Reaper to promote DIAP1 degradation, suggesting that co-localization of DIAP and Reaper at a membrane surface is critical for the induction of DIAP degradation. Collectively, these data provide a specific function for the GH3 domain in conferring protein-lipid interactions, demonstrate that both Reaper binding and mitochondrial localization are required for accelerated IAP degradation, and suggest that membrane localization per se contributes to DIAP1 auto-ubiquitination and degradation.     

A collective form of cell death requires homeodomain interacting protein kinase

We examined post-eclosion elimination of the Drosophila wing epithelium in vivo where collective "suicide waves" promote sudden, coordinated death of epithelial sheets without a final engulfment step. Like apoptosis in earlier developmental stages, this unique communal form of cell death is controlled through the apoptosome proteins, Dronc and Dark, together with the IAP antagonists, Reaper, Grim, and Hid. Genetic lesions in these pathways caused intervein epithelial cells to persist, prompting a characteristic late-onset blemishing phenotype throughout the wing blade. We leveraged this phenotype in mosaic animals to discover relevant genes and establish here that homeodomain interacting protein kinase (HIPK) is required for collective death of the wing epithelium. Extra cells also persisted in other tissues, establishing a more generalized requirement for HIPK in the regulation of cell death and cell numbers.     

Grim stimulates Diap1 poly-ubiquitination by binding to UbcD1

Diap1 is an essential Drosophila cell death regulator that binds to caspases and inhibits their activity. Reaper, Grim and Hid each antagonize Diap1 by binding to its BIR domain, activating the caspases and eventually causing cell death. Reaper and Hid induce cell death in a Ring-dependent manner by stimulating Diap1 auto-ubiquitination and degradation. It was not clear that how Grim causes the ubiquitination and degradation of Diap1 in Grim-dependent cell death. We found that Grim stimulates poly-ubiquitination of Diap1 in the presence of UbcD1 and that it binds to UbcD1 in a GST pull-down assay, so presumably promoting Diap1 degradation. The possibility that dBruce is another E2 interacting with Diap1 was examined. The UBC domain of dBruce slightly stimulated poly-ubiquitination of Diap1 in Drosophila extracts but not in the reconstitution assay. However Grim did not stimulate Diap1 poly-ubiquitination in the presence of the UBC domain of dBruce. Taken together, these results suggest that Grim stimulates the poly-ubiquitination and presumably degradation of Diap1 in a novel way by binding to UbcD1 but not to the UBC domain of dBruce as an E2.     

Inhibiting the mitochondrial fission machinery does not prevent Bax/Bak-dependent apoptosis

Apoptosis, induced by a number of death stimuli, is associated with a fragmentation of the mitochondrial network. These morphological changes in mitochondria have been shown to require proteins, such as Drp1 or hFis1, which are involved in regulating the fission of mitochondria. However, the precise role of mitochondrial fission during apoptosis remains elusive. Here we report that inhibiting the fission machinery in Bax/Bak-mediated apoptosis, by down-regulating of Drp1 or hFis1, prevents the fragmentation of the mitochondrial network and partially inhibits the release of cytochrome c from the mitochondria but fails to block the efflux of Smac/DIABLO. In addition, preventing mitochondrial fragmentation does not inhibit cell death induced by Bax/Bak-dependent death stimuli, in contrast to the effects of Bcl-xL or caspase inhibition. Therefore, the fission of mitochondria is a dispensable event in Bax/Bak-dependent apoptosis.     

Inhibition of translation and induction of apoptosis by Bunyaviral nonstructural proteins bearing sequence similarity to reaper

Members of the California serogroup of bunyaviruses (family Bunyaviridae) are the leading cause of pediatric viral encephalitis in North America. Significant cell death is observed as part of the infection pathology. We now report that a Bunyaviral nonstructural protein termed NSs shows sequence similarity to Reaper, a proapoptotic protein from Drosophila. Although NSs proteins lack the Reaper N-terminal motif critical for IAP inhibition, they do retain other functions of Reaper that map to conserved C-terminal regions. Like Reaper, NSs proteins induce mitochondrial cytochrome c release and caspase activation in cell-free extracts and promote neuronal apoptosis and mortality in a mouse model. Independent of caspase activation, Bunyavirus NSs proteins also share with Reaper the ability to directly inhibit cellular protein translation. We have found that the shared capacity to inhibit translation and induce apoptosis resides in common sequence motifs present in both Reaper and NSs proteins. Data presented here suggest that NSs induce apoptosis through a mechanism similar to that used by Reaper, as both proteins bind to an apoptotic regulator called Scythe and can relieve Scythe inhibition of Hsp70. Thus, bunyavirus NSs proteins have multiple Reaper-like functions that likely contribute to viral pathogenesis by promoting cell death and/or inhibiting cellular translation.     

6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used neurotoxin to study Parkinson's disease. Herein we studied the potential effects of 6-OHDA on mitochondrial morphology in SH-SY5Y neuroblastoma cells. By immunofluorescence and time-lapse fluorescence microscopy we demonstrated that 6-OHDA induced profound mitochondrial fragmentation in SH-SY5Y cells, an event that was similar to mitochondrial fission induced by overexpression of Fis1p, a membrane adaptor for the dynamin-related protein 1 (DLP1/Drp1). 6-OHDA failed to induce any changes in peroxisome morphology. Biochemical experiments revealed that 6-OHDA-induced mitochondrial fragmentation is an early event preceding the collapse of the mitochondrial membrane potential and cytochrome c release in SH-SY5Y cells. Silencing of DLP1/Drp1, which is involved in mitochondrial and peroxisomal fission, prevented 6-OHDA-induced fragmentation of mitochondria. Furthermore, in cells silenced for Drp1, 6-OHDA-induced cell death was reduced, indicating that a block in mitochondrial fission protects SH-SY5Y cells against 6-OHDA toxicity. Experiments in mouse embryonic fibroblasts deficient in Bax or p53 revealed that both proteins are not essential for 6-OHDA-induced mitochondrial fragmentation. Our data demonstrate for the first time an involvement of mitochondrial fragmentation and Drp1 function in 6-OHDA-induced apoptosis.     

Structural analysis of a functional DIAP1 fragment bound to grim and hid peptides

The inhibitor of apoptosis protein DIAP1 suppresses apoptosis in Drosophila, with the second BIR domain (BIR2) playing an important role. Three proteins, Hid, Grim, and Reaper, promote apoptosis, in part by binding to DIAP1 through their conserved N-terminal sequences. The crystal structures of DIAP1-BIR2 by itself and in complex with the N-terminal peptides from Hid and Grim reveal that these peptides bind a surface groove on DIAP1, with the first four amino acids mimicking the binding of the Smac tetrapeptide to XIAP. The next 3 residues also contribute to binding through hydrophobic interactions. Interestingly, peptide binding induces the formation of an additional alpha helix in DIAP1. Our study reveals the structural conservation and diversity necessary for the binding of IAPs by the Drosophila Hid/Grim/Reaper and the mammalian Smac proteins.