Drosophila caspase DRONC is required for specific developmental cell death pathways and stress-induced apoptosis

Proteases of the caspase family play key roles in the execution of apoptosis. In Drosophila there are seven caspases, but their roles in cell death have not been studied in detail due to a lack of availability of specific mutants. Here, we describe the generation of a specific mutant of the Drosophila gene encoding DRONC, the only caspase recruitment domain (CARD) containing apical caspase in the fly. dronc mutants are pupal lethal and our studies show that DRONC is required for many forms of developmental cell deaths and apoptosis induced by DNA damage. Furthermore, we demonstrate that DRONC is required for the autophagic death of larval salivary glands during metamorphosis, but not for histolysis of larval midguts. Our results indicate that DRONC is involved in specific developmental cell death pathways and that in some tissues, effector caspase activation and cell death can occur independently of DRONC.     

Cell survival and proliferation in Drosophila S2 cells following apoptotic stress in the absence of the APAF-1 homolog, ARK, or downstream caspases

In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, as its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. Here, we further dissect the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. We found that the induction of apoptosis was dependent in each case on expression of ARK and DRONC and surviving cells continued to proliferate. We noted a difference in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these had dramatic effects to sustain cell survival following depletion of DIAP1, but had only minor effects following cellular stress. Our results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted.     

Apoptosis-induced compensatory proliferation. The Cell is dead. Long live the Cell!

In multi-cellular organisms, activation of apoptosis can trigger compensatory proliferation in surrounding cells to maintain tissue homeostasis. Genetic studies in Drosophila have indicated that distinct mechanisms of compensatory proliferation are employed in apoptotic tissues of different developmental states. In proliferating eye and wing tissues, the initiator caspase Dronc coordinates cell death and compensatory proliferation through the Jun N-terminal kinase and p53. The mitogens Decapentaplegic and Wingless are induced in this process. By contrast, in differentiating eye tissues, the effector caspases DrICE and Dcp-1 activate the Hedgehog signaling pathway to induce compensatory proliferation. In this review, we summarize these findings and discuss how activation of apoptosis is linked to the process of compensatory proliferation. The developmental and pathological relevance of compensatory proliferation is also discussed.     

A biochemical analysis of the activation of the Drosophila caspase DRONC

The activation of caspases is the principal event in the execution of apoptosis. Initiator caspases are activated through an autocatalytic mechanism often involving dimerisation or oligomerisation. In Drosophila, the only initiator caspase DRONC, is tightly inhibited by DIAP1 and removal of DIAP1 permits activation of DRONC by the Drosophila Apaf-1-related killer, ARK. ARK is proposed to facilitate DRONC oligomerisation and autoprocessing at residue E352. This study examines whether autoprocessing of DRONC is required for its activation and for DRONC-mediated cell death. Using purified recombinant proteins, we show here that while DRONC autocleaves at residue E352, mutation of this site did not abolish enzyme activation, DRICE-induced cleavage of DRONC or DRONC-mediated activation of DRICE. We performed a detailed mutational analysis of DRONC cleavage sites and show that overexpression of DRONC cleavage mutants in Drosophila cells retain pro-apoptotic activity. Using an in vitro cell-free assay, we found ARK alone did not activate DRONC and demonstrate a requirement for an additional cytosolic factor in ARK-mediated DRONC activation. These results suggest that, similar to mammalian caspase-2 and caspase-9, the initial cleavage of DRONC is not essential for its activation and suggest a mechanism of ARK-mediated DRONC activation different from that proposed previously.     

Distinct mechanisms of apoptosis-induced compensatory proliferation in proliferating and differentiating tissues in the Drosophila eye

In multicellular organisms, apoptotic cells induce compensatory proliferation of neighboring cells to maintain tissue homeostasis. In the Drosophila wing imaginal disc, dying cells trigger compensatory proliferation through secretion of the mitogens Decapentaplegic (Dpp) and Wingless (Wg). This process is under control of the initiator caspase Dronc, but not effector caspases. Here we show that a second mechanism of apoptosis-induced compensatory proliferation exists. This mechanism is dependent on effector caspases which trigger the activation of Hedgehog (Hh) signaling for compensatory proliferation. Furthermore, whereas Dpp and Wg signaling is preferentially employed in apoptotic proliferating tissues, Hh signaling is activated in differentiating eye tissues. Interestingly, effector caspases in photoreceptor neurons stimulate Hh signaling which triggers cell-cycle reentry of cells that had previously exited the cell cycle. In summary, dependent on the developmental potential of the affected tissue, different caspases trigger distinct forms of compensatory proliferation in an apparent nonapoptotic function.     

The Drosophila caspase DRONC cleaves following glutamate or aspartate and is regulated by DIAP1, HID, and GRIM

The caspase family of cysteine proteases plays important roles in bringing about apoptotic cell death. All caspases studied to date cleave substrates COOH-terminal to an aspartate. Here we show that the Drosophila caspase DRONC cleaves COOH-terminal to glutamate as well as aspartate. DRONC autoprocesses itself following a glutamate residue, but processes a second caspase, drICE, following an aspartate. DRONC prefers tetrapeptide substrates in which aliphatic amino acids are present at the P2 position, and the P1 residue can be either aspartate or glutamate. Expression of a dominant negative form of DRONC blocks cell death induced by the Drosophila cell death activators reaper, hid, and grim, and DRONC overexpression in flies promotes cell death. Furthermore, the Drosophila cell death inhibitor DIAP1 inhibits DRONC activity in yeast, and DIAP1's ability to inhibit DRONC-dependent yeast cell death is suppressed by HID and GRIM. These observations suggest that DRONC acts to promote cell death. However, DRONC activity is not suppressed by the caspase inhibitor and cell death suppressor baculovirus p35. We discuss possible models for DRONC function as a cell death inhibitor.     

Down-regulation of DIAP1 triggers a novel Drosophila cell death pathway mediated by Dark and DRONC

Members of the inhibitor of apoptosis protein (IAP) family can inhibit caspases and cell death in a variety of insect and vertebrate systems. Drosophila IAP1 (DIAP1) inhibits cell death to facilitate normal embryonic development. Here, using RNA interference, we showed that down-regulation of DIAP1 is sufficient to induce cell death in Drosophila S2 cells. Although this cell death process was accompanied by elevated caspase activity, this activation was not essential for cell death. We found that DIAP1 depletion-induced cell death was strongly suppressed by a reduction in the Drosophila caspase DRONC or the Drosophila apoptotic protease-activating factor-1 (Apaf-1) homolog, Dark. RNA interference studies in Drosophila embryos also demonstrated that the action of Dark is epistatic to that of DIAP1 in this cell death pathway. The cell death caused by down-regulation of DIAP1 was accelerated by overexpression of DRONC and Dark, and a caspase-inactive mutant form of DRONC could functionally substitute the wild-type DRONC in accelerating cell death. These results suggest the existence of a novel mechanism for cell death signaling in Drosophila that is mediated by DRONC and Dark.     

The Drosophila caspase DRONC is regulated by DIAP1

We have isolated the recently identified Drosophila caspase DRONC through its interaction with the effector caspase drICE. Ectopic expression of DRONC induces cell death in Schizosaccharomyces pombe, mammalian fibroblasts and the developing Drosophila eye. The caspase inhibitor p35 fails to rescue DRONC-induced cell death in vivo and is not cleaved by DRONC in vitro, making DRONC the first identified p35-resistant caspase. The DRONC pro-domain interacts with Drosphila inhibitor of apoptosis protein 1 (DIAP1), and co-expression of DIAP1 in the developing Drosophila eye completely reverts the eye ablation phenotype induced by pro-DRONC expression. In contrast, DIAP1 fails to rescue eye ablation induced by DRONC lacking the pro-domain, indicating that interaction of DIAP1 with the pro-domain of DRONC is required for suppression of DRONC-mediated cell death. Heterozygosity at the diap1 locus enhances the pro-DRONC eye phenotype, consistent with a role for endogenous DIAP1 in suppression of DRONC activation. Both heterozygosity at the dronc locus and expression of dominant-negative DRONC mutants suppress the eye phenotype caused by reaper (RPR) and head involution defective (HID), consistent with the idea that DRONC functions in the RPR and HID pathway.     

Baculovirus caspase inhibitors P49 and P35 block virus-induced apoptosis downstream of effector caspase DrICE activation in Drosophila melanogaster cells

Baculoviruses induce widespread apoptosis in invertebrates. To better understand the pathways by which these DNA viruses trigger apoptosis, we have used a combination of RNA silencing and overexpression of viral and host apoptotic regulators to identify cell death components in the model system of Drosophila melanogaster. Here we report that the principal effector caspase DrICE is required for baculovirus-induced apoptosis of Drosophila DL-1 cells as demonstrated by RNA silencing. proDrICE was proteolytically cleaved and activated during infection. Activation was blocked by overexpression of the cellular inhibitor-of-apoptosis proteins DIAP1 and SfIAP but not by the baculovirus caspase inhibitor P49 or P35. Rather, the substrate inhibitors P49 and P35 prevented virus-induced apoptosis by arresting active DrICE through formation of stable inhibitory complexes. Consistent with a two-step activation mechanism, proDrICE was cleaved at the large/small subunit junction TETD(230)-G by a DIAP1-inhibitable, P49/P35-resistant protease and then at the prodomain junction DHTD(28)-A by a P49/P35-sensitive protease. Confirming that P49 targeted DrICE and not the initiator caspase DRONC, depletion of DrICE by RNA silencing suppressed virus-induced cleavage of P49. Collectively, our findings indicate that whereas DIAP1 functions upstream to block DrICE activation, P49 and P35 act downstream by inhibiting active DrICE. Given that P49 has the potential to inhibit both upstream initiator caspases and downstream effector caspases, we conclude that P49 is a broad-spectrum caspase inhibitor that likely provides a selective advantage to baculoviruses in different cellular backgrounds.     

The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells

In Drosophila, activation of the apical caspase DRONC requires the apoptotic protease-activating factor homologue, DARK. However, unlike caspase activation in mammals, DRONC activation is not accompanied by the release of cytochrome c from mitochondria. Drosophila encodes two cytochrome c proteins, Cytc-p (DC4) the predominantly expressed species, and Cytc-d (DC3), which is implicated in caspase activation during spermatogenesis. Here, we report that silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells. We find that loss of function mutations in dc3 and dc4, do not affect caspase activation during Drosophila development and that ectopic expression of DC3 or DC4 in Drosophila cells does not induce caspase activation. In cell-free studies, recombinant DC3 or DC4 failed to activate caspases in Drosophila cell lysates, but remarkably induced caspase activation in extracts from human cells. Overall, our results argue that DARK-mediated DRONC activation occurs independently of cytochrome c.     

Identification of a Drosophila melanogaster ICE/CED-3-related protease, drICE.

Cysteine proteases of the ICE/CED-3 family (caspases) are required for the execution of programmed cell death (PCD) in a wide range of multicellular organisms. Caspases are implicated in the execution of apoptosis in Drosophila melanogaster by the observation that expression of baculovirus p35, a caspase inhibitor, blocks cell death in vivo in Drosophila. We report here the identification and characterization of drICE, a D. melanogaster caspase. We show that overexpression of drICE sensitizes Drosophila cells to apoptotic stimuli and that expression of an N-terminally truncated form of drICE rapidly induces apoptosis in Drosophila cells. Induction of apoptosis by rpr overexpression or by cycloheximide or etoposide treatment of Drosophila cells results in proteolytic processing of drICE. We further show that drICE is a cysteine protease that cleaves baculovirus p35 and Drosophila lamin DmO in vitro and that drICE is expressed at all the stages of Drosophila development at which PCD can be induced. Taken together, these results strongly argue that drICE is an apoptotic caspase that acts downstream of rpr. drICE is therefore the first unequivocal link between the molecular machinery of Drosophila cell death and the conserved machinery of Caenorhabditis elegans and vertebrates. Identification of drICE should facilitate the elucidation of upstream regulators and downstream targets of caspases by genetic screening.     

Baculovirus apoptotic suppressor P49 is a substrate inhibitor of initiator caspases resistant to P35 in vivo

Caspases play a critical role in the execution of metazoan apoptosis and are thus attractive therapeutic targets for apoptosis-associated diseases. Here we report that baculovirus P49, a homolog of pancaspase inhibitor P35, prevents apoptosis in invertebrates by inhibiting an initiator caspase that is P35 insensitive. Consequently P49 blocked proteolytic activation of effector caspases at a unique step upstream from that affected by P35 but downstream from inhibitor of apoptosis Op-IAP. Like P35, P49 was cleaved by and stably associated with its caspase target. Ectopically expressed P49 blocked apoptosis in cultured cells from a phylogenetically distinct organism, Drosophila melanogaster. Furthermore, P49 inhibited human caspase-9, demonstrating its capacity to affect a vertebrate initiator caspase. Thus, P49 is a substrate inhibitor with a novel in vivo specificity for a P35-insensitive initiator caspase that functions at an evolutionarily conserved step in the caspase cascade. These data indicate that activated initiator caspases provide another effective target for apoptotic intervention by substrate inhibitors.     

The Drosophila DIAP1 protein is required to prevent accumulation of a continuously generated,processed form of the apical caspase DRONC

Although loss of the inhibitor of apoptosis (IAP) protein DIAP1 has been shown to result in caspase activation and spontaneous cell death in Drosophila cells and embryos, the point at which DIAP1 normally functions to inhibit caspase activation is unknown. Depletion of the DIAP1 protein in Drosophila S2 cells or the Sf-IAP protein in Spodoptera frugiperda Sf21 cells by RNA interference (RNAi) or cycloheximide treatment resulted in rapid and widespread caspase-dependent apoptosis. Co-silencing of dronc or dark largely suppressed this apoptosis, indicating that DIAP1 is normally required to inhibit an activity dependent on these proteins. Silencing of dronc also inhibited DRICE processing following stimulation of apoptosis, demonstrating that DRONC functions as an apical caspase in S2 cells. Silencing of diap1 or treatment with UV light induced DRONC processing, which occurred in two steps. The first step appeared to occur continuously even in the absence of an apoptotic signal and to be dependent on DARK, because full-length DRONC accumulated when dark was silenced in non-apoptotic cells. In addition, treatment with the proteasome inhibitor MG132 resulted in accumulation of this initially processed form of DRONC, but not full-length DRONC, in non-apoptotic cells. The second step in DRONC processing was observed only in apoptotic cells. These results indicate that the initial step in DRONC processing occurs continuously via a DARK-dependent mechanism in Drosophila cells and that DIAP1 is required to prevent excess accumulation of this first form of processed DRONC, presumably through its ability to act as a ubiquitin-protein ligase.     

drICE is an essential caspase required for apoptotic activity in Drosophila cells.

Caspases are involved in the execution of cell death in all multicellular organisms so far studied, including the nematode worm, fruit fly and vertebrates. While Caenorhabditis elegans has only a single identified caspase, CED-3, whose activity is absolutely required for all developmental programmed cell deaths, most mammalian cell types express multiple caspases with varying specificities. The fruit fly Drosophila melanogaster is genetically tractable, less complex than vertebrates and possesses two known caspases, DCP-1 and drICE. The fly may therefore provide a good model system for examining the hierarchy and relative roles of individual caspases in the execution of apoptosis. We have examined the role of drICE in in vitro apoptosis of the D.melanogaster cell line S2. We show that cytoplasmic lysates made from S2 cells undergoing apoptosis induced by either reaper (rpr) expression or cycloheximide treatment contain a caspase activity with DEVD specificity which can cleave p35, lamin DmO, drICE and DCP-1 in vitro, and which can trigger chromatin condensation in isolated nuclei. Using antibodies specific to drICE, we show that immunodepletion of drICE from these lysates is sufficient to remove most measurable in vitro apoptotic activity, and that re-addition of exogenous drICE to such immunodepleted lysates restores apoptotic activity. We conclude that, at least in S2 cells, drICE can be the sole caspase effector of apoptosis.     

The role of cytochrome c in caspase activation in Drosophila melanogaster cells

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. We demonstrate here that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals.     

The analysis of biochemical markers of MCF-7 cells apoptosis induced by recombinant analog of lactaptin

Earlier we isolated and characterized human milk pro-apoptotic peptide - lactaptin and generated its engineered analog - RL2. It was shown that both lactaptin and RL2 are capable to induce apoptotic death of MCF-7 cells. In this report we have analyzed biochemical markers of RL2 induced MCF-7 apoptosis. The activation of initiator and effector caspases as well as mitochondrial membrane potential and cytoplasm membrane changes were analyzed using flow cytometry and Western-blot methods. We have found that RL2 induced apoptotic death of MCF-7 cells was accompanied by PS exposure on the plasma membrane surface. It also was shown that RL2 has induced dissipation of mitochondrial membrane potential and resulted in activation of initiator caspases 8, 9 and effector caspase 7.