Activation mechanism and substrate specificity of the Drosophila initiator caspase DRONC

Drosophila Nedd2-like caspase (DRONC), an initiator caspase in Drosophila melanogaster and ortholog of human caspase-9, is cleaved during its activation in vitro and in vivo. We show that, in contrast to conclusions from previous studies, cleavage is neither necessary nor sufficient for DRONC activation. Instead, our data suggest that DRONC is activated by dimerization, a mechanism used by its counterparts in humans. Subsequent cleavage at Glu352 stabilizes the active dimer. Since cleavage is at a Glu residue, it has been proposed that DRONC is a dual Asp- and Glu-specific caspase. We used positional-scanning peptide libraries to define the P1-P4 peptide sequence preferences of DRONC, and show that it is indeed equally active on optimized tetrapeptides containing either Asp or Glu in P1. Furthermore, mutagenesis reveals that Asp and Glu residues are equally tolerated at the primary autoprocessing site of DRONC itself. However, when its specificity is tested on a natural substrate, the Drosophila executioner caspase DRICE, a clear preference for Asp emerges. The formerly proposed Glu preference is thus incorrect. DRONC does not differentiate between Asp and Glu in poor substrates, but prefers Asp when tested on a good substrate.     

Dronc caspase exerts a non-apoptotic function to restrain phospho-Numb-induced ectopic neuroblast formation in Drosophila

Drosophila neuroblasts have served as a model to understand how the balance of stem cell self-renewal versus differentiation is achieved. Drosophila Numb protein regulates this process through its preferential segregation into the differentiating daughter cell. How Numb restricts the proliferation and self-renewal potentials of the recipient cell remains enigmatic. Here, we show that phosphorylation at conserved sites regulates the tumor suppressor activity of Numb. Enforced expression of a phospho-mimetic form of Numb (Numb-TS4D) or genetic manipulation that boosts phospho-Numb levels, attenuates endogenous Numb activity and causes ectopic neuroblast formation (ENF). This effect on neuroblast homeostasis occurs only in the type II neuroblast lineage. We identify Dronc caspase as a novel binding partner of Numb, and demonstrate that overexpression of Dronc suppresses the effects of Numb-TS4D in a non-apoptotic and possibly non-catalytic manner. Reduction of Dronc activity facilitates ENF induced by phospho-Numb. Our findings uncover a molecular mechanism that regulates Numb activity and suggest a novel role for Dronc caspase in regulating neural stem cell homeostasis.     

The CARD-carrying caspase Dronc is essential for most, but not all, developmental cell death in Drosophila

The initiator caspase Dronc is the only Drosophila caspase that contains a caspase activation and recruitment domain (CARD). Although Dronc has been implicated as an important effector of apoptosis, the genetic function of dronc in normal development is unclear because dronc mutants have not been available. In an EMS mutagenesis screen, we isolated four point mutations in dronc that recessively suppress the eye ablation phenotype caused by eye-specific overexpression of hid. Homozygous mutant dronc animals die during pupal stages; however, at a low frequency we obtained homozygous adult escapers. These escapers have additional cells in the eye and wings that are less transparent and slightly curved down. We determined that this is due to lack of apoptosis. Our analyses of dronc mutant embryos suggest that dronc is essential for most apoptotic cell death during Drosophila development, but they also imply the existence of a dronc-independent cell death pathway. We also constructed double mutant flies for dronc and the apoptosis inhibitor diap1. dronc mutants can rescue the ovarian degeneration phenotype caused by diap1 mutations, confirming that dronc acts genetically downstream of diap1.     

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.     

Compensatory proliferation induced by cell death in the Drosophila wing disc requires activity of the apical cell death caspase Dronc in a nonapoptotic role

Achieving proper organ size requires a balance between proliferation and cell death. For example, at least 40%-60% of cells in the Drosophila wing disc can be lost, yet these discs go on to give rise to normal-looking adult wings as a result of compensatory proliferation. The signals that drive this proliferation are unknown. One intriguing possibility is that they derive, at least in part, from the dying cells. To explore this hypothesis, we activated cell death signaling in specific populations of cells in the developing wing but prevented these cells from dying through expression of the baculovirus p35 protein, which inhibits the activity of effector caspases that mediate apoptosis. This allowed us to uncouple the activation steps of apoptosis from death itself. Here we report that stimulation of cell death signaling in the wing disc-in the absence of cell death-results in increased proliferation and ectopic expression of Wingless, a known mitogen in the wing. Activation of the apical cell death caspase Dronc is necessary and sufficient to drive both of these processes. Our results demonstrate an unanticipated function, the nonautonomous induction of proliferation, of an apical cell death caspase. This activity is likely to contribute to tissue homeostasis by promoting local compensatory proliferation in response to cell death. We speculate that dying cells may communicate cell fate or behavior instructions to their neighbors in other contexts as well.     

Metabolic regulation of Drosophila apoptosis through inhibitory phosphorylation of Dronc

Apoptosis ensures tissue homeostasis in response to developmental cues or cellular damage. Recently reported genome‐wide RNAi screens have suggested that several metabolic regulators can modulate caspase activation in Drosophila. Here, we establish a previously unrecognized link between metabolism and Drosophila apoptosis by showing that cellular NADPH levels modulate the initiator caspase Dronc through its phosphorylation at S130. Depletion of NADPH removed this inhibitory phosphorylation, resulting in the activation of Dronc and subsequent cell death. Conversely, upregulation of NADPH prevented Dronc‐mediated apoptosis upon DIAP1 RNAi or cycloheximide treatment. Furthermore, this CaMKII‐mediated phosphorylation of Dronc hindered Dronc activation, but not its catalytic activity. Blockade of NADPH production aggravated the death‐inducing activity of Dronc in specific neurons, but not in the photoreceptor cells of the eyes of transgenic flies; similarly, non‐phosphorylatable Dronc was more potent than wild type in triggering specific neuronal apoptosis. Our observations reveal a novel regulatory circuitry in Drosophila apoptosis, and, as NADPH levels are elevated in cancer cells, also provide a genetic model to understand aberrations in cancer cell apoptosis resulting from metabolic alterations.     

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.     

Death receptor-induced activation of initiator caspase 8 is antagonized by serine/threonine kinase PAK4

Normal cell growth requires a precisely controlled balance between cell death and survival. This involves activation of different types of intracellular signaling cascades within the cell. While some types of signaling proteins regulate apoptosis, or programmed cell death, other proteins within the cell can promote survival. The serine/threonine kinase PAK4 can protect cells from apoptosis in response to several different types of stimuli. As is the case for other members of the p21-activated kinase (PAK) family, one way that PAK4 may promote cell survival is by phosphorylating and thereby inhibiting the proapoptotic protein Bad. This leads in turn to the inhibition of effector caspases such as caspase 3. Here we show that in response to cytokines which activate death domain-containing receptors, such as the tumor necrosis factor and Fas receptors, PAK4 can inhibit the death signal by a different mechanism. Under these conditions, PAK4 inhibits apoptosis early in the caspase cascade, antagonizing the activation of initiator caspase 8. This inhibition, which does not require PAK4's kinase activity, may involve inhibition of caspase 8 recruitment to the death domain receptors. This role in regulating initiator caspases is an entirely novel role for the PAK proteins and suggests a new mechanism by which these proteins promote cell survival.     

A ubiquitin ligase complex regulates caspase activation during sperm differentiation in Drosophila

In both insects and mammals, spermatids eliminate their bulk cytoplasm as they undergo terminal differentiation. In Drosophila, this process of dramatic cellular remodeling requires apoptotic proteins, including caspases. To gain further insight into the regulation of caspases, we screened a large collection of sterile male flies for mutants that block effector caspase activation at the onset of spermatid individualization. Here, we describe the identification and characterization of a testis-specific, Cullin-3–dependent ubiquitin ligase complex that is required for caspase activation in spermatids. Mutations in either a testis-specific isoform of Cullin-3 (Cul3_Testis), the small RING protein Roc1b, or a Drosophila orthologue of the mammalian BTB-Kelch protein Klhl10 all reduce or eliminate effector caspase activation in spermatids. Importantly, all three genes encode proteins that can physically interact to form a ubiquitin ligase complex. Roc1b binds to the catalytic core of Cullin-3, and Klhl10 binds specifically to a unique testis-specific N-terminal Cullin-3 (TeNC) domain of Cul3_Testis that is required for activation of effector caspase in spermatids. Finally, the BIR domain region of the giant inhibitor of apoptosis–like protein dBruce is sufficient to bind to Klhl10, which is consistent with the idea that dBruce is a substrate for the Cullin-3-based E3-ligase complex. These findings reveal a novel role of Cullin-based ubiquitin ligases in caspase regulation.     

Oligomerization is a general mechanism for the activation of apoptosis initiator and inflammatory procaspases

Proteolytic activation of initiator procaspases is a crucial step in the cellular commitment to apoptosis. Alternative models have been postulated for the activation mechanism, namely the oligomerization or induced proximity model and the allosteric regulation model. While the former holds that procaspases become activated upon proper oligomerization by an adaptor protein, the latter states that the adaptor is an allosteric regulator for procaspases. The allosteric regulation model has been applied for the activation of procaspase-9 by apoptotic protease-activating factor (Apaf-1) in an oligomeric complex known as the apoptosome. Using approaches that allow for controlled oligomerization, we show here that aggregation of multiple procaspase-9 molecules can induce their activation independent of the apoptosome. Oligomerization-induced procaspase-9 activation, both within the apoptosome and in artificial systems, requires stable homophilic association of the protease domains, raising the possibility that the function of Apaf-1 is not only to oligomerize procaspase-9 but also to maintain the interaction of the caspase-9 protease domain after processing. In addition, we provide biochemical evidence that other apoptosis initiator caspases (caspase-2 and -10) as well as a procaspase involved in inflammation (murine caspase-11) are also activated by oligomerization. Thus, oligomerization of precursor molecules appears to be a general mechanism for the activation of both apoptosis initiator and inflammatory procaspases.     

Molecular mechanism of Reaper-Grim-Hid-mediated suppression of DIAP1-dependent Dronc ubiquitination

The inhibitor of apoptosis protein DIAP1 inhibits Dronc-dependent cell death by ubiquitinating Dronc. The pro-death proteins Reaper, Hid and Grim (RHG) promote apoptosis by antagonizing DIAP1 function. Here we report the structural basis of Dronc recognition by DIAP1 as well as a novel mechanism by which the RHG proteins remove DIAP1-mediated downregulation of Dronc. Biochemical and structural analyses revealed that the second BIR (BIR2) domain of DIAP1 recognizes a 12-residue sequence in Dronc. This recognition is essential for DIAP1 binding to Dronc, and for targeting Dronc for ubiquitination. Notably, the Dronc-binding surface on BIR2 coincides with that required for binding to the N termini of the RHG proteins, which competitively eliminate DIAP1-mediated ubiquitination of Dronc. These observations reveal the molecular mechanisms of how DIAP1 recognizes Dronc, and more importantly, how the RHG proteins remove DIAP1-mediated ubiquitination of Dronc.     

The two Drosophila cytochrome C proteins can function in both respiration and caspase activation

Cytochrome C has two apparently separable cellular functions: respiration and caspase activation during apoptosis. While a role of the mitochondria and cytochrome C in the assembly of the apoptosome and caspase activation has been established for mammalian cells, the existence of a comparable function for cytochrome C in invertebrates remains controversial. Drosophila possesses two cytochrome c genes, cyt-c-d and cyt-c-p. We show that only cyt-c-d is required for caspase activation in an apoptosis-like process during spermatid differentiation, whereas cyt-c-p is required for respiration in the soma. However, both cytochrome C proteins can function interchangeably in respiration and caspase activation, and the difference in their genetic requirements can be attributed to differential expression in the soma and testes. Furthermore, orthologues of the apoptosome components, Ark (Apaf-1) and Dronc (caspase-9), are also required for the proper removal of bulk cytoplasm during spermatogenesis. Finally, several mutants that block caspase activation during spermatogenesis were isolated in a genetic screen, including mutants with defects in spermatid mitochondrial organization. These observations establish a role for the mitochondria in caspase activation during spermatogenesis.     

Characterization of the Drosophila caspase, DAMM

Caspases are main effectors of apoptosis in metazoans. Genome analysis indicates that there are seven caspases in Drosophila, six of which have been previously characterized. Here we describe the cloning and characterization of the last Drosophila caspase, DAMM. Similar to mammalian effector caspases, DAMM lacks a long prodomain. We show that the DAMM precursor, along with the caspases DRONC and DECAY, is partially processed in cells undergoing apoptosis. Recombinant DAMM produced in Escherichia coli shows significant catalytic activity on a pentapeptide caspase substrate. Low levels of damm mRNA are ubiquitously expressed in Drosophila embryos during early stages of development. Relatively high levels of damm mRNA are detected in larval salivary glands and midgut, and in adult egg chambers. Ectopic expression of DAMM in cultured cells induces apoptosis, and similarly, transgenic overexpression of DAMM, but not of a catalytically inactive DAMM mutant, in Drosophila results in a rough eye phenotype. We demonstrate that expression of the catalytically inactive DAMM mutant protein significantly suppresses the rough eye phenotype due to the overexpression of HID, suggesting that DAMM may be required in a hid-mediated cell death 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.     

Mechanisms of caspase activation

The core effectors of apoptosis encompass proteolytic enzymes of the caspase family, which reside as latent precursors in most nucleated metazoan cells. A majority of studies on apoptosis are based on the assumption that caspase precursors are activated by cleavage, a common mechanism for most protease zymogen activations. Although this appears to be true for the executioner caspases, recent research points to a distinct activation mechanism for the initiator caspases that trigger the apoptotic pathways. This mechanism is proximity-induced dimerization without cleavage, and its elucidation has led to the revision of concepts of feedback regulation of apoptosis.     

Non‐apoptotic caspase activation preserves Drosophila intestinal progenitor cells in quiescence

Caspase malfunction in stem cells often precedes the appearance and progression of multiple types of cancer, including human colorectal cancer. However, the caspase‐dependent regulation of intestinal stem cell properties remains poorly understood. Here, we demonstrate that Dronc, the Drosophila ortholog of caspase‐9/2 in mammals, limits the number of intestinal progenitor cells and their entry into the enterocyte differentiation programme. Strikingly, these unexpected roles for Dronc are non‐apoptotic and have been uncovered under experimental conditions without epithelial replenishment. Supporting the non‐apoptotic nature of these functions, we show that they require the enzymatic activity of Dronc, but are largely independent of the apoptotic pathway. Alternatively, our genetic and functional data suggest that they are linked to the caspase‐mediated regulation of Notch signalling. Our findings provide novel insights into the non‐apoptotic, caspase‐dependent modulation of stem cell properties that could improve our understanding of the origin of intestinal malignancies.     

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.     

Interdimer processing and linearity of procaspase-3 activation. A unifying mechanism for the activation of initiator and effector caspases

Caspase activation during apoptosis occurs in a cascade from the initiator caspase(s) (e.g. caspase-8) to the effector caspases (e.g. caspase-3), which ensures the generation of large amounts of active caspases to dismantle cells. However, the mechanism that safeguards against inadvertent caspase activation is not well understood. Previous studies have suggested that the activation of procaspase-8 is mediated by cross-cleavage of precursor dimers, formed upon apoptosis induction, which are not only enzymatically competent but also highly susceptible to cleavage, and that procaspase-8 activation is a linear process without self-amplification. Effector procaspases constitutively exist as dimers and their activation is started by trans-cleavage by an initiator caspase followed by autocleavage of effector caspases. Here we show that the dimerization of caspase-3 molecules through their protease domains is required for their processing by initiator caspases. The subsequent autoprocessing takes place through cleavage between the dimeric intermediates. Moreover, mature caspase-3 fails to process its own precursor. Thus, despite a marked difference in the generation of active intermediates, the activation of initiator and effector caspases shares the features of interdimer cleavage and lack of self-amplification. These features may be important in preventing accidental cell death.