Defining the core apoptosis pathway in the mosquito disease vector Aedes aegypti: the roles of iap1, ark, dronc, and effector caspases

To date, our knowledge of apoptosis regulation in insects comes almost exclusively from the model organism Drosophila melanogaster. In contrast, despite the identification of numerous genes that are presumed to regulate apoptosis in other insects based on sequence homology, little has been done to examine the molecular pathways that regulate apoptosis in other insects, including medically important disease vectors. In D. melanogaster, the core apoptosis pathway consists of the caspase negative regulator DIAP1, IAP antagonists, the initiator caspase Dronc and its activating protein Ark, and the effector caspase DrICE. Here we have studied the functions of several genes from the mosquito disease vector Aedes aegypti that share homology with the core apoptosis genes in D. melanogaster. Silencing of the iap1 gene in the A. aegypti cell line Aag2 caused spontaneous apoptosis, indicating that IAP1 plays a role in cell survival similar to that of DIAP1. Silencing A. aegypti ark or dronc completely inhibited apoptosis triggered by several different apoptotic stimuli. However, individual silencing of the effector caspases CASPS7 or CASPS8, which are the closest relatives to DrICE, only partially inhibited apoptosis, and silencing both CASPS7 and CASPS8 together did not have a significant additional effect. Our results suggest that the core pathway that regulates apoptosis in A. aegypti is similar to that of D. melanogaster, but that more than one effector caspase is involved in apoptosis in A. aegypti. This is interesting in light of the fact that the caspase family has expanded in mosquitoes compared to D. melanogaster.     

Degradation of DIAP1 by the N-end rule pathway is essential for regulating apoptosis

Some members of the inhibitor of apoptosis (IAP) protein family block apoptosis by binding to and neutralizing active caspases. We recently demonstrated that a physical association between IAP and caspases alone is insufficient to regulate caspases in vivo and that an additional level of control is provided by IAP-mediated ubiquitination of both itself and the associated caspases. Here we show that Drosophila IAP 1 (DIAP1) is degraded by the 'N-end rule' pathway and that this process is indispensable for regulating apoptosis. Caspase-mediated cleavage of DIAP1 at position 20 converts the more stable pro-N-degron of DIAP1 into the highly unstable, Asn-bearing, DIAP1 N-degron of the N-end rule degradation pathway. Thus, DIAP1 represents the first known metazoan substrate of the N-end rule pathway that is targeted for degradation through its amino-terminal Asn residue. We demonstrate that the N-end rule pathway is required for regulation of apoptosis induced by Reaper and Hid expression in the Drosophila melanogaster eye. Our data suggest that DIAP1 instability, mediated through caspase activity and subsequent exposure of the N-end rule pathway, is essential for suppression of apoptosis. We suggest that DIAP1 safeguards cell viability through the coordinated mutual destruction of itself and associated active caspases.     

IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms

Some members of the inhibitor of apoptosis (IAP) family suppress apoptosis by neutralizing caspases. The current model suggests that all caspase-regulatory IAPs function as direct enzyme inhibitors, blocking effector caspases by binding to their catalytically active pockets. Here we show that IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms. Whereas XIAP binds directly to the active-site pockets of effector caspases, we find that regulation of effector caspases by Drosophila IAP1 (DIAP1) requires an evolutionarily conserved IAP-binding motif (IBM) at the neo-amino terminus of the large caspase subunit. Remarkably, unlike XIAP, DIAP1-sequestered effector caspases remain catalytically active, suggesting that DIAP1 does not function as a bona fide enzyme inhibitor. Moreover, we demonstrate that the mammalian IAP c-IAP1 interacts with caspase-7 in an exclusively IBM-dependent, but active site pocket-independent, manner that is mechanistically similar to DIAP1. The importance of IBM-mediated regulation of effector-caspases in vivo is substantiated by the enhanced apoptotic potency of IBM-mutant versions of drICE, DCP-1 and caspase-7.     

Flock house virus induces apoptosis by depletion of Drosophila inhibitor-of-apoptosis protein DIAP1

The molecular mechanisms by which RNA viruses induce apoptosis and apoptosis-associated pathology are not fully understood. Here we show that flock house virus (FHV), one of the simplest RNA viruses (family, Nodaviridae), induces robust apoptosis of permissive Drosophila Line-1 (DL-1) cells. To define the pathway by which FHV triggers apoptosis in this model invertebrate system, we investigated the potential role of Drosophila apoptotic effectors during infection. Suggesting the involvement of host caspases, the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluromethylketone (z-VAD-fmk) prevented FHV-induced cytopathology and prolonged cell survival. RNA interference-mediated ablation of the principal Drosophila effector caspase DrICE or its upstream initiator caspase DRONC prevented FHV-induced apoptosis and demonstrated direct participation of this intrinsic caspase pathway. Prior to the FHV-induced activation of DrICE, the intracellular level of inhibitor-of-apoptosis (IAP) protein DIAP1, the principal caspase regulator in Drosophila melanogaster, was dramatically reduced. DIAP1 was depleted despite z-VAD-fmk-mediated caspase inhibition during infection, suggesting that the loss of DIAP1 was caused by an upstream FHV-induced signal. The RNA interference-mediated knockdown of DIAP1 caused rapid and uniform apoptosis of DL-1 cells and thus indicated that DIAP1 depletion is sufficient to trigger apoptosis. Confirming this conclusion, the elevation of intracellular DIAP1 levels in stable diap1-transfected cells blocked caspase activation and prevented FHV-induced apoptosis. Collectively, our findings suggest that DIAP1 is a critical sensor of virus infection, which upon virus-signaled depletion relieves caspase inhibition, which subsequently executes apoptotic death. Thus, our study supports the hypothesis that altering the level or the activity of cellular IAP proteins is a general mechanism by which RNA viruses trigger apoptosis.     

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.     

The Drosophila inhibitor of apoptosis (IAP) DIAP2 is dispensable for cell survival, required for the innate immune response to gram-negative bacterial infection, and can be negatively regulated by the reaper/hid/grim family of IAP-binding apoptosis inducers

Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeat domains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. We report the generation of diap2 null mutants. These flies are viable and show no defects in developmental or stress-induced apoptosis. Instead, DIAP2 is required for the innate immune response to Gram-negative bacterial infection. DIAP2 promotes cytoplasmic cleavage and nuclear translocation of the NF-kappaB homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways.     

Hid,Rpr and Grim negatively regulate DIAP1 levels through distinct mechanisms

Inhibitor of apoptosis (IAP) proteins suppress apoptosis and inhibit caspases. Several IAPs also function as ubiquitin-protein ligases. Regulators of IAP auto-ubiquitination, and thus IAP levels, have yet to be identified. Here we show that Head involution defective (Hid), Reaper (Rpr) and Grim downregulate Drosophila melanogaster IAP1 (DIAP) protein levels. Hid stimulates DIAP1 polyubiquitination and degradation. In contrast to Hid, Rpr and Grim can downregulate DIAP1 through mechanisms that do not require DIAP1 function as a ubiquitin-protein ligase. Observations with Grim suggest that one mechanism by which these proteins produce a relative decrease in DIAP1 levels is to promote a general suppression of protein translation. These observations define two mechanisms through which DIAP1 ubiquitination controls cell death: first, increased ubiquitination promotes degradation directly; second, a decrease in global protein synthesis results in a differential loss of short-lived proteins such as DIAP1. Because loss of DIAP1 is sufficient to promote caspase activation, these mechanisms should promote apoptosis.     

Drosophila Omi, a mitochondrial-localized IAP antagonist and proapoptotic serine protease

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.     

DIAP2 functions as a mechanism-based regulator of drICE that contributes to the caspase activity threshold in living cells

In addition to their well-known function in apoptosis, caspases are also important in several nonapoptotic processes. How caspase activity is restrained and shut down under such nonapoptotic conditions remains unknown. Here, we show that Drosophila melanogaster inhibitor of apoptosis protein 2 (DIAP2) controls the level of caspase activity in living cells. Animals that lack DIAP2 have higher levels of drICE activity. Although diap2-deficient cells remain viable, they are sensitized to apoptosis following treatment with sublethal doses of x-ray irradiation. We find that DIAP2 regulates the effector caspase drICE through a mechanism that resembles the one of the caspase inhibitor p35. As for p35, cleavage of DIAP2 is required for caspase inhibition. Our data suggest that DIAP2 forms a covalent adduct with the catalytic machinery of drICE. In addition, DIAP2 also requires a functional RING finger domain to block cell death and target drICE for ubiquitylation. Because DIAP2 efficiently interacts with drICE, our data suggest that DIAP2 controls drICE in its apoptotic and nonapoptotic roles.     

Jafrac2 is an IAP antagonist that promotes cell death by liberating Dronc from DIAP1

Members of the Inhibitor of Apoptosis Protein (IAP) family are essential for cell survival in Drosophila and appear to neutralize the cell death machinery by binding to and ubiquitylating pro-apoptotic caspases. Cell death is triggered when "Reaper-like" proteins bind to IAPs and liberate caspases from IAPs. We have identified the thioredoxin peroxidase Jafrac2 as an IAP-interacting protein in Drosophila cells that harbours a conserved N-terminal IAP-binding motif. In healthy cells, Jafrac2 resides in the endoplasmic reticulum but is rapidly released into the cytosol following induction of apoptosis. Mature Jafrac2 interacts genetically and biochemically with DIAP1 and promotes cell death in tissue culture cells and the Drosophila developing eye. In common with Rpr, Jafrac2-mediated cell death is contingent on DIAP1 binding because mutations that abolish the Jafrac2-DIAP1 interaction suppress the eye phenotype caused by Jafrac2 expression. We show that Jafrac2 displaces Dronc from DIAP1 by competing with Dronc for the binding of DIAP1, consistent with the idea that Jafrac2 triggers cell death by liberating Dronc from DIAP1-mediated inhibition.     

Pro-apoptotic cell death genes, <Emphasis Type="Italic">hid</Emphasis> and <Emphasis Type="Italic">reaper</Emphasis>, from the tephritid pest species, <Emphasis Type="Italic">Anastrepha suspensa</Emphasis>

Pro-apoptotic proteins from the reaper, hid, grim (RHG) family are primary regulators of programmed cell death in Drosophila due to their antagonistic effect on inhibitor of apoptosis (IAP) proteins, thereby releasing IAP-inhibition of caspases that effect apoptosis. Using a degenerate PCR approach to conserved domains from the 12 Drosophila species, we have identified the first reaper and hid orthologs from a tephritid, the Caribfly Anastrepha suspensa. As-hid is the first identified non-drosophilid homolog of hid, and As-rpr is the second non-drosophilid rpr homolog. Both genes share more than 50% amino acid sequence identity with their Drosophila homologs, suggesting that insect pro-apoptotic peptides may be more conserved than previously anticipated. Importantly, both genes encode the conserved IBM and GH3 motifs that are key for IAP-inhibition and mitochondrial localization. Functional verification of both genes as cell death effectors was demonstrated by cell death assays in A. suspensa embryonic cell culture, as well as in heterologous Drosophila melanogaster S2 cells. Notably, heterologous cell death activity was found to be higher for Anastrepha genes than their Drosophila counterparts. In common with the Drosophila cognates, As-hid and As-rpr negatively regulated the Drosophila inhibitor of apoptosis (DIAP1) gene to promote apoptosis, and both genes when used together effected increased cell death activity, indicating a co-operative function for As-hid and As-rpr. We show that these tephritid cell death genes are functional and potent as cell death effectors, and could be used to design improved transgenic lethality systems for insect population control.     

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.     

Dissection of DIAP1 functional domains via a mutant replacement strategy

Inhibitor of apoptosis proteins (IAPs) act as endogenous inhibitors of active caspases. Drosophila IAP1 (DIAP1) activity is required to keep cells from undergoing apoptosis. The central cell death regulators Reaper and Hid induce apoptosis very rapidly by inhibiting DIAP1 function. We have developed a system for replacing endogenous DIAP1 with mutant forms of the protein, allowing us to examine the roles of various domains of the protein in living and dying cells. We found that DIAP1 is cleaved by a caspase early after the initiation of apoptosis. This cleavage is required for DIAP1 degradation, but Rpr and Hid can still initiate apoptosis in the absence of cleavage. The cleavage of DIAP1 promotes DIAP1 degradation in a manner dependent on the function of the ubiquitin ligase function of the DIAP1 ring domain. This ring domain function is required for Hid-induced apoptosis. We propose a model that synthesizes our data with those of other laboratories and provide a consistent model for DIAP1 function in living and dying cells.     

IAP-antagonists exhibit non-redundant modes of action through differential DIAP1 binding

The Drosophila inhibitor of apoptosis protein DIAP1 ensures cell viability by directly inhibiting caspases. In cells destined to die this IAP-mediated inhibition of caspases is overcome by IAP-antagonists. Genetic evidence indicates that IAP-antagonists are non-equivalent and function synergistically to promote apoptosis. Here we provide biochemical evidence for the non-equivalent mode of action of Reaper, Grim, Hid and Jafrac2. We find that these IAP-antagonists display differential and selective binding to specific DIAP1 BIR domains. Consistently, we show that each DIAP1 BIR region associates with distinct caspases. The differential DIAP1 BIR interaction seen both between initiator and effector caspases and within IAP-antagonist family members suggests that different IAP-antagonists inhibit distinct caspases from interacting with DIAP1. Surprisingly, we also find that the caspase-binding residues of XIAP predicted to be strictly conserved in caspase-binding IAPs, are absent in DIAP1. In contrast to XIAP, residues C-terminal to the DIAP1 BIR1 domain are indispensable for caspase association. Our studies on DIAP1 and caspases expose significant differences between DIAP1 and XIAP suggesting that DIAP1 and XIAP inhibit caspases in different ways.     

Identification of mammalian mitochondrial proteins that interact with IAPs via N-terminal IAP binding motifs

Direct IAP binding protein with low pI/second mitochondrial activator of caspases, HtrA2/Omi and GstPT/eRF3 are mammalian proteins that bind via N-terminal inhibitor of apoptosis protein (IAP) binding motifs (IBMs) to the baculoviral IAP repeat (BIR) domains of IAPs. These interactions can prevent IAPs from inhibiting caspases, or displace active caspases, thereby promoting cell death. We have identified several additional potential IAP antagonists, including glutamate dehydrogenase (GdH), Nipsnap 3 and 4, CLPX, leucine-rich pentatricopeptide repeat motif-containing protein and 3-hydroxyisobutyrate dehydrogenase. All are mitochondrial proteins from which N-terminal import sequences are removed generating N-terminal IBMs. Whereas most of these proteins have alanine at the N-terminal position, as observed for previously described antagonists, GdH has an N-terminal serine residue that is essential for X-linked IAP (XIAP) interaction. These newly described IAP binding proteins interact with XIAP mainly via BIR2, with binding eliminated or significantly reduced by a single point mutation (D214S) within this domain. Through this interaction, many are able to antagonise XIAP inhibition of caspase 3 in vitro.     

Drosophila IKK-related kinase regulates nonapoptotic function of caspases via degradation of IAPs

Caspase activation has been extensively studied in the context of apoptosis. However, caspases also control other cellular functions, although the mechanisms regulating caspases in nonapoptotic contexts remain obscure. Drosophila IAP1 (DIAP1) is an endogenous caspase inhibitor that is crucial for regulating cell death during development. Here we describe Drosophila IKK-related kinase (DmIKKvarepsilon) as a regulator of caspase activation in a nonapoptotic context. We show that DmIKKvarepsilon promotes degradation of DIAP1 through direct phosphorylation. Knockdown of DmIKKvarepsilon in the proneural clusters of the wing imaginal disc, in which nonapoptotic caspase activity is required for proper sensory organ precursor (SOP) development, stabilizes endogenous DIAP1 and affects Drosophila SOP development. Our results demonstrate that DmIKKvarepsilon is a determinant of DIAP1 protein levels and that it establishes the threshold of activity required for the execution of nonapoptotic caspase functions.     

Systematic in vivo RNAi analysis of putative components of the Drosophila cell death machinery

Despite the identification of numerous key players of the cell death machinery, little is known about their physiological role. Using RNA interference (RNAi) in vivo, we have studied the requirement of all Drosophila caspases and caspase-adaptors in different paradigms of apoptosis. Of the seven caspases, Dronc, drICE, Strica and Decay are rate limiting for apoptosis. Surprisingly, Hid-mediated apoptosis requires a broader range of caspases than apoptosis initiated by loss of the caspase inhibitor DIAP1, suggesting that Hid causes apoptosis not only by antagonizing DIAP1 but also by activating DIAP1-independent caspase cascades. While Hid killing requires Strica, Decay, Dronc/Dark and drICE, apoptosis triggered by DIAP1 depletion merely relied upon Dronc/Dark and drICE. Furthermore, we found that overexpression of DIAP2 can rescue diap1-RNAi-mediated apoptosis, suggesting that DIAP2 regulates caspases directly. Consistently, we show that DIAP2 binds active drICE. Since DIAP2 associates with Hid, we propose a model whereby Hid co-ordinately targets both DIAP1 and DIAP2 to unleash drICE.     

The vitelline membranes of Aedes aegypti and Drosophila melanogaster: A comparative review

Summary

The insect eggshell provides a model system for the study of gene regulation because several proteins are synthesized in an ordered spatial and temporal pattern within a single tissue, the follicular epithelium. Progress is being made towards an understanding of Aedes aegypti and Drosophila melanogaster vitelline membrane formation. The vitelline membrane is the innermost layer of the eggshells of A. aegypti and D. melanogaster. Genes encoding three A. aegypti and four D. melanogaster vitelline membrane proteins have been cloned and sequenced. Significant similarity is observed between the A. aegypti and D. melanogaster vitelline membrane genes. Both families contain highly conserved regions of 34 and 38 amino acids in A. aegypti and D. melanogaster respectively. The protein composition of the two families are both rich in proline and alanine, but differ in their serine and histidine compositions. The regulation of vitelline membrane gene expression in A. aegypti and D. melanogaster is compared.     

The insect caspases

Developmental and tissue homeostasis is a delicate balance between cell proliferation and cell death. The activation of caspases, a conserved family of cysteine proteases, is a main event in the initiation and execution of programmed cell death. While caspases have been characterized from many organisms, comparatively little is known about insect caspases. In Drosophila melanogaster, seven caspases have been characterized; three initiators and four effectors. In mosquitoes, several putative caspases have been identified in the genomes of Aedes aegypti and Anopheles gambiae. A small number of caspases have been identified in the Lepidoptera, the flour beetle, Tribolium castaneum, and the pea aphid, Acyrthosiphon pisum. The availability of new insect genome sequences will provide a unique opportunity to examine the caspase family across an evolutionarily diverse phylum and will provide valuable insights into their function and regulation.     

IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis

XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.