InteractiveFly: GeneBrief

Death-associated inhibitor of apoptosis 2: Biological Overview | References

Gene name - Death-associated inhibitor of apoptosis 2

Synonyms -

Cytological map position - 52D2-52D2

Function - signaling

Keywords - anti-apoptosis, antimicrobial humoral response

Symbol - Diap2

FlyBase ID: FBgn0015247

Genetic map position - 2R:11,819,675..11,822,330 [-]

Classification - Baculoviral inhibition of apoptosis protein repeat domain

Cellular location - cytoplasmic

NCBI link: EntrezGene

Diap2 orthologs: Biolitmine
Recent literature
Guntermann, S., Fraser, B., Hazes, B. and Foley, E. (2015). Independent proteolytic activities control the stability and size of Drosophila Inhibitor of apoptosis 2 Protein. J Innate Immun [Epub ahead of print]. PubMed ID: 25968339
The Drosophila immune deficiency pathway defends many bacterial pathogens and bears striking molecular similarities to the mammalian tumor necrosis factor signal transduction pathway. Orthologous inhibitors of apoptosis ubiquitin ligases act at a proximal stage of both responses to coordinate the assembly of signal transduction platforms that shape host immune responses. This study examined the molecular basis for inhibitor of apoptosis 2 protein regulation in the immune deficiency pathway. These studies identified two distinct proteolytic events that determine the stability and composition of cellular inhibitor of apoptosis 2 protein pools. Apoptotic caspase activity cleaves inhibitor of apoptosis 2 at an N-terminal aspartate to generate a truncated protein that retains the ability to interact with immune deficiency pathway members. It was also showed that a C-terminal ubiquitin ligase activity within inhibitor of apoptosis 2 directs the proteasomal destruction of full-length and truncated inhibitor of apoptosis 2 isoforms. These studies add to the appreciation of the regulation of innate immunity and suggest potential links between apoptotic caspases and innate defenses.
Aalto, A. L., Mohan, A. K., Schwintzer, L., Kupka, S., Kietz, C., Walczak, H., Broemer, M. and Meinander, A. (2019). M1-linked ubiquitination by LUBEL is required for inflammatory responses to oral infection in Drosophila. Cell Death Differ 26(5): 860-876. PubMed ID: 30026495
Post-translational modifications such as ubiquitination play a key role in regulation of inflammatory nuclear factor-kappaB (NF-kappaB) signalling. The Drosophila IkappaB kinase gamma (IKKgamma) Kenny is a central regulator of the Drosophila Imd pathway responsible for activation of the NF-kappaB Relish. This study found that Drosophila E3 ligase and HOIL-1L interacting protein (HOIP) orthologue linear ubiquitin E3 ligase (LUBEL) catalyses formation of M1-linked linear ubiquitin (M1-Ub) chains in flies in a signal-dependent manner upon bacterial infection. Upon activation of the Imd pathway, LUBEL modifies Kenny with M1-Ub chains. Interestingly, the LUBEL-mediated M1-Ub chains seem to be targeted both directly to Kenny and to K63-linked ubiquitin chains conjugated to Kenny by DIAP2. This suggests that DIAP2 and LUBEL work together to promote Kenny-mediated activation of Relish. LUBEL-mediated M1-Ub chain formation is required for flies to survive oral infection with Gram-negative bacteria, for activation of Relish-mediated expression of antimicrobial peptide genes and for pathogen clearance during oral infection. Interestingly, LUBEL is not required for mounting an immune response against systemic infection, as Relish-mediated antimicrobial peptide genes can be expressed in the absence of LUBEL during septic injury. Finally, transgenic induction of LUBEL-mediated M1-Ub drives expression of antimicrobial peptide genes and hyperplasia in the midgut in the absence of infection. This suggests that M1-Ub chains are important for Imd signalling and immune responses in the intestinal epithelia, and that enhanced M1-Ub chain formation is able to drive chronic intestinal inflammation in flies.
Kietz, C., Mohan, A. K., Pollari, V., Tuominen, I. E., Ribeiro, P. S., Meier, P. and Meinander, A. (2021). Drice restrains Diap2-mediated inflammatory signalling and intestinal inflammation. Cell Death Differ. PubMed ID: 34262145
The Drosophila IAP protein, Diap2, is a key mediator of NF-κB signalling and innate immune responses. Diap2 is required for both local immune activation, taking place in the epithelial cells of the gut and trachea, and for mounting systemic immune responses in the cells of the fat body. This study has found that transgenic expression of Diap2 leads to a spontaneous induction of NF-κB target genes, inducing chronic inflammation in the Drosophila midgut, but not in the fat body. Drice is a Drosophila effector caspase known to interact and form a stable complex with Diap2. This complex formation was found to induce its subsequent degradation, thereby regulating the amount of Diap2 driving NF-κB signalling in the intestine. Concordantly, loss of Drice activity leads to accumulation of Diap2 and to chronic intestinal inflammation. Interestingly, Drice does not interfere with pathogen-induced signalling, suggesting that it protects from immune responses induced by resident microbes. Accordingly, no inflammation was detected in transgenic Diap2 flies and Drice-mutant flies reared in axenic conditions. Hence, this study shows that Drice, by restraining Diap2, halts unwanted inflammatory signalling in the intestine.


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. This paper reports 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 IAP-binding motif (IBM)-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways (Huh, 2007).

The inhibitor of apoptosis (IAP) family proteins contain one or more repeats of an ~70-amino acid motif known as a baculovirus IAP repeat (BIR), which mediates interactions with multiple death activators and plays an essential role in the ability of these proteins to inhibit cell death. IAPs also contain a C-terminal RING E3 ubiquitin ligase domain that can target bound proteins, as well as the IAP itself, for ubiquitination and in some cases degradation. The Drosophila genome encodes two BIR and RING domain-containing IAP family members, DIAP1 and DIAP2, and ectopic expression of either protein inhibits apoptosis (Hay, 1995; Hay, 2000; Leulier, 2006b). DIAP1 is required continuously in many cells to inhibit the apical caspase Dronc and effector caspases activated by Dronc, such as Drice. Critical interactions between caspases and DIAP1 are mediated by a surface groove within each DIAP1 BIR domain and short IAP-binding motifs (IBM) present in Dronc or Drice. Apoptosis in the fly can be induced by expression of proteins such as Reaper, Hid, Grim, Sickle, and Jafrac2 (the RHG proteins). Each of these proteins contains an N-terminal IBM that mediates competitive binding to DIAP1 through the same BIR surface grooves that are required for DIAP1-caspase interactions. RHG proteins can also promote ubiquitin-dependent degradation of DIAP1. Both activities have the effect of liberating active caspases, resulting in apoptosis. IAPs that inhibit apoptosis, as well as inhibitory RHG counterparts, are also found in mammals (Huh, 2007).

Several observations have suggested that DIAP2 might also be an important apoptosis inhibitor. DIAP2 can bind Rpr and Hid (Vucic, 1997; Vucic, 1998; Leulier, 2006b; Doumanis, 2001) and the caspases Drice (Leulier, 2006b) and Strica (Doumanis, 2001). Overexpression of DIAP2 can also inhibit Rpr- and Hid-dependent apoptosis, developmental apoptosis in the eye (Hay, 1995), as well as apoptosis associated with decreased levels of diap1 (Leulier, 2006b). In addition, RNAi-mediated knockdown of DIAP2 in the S2 cell line has been reported to result in increased susceptibility to stress-induced apoptosis (Zimmermann, 2001). RNAi of diap2 in larvae and pupae has generated conflicting results. One group reported no effect of heat shock-induced expression of diap2 dsRNA on developmental cell death or viability (Yin, 2004), and second group reported organismal lethality in response to ubiquitous diap2 dsRNA expression (Kleino, 2005). Finally, several RNAi-based studies in S2 cells have also provided evidence that DIAP2 is required for the innate immune response to Gram-negative bacteria infection (Kleino, 2005; Gesellchen, 2005). However, these studies came to conflicting conclusions regarding the site of action for DIAP2 and involved primarily use of a single long time immortalized cell line (Huh, 2007).

Activation of apoptosis and the innate immune response both require tight control, and deregulation can lead to cancer, neurodegenerative diseases, immunodeficiency, or chronic inflammation. Molecular links between apoptotic and immune signaling pathways may exist, and DIAP2 is an interesting candidate protein to act as a point of convergence. Questions regarding the role of DIAP2 as a cell death and/or immune regulator can best be approached through the characterization of deletion mutant animals. In contrast with previous work, this study shows that diap2 mutants are viable and do not show increased sensitivity to a number of different cell death stimuli. Instead, DIAP2 is required for immune deficiency signaling in response to Gram-negative bacteria infection. DIAP2 promotes but is not absolutely essential for Relish cleavage. Nonetheless, loss of DIAP2 is associated with a profound defect in Relish nuclear translocation and antimicrobial peptide (AMP) expression. Together, these results suggest that DIAP2 regulates Relish function at several points. Interestingly, DIAP2 levels could be dramatically reduced in cells expressing the apoptosis inducers Rpr or Hid, suggesting an hypothesis in which DIAP2 acts as a point of convergence between immune and apoptotic signaling. Binding of death-inducing RHG proteins may act as a safety device to prevent unwanted chronic inflammation in the context in which massive cell death occurs during development or in response to environmental stress. Other IBM proteins may regulate DIAP2-dependent immune responses in other contexts (Huh, 2007).

DIAP1 and DIAP2 both inhibit apoptosis when overexpressed, and DIAP1 is essential for the survival of many cells. This study has shown that endogenous diap2 does not function as a major regulator of apoptosis. DIAP2 expression does not inhibit the activity of multiple caspases, including the key apoptosis activators Drice and Dronc. In addition, animals in which diap2 is deleted are viable, healthy, fertile, and as resistant as wild type animals to a number of stresses. The fact that DIAP2 can inhibit apoptosis when overexpressed probably reflects, at least in part, its ability to sequester and/or degrade apoptosis inducers that also target DIAP1, such as the RHG proteins. This study shows that DIAP2, perhaps functioning as a E3 ubiquitin ligase, is required for the IMD-mediated humoral response to Gram-negative bacterial infection, with DIAP2 being required for normal levels of Relish cleavage and nuclear translocation. DIAP2 may also function at other steps in the IMD pathway, although it is not absolutely required once Relish has undergone cleavage, at least when the cleaved version of Relish is expressed directly. Finally, modest but significant increases are seen in the immune response when the endogenous DIAP2 genetic dosage is increased, and a strong down-regulation of DIAP2 levels is seen in the presence of the apoptosis inducers Rpr and Hid. These observations suggest models in which IMD signaling is sensitive to the levels of DIAP2, and IBM-containing proteins regulate DIAP2 function during apoptosis and perhaps other contexts, through interactions with its BIR domains (Huh, 2007).

The observations, derived from in vivo studies of deletion mutants, stand in contrast to several recent reports, in which down-regulation of diap2 was brought about by using long dsRNA to induce RNAi-dependent degradation of diap2 mRNA. RNAi of diap2 was reported to sensitize S2 cells to several cell death activators (Zimmermann, 2002) and to result in pupal lethality when expressed ubiquitously (Kleino, 2005). Differences between these results and the current study are most simply explained as a result of RNAi off-targeting of unknown transcripts required for cell health and organismal viability in the earlier works. The ~500-bp dsRNAs used in the above-mentioned studies have the potential to be diced into many different 21-bp fragments for loading into the RISC complex and subsequent use as guide sequences. A number of recent reports have shown that off-targeting is a frequent occurrence and can result in the production of toxic phenotypes. The basis for this has been made clear through experiments demonstrating that a single RISC-bound guide sequence can target many mRNA sequences, which need be only partially complementary to the guide strand (Huh, 2007).

Two groups, again using long dsRNA to target diap2 in S2 cells, have also recently reported that diap2 is required for the IMD response. One group concluded that diap2 acted upstream of or at the level of TAK1 (Gesellchen, 2005). This conclusion was based, in part, on the observation that reduction of diap2 resulted in decreased expression of the dTAK1- and JNK-dependent early response gene puckered following immune challenge. In contrast, this study found that expression of puckered, and a second early response gene, CG13482, were induced normally in animal mutants for diap2, making this site of action unlikely. How can these different observations be reconciled? One possibility is that differences in the systems are important. This study measured immune responses in the context of the entire organism. In doing this the observations reflect the summed action of (and potentially cross-talk between) multiple cell types and signaling pathways. In contrast, cell line-based studies are, by their nature, much more focused. Thus, it seems not unreasonable to postulate that while Tak-dependent activation of JNK and/or JNK target genes may be sensitive to levels of diap2 in one cell type, a different pathway may dominate in the intact organism. Alternatively, the effects of RNAi-mediated knockdown of diap2 on puckered expression in S2 cells may again reflect off-targeting of unknown transcripts by long dsRNA (Huh, 2007).

In contrast to Gesellchen (2005), Kleino (2005) proposed DIAP2 functions in the IMD pathway downstream of Relish cleavage because that study saw no effect of diap2 RNAi on Relish cleavage in S2 cells. This difference from the current observations, in which loss of diap2 resulted in a significant decrease in Relish cleavage, may indicate differences in systems. However, it is thought more likely to reflect the difficulty in generating null mutant phenotypes for diap2 using RNAi. Gesellchen and Kleino did find that expression of pre-cleaved Relish was able to activate the immune response (and thus presumably able to translocate into the nucleus) in cells treated with diap2 dsRNA. Importantly, this study observed a similar ability of precleaved Relish to activate transcription of AMP in the complete absence of diap2 in flies (Huh, 2007).

What do these observations say about the site(s) of DIAP2 action? The current observations demonstrate that DIAP2 plays a role in bringing about Relish cleavage, which is required for Relish nuclear translocation and function (Stoven, 2000). Does DIAP2 function at other steps as well? The fact that loss of DIAP2 had a greater effect on nuclear translocation of endogenous Relish and AMP production than it did on Relish cleavage is consistent with the hypothesis that it may. Expression of pre-cleaved Relish bypassed the requirement for DIAP2 in AMP production, suggesting that DIAP2 is not absolutely required for Relish nuclear translocation or function. In addition, no nuclear translocation of DIAP2 was observed in response to immune challenge. However, these observations do not exclude the possibility that the levels of pre-cleaved Relish generated in the fat body by heat shock are so high that Relish enters the nucleus through unphysiological pathways. For example, perhaps overexpression of pre-cleaved Relish titrates out an inhibitor of Relish nuclear translocation/function that is normally removed by DIAP2. It is also worth considering that pre-cleaved Relish expressed from a transgene may not be the same as processed Relish generated in response to IMD signaling. For example, endogenous Relish is phosphorylated by the IKK complex in response to IMD signaling and cleaved by Dredd. Processed endogenous Relish (but perhaps not transgene-expressed pre-cleaved Relish) may exist as a part of a complex and/or interact with factors that inhibit nuclear import unless DIAP2 promotes their removal. These hypotheses can best be explored through the identification of proteins that bind endogenous cleaved Relish and DIAP2. Finally, what is the significance of DIAP2-independent cleavage of Relish? Perhaps DIAP2 is not absolutely essential, mechanistically, for Relish cleavage (for example, if it acts as an inhibitor of an inhibitor of cleavage, some Relish may escape the inhibitor and thus be cleaved). Alternatively, other related E3s such as DIAP1 may be able to partially substitute for DIAP2 function (at least when DIAP2 is completely missing), an issue that requires further exploration. Regardless, the observation that loss of DIAP2 prevents a significant fraction of Relish cleavage and that DIAP2 is required for Relish-dependent AMP expression demonstrates that DIAP2 is a critical regulator of Relish function (Huh, 2007).

How does DIAP2 regulate Relish cleavage? It is unlikely that DIAP2 functions as an inhibitor of the caspase Dredd since DIAP2 does not inhibit other tested caspases, and Dredd, like DIAP2, is required for activation of the IMD pathway. However, it is worth noting that several mammalian IAPs that inhibit apoptosis when overexpressed, cIAP1 and cIAP2, bind active caspases even though they do not function as caspase inhibitors (Eckelman, 2005). Binding of DIAP2 to Drice and Strica has been reported (Leulier, 2006b). Binding between DIAP2 and Dredd has not been reported (it is not clear it has been searched for). Thus, it remains possible that interactions between Dredd and DIAP2, perhaps involving ubiquitination by DIAP2, positively regulate Dredd activation, activity, or substrate targeting. Roles for E3 ligase activity of DIAP2 in the IMD pathway activation (upstream and/or downstream of Relish cleavage) are suggested by several observations. Expression of a RING domain point mutant version of diap2, diap27.4C472Y, in the diap2E151 mutant background failed to rescue Relish cleavage and IMD signaling. In contrast, expression of this same mutant protein in a wild type background suppressed the IMD response, suggesting dominant negative activity. In mammals, ubiquitination exerts degradation-dependent effects on immunity through removal of IkappaB and processing of NF-kappaB. In contrast, activation of Tak1 and the IKK complex require ubiquitination in degradation-independent roles. Drosophila counterparts of Tak1 and the IKK complex are also essential for Relish processing. However, several observations suggest that diap2 does not act at the level of dTAK1. First, as noted above, expression of puckered and CG13482, immediate transcriptional targets of the Tak1-JNK signaling module, are not affected in diap2 mutant flies. Second, ectopic expression or down-regulation of diap2 in the fly eye fails to influence Tak1-dependent cell killing, which is JNK-dependent. Recent observations suggest that activation of the IKK complex requires the Drosophila ubiquitin-carrier proteins Bendless (Ubc13) and dUEV1a, along with a yet to be identified E3 ubiquitin ligase (Zhou, 2005). Both Ubc13 (MAALTP) and dUEV1a (MANTSS) contain potential IBM motifs at their N termini. It will be interesting to determine whether DIAP2 binds one or both these proteins and participates in this process (Huh, 2007).

Careful control over the intensity and timing of an immune response is important, because hyperactivation of the immune system induces fitness costs with respect to other aspects of the organism life cycle such as fertility and longevity. Hyperactivation can also lead to the induction of tissue inflammation and cell death. In the context of these considerations, it is interesting that modest increases in the genetic dose of diap2 resulted in an enhanced immune response, whereas expression of cell death activators such as Rpr or Hid brought about DIAP2 removal. Rpr and Hid, as well as other members of the RHG family, are induced or released from a sequestering environment in response to stresses that can lead to cell death and/or tissue damage. It is speculated that in binding DIAP2 as well as DIAP1, these proteins accomplish two goals at once. They induce cell death through inhibition of DIAP1 and prevent activation of an immune response, which requires cleavage of Relish by the caspase Dredd, a process likely to be stimulated by loss of DIAP1 (a caspase inhibitor) and/or the activation of other caspases downstream of this loss. In other words, RHG protein binding to DIAP2 BIR domains may function in some contexts as a safety lock, down-regulating DIAP2 function, thereby preventing an inappropriate immune response in contexts in which there are high levels of cell death (perhaps during metamorphosis or larval fat body histolysis) or cell-damaging stress .This model is speculative, but it is testable and provides an explanation (perhaps partial) for the observation that unlike necrotic cell death, which often leads to inflammation, apoptotic cell death is characterized by a lack of inflammation, and sometimes even immunosuppression. Evidence consistent with such a model comes from the observation that activation of apoptosis effectors directly (by decreasing DIAP1), rather than through expression of RHG proteins (which would also down-regulate DIAP2), resulted in an increased immune response (Huh, 2007).

Related, alternative models can also be considered. For example, caspase cleavage exposes IBM-like motifs in many proteins. If this occurs in response to Dredd activation in the insect fat body, it could serve as a form of negative feedback, creating proteins that bind DIAP2 BIRs, displacing bound proteins required for the immune response, and/or promoting changes in DIAP2 localization or stability. Target genes activated by the immune response might play a similar role. RNAi in S2 cells of several transcripts encoding proteins with N-terminal sequences similar to those of known IAP-binding proteins or peptides results in increased immune activation, suggesting these as candidate negative regulators of DIAP2. IBM domain proteins might also play positive roles in immune regulation. In the context of this possibility, it is interesting to note that binding of the mammalian IBM protein Smac/Diablo promotes XIAP stabilization rather than degradation. The hypothesis that IBM proteins regulate DIAP2 activity, in its capacity as an essential component of the innate immune system, is speculative but is testable. Given that IBM domain proteins function as evolutionarily conserved regulators of cell death by displacing IAP-bound proteins and promoting IAP degradation and that DIAP2 is able to bind proteins with these same motifs, it would be surprising if similar regulatory mechanisms were not utilized in the immune system (Huh, 2007).

It is noted that recent report also described the characterization of Drosophila IAP2 mutant flies (Leulier, 2006b). That study placed DIAP2 function downstream or in parallel to Relish because diap2 mutant phenotypes were not rescued by ectopic expression of full-length Relish. However, since DIAP2 plays a significant role in promoting Relish cleavage (Huh, 2007), a failure of full-length Relish (which requires cleavage) to rescue immune responsiveness in diap2 mutants is not surprising (Huh, 2007).

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. This study shows that Drosophila 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. DIAP2 was found to regulate 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. The 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, these data suggest that DIAP2 controls drICE in its apoptotic and nonapoptotic roles (Ribeiro, 2007).

Caspases are best known for their role in executing apoptosis, however, they also play important signaling roles in nonapoptotic processes, such as regulation of actin dynamics, innate immunity, and cell proliferation, differentiation, and survival. Under such conditions, caspases are activated without killing the cell. Given the irreversible nature of caspase-mediated proteolysis, caspases' activation and activity is subject to complex regulation. After zymogen activation, caspase activity can be controlled by various cellular and viral inhibitors. Interestingly, the viral inhibitors CrmA and p35 neutralize caspases through a fundamentally different mechanism than cellular inhibitors such as the inhibitor of apoptosis (IAP) proteins (Salvesen, 2002). CrmA and p35 function as 'suicide substrates,' whereby cleavage of CrmA and p35 is required to block the caspase. Initially, CrmA and p35 are cleaved through a mechanism that resembles substrate hydrolysis. However, during the reaction, the protease's catalytic machinery is trapped via covalent linkage (Stennicke, 2006). This strategy is referred to as 'mechanism-based' inhibition because it relies on the caspase's catalytic property. So far, cellular-derived mechanism-based caspase inhibitors have not been identified (Ribeiro, 2007).

IAPs are defined by the presence of one to three copies of the baculovirus IAP repeat (BIR) domain, which functions as a protein interaction module. In addition, certain but not all IAPs also carry a C-terminal RING finger domain, which provides these molecules with E3 ubiquitin-protein ligase activity. In Drosophila, DIAP1-mediated inhibition of caspases is indispensable for cell survival. Mutations that abrogate the physical association of DIAP1 with the effector caspases drICE or DCP-1 or the initiator caspase Dronc cause spontaneous caspase activation and cell death (Ribeiro, 2007).

The involvement of the second Drosophila IAP (DIAP2) in regulating caspases is less clear. Overexpression studies suggest that DIAP2 can suppress cell death induced by the IAP antagonists reaper (Rpr) and head involution defective (Hid; Hay, 1995) and suppress apoptosis induced by diap1-RNAi (Leulier, 2006a). Because diap1-RNAi-mediated death is independent of IAP antagonists, this suggests that DIAP2 can neutralize caspases. Of the seven D. melanogaster caspases, DIAP2 binds only to the effector caspase drICE and the atypical caspase Strica (Doumanis, 2001). In contrast, DIAP1 exhibits much broader caspase selectivity because it inhibits Dronc, drICE, and DCP-1. Based on the domain architecture and sequence alignments, DIAP2 is the closest homologue to mammalian IAPs. In contrast, DIAP1 is more related to IAPs from insect viruses. Like the mammalian x-linked IAP (XIAP), DIAP2 carries three BIR domains and a C-terminal RING finger. XIAP blocks apoptosis by acting as a potent enzymatic inhibitor of caspase-3, -7, and -9 (Shi, 2002). Residues located immediately upstream of XIAP's BIR2 domain directly bind to the active site pockets of caspase-3 and -7, thereby obstructing substrate entry. Importantly, XIAP is not cleaved by caspases because it binds to the catalytic pockets of effector caspases in a reverse orientation. Thus, XIAP relies on allosteric mechanisms to block effector caspase activity. XIAP's ability to suppress effector caspases depends on the side chain of Asp148 and, to a lesser extent, Val146. It is predicted that Asp148 must be conserved in all IAPs that neutralize effector caspases. Consistently, Asp148 is conserved in cIAP1 and 2. Intriguingly, DIAP2 also shows homology to XIAP's Val146 and Asp148, as it carries equivalent residues at Val98 and Asp100 (Silke, 2001). In contrast, these residues are absent in DIAP1 (Ribeiro, 2007).

Of all the IAPs, XIAP appears to be the only IAP that potently inhibits caspases in vitro (Eckelman, 2006). Other IAPs, such as cIAP1 and 2, are poor inhibitors of caspases under these conditions. cIAP1 and 2 may rely on their E3 ligase activity to block caspases in vivo, making them caspase regulators rather than inhibitors. Although DIAP1 is a relatively good inhibitor of D. melanogaster caspases in vitro, DIAP1-caspase physical association alone is insufficient to neutralize caspases in the fly. In addition to binding, DIAP1 uses two independent mechanisms to regulate caspases. One relies on the E3 ubiquitin protein ligase activity of DIAP1's own RING finger, whereas the other, the 'N-end' rule, functions independently of this domain. The RING finger of DIAP1 is required to target the proform of the initiator caspase Dronc for ubiquitylation and inactivation. In contrast, active effector caspases seem to be neutralized through a mechanism that involves both the RING finger domain as well as the N-end rule degradation machinery that is recruited by DIAP1 (Ribeiro, 2007 and references therein).

In Drosophila, developmentally regulated apoptosis is induced by the IAP antagonists Rpr, Grim, and Hid. Embryos lacking rpr, grim, and hid are virtually devoid of apoptosis and die at the end of embryogenesis with accumulation of supernumeral cells. The current model suggests that Rpr, Grim, and Hid induce apoptosis by binding to the BIR domains of DIAP1, thereby liberating caspases from IAP inhibition. Furthermore, IAP antagonists also deplete DIAP1 protein levels by promoting its degradation and cause mitochondrial permeability. Although Rpr, Grim, and Hid efficiently antagonize DIAP1, they also associate with DIAP2 (Vucic, 1997; Vucic, 1998; Leulier, 2006b). This implies that DIAP2 contributes to the overall antiapoptotic threshold of a cell, a view that is supported by the notion that RNAi-mediated knockdown of DIAP2 sensitizes cultured cells to stress-induced death (Zimmermann, 2002). However, analyses using diap2 mutant flies have failed to expose any involvement of DIAP2 in regulating programmed cell death. Instead, they have shown that DIAP2 is required for NF kappaB activation during the innate immune response in D. melanogaster (Gesellchen, 2005; Kleino, 2005; Leulier, 2006b; Huh, 2007; Ribeiro, 2007 and references therein).

This study used the fluorescence resonance energy transfer (FRET)-based caspase-3 indicator SCAT3 to examine DIAP2's role in regulating caspase activity in vivo. Using diap2 mutant animals, it was found that these animals harbor significantly increased levels of drICE activity. Consistent with higher levels of active caspases, diap2 mutant cells are sensitized to apoptosis after exposure to sublethal doses of x-ray irradiation. Furthermore, DIAP2 tightly associates with the effector caspase drICE and DIAP2-mediated drICE inhibition requires DIAP2 cleavage at Asp100. The data are consistent with the notion that after cleavage, the caspase forms a covalent adduct with DIAP2, which results in the stabilization of the DIAP2-caspase complex. This mode of caspase binding is similar to that of p35 or CrmA. However, in addition to the mechanism-based trapping, DIAP2 also requires a functional RING finger domain to neutralize drICE-mediated cell death. Consistently, it was found that DIAP2 robustly ubiquitylates drICE in vivo (Ribeiro, 2007).

The data suggest that DIAP2 is a caspase regulator that contributes to the caspase activity threshold. Several lines of evidence support this view. First, loss of diap2 causes an increase in basal levels of caspase activity in vivo. Consistent with this increase in basal levels of caspase activity, diap2 mutant cells are sensitized to sublethal doses of x-ray irradiation, and, in tissue culture cells, depletion of DIAP2 by RNAi sensitizes cells to treatment with chemotherapeutic drugs (Zimmermann, 2002). Second, DIAP2 overexpression in the developing fly eye efficiently suppresses cell death triggered by diap1 RNAi. Importantly, diap1 RNAi causes spontaneous and unrestrained caspase activation and cell death that occurs independently of the action of IAP antagonists (Leulier, 2006a). The efficiency with which DIAP2 rescues the diap1 RNAi phenotype is highly reminiscent of that of p35. Both DIAP2 and p35 rescue the diap1 RNAi eye size and pigmentation to an apparent normal morphology but fail to restore the formation of bristles (Leulier, 2006a). The notion that DIAP2 expression phenocopies the expression of p35, in the absence of any involvement of IAP antagonists, suggests that DIAP2 can function as a direct caspase inhibitor for a p35-sensitive caspase. Third, DIAP2 physically interacts with the effector caspase drICE and suppresses drICE-mediated cell death. Fourth, endogenous DIAP2 is readily cleaved by caspases, which indicates that DIAP2 encounters caspases in vivo. Moreover, after induction of apoptosis, the timing and extent of DIAP2 cleavage is highly reminiscent to that of DIAP1, which suggests that cleavage of DIAP2 is a relatively early event during apoptosis. Finally, IAP antagonists bind to DIAP1 and 2 with similar efficiencies. Consistent with the view that the caspase inhibitory activity of both IAPs is antagonized by IAP antagonists, it was found that Hid blocks DIAP1 and DIAP2 from binding to drICE. Together, these data support a model in which DIAP1 and 2 coordinately control drICE, thereby blocking the amplification of the caspase cascade. Accordingly, DIAP2 contributes to the apoptotic threshold by regulating drICE. However, DIAP1 controls initiation of apoptosis, as it is the sole IAP that regulates the initiator caspase Dronc and, with it, the formation of the Dronc/Dark apoptosome. Ultimately, programmed cell death is induced when IAP antagonists such as Hid synchronously bind to DIAP1 and 2, allowing apoptosome formation and unhindered amplification of the caspase cascade (Ribeiro, 2007).

Although DIAP2 seems to be a bona fide regulator of drICE, its loss does not cause spontaneous apoptosis. This is most likely caused by its epistatic position in the caspase cascade and its restricted specificity for caspases (see DIAP2's epistatic position in the caspase cascade). Because DIAP2 exclusively regulates drICE and does not bind other caspases (Leulier, 2006b), its loss merely causes an increase in caspase activity, presumably because of a small amount of uninhibited active drICE. Under such conditions, drICE activation remains regulated because it requires proteolytic input from Dronc, which, in turn, is under the control of DIAP1. Although loss of DIAP2 results in sublethal levels of active caspases, RNAi-mediated depletion of DIAP1 causes strong, apoptosome-driven activation of downstream effector caspases, which leads to the amplification of the proteolytic signal and death of the cell. Under diap1 RNAi conditions, levels of endogenous DIAP2 seem to be insufficient to suppress the amount of active caspases generated. But, if the levels of DIAP2 are increased, it efficiently blocks diap1 RNAi-mediated cell death. However, when programmed cell death is triggered through the activation of IAP antagonists, both IAPs are targeted equally, thereby releasing the inhibition of the cell death machinery. Thus, controlled activation of caspases and cell survival are ensured by both DIAP1 and 2 (Ribeiro, 2007).

The notion that diap2 mutant animals have increased levels of caspase activity indicates that cells can generate and tolerate a certain amount of activated caspases without undergoing apoptosis. This is consistent with the notion that caspases fulfill important signaling functions independent of executing cell death. For instance, DmIKKepsilon activation was recently found to reduce DIAP1 protein levels, thereby allowing activation of sublethal levels of Dronc, which, in turn, is required for the proper development of sensory organ precursor cells (Kuranaga, 2006). Thus, modulating caspase activity in a selective, transient, and possibly spatially restricted manner is likely to be a widespread phenomenon that allows caspases to take part in signaling functions without jeopardizing cell viability. Concerted reduction of DIAP1 and 2 protein levels, or their controlled and selective inhibition, may therefore allow controlled activation of caspases and caspase-mediated signaling. The cellular concentration of IAPs would thereby set a cell type-specific threshold for caspase activation and activity. In DIAP2 mutants, however, cells are left in an unbalanced, sensitized state as they accumulate higher than normal levels of active caspases. Accordingly, diap2 mutant third instar larvae are sensitized to apoptosis when subjected to sublethal doses of x-ray irradiation. This sensitized state is only visualized by exposing diap2 mutant animals to sublethal insults. When strong proapoptotic stimuli are used, such as 40 Gy of x-ray irradiation (Huh, 2007) or overexpression of Rpr, Hid, and Grim, diap2 mutant animals display the same cell death phenotypes as WT counterparts (Huh, 2007). Under these strong proapoptotic conditions, IAP antagonists neutralize both IAPs, causing full activation and amplification of the proteolytic caspase cascade. Thus, the contribution of the sublethal amount of active caspases in diap2 mutants seems to be masked by the higher concentration of active caspases generated under such apoptotic conditions (Ribeiro, 2007).

This molecular characterization has uncovered an unexpected mechanism through which DIAP2 restrains the target enzyme drICE. Surprisingly, DIAP2 acts as a pseudosubstrate that, after cleavage, seems to trap the active caspase via a covalent linkage between DIAP2 and the catalytic machinery of drICE. Mutation of the DIAP2 caspase cleavage site abrogates its ability to bind drICE and suppress drICE-mediated cell death. It is unusual for proteins that regulate enzymes to use a mechanism-based strategy, and most types avoid the catalytic machinery by simply blocking the substrate cleft in a lock and key strategy. The cleavage of DIAP2 distinguishes it from all natural inhibitors with the exception of p35, CrmA, serpins, and α-macroglobulins. Serpins and p35 both require cleavage to function as protease inhibitors. p35 arrests proteolysis at the thioacyl intermediate stage, which results in a covalent adduct between p35 and the caspase. In particular, the cleavage site residue Asp87 of p35 links up with the catalytic cysteine of the caspase (Riedl, 2001). Thus, cleaved p35 locks the catalytic machinery of the caspase in a nonproductive inactive configuration. The exquisite sensitivity of the DIAP2-drICE complex to strong nucleophiles such as NH2OH or DTT is diagnostic of a stable thioester adduct (Riedl, 2001) between DIAP2 and drICE. Nevertheless, it remains possible that this complex could form because of a disulfide bridge between DIAP2 and the catalytic thiol of drICE. However, the requirement of DIAP2's Asp100 for drICE binding suggests that Asp100 is involved in forming a thioester covalent adduct with the caspase active site Cys. Ultimately, crystal structure or mass spectrometric analysis will be required to identify the chemical nature of this complex. However, the relatively harsh conditions required for sample preparations for mass spectrometric analysis combined with the labile nature of thiol esters will make it difficult to identify a fragment containing a covalent linkage between DIAP2 and drICE. Collectively, the data suggest that the DIAP2-drICE complex is stabilized by a covalent linkage (Ribeiro, 2007).

Despite the notion that p35 and DIAP2 both occupy and produce a covalent linkage with the active site of caspases, the modes of interaction and mechanism of inhibition are very different. Unlike p35, DIAP2 requires additional domains for caspase binding and inhibition. First, DIAP2 needs to associate with drICE through a bimodular interaction, whereby the BIR3 domain binds to the IBM of drICE and the 97-Ser-Val-Val-Asp-100 region of DIAP2 occupies the catalytic pocket of the caspase. Each motif on its own is insufficient for caspase binding as mutation of either the BIR3 or cleavage site interferes with complex formation. Second, physical interaction with drICE is not sufficient to block caspase activity. In addition, DIAP2 requires a functional RING finger domain to regulate drICE-mediated cell death. This is evident because a DIAP2 RING finger mutant fails to suppress cell death induced by RNAi-mediated depletion of DIAP1. Moreover, DIAP2 fails to suppress drICE in vitro. In this respect, DIAP2 functions as mechanism-based regulator (relying on the enzyme's catalytic property to trap it) rather than inhibitor. After capture, DIAP2 targets drICE for ubiquitylation (Ribeiro, 2007).

An important question is why DIAP2 does not function as an inhibitor like p35 if it also establishes a covalent linkage with the catalytic active site cysteine of the caspase. One possibility is that DIAP2 and p35 differ in their ability to protect the highly labile thioester bond from hydrolysis. This view is supported by the notion that the DIAP2-drICE interaction is significantly more sensitive to nucleophile attack than p35-drICE. Thus, in the situation of DIAP2, the thioester linkage with drICE may simply help to stabilize the caspase interaction, whereas in p35-drICE, it is used to irreversibly inhibit the protease. The more labile nature of the DIAP2-drICE complex may allow regulation, which cannot occur with p35. Moreover, p35 inhibits all effector caspases, whereas DIAP2 only regulates drICE. The observation that the nontarget caspase DCP-1 can also cleave DIAP2 but is not inhibited raises the possibility that DCP-1 controls the level of functional DIAP2 through proteolytic inactivation. Examples of such regulation are manifold and include the proteolytic inactivation of serpins by nontarget proteases. Because DIAP2 tightly interacts with drICE, it is likely to control its apoptotic and nonapoptotic functions (Ribeiro, 2007).

The Drosophila Inhibitor of Apoptosis Protein DIAP2 functions in innate immunity and is essential to resist gram-negative bacterial infection

The founding member of the inhibitor of apoptosis protein (IAP) family was originally identified as a cell death inhibitor. However, recent evidence suggests that IAPs are multifunctional signaling devices that influence diverse biological processes. To investigate the in vivo function of Drosophila melanogaster IAP2, diap2 null alleles were generated. diap2 mutant animals develop normally and are fully viable, suggesting that diap2 is dispensable for proper development. However, these animals were acutely sensitive to infection by gram-negative bacteria. In Drosophila, infection by gram-negative bacteria triggers the innate immune response by activating the immune deficiency (imd) signaling cascade, a NF-kappaB-dependent pathway that shares striking similarities with the pathway of mammalian tumor necrosis factor receptor 1 (TNFR1). diap2 mutant flies failed to activate NF-kappaB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumbed to bacterial infection. Genetic epistasis analysis places diap2 downstream of or in parallel to imd, Dredd, Tak1, and Relish. Therefore, DIAP2 functions in the host immune response to gram-negative bacteria. In contrast, it was found that the Drosophila TNFR-associated factor (Traf) family member Traf2 is dispensable in resistance to gram-negative bacterial infection. Taken together, genetic data identify DIAP2 as an essential component of the Imd signaling cascade, protecting the organism from infiltrating microbes (Leulier, 2006b).

The Drosophila innate immune response relies mainly on the differential expression of a variety of small peptides with antimicrobial activities. Depending on the infiltrating microbe, Drosophila selectively activates two distinct signaling pathways. While infections by fungi or gram-positive bacteria stimulate the Toll pathway, infection by gram-negative bacteria triggers the activation of the immune deficiency (Imd) signaling cascade. Activation of both Toll- and Imd signaling results in the activation of NF-kappaB-like transcription factors leading to the expression of specific sets of antimicrobial peptides. This study demonstrates through mutation analysis that DIAP2 plays a pivotal role in the Drosophila innate immune response (Leulier, 2006b).

In vivo, DIAP2 is indispensable for Imd-mediated expression of antibacterial peptide genes. Like known mutants of the Imd pathway, flies with a mutation in the diap2 gene fail to induce expression of Attacin-A, Cecropin-A1, Defensin, Diptericin, Drosocin and Metchikowin, and mount an efficient immune reaction in response to infection by Gram-negative bacteria. Consequently, diap2 mutant flies succumb to Gram-negative bacterial infection. In contrast, such flies mount a normal Toll-dependent immune response and are resistant to infection by fungi and Gram-positive bacteria. The diap2 null mutant phenotype, therefore, demonstrates that DIAP2 is an essential component of the Imd pathway. Thus, these data are consistent with recent RNAi studies that have implicated diap2 in the Imd pathway (Leulier, 2006b).

DIAP2 is a member of the evolutionarily conserved IAP protein family. IAPs are classified by the presence of the Baculovirus IAP Repeat (BIR) domain through which they interact with various 'client' proteins. Genetic analysis of the Drosophila IAP DIAP1 has provided some of the most compelling insights into the in vivo function of this protein family. DIAP1, the first and most extensively studied Drosophila IAP, is essential for cell survival and acts as a potent caspase inhibitor. Mutations that abrogate physical association of DIAP1 with caspases cause widespread and unrestrained caspase activation leading to cell and organismal death. In contrast to diap1, diap2 null-mutants do not show an apparent cell death phenotype and develop normally. This is unexpected because both these IAPs interact with caspases and IAP-antagonists with similar affinities. Moreover, when overexpressed, DIAP2 can rescue diap1-RNAi-mediated apoptosis, suggesting that DIAP2 can functionally substitute for DIAP1 in its ability to regulate caspases. Nevertheless, diap2 mutant animals do not show any apparent apoptosis related phenotypes during development. However, these animals appear to be sensitised to Reaper-mediated killing in the eye. The lack of any apparent gross developmental phenotype may be due to sufficiently high levels of DIAP1 protein that may thwart unscheduled caspase activation in response to loss of DIAP2. In this respect it is noteworthy that during embryonic development the levels of diap1 mRNA dramatically exceed those of diap2 (17 fold difference). Moreover, similarly to cIAP2 knock-out mice, where a cell death phenotype is revealed only after LPS challenge, phenotypic manifestation may only become apparent under certain conditions or in selective tissues. In agreement with this notion, RNAi-mediated depletion of DIAP2 has no effect on cell viability in unchallenged tissue culture cells, but significantly sensitizes S2 cells to stress-induced apoptosis (Leulier, 2006b).

Although IAPs have originally been identified as apoptosis inhibitors, recent evidence suggests that IAPs are multifunctional signalling devices that, depending on the protein they interact with, influence diverse biological processes. In this respect, it is noteworthy that IAPs also carry C-terminal RING finger domains providing them with E3 ubiquitin-protein ligase, and hence, signalling activity. Thus, in addition to inhibiting apoptosis, IAPs also fulfil functions that operate independently of their ability to control caspases and cell death. Therefore, BIR-containing proteins are more precisely referred to as BIRCs rather than IAPs. Consistent with the notion that BIRCs are multifunctional proteins, the mammalian c-IAP1 and c-IAP2 bind to caspases as well as RIP1 and TRAF320 2, two components of the TNF receptor signalling complex. c-IAP1 or c-IAP2, or both, can promote ubiquitylation and degradation of TRAF2, RIP1 and NF-kappaB kinase (IKKγ)/NF-kappaB essential modulator (NEMO). Hence, these BIRC proteins are thought to modulate the response to TNF. More recently, another BIRC protein was identified as a important regulator of innate immune surveillance in mammals. BIRC1e (NAIP5) was found to control the intracellular pathogen Legionella pneumophila, a Gram-negative microbe that causes severe bacterial pneumonia known as Legionnaires' disease. BIRC1e protects infected host macrophages by restricting intracellular replication of this pathogen (Leulier, 2006b).

The Drosophila BIRC protein DIAP2 is similarly required for innate immune responses and the resistance to Gram-negative bacterial infection. diap2 null mutants become highly susceptible to Gram-negative bacteria and fail to induce antibacterial peptide gene expression. Intriguingly, the Imd pathway, which is required for antibacterial peptide gene expression in response to Gram-negative microbes, shares significant similarities with the TNFR1 signalling cascade. The notion that the BIRC proteins c-IAP1, c-IAP2 and DIAP2 are core components of the TNFR1- and Imd-pathway, respectively, further reinforces the parallels between the mammalian TNFR1 pathway and the one of Imd of Drosophila, pointing to an evolutionary conservation of these pathways in NF-kappaB activation. Moreover, both pathways seem to rely on ubiquitin-mediated protein modifications. As in human cells, where activation of TAK1 and IKK requires the E2 ubiquitin-conjugating enzyme complex Ubc13/UEV1A, Drosophila Ubc13(Bendless)/UEV1A are similarly required for activating Tak1 and the Drosophila IKK complex. Moreover, recent RNAi data from cultured cells suggest that Drosophila Tab contributes to Imd signalling, although this still awaits in vivo validation. Therefore, similar to the TNFR1 pathway, ubiquitin-mediated protein modification is likely to activate the Tak1/Tab complex via Tab's ability to bind to Lys63-linked polyubiquitin chains, thereby recruiting Tak1 to activator platforms. In contrast to the E2 ubiquitin-conjugating enzyme complex, little is known about the nature of the E3 ubiquitin-ligase of the Imd pathway. While TRAF2 is crucial for Ubc13/UEV1A-mediated ubiquitylation in the mammalian TNFR1 pathway, it seems that for Imd signalling Traf2, the TRAF2/6 orthologue in flies, is not a critical component. Traf2 null mutation did not completely block NF-kappaB activation in Drosophila. Moreover, the data clearly indicate that Traf2 mutant flies are fully competent to mount an immune response that resists Gram-negative bacterial infection. Hence, Traf2 appears not to be essential for an effective Imd-mediated immune response. Since the Drosophila genome encodes at least three TRAF family members, it is possible that the loss of Traf2 function is complemented by other TRAF family members. Alternatively, other signalling pathways that bypass Traf2 to transmit the infection signal to NF-kappaB may exist in Drosophila. In agreement with this notion, RNAi-mediated knock-down of all three Drosophila TRAFs also did not abrogate Imd signalling in S2 cells. Thus, an E3 ubiquitin ligase different to, or in addition of Traf2 may be responsible for Imd signalling in Drosophila. Since DIAP2 carries a RING finger domain, it represents a likely candidate (Leulier, 2006b).

The genetic epistatic analysis places diap2 downstream or in parallel of imd, Dredd, Tak1 and Relish. Over-expression of imd, Dredd, Tak1 and Relish failed to induce Diptericin expression in diap2 mutant animals; in wild-type animals, enforced expression of these genes (in the absence of any infection) resulted in reproducible Diptericin induction. Intriguingly, Diptericin induction following enforced expression of imd and Dredd is also blocked in Tak1 mutant animals, indicating that both DIAP2 and Tak1 are required downstream of Dredd. In contrast, kenny and ird5 seem not to be required for Diptericin induction when Dredd is over-expressed. A recent report indicates that Relish cleavage and nuclear translocation on its own is not sufficient for Diptericin expression, and that, at least in vivo, a further cooperative input from the JNK signalling pathway is required. According to this scenario the Imd signalling pathway bifurcates at the level of Tak1, with Tak1 activating the NF-kappaB as well as JNK signalling branch, both of which are required for expression of antibacterial peptide genes in the fat body. In light of this model, the observation that diap2 acts genetically downstream of imd, Dredd, Tak1 and Relish may indicate that DIAP2 functions at the level of Tak1. This view is in agreement with recent reports from Drosophila tissue culture cells, which suggest that DIAP2 is required for Tak1-mediated JNK activation. In this respect, DIAP2 functions at the same epistatic position as the putative E3 ubiquitin ligase of the Imd pathway. Future biochemical experiments will be required to test whether DIAP2 is indeed the E3 ubiquitin ligase that functions together with Ubc13/UEV1A to stimulate Tak1 (Leulier, 2006b).

Although the underlying mechanism for the impaired induction of antibacterial peptide gene expression by loss of DIAP2 remains to be defined, the genetic observations made in this study are likely to have relevance not only for innate immune responses in Drosophila but also for TNFR1 signalling in mammals. While in flies DIAP2 is indispensable for Imd signalling, genetic studies in mice have, so far, failed to uncover a physiological role for c-IAP1 and c-IAP2 in TNFR1 signalling. Since c-iap1 knock out mice carry significantly elevated levels of c-IAP2 protein, it is feasible that the increased c-IAP2 levels functionally compensate for the loss of c-IAP1. Consistently, mammalian IAPs have been reported to be under strict homeostatic control by regulating each other's protein levels, which provides a mechanistic explanation for the crosstalk among IAPs. Thus, in mammals, double knock out mice lacking both c-iap1 and c-iap2 genes will be required to study the role of c-IAP1 and c-IAP2 in TNFR1 signalling. Therefore, Drosophila, where redundancies and compensatory mechanisms are less problematic, provides an ideal model system to study caspase-independent functions of IAPs in an in vivo setting (Leulier, 2006b).

Caspase-Mediated Cleavage, IAP binding, and ubiquitination: linking three mechanisms crucial for Drosophila NF-kappaB signaling

Innate immune responses are critical for the immediate protection against microbial infection. In Drosophila, infection leads to the rapid and robust production of antimicrobial peptides through two NF-kappaB signaling pathways - IMD and Toll. The IMD pathway is triggered by diaminopimelic (DAP)-type peptidoglycan, common to most Gram-negative bacteria. Signaling downstream from the peptidoglycan (PGN) receptors is thought to involve K63 ubiquitination and caspase-mediated cleavage, but the molecular mechanisms remain obscure. This study shows that PGN stimulation causes caspase-mediated cleavage of the Imd protein, exposing a highly conserved IAP-binding motif (IBM) at its neo-N terminus. A functional IBM is required for the association of cleaved IMD with the ubiquitin E3-ligase DIAP2. Through its association with DIAP2, IMD is rapidly conjugated with K63-linked polyubiquitin chains. These results mechanistically connect caspase-mediated cleavage and K63 ubiquitination in immune-induced NF-kappaB signaling (Paquette, 2010).

Activation of the Drosophila IMD pathway by DAP-type peptidoglycan (PGN) leads to the robust and rapid production of a battery of antimicrobial peptides (AMPs) and other immune-responsive genes. Two peptidoglycan recognition protein (PGRP) receptors are responsible for the recognition of DAP-type PGN, the cell surface receptor PGRP-LC and the cytosolic receptor PGRP-LE (Kaneko, 2006). DAP-type PGN binding causes these receptors to multimerize or cluster (Chang, 2006; Lim, 2006), triggering signal transduction. IMD signaling culminates in activation of the NF-κB precursor Relish and transcriptional induction of AMP genes (Paquette, 2010).

Currently, the molecular mechanisms linking these PGN-binding receptors and activation of Relish remain unclear. Genetic experiments suggest that the most receptor-proximal component of the pathway is the imd protein, while the MAP3 kinase TAK1 appears to function downstream. In turn, TAK1 is required for activation of the Drosophila IKK complex, which is essential for the immune-induced cleavage and activation of the NF-κB precursor Relish, the key transcription factor required for immune-responsive AMP gene expression. In addition to NF-κB signaling, TAK1 also mediates immune-induced JNK signaling (Paquette, 2010 and references therein).

Other major components in the IMD pathway include the caspase-8-like DREDD and its adaptor FADD. RNAi-based studies suggest that these proteins have two distinct roles in IMD pathway signaling, one relatively early in the cascade and the second further downstream. Using RNAi, DREDD and FADD were shown to be required for immune-induced activation of the IKK complex (Zhou, 2005). These data suggested that DREDD and FADD function downstream of IMD but upstream of TAK1; however, it was not established if this upstream role for DREDD involves its protease activity. In its second role, DREDD is thought to proteolytically cleave Relish (Paquette, 2010).

In addition to the components outlined above, several studies have suggested that ubiquitination plays a critical role in the IMD signaling cascade. Recently, Drosophila inhibitor of apoptosis 2 (DIAP2) was shown to be a crucial component of the IMD pathway (Gesellchen, 2005; Huh, 2007; Kleino, 2005; Leulier, 2006). Typical of IAP proteins, DIAP2 has three N-terminal BIR domains, which are involved in interactions with proteins carrying conserved IAP-binding motifs (IBMs). In addition, some IAPs, including DIAP2, carry a C-terminal RING finger domain that provides these proteins with ubiquitin E3-ligase activity. Although it is unclear where in the pathway DIAP2 functions, one study showed that the RING finger is indispensable for its role in the immune response, suggesting it operates as an E3-ubiquitin ligase. Also, Zhou (2005) showed, using RNAi-based approaches, that the E2-ubiquitin-conjugating enzymes Uev1a and Ubc13 (bendless) are critical components of the IMD pathway. Notably, Ubc13 and Uev1a function together in a complex to generate K63-linked polyubiquitin chains. K63-polyubiquitin chains are not linked to proteasomal degradation but instead are thought to play regulatory roles. However, no K63-ubiquitinated target protein(s) has been identified in the IMD pathway. Although no connection between DIAP2 and the Bend/Uev1a E2 complex has been established, one attractive scenario is that DIAP2 functions as an E3 together with the Bend-Uev1a E2 complex (Paquette, 2010).

The imd1 allele is a strong hypomorphic mutation that impairs innate immune responses. Surprisingly, this allele encodes a conservative amino acid substitution, alanine (A) to valine (V) at position 31, and is positioned in a region with no obvious structural motifs. The reason for the strong hypomorphic phenotype associated with the A31V substitution remains unclear. This work, demonstrates that imd protein is rapidly cleaved following PGN stimulation. Cleavage requires the caspase DREDD and occurs at caspase recognition motif 27LEKD/A31, creating a neo-N terminus at A31 that is critical for the immune-induced association of IMD with DIAP2. Substitution of the neo-N terminus with valine, as in imd1, disrupts the IMD-DIAP2 interaction. Moreover, once associated with DIAP2, cleaved IMD is rapidly K63-polyubiquitinated. Together, these data resolve a number of outstanding questions in IMD signal transduction and present a clear molecular mechanism linking caspase-mediated cleavage to NF-κB activation (Paquette, 2010).

Previous work has demonstrated that the caspase-8-like protease DREDD and its binding partner FADD are required upstream in the IMD pathway, at a position similar to Ubc13 and Uev1a (Zhou, 2005). However, it was not clear from these studies if the protease activity of DREDD is also required in this role upstream in the IMD pathway. This study shows that upon immune stimulation the imd protein is rapidly cleaved in a DREDD- and FADD-dependent manner. In fact, expression of DREDD, without immune stimulation, is sufficient to cause IMD cleavage. A caspase recognition site was identified in IMD, with cleavage predicted to occur after aspartate 30. Substitution of this residue with alanine prevents signal-induced cleavage and creates a dominant-negative allele of imd. This putative cleavage site in IMD (27LEKD/A31) is similar to the Relish cleavage site (542LQHD/G546), consistent with the notion that both proteins are cleaved by the same protease. Likewise, when IMD cleavage was blocked by caspase inhibitors, IMD was no longer ubiquitinated. Alignment of imd protein sequences from 12 Drosophila species and the Anopheles mosquito showed that the cleavage site is highly conserved (LEKD or LETD in all cases). These findings strongly argue that IMD cleavage after position 30 is mediated by DREDD and that this cleavage is critical for further downstream signaling events (Paquette, 2010).

Cleavage of IMD exposes a highly conserved IBM, which then binds the BIR2/3 domains of DIAP2. In the context of programmed cell-death regulation, these IBM motifs are best defined by their neo-N terminal alanine as well as the proline at position 3, both of which are also present in cleaved IMD, supporting the notion that IMD includes an IBM starting at position 31. The notion that IMD carries an IBM also provides a molecular explanation for the hypomorphic phenotype observed in the imd1 mutant, which carries a valine substitution for this alanine at position 1 of cleaved IMD. Although several IAP proteins have been implicated in mammalian innate immune/NF-κB signaling, the significance of their associated BIR domains, as well as their possible binding to proteins with exposed IBMs, has remained largely unexplored. This study shows that the BIR/IBM association plays a crucial role in innate immune NF-κB signaling in Drosophila. These findings present a unique role for the BIR-IBM interaction module outside of the cell-death arena (Paquette, 2010).

Furthermore, characterization of signaling in the imd1, diap2, dredd, and PGRP-LC/LE mutant flies provides critical in vivo verification of the cell-culture data and leads to a proposed model. In particular, the molecular mechanism suggests that immune stimulation leads to the DREDD-dependent cleavage of IMD, perhaps by recruiting IMD, FADD, and DREDD to a receptor complex. Consistent with this aspect of the model, dredd mutants and receptor mutants failed to cleave (or ubiquitinate IMD) following infection. Once cleaved, the exposed IBM of IMD interacts with BIR2 and BIR3 of DIAP2. Currently, it is not known precisely where in the cell the IMD/DIAP2 association occurs. Once associated with DIAP2, cleaved IMD is rapidly K63 ubiquitinated. As the RING-mutated version of diap2 failed to support IMD ubiquitination in flies, DIAP2 likely functions as the E3 for this reaction. Furthermore, the imd1 allele, which fails to interact with DIAP2 because of a mutation in the IBM, demonstrates the critical nature of the IMD-DIAP2 interaction for innate immune signaling. Consistent with the notion that cleavage precedes ubiquitination, mutants that fail to generate ubiquitinated IMD (i.e., diap2 and imd1) actually accumulate more cleaved IMD than is observed in wild-type flies. Presumably, in wild-type animals, cleaved IMD is efficiently ubiquitinated and thus is difficult to detect in assays. In contrast, dredd mutants or mutants lacking the key immunoreceptors (PGRP-LC/LE) failed to cleave and ubiquitinate IMD, consistent cell-culture data (Paquette, 2010).

Previous work has suggested that ubiquitination plays a critical role in IMD signaling in the Drosophila immune response. However, the molecular target(s) of ubiquitination and the mechanisms of its activation have remained elusive. The data presented in this study indicate that DIAP2 functions as the E3-ligase in the IMD pathway, a function usually attributed to the TRAF or, more recently, cIAP proteins in mammalian NF-κB signaling pathways (Bertrand, 2009). The E2 complex of Bend and Uev1a also appears to be involved in IMD ubiquitination. RNAi targeting of these K63-ubiquitinating enzymes reproducibly decreases IMD ubiquitination and the induction of target genes; however, the degree of inhibition is variable and never complete (Zhou, 2005). This study show that a third E2 enzyme, Effete, the Drosophila Ubc5 homolog, also plays a vital role in ubiquitination of IMD. RNAi treatment targeting Effete, in concert with Uev1a and/or Bendless reproducibly eliminated IMD ubiquitination and the induction of Diptericin (Paquette, 2010).

Several lines of evidence argue that IMD is the critical target for K63 ubiqutination in this pathway. First, IMD is by far the most robustly modified component that identified, and the only one in which modifications can be detected in whole animals. Second, the protein produced as a result of the imd1 mutation, which does not signal, is also not ubiquitinated. Third, a deletion mutant, IMDΔ5, is present that is not ubiquitinated and fails to signal. Finally, Thevenon (2009) recently identified the Drosophila ubiquitin-specific protease, USP36, as a negative regulator of IMD ubiquitination. Functionally, USP36 is able to remove K63-polyubiquitin chains from IMD, promoting K48-mediated polyubiquitination and degradation of IMD. Consistent with the current model, animals which overexpress USP36 show decreased levels of IMD ubiquitination and reduced IMD pathway activation as monitored by Diptericin RNA expression, and are susceptible to bacterial infection. Together, these data strongly argue that IMD is the critical substrate for K63-polyubiqutination in IMD pathway signaling, although other proteins may also be conjugated to lesser degree (as shown in this study for DIAP2) and could potentially substitute for IMD as the platform for ubiquitin conjugation. Interestingly, Xia (2009) recently showed that unanchored K63-polyubiquitin chains (i.e., ubiquitin chains that are not conjugated to a target substrate) are sufficient to activate the mammalian TAK1 and IKK kinase complexes. Furthermore, unanchored polyubiquitin chains are produced after stimulation of HEK cells with IL-1β (Xia, 2009). Thus, the presence (or absence) of K63-polyubiquitin chains may be more important than their conjugation substrate (Paquette, 2010).

K63-polyubiquitin chains are likely to serve as scaffolds to recruit the key kinases TAK1 and IKK in the IMD pathway. Both of these kinases include regulatory subunits with highly conserved K63-polyubiquitin binding domains. Drosophila TAB2, which complexes with TAK1, and the IKKγ subunit are predicted to contain conserved K63-polyubiquitin-binding domains. Thus, it is hypothesized that K63-polyubiquitin chains will recruit both the TAB2/TAK1 complex and the IKK complex, creating a local environment for optimal kinase activation and signal transduction; however, this aspect of the model is still speculative (Paquette, 2010).

Although mammalian caspase-8 and FADD are best known for their role in apoptosis, a growing body of literature indicates that these factors, along with RIP1 (which has some homology to IMD), also function in RIG-I signaling to NF-κB. In addition, caspase-8 has been implicated in NF-κB signaling in B cell, T cell, and LPS signaling. Cells, from mice or humans, lacking caspase-8 have defects in immune activation, cytokine production, and nuclear translocation of NF-κB p50/p65. Furthermore, recent evidence also shows that during mammalian NOD signaling the RIP2 protein is ubiquitinated in a cIAP1/2-dependent manner. Given that Drosophila homologs of RIP1, FADD cIAP1/2, and caspase-8 also function in the IMD pathway, the results presented in this study may help elucidate the mechanism by which these factors function in these mammalian immune signaling pathways (Paquette, 2010).

Innate immune signaling in Drosophila is regulated by TGFbeta-activated kinase (Tak1)-triggered ubiquitin editing

Coordinated regulation of innate immune responses is necessary in all metazoans. In Drosophila, the Imd pathway detects gram-negative bacterial infections through recognition of DAP-type peptidoglycan and activation of the NF-kappaB precursor Relish, which drives robust antimicrobial peptide (AMP) gene expression. Imd is a receptor-proximal adaptor protein homologous to mammalian RIP1 that is regulated by proteolytic cleavage and K63-polyubiquitination. However, the precise events and molecular mechanisms that control the post-translational modification of Imd remain unclear. This study demonstrates that Imd is rapidly K63-polyubiquitinated at lysine residues 137 and 153 by the sequential action of two E2 enzymes, Ubc5 (Effete) and Ubc13 (Bendless)-Uev1a, in conjunction with the E3 ligase Diap2. K63-ubiquitination activates the TGFβ-activated kinase (Tak1), which feeds back to phosphorylate Imd, triggering the removal of K63-chains and the addition of K48-polyubiquitin. This ubiquitin editing process results in the proteosomal degradation of Imd, which is proposed to function to restore homeostasis to the Drosophila immune response (Chen, 2017).

Previous work has demonstrated that Imd is cleaved, K63-polyubiquitinated and phosphorylated upon immune stimulation (Paquette, 2010). While this earlier study did not find K48- polyubiquitin chains, others have reported evidence of both K63- and K48-Imd modifications (Thevenon, 2009). However, the overall dynamics of and interconnections between these IMD post-translational modifications remained unclear. This study showed that peptidoglycan (PGN) from bacterial cell walls stimulation of S2 cells leads to five different Imd modifications: proteolytic cleavage, K63-polyubiquitination, phosphorylation, K63-deubiquitination and K48-polyubiquitination, which leads to degradation of Imd through proteasome. These immune triggered signaling events are robust and incredibly rapid, with Imd cleavage and K63-polyubiquitination occurring as early as 2 minutes after PGN stimulation. While K63-modification peaks early and then steadily declines, K48-conjugation appears later, along with phosphorylation, and declines in proteasome-dependent manner. These kinetics argue that Imd is sequentially conjugated with K63 then K48 ubiquitin, so-called ubiquitin editing, as has been reported for IRAK1 and RIP1 in mammalian innate immune signaling pathways (Chen, 2017).

In addition to ubiquitination, two slow-migrating species of Imd were detected and shown to be phosphorylated forms. Judging by their size, these two phospho-forms appear to be derived from either full-length Imd (upper) or cleaved Imd (lower). Interestingly, persistence of phosphorylated Imd was observed in the proteasome-inhibited samples, suggesting that Imd is both K48-polyubiquitinated and phosphorylated before entering proteasome. Tak1 is required for these phosphorylation events as well as for ubiquitin editing, demonstrating key role for this MAP3K in a negative feedback loop (Chen, 2017).

Conjugation of ubiquitin usually occurs on lysine side chains of target proteins, and mass spectrometry of immuno-purified endogenously expressed Imd identified K137 and K153 as the sites of ubiquitin linkage. Note, the mass spec analysis includes 50% coverage of Imd, and a cluster of four lysine residues at the very C-terminus were not observed. Substitution of K137 and K153 residue with Arginine prevented signal- induced ubiquitination of Imd in S2 cells and reduced expression of the AMP gene Diptericin in both cells and flies. These results demonstrate that both lysine residues are required for K63- polyubiquitination and downstream signaling events. In S2 cells, mutation of single lysine residue led to a partial reduction of K63- ubiquitination and a partial reduction of AMP gene induction. Surprisingly, single lysine mutation did not correspondingly reduce Imd K48- polyubiquitination, while the double lysine mutation completely blocked it. These results suggest that even the reduced signal, mediated by a single K63-chain, is sufficient to trigger a robust feedback response with K48-chain formation. On the other hand, a complete block in K63-chains prevents Tak1 activation, which in turn fails to promote the ubiquitin editing of Imd. These findings are consistent with results observed with knockdown of Ubc5, Ubc13 and Uev1a. Ubc5 depletion prevented all K63 ubiquitination and signaling (as measured by Diptericin induction), and subsequent K48 modification was absent, while the Ubc13 and/or Uev1a knockdown showed residual K63 chains and greatly reduced Diptericin expression but robust K48- ubiquitination. These results also suggest that K48-polyubiquitination may occur on lysine residues beyond K137 and K153, although more detailed mass spectrometric analyses is required to map these sites more thoroughly (Chen, 2017).

On the other hand, only double lysine mutation leads to significant reduction of AMP expression in adult flies, and this is reduction is not as robust as in cultured cells. This pattern suggests that activation of the NF-κB protein Relish and its transcriptional targets do not solely rely on ubiquitination of Imd K137/153. Other possible ubiquitination targets include the upstream caspase Dredd, which has been shown to be critical for signaling (26), or the E3 ligase Diap2. This redundancy may represent multiple parallel mechanisms that contribute to the NF-κB activation. Furthermore, tissue specific immune differences may contribute to the discrepancy in Diptericin induction between macrophage-derived S2 cells and whole flies, in which the fat body rather than hemocytes is the major organ for inducible expression of AMPs (Chen, 2017).

Previous work has suggested that Diap2 is the E3 ligase for Imd ubiquitination. With the advantage of ubiquitin linkage specific antibodies, data presented in this study show that Diap2 is required for Imd K63-polyubiquitination and signaling, as measured by induction level of Diptericin. Moreover, the accumulation of cleaved but non-ubiquitinated Imd, in the Diap2 depleted cells and flies, provides further evidence that ubiquitination is downstream of Imd cleavage and highlights the role of Diap2 as the critical E3 in the K63- modification of Imd. In addition, Imd is no longer K48-modified when Diap2 is removed, suggesting that either Diap2 is also involved in K48- conjugation, or the failure of K63- polyubiquitination leads to the loss of K48- polyubiquitination. Given the role of Tak1 in this ubiquitin editing event, the latter hypothesis is favored, and another E3 is likely involved in the K48 conjugation (Chen, 2017).

In addition to E3 ligases, E2 ubiquitin conjugating enzymes are the other key factors in the ubiquitin conjugation reaction. Previously work has shown that Ubc5 and Ubc13-Uev1a are all involved in Imd ubiquitination. However, the mechanism by which these E2s collaborated was unclear. Results from in vitro reconstituted ubiquitination assays suggested a two-step reaction model for ubiquitin conjugation with different E2s. In particular, it was shown that some E2s, such as Ubc5, are effective at the initial ubiquitination of substrates but are ineffective at generating long chains, while other E2s, like Ubc13/Uev1a, are efficient at generating long ubiquitin chains but fail to conjugate substrate proteins. Thus, these two types of E2s can work together to generate long ubiquitin chains conjugated to target proteins. It is proposed that Imd undergoes ubiquitin chain initiation and elongation catalyzed by two separate E2s. Once cleaved, Imd interacts with Diap2 through its BIR repeats and is first modified by Ubc5-mediated substrate ubiquitination on lysines 137 and K153. Subsequently, the E2 complex of Ubc13-Uev1a pairs with an E3 (possibly Diap2 although another unidentifed E3 is not excluded) and switches the reaction to chain elongation mode, during which additional ubiquitin molecules are attached to the substrate-linked ubiquitin in a K63-specific manner. In the absence of Ubc13/Uev1a, Ubc5 alone is still able to elongate the polyubiquitin chains, but less efficiently and with unknown linkages (Chen, 2017).

Induction of Diptericin expression generally tracks with the K63-polyubiquitination signal (but not the total Ub signal) observed in various E2 knockdown cells. The one exception is the samples in which Ubc13 and Uev1a are both knocked down, and Ubc5 is still available. These samples display a similar K63 intensity as the single Ubc13 or Uev1a RNAi lanes, but Diptericin induction is lower, close to background levels. Ubc5 alone is known to conjugate ubiquitin without linkage specificity, a random polyubiquitin chain that consist of all seven types lysine linkage. Since the K63 antibody recognizes K63-linked diubiquitin, it is possible that Ubc5-mediated random ubiquitin chain elongation generates some K63 di-ubiquitin linkages which are detected by this antibody, but are unable support signaling due to their altered topology and limited amounts of K63-linkages. More detailed biochemical characterization of these Ubc5-catalyzed chains is required to confirm this hypothesis (Chen, 2017).

K48 modification of Imd shows a subtle difference relative to the K63 chains. Again, knock down of Ubc5 causes complete blocking of K48 conjugation, but depletion of Ubc13/Uev1a has no effect. The failure of K48 modification in the Ubc5 depleted cells may have two possible underlying causes. Firstly, the complete lack of K63 chains will fail to activate Tak1 and the subsequent ubiquitin editing feedback loop, while the Ubc13 and/or Uev1a RNAi display some residual K63 activity and thus can trigger Tak1 and the feedback response. Alternatively, Ubc5 might be directly required for Imd K48-polyubiquitination as shown in the degradation of proteins in multiple Drosophila pathways including eye development, maintenance of germline stem cells and apoptosis. These are not mutually exclusive possibilities (Chen, 2017).

Phosphorylation of Imd appears to be a major regulator of these ubiquitin-editing events. Knockdown of Tak1 prevents Imd phosphorylation in S2 cells and in adult flies. Moreover, immune-purified Tak1 can directly phosphorylate recombinant Imd in vitro, while neither JNKK nor IKK are required for phosphorylation of cleaved Imd, strongly arguing that Tak1 directly modifies Imd. RNAi-depletion or drug inhibition of Tak1 prevents K63-deubiquitination and the subsequent K48-polyubiquitination/proteasome-mediated degradation, leading to accumulation of cleaved but unphosphorylated Imd, presumably an intermediate during chain editing. From these results, it is inferred that Tak1-mediated phosphorylation of Imd is required for ubiquitin editing. Future studies will reveal the underlying mechanisms by which phosphorylation triggers K63-deubiquitination and K48 chain conjugation. Nonetheless, these results are consistent with earlier reports of Imd regulation by K63-deubiquitination and degradation (Chen, 2017).

Considering the results presented in this studdy together with earlier studies, the following model of Imd signal activation and subsequent down-regulation is proposed. One of the earliest events after PGN-stimulation is the rapid cleavage of Imd by the caspase-8 homolog DREDD at D30. Cleaved Imd then interacts with BIR-repeats of the E3 ligase Diap2 and is K63-polyubiquitinated through the sequential action of Ubc5, for substrate conjugation, and Ubc13-Uev1a, for catalyzing long K63 chains. These K63-polyubiquitin chains are then likely to activate the Tak1/Tab2 kinase complex through the conserved K63-binding motif in Tab2, which in turns signals through IKK complex to activate the NF-κB precursor Relish. Relish is central for the robust induction of AMP gene transcription. Meanwhile, Tak1 also mediates a retrograde signal that phosphorylates Imd and triggers ubiquitin editing, and leads to the degradation of Imd through proteasome. This regulatory interaction between Tak1 and Imd represents a novel homeostatic loop whereby the Drosophila immune response is rapidly activated but also quickly shutdown. Future studies are necessary to determine function of this feedback loop relative to other feedback mechanisms reported for the Imd pathway (Chen, 2017).

Female-biased upregulation of insulin pathway activity mediates the sex difference in Drosophila body size plasticity

Nutrient-dependent body size plasticity differs between the sexes in most species, including mammals. Previous work in Drosophila showed that body size plasticity was higher in females, yet the mechanisms underlying increased female body size plasticity remain unclear. This study discovered that a protein-rich diet augments body size in females and not males because of a female-biased increase in activity of the conserved insulin/insulin-like growth factor signaling pathway (IIS). This sex-biased upregulation of IIS activity was triggered by a diet-induced increase in stunted mRNA in females, and required Drosophila insulin-like peptide 2, illuminating new sex-specific roles for these genes. Importantly, this study shows that sex determination gene transformer promotes the diet-induced increase in stunted mRNA via transcriptional coactivator Spargel to regulate the male-female difference in body size plasticity. Together, these findings provide vital insight into conserved mechanisms underlying the sex difference in nutrient-dependent body size plasticity (Millington, 2021).

In many animals, body size plasticity in response to environmental factors such as nutrition differs between the sexes. While past studies have identified mechanisms underlying nutrient-dependent growth in a mixed-sex population, and revealed factors that promote sex-specific growth in a single nutritional context, the mechanisms underlying the sex difference in nutrient-dependent body size plasticity remain unknown. This study showed that females have higher phenotypic plasticity compared with males when reared on a protein-rich diet, and elucidated the molecular mechanisms underlying the sex difference in nutrient-dependent body size plasticity in this context. The data suggests a model in which high levels of dietary protein augment female body size by stimulating an increase in IIS activity, where a requirement was identified for dilp2 and stunted (sun) in promoting this nutrient-dependent increase in IIS activity. Importantly, it was discovered that tra is the factor responsible for stimulating sun mRNA levels and IIS activity in a protein-rich context, revealing a novel role for sex determination gene tra in regulating phenotypic plasticity. Mechanistically, tra enhanced sun mRNA levels and body size in protein-rich conditions via transcriptional coactivator Srl, identifying Srl as one link between tra and the nutrient-dependent regulation of gene expression. Together, these findings provide new insight into how Drosophila females achieve increased nutrient-dependent body size plasticity compared with males (Millington, 2021).

One key feature of this increased phenotypic plasticity in females was a female-biased increase in IIS activity in a protein-rich context. This reveals a previously unrecognized sex difference in the coupling between IIS activity and dietary protein. In females, there was tight coupling between increased nutrient input and enhanced IIS activity across a wide protein concentration range in all control genotypes. In males, this close coordination between dietary protein and IIS activity was weaker in a protein-rich context. The data shows that sex-biased nutrient-dependent change to IIS activity during development is physiologically significant, as it supports an increased rate of growth and consequently larger body size in females but not in males raised on a protein-rich diet. In future studies, it will be important to determine whether the sex difference in coupling between nutrients and IIS activity exists in other contexts. For example, previous studies on the extension of life span by dietary restriction have shown that male and female flies differ in the concentration of nutrients that produces the maximum life span extension, and in the magnitude of life span extension produced by dietary restriction. Similar sex-specific effects of dietary restriction and reduced IIS on life span have also been observed in mice and humans. Future studies will be needed to determine whether a male-female difference in coupling between nutrients and IIS activity account for these sex-specific life span responses to dietary restriction. Indeed, given that sex differences have been reported in the risk of developing diseases associated with overnutrition and dysregulation of IIS activity such as obesity and type 2 diabetes, more detailed knowledge of the male-female difference in coupling between nutrients and IIS activity in other models may provide insights into this sex-biased risk of disease (Millington, 2021).

In addition to revealing a sex difference in the nutrient-dependent upregulation of IIS activity, the data identified a female-specific requirement for dilp2 and sun in mediating the diet-induced increase in IIS activity in a protein-rich context. While previous studies have shown that both dilp2 and sun positively regulate body size, this study describes new sex-specific roles for dilp2 and sun in nutrient-dependent phenotypic plasticity. Elegant studies have shown that sun is a secreted factor that stimulates Dilp2 release from the IPCs. Together with the current data, this suggests a model in which females are able to achieve a larger body size in a protein-rich diet because they have the ability to upregulate sun mRNA levels, whereas males do not. Indeed, this study shows that higher sun mRNA levels are sufficient to augment body size. This model aligns well with findings from two previous studies on Dilp2 secretion in male and female larvae. The first study, which raised larvae on a protein-rich diet equivalent to the 2Y diet (high protein), found increased Dilp2 secretion in females compared to males. The second study, which raised larvae on a diet equivalent to the 1Y diet (low protein), found no sex difference in Dilp2 secretion and no effects of dilp2 loss on body size. Thus, while these previous studies differed in their initial findings on a sex difference in Dilp2 secretion, the current data reconcile these minor differences by identifying context-dependent effects of dilp2 on body size. It is important to note that absolute confirmation of a sex difference in hemolymph Dilp2 levels will be needed in future studies because the body size plasticity defects in the dilp2-HF strain precluded its use as a tool to quantify circulating Dilp2 levels in this study. Future studies will also need to determine whether these sex-specific and context-dependent effects of dilp2 are observed in other phenotypes regulated by dilp2 and other dilp genes. For example, flies carrying mutations in dilp genes show changes to aging, metabolism, sleep, and immunity, among other phenotypes. Further, it will be interesting to determine whether the sex-specific regulation of sun is observed in any other contexts, and whether it will influence sex differences in phenotypes associated with altered IIS activity, such as life span (Millington, 2021).

While these findings on sun and dilp2 provide mechanistic insight into the molecular basis for the larger body size of females reared on a protein-rich diet, a key finding from this study was the identification of sex determination gene tra as the factor that confers plasticity to females. Normally, nutrient-dependent body size plasticity is higher in females than in males in a protein-rich context. In females lacking a functional Tra protein, however, this increased nutrient-dependent body size plasticity was abolished. In males, which normally lack a functional Tra protein, ectopic Tra expression conferred increased nutrient-dependent body size plasticity. While a previous study showed that on the 2Y diet Tra promotes Dilp2 secretion , the current study extends this finding in two ways: by identifying sun as one link between Tra, Dilp2, and changes to IIS activity; and by showing that Tra regulates sun mRNA via conserved transcriptional coactivator Srl. While previous studies discovered Srl as the factor that promotes sun mRNA levels in response to dietary protein in a mixed-sex larval population, the current findings reveal a previously unrecognized sex-specific role for Srl in regulating transcription. Because loss of Tra reduces Srl transcriptional activity, this new link between Tra and Srl suggests an additional way in which Tra may impact gene expression beyond its canonical downstream targets dsx and fru. While this builds on recent studies that reveal a number of additional Tra-regulated genes, it will be important to determine whether these additional Tra-regulated genes including sun represent direct targets of Tra/Srl. Future studies will also be needed to elucidate how Tra impacts Srl transcriptional activity in a context-dependent manner. However, uncovering a connection between a sex determination gene and a key regulator of genes involved in mitochondrial function suggests an additional mechanism that may contribute to sex differences in phenotypes affected by mitochondrial function (e.g., lifespan). In addition, it will be critical to explore how the presence of Tra allows an individual to couple dietary protein with body size. Because the tra locus is regulated both by alternative splicing and transcription, and Tra protein is regulated by phosphorylation, this study highlights the importance of additional studies on the regulation of the tra genomic locus and Tra protein throughout development to gain mechanistic insight into its effects on nutrient-dependent body size plasticity (Millington, 2021).

While the main outcome of this work was to reveal the molecular mechanisms that regulate the sex difference in nutrient-dependent body size plasticity, this study also provides some insight into how genes that contribute to nutrient-dependent body size plasticity affect female fecundity and male fertility. The findings align well with previous studies demonstrating that increased nutrient availability during development and a larger female body size confers increased ovariole number and fertility, as females lacking either dilp2 or fat body-derived sun were unable to augment egg production in a protein-rich context. Given that previous studies demonstrate IIS activity influences germline stem cells in the ovary in adult flies, there is a clear reproductive benefit that arises from the tight coupling between nutrient availability, IIS activity, and body size in females. In males, however, the relationship between fertility and body size remains less clear. While larger males are more reproductively successful both in the wild and in laboratory conditions. Given that this study revealed no significant increase in the number of progeny produced by larger males, the fertility benefits that accompany a larger body size in males may be context-dependent. For example, a larger body size increases the ability of males to outcompete smaller males. Thus, in crowded situations, a bigger body may provide significant fertility gains. However, in conditions where nutrients are limiting, an imbalance in the allocation of energy from food to growth rather than to reproduction may decrease fertility. Future studies will need to resolve the relationship between body size and fertility in males, as this will suggest the ultimate reason(s) for the sex difference in nutrient-dependent body size plasticity (Millington, 2021).

Insulin signaling promotes germline proliferation in C. elegans

Cell proliferation must be coordinated with cell fate specification during development, yet interactions among pathways that control these two critical aspects of development are not well understood. The coordination of cell fate specification and proliferation is particularly crucial during early germline development, when it impacts the establishment of stem/progenitor cell populations and ultimately the production of gametes. In C. elegans, insulin/IGF-like receptor (IIR) signaling has been implicated in fertility, but the basis for the fertility defect had not been previously characterized. This study found that IIR signaling is required for robust larval germline proliferation, separate from its well-characterized role in preventing dauer entry. IIR signaling stimulates the larval germline cell cycle. This activity is distinct from Notch signaling, occurs in a predominantly germline-autonomous manner, and responds to somatic activity of ins-3 and ins-33, genes that encode putative insulin-like ligands. IIR signaling in this role acts through the canonical PI3K pathway, inhibiting DAF-16/FOXO. However, signaling from these ligands does not inhibit daf-16 in neurons nor in the intestine, two tissues previously implicated in other IIR roles. These data are consistent with a model in which: (1) under replete reproductive conditions, the larval germline responds to insulin signaling to ensure robust germline proliferation that builds up the germline stem cell population; and (2) distinct insulin-like ligands contribute to different phenotypes by acting on IIR signaling in different tissues (Michaelson, 2010).

Ubiquitylation of the initiator caspase DREDD is required for innate immune signalling

Caspases have been extensively studied as critical initiators and executioners of cell death pathways. However, caspases also take part in non-apoptotic signalling events such as the regulation of innate immunity and activation of nuclear factor-κB (NF-κB). How caspases are activated under these conditions and process a selective set of substrates to allow NF-κB signalling without killing the cell remains largely unknown. This study shows that stimulation of the Drosophila pattern recognition protein PGRP-LCx induces DIAP2-dependent polyubiquitylation of the initiator caspase DREDD. Signal-dependent ubiquitylation of DREDD is required for full processing of IMD, NF-κB/Relish and expression of antimicrobial peptide genes in response to infection with Gram-negative bacteria. The results identify a mechanism that positively controls NF-κB signalling via ubiquitin-mediated activation of DREDD. The direct involvement of ubiquitylation in caspase activation represents a novel mechanism for non-apoptotic caspase-mediated signalling (Meinander, 2012).

The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors

The innate immune system needs to distinguish between harmful and innocuous stimuli to adapt its activation to the level of threat. How Drosophila mounts differential immune responses to dead and live Gram-negative bacteria using the single peptidoglycan receptor PGRP-LC is unknown. This study describes rPGRP-LC, an alternative splice variant of PGRP-LC that selectively dampens immune response activation in response to dead bacteria. rPGRP-LC-deficient flies cannot resolve immune activation after Gram-negative infection and die prematurely. The alternative exon in the encoding gene, here called rPGRP-LC, encodes an adaptor module that targets rPGRP-LC to membrane microdomains and interacts with the negative regulator Pirk and the ubiquitin ligase DIAP2. rPGRP-LC-mediated resolution of an efficient immune response requires degradation of activating and regulatory receptors via endosomal ESCRT sorting. It is proposed that rPGRP-LC selectively responds to peptidoglycans from dead bacteria to tailor the immune response to the level of threat (Neyen, 2016).

PGRP-LC has a clear role as the major signaling receptor sensing Gram-negative bacteria in flies, but its contribution to the resolution phase once bacteria are killed and release polymeric PGN has remained elusive. This study has uncovered a regulatory isoform of LC (rLC) that adjusts the immune response to the level of threat. rLC specifically downregulates IMD pathway activation in response to polymeric PGN, a hallmark of efficient bacterial killing. The data are consistent with a model whereby the presence of rLC leads to efficient endocytosis of LC and termination of signaling via the ESCRT pathway. Trafficking-mediated shutdown of LC-dependent signaling ensures that LC receptors are switched off once the balance is tipped in favor of ligands signifying dead bacteria, allowing Drosophila to terminate a successful immune response. Failure to do so results in over-signaling, leading to the death of the host despite bacterial clearance. Consistent with this model, defects were found in endosome maturation and in the formation of MVBs enhance immune activation and prevent immune resolution. In addition to regulating LC signaling via the ESCRT machinery, rLC can also inhibit LC signaling by forming signaling-incompetent rLC-LC heterodimers or rLC-rLC homodimers (Neyen, 2016).

Recent evidence from vertebrates also implicates the ESCRT machinery in suppressing spurious NF-κB activation: the TNFR superfamily member lymphotoxin-β receptor, which activates a signaling cascade that is functionally similar to IMD signaling, is degraded in an ESCRT-dependent manner in zebrafish and human cells. Thus ESCRT-mediated clearance of receptors upstream of NF-κB seems well conserved throughout evolution (Neyen, 2016).

Internalization of receptor-ligand complexes raises the question of whether peptidoglycan is fully degraded in the endolysosomal compartment or fragmented and released into the cytosol for sensing by PGRP-LE, as is the case for peptidoglycan sensing by cytoplasmic NOD2 receptors in mammalian cells. Mechanistic coupling of LC-dependent peptidoglycan endocytosis and PGRP-LE-dependent cytosolic sensing of exported peptidoglycan fragments would help explain the partial cooperation between the two receptors (Neyen, 2016).

Molecularly, rLC is characterized by a cytosolic PHD domain predicted to bind to phosphoinositides. The PHD domain targets rLC were found to be distinct membrane domains but it cannot be excluded that this localization relies on additional protein-protein interactions. Furthermore, the PHD domain also mediates binding of rLC to the cytosolic regulator Pirk and the ubiquitin ligase DIAP2. The combined capability to control membrane localization and to recruit downstream signaling modulators is reminiscent of the 'sorting-signaling adaptor paradigm' that is emerging for mammalian PRRs. Sorting adaptors are cytosolic signaling components with phosphoinositide-binding domains that are selectively recruited to defined subcellular locations and thereby shape the signaling output of the receptors they interact with. In vertebrate immune signaling, bacterial sensing modules (for example, TLRs), lipid-binding sorting modules (for example, TIRAP or TRIF) and signaling modules (for example, MyD88 and TRAM) are carried on separate molecules and assemble via transient interactions. Drosophila MyD88 combines sorting and signaling functions in a single molecule, bypassing the need for TIRAP. Notably, rLC merges features of sensing and signaling receptors and sorting adaptors into a single molecule. The fact that Drosophila rLC has no immediate homologs in vertebrates with PGN-sensing and PGN-signaling pathways suggests evolutionary uncoupling of sensing and sorting domains, possibly to increase the spectrum of signaling by combinatorial recruitment of adaptors to sensing receptors (Neyen, 2016).


Search PubMed for articles about Drosophila Diap2

Bertrand, M. J., et al. (2009). Cellular inhibitors of apoptosis cIAP1 and cIAP2 are required for innate immunity signaling by the pattern recognition receptors NOD1 and NOD2. Immunity 30: 789-801. PubMed ID: 19464198

Chang, C. I., et al. (2006). Deisenhofer, Structure of trachael cytotoxin in complex with a heterodimeric pattern-recognition receptor. Science 311: 1761-1764. PubMed ID: 16556841

Chen, L., Paquette, N., Mamoor, S., Rus, F., Nandy, A., Leszyk, J., Shaffer, S. A. and Silverman, N. (2017). Innate immune signaling in Drosophila is regulated by TGFbeta-activated kinase (Tak1)-triggered ubiquitin editing. J Biol Chem [Epub ahead of print]. PubMed ID: 28377500

Doumanis, J., Quinn, L., Richardson, H., and Kumar, S. (2001). STRICA, a novel Drosophila melanogaster caspase with an unusual serine/threonine-rich prodomain, interacts with DIAP1 and DIAP2. Cell Death Differ. 8: 387-394. PubMed ID: 11550090

Eckelman, B. P. and Salvesen, G. S. (2005). The human anti-apoptotic proteins cIAP1 and cIAP2 bind but do not inhibit caspases. J. Biol. Chem. 281: 3254-3260. PubMed ID: 16339151

Gesellchen, V., Kuttenkeuler, D., Steckel, M., Pelte, N. and Boutros, M. (2005). An RNA interference screen identifies Inhibitor of Apoptosis Protein 2 as a regulator of innate immune signalling in Drosophila. EMBO Rep. 6: 979-984. PubMed ID: 16170305

Hay, B. A., Wassarman, D. A. and Rubin, G. M. (1995). Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell 83(7): 1253-62. 8548811

Hay, B. A. (2000). Understanding IAP function and regulation: a view from Drosophila. Cell Death Differ. 7: 1045-56. PubMed ID: 11139277

Huh, J. R., et al (2007). 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. J. Biol. Chem. 282: 2056-2068. PubMed ID: 17068333

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Biological Overview

date revised: 12 December 2016

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