NF-kappaB/Rel-mediated regulation of the neural fate in Drosophila

Two distinct roles are described for Dorsal, Dif and Relish, the three NF-kappaB/Rel proteins of Drosophila, in the development of the peripheral nervous system. First, these factors regulate transcription of scute during the singling out of sensory organ precursors from clusters of cells expressing the proneural genes achaete and scute. This effect is possibly mediated through binding sites for NF-kappaB/Rel proteins in a regulatory module of the scute gene required for maintenance of scute expression in precursors as well as repression in cells surrounding precursors. Second, genetic evidence suggests that the receptor Toll-8, Relish, Dif and Dorsal, and the caspase Dredd pathway are active over the entire imaginal disc epithelium, but Toll-8 expression is excluded from sensory organ precursors. Relish promotes rapid turnover of transcripts of the target genes scute and asense through an indirect, post-transcriptional mechanism. It is proposed that this buffering of gene expression levels serves to keep the neuro-epithelium constantly poised for neurogenesis (Ayyar, 2007).

The results suggest a dual role for the NF-kappaB/Rel proteins of Drosophila in the formation of SOPs. First, they could be recruited directly to the sc promoter and regulate transcription. The SOP enhancer of sc, required for auto-regulation of sc in the SOPs, contains α boxes (ACTAGA), consensus sequences for NF-kappaB/Rel. Evidence has been obtained for a role of these sequences in both activation and repression of sc. Expression of Rel-VP16, a potent transcriptional activator form of Relish, is able to ectopically activate a reporter gene containing the intact sc SOP enhancer but not one in which the α3 box is mutated. So activation in this experimental situation requires the presence of an intact α3 site. The experiment does not rule out indirect effects, so further work is required to verify whether activation is direct. It is suggested the NF-kappaB/Rel proteins participate in activation and repression of transcription of sc, a hypothesis consistent with dl, Dif and Rel mutant phenotypes of additional as well as missing bristles. Second, unexpected role is described of Rel in mRNA turnover of sc, ase and sens, neuronal genes required to specify and/or maintain the neuronal fate of SOP cells. In Rel mutants, transcripts of sc, ase and sens accumulate due to increased transcript stability. Therefore in the wild type, Relish promotes rapid mRNA turnover, presumably indirectly through an unidentified transcriptional target. A similar phenotype is observed in Toll-8 mutants, which furthermore, interact genetically with Rel mutants. Transcripts for Rel are reduced in the Toll-8 mutant suggesting a role for Toll-8 in maintaining the levels of Rel transcript. This might be the reason for the genetic interaction (Ayyar, 2007).

A number of differences are apparent between mutants of the three NF-kappaB/Rel-encoding genes of Drosophila. Mutants triply homo- or hetero-zygous have a normal complement of bristles, while single homo- or hetero-zygous animals have either additional or missing bristles. This suggests possible opposing functions for these genes. Furthermore bristle phenotypes due to loss or gain of function differ in detail between the three mutants. Together these results point to the importance of the stoichiometric relationships between the three NF-kappaB/Rel proteins and raise the possibility that different Dorsal/Dif/Relish homo- or hetero-dimers may have distinct binding sites and therefore different targets. This merits further investigation (Ayyar, 2007).

If NF-kappaB/Rel proteins both activate and repress sc, then they are expected to activate in SOP cells and repress in cells of the proneural clusters not chosen to be SOPs. Two possible ways that this could occur are discussed. First, activation in the SOP may rely on high levels of proneural protein and low levels of NF-kappaB/Rel protein; conversely repression may require low levels of proneural and high levels of NF-kappaB/Rel protein. Notch-mediated lateral inhibition results in high levels of Sc in the SOP and lower levels in surrounding cells. Toll-8 expression is excluded from SOP cells suggesting, that, if Toll-8 affects NF-kappaB/Rel activity, there would be lower levels of NF-kappaB/Rel in SOPs. NF-kappaB has been shown to activate transcription even without stimulus if IkappaB levels are low enough to allow NF-kappaB-dependent gene expression in the basal state. Interestingly, it has been shown that low levels of Dorsal can act synergistically with bHLH proteins to activate target genes in the embryo. This depends on direct association of Dorsal and bHLH proteins and cooperative binding to closely linked binding sites for the two respective proteins. Furthermore cooperative binding for Sc and Dorsal has been demonstrated. In the sc SOP enhancer one of the alpha boxes is indeed close to an E box, so perhaps high levels of Sc and low levels of NF-kappaB/Rel combine to activate transcription in the SOP. Two observations are consistent with this hypothesis: Rel-VP16 was able to ectopically activate sc-SOPE-lacZ only at sites where ac and sc are expressed and, after over-expression of NF-kappaB/Rel proteins, bristles are generally missing on the lateral notum (where Toll-8 levels are high), whereas ectopic bristles are found on the medial notum (where Toll-8 levels are low) (Ayyar, 2007).

A second means by which NF-kappaB/Rel proteins could act differently in SOP and in non-SOP cells, may be the presence/absence of co-factors. It has been shown that Dorsal can be converted from an activator to a repressor by association with the co-repressor Groucho. This bi-functionality is attributable to the fact that Dl only weakly interacts with Gro. During embryogenesis both Cut and Dead ringer bind an AT-rich silencer sequence, AT2, present in target genes of Dorsal and both Dorsal and Dead ringer bind the co-repressor Groucho and recruit it to DNA. A similar AT-rich sequence (the β box) is present in the sc SOP enhancer. Furthermore repression of sc by the E(spl) proteins, targets of Notch signalling in non-SOP cells, is already known to require the activity of Groucho (Ayyar, 2007).

Transcripts for sc, ase and sens (and GFP) accumulate in Rel and Toll-8 mutants as a result of increased transcript stability. Transcript stability correlates with the presence of a six or seven nucleotide motif in the transcribed sequence of these genes. The motif is present in sc, ase and sens, but not ac the transcription of which is unaffected in Rel mutants. The motif is almost identical to the heptamer in MyoD and Sox9 that is associated with transcript stability after inhibition of NF-kappaB/Rel signalling in C2C12 cells. A sc mutant with a truncated sc transcript lacking one of the two motifs present in the coding sequence of this gene, has a phenotype similar to Rel and Toll-8 mutants and an increase in sc mRNA. It has been suggested that increased stability of the transcripts rather than increased transcription underlies this phenotype. The presence of the heptamer is noted in a number of genes involved in sensory organ patterning suggesting possible regulation by NF-kappaB/Rel of a battery of genes in the imaginal epithelium. A similar motif is present in other vertebrate targets of NF-kappaB/Rel. Post-transcriptional regulation of target genes by NF-kappaB/Rel could therefore be an ancient feature common to Drosophila and mammals and possibly even jellyfish. It has been suggest that an unknown factor, presumably a transcriptional target of NF-kappaB/Rel, regulates messenger turnover through association with this sequence. In Rel and Toll-8 mutants the accumulated transcripts are not translated. This must be an effect of the mutants because ectopic expression in wild-type flies allows translation and ectopic bristle formation (Ayyar, 2007).

Promotion of a rapid turnover of transcripts of neuronal genes presumably does not take place in the SOPs where high levels of the protein products of these genes are required. Accordingly Toll-8 expression is extinguished in the SOPs after their formation. Factors specific to the SOP presumably allow translation of the transcripts. It is therefore suggested that high levels of Relish provided by Toll-8 in non-SOP cells might be required for post-transcriptional regulation of neuronal genes (Ayyar, 2007).

In wild-type animals expression of neuronal precursor genes such as sens and ase is restricted to SOPs where they are activated by high levels of Ac and Sc. The results suggest that they are in fact expressed over the entire neuro-epithelium but that mRNA turnover is rapid due to NF-kappaB/Rel activity. Activation of ac-sc in proneural clusters would counteract the effects of NF-kappaB/Rel to allow selection of SOPs. After selection of SOPs for the large sensory bristles is finished, Toll-8 expression is maintained in the epithelium, suggesting that high levels of NF-kappaB/Rel are still required for continued transcript turnover. Continuous buffering of neuronal gene expression presumably continues until the next round of neurogenesis that takes place after pupariation when precursors for the small bristles form. Therefore it is hypothesized that NF-kappaB/Rel plays a subtle role in maintaining steady state levels of expression of many genes required for neural development. The maintenance of low levels of expression of neuronal genes would keep the tissue poised for neurogenesis that takes place in repeated rounds. Perhaps low levels of expression of neuronal genes are characteristic of neuro-epithelia in general (Ayyar, 2007).

The hypothesis concerning the dual role of NF-kappaB/Rel in neurogenesis in Drosophila is as follows. The neuro-epithelium of the imaginal discs expresses neuronal genes. Prior to development of SOPs, high levels of Toll-8 maintain high levels of Rel and result in nuclear accumulation of NF-kappaB/Rel. Through an unknown transcriptional target(s), Relish promotes rapid turnover of neuronal transcripts by a post-transcriptional mechanism. This might be mediated by a specific sequence in the coding regions of target genes. Activation of ac and sc in proneural clusters by regulatory proteins of the notal prepattern counteracts the effects of Relish. After singling out of SOPs by Notch-mediated lateral inhibition, Toll-8 expression ceases in the SOPs. Reduced levels of signal uncover a trans-activator function for NF-kappaB/Rel that, synergistically with Sc, helps to maintain high levels of sc expression in the SOP, possibly through direct binding to consensus sequences in the sc SOP enhancer. The NF-kappaB/Rel proteins may also directly repress sc in non-SOP cells of the proneural clusters. It remains to be seen to what extent each of the three proteins participates in these two processes (Ayyar, 2007).

Drosophila Calcineurin, acting upstream of relish, promotes induction of innate immune responses

The sophisticated adaptive immune system of vertebrates overlies an ancient set of innate immune-response pathways, which have been genetically dissected in Drosophila. Although conserved regulatory pathways have been defined, calcineurin, a Ca2+-dependent phosphatase, has not been previously implicated in Drosophila immunity. Calcineurin activates mammalian immune responses by activating the nuclear translocation of the vertebrate-specific transcription factors NFAT1-4. In Drosophila, infection with gram-negative bacteria promotes the activation of the Relish transcription factor through the Imd pathway. The activity of this pathway in the larva is modulated by nitric oxide (NO). This study shows that the input by NO is mediated by calcineurin. Pharmacological inhibition of calcineurin suppressed the Relish-dependent gene expression that occurs in response to gram-negative bacteria or NO. One of the three calcineurin genes in Drosophila, CanA1, mediates NO-induced nuclear translocation of Relish in a cell-culture assay. A CanA1 RNA interference (RNAi) transgene suppressed immune induction in larvae upon infection or upon treatment with NO donors, whereas a gain-of-function CanA1 transgene activated immune responses in untreated larvae. Interestingly, CanA1 RNAi in hemocytes but not the fat body was sufficient to block immune induction in the fat body. Thus, CanA1 provides an additional input into Relish-promoted immune responses and functions in hemocytes to promote a tissue-to-tissue signaling cascade required for robust immune response (Dijkers, 2007).

Altogether, these findings show that calcineurin contributes to innate immune responses and conveys an NO signal that activates AMP production in the Drosophila larva. The bacteria (Ecc15) fed to the larvae remain confined to the gut but nonetheless induce responses in the fat body. Because infection induces NOS in the gut, NO produced in the gut might signal to hemocytes, which then induce responses in the fat body. This proposal is supported by previous demonstrations that NOS contributes to immune induction and that Domino mutant larvae, which have a severe reduction of hemocytes (among other defects), fail to induce Dipt in response to NO or to natural infection. Furthermore, psidin gene function in hemocytes promotes fat-body expression of AMPs (Brennan, 2007). The demonstration that CanA1 is required in hemocytes for the immune response in the fat body provides further support of this proposal. In this model, the response of the hemocyte to NO is independent of Imd, like the response of S2 cells, whereas robust induction of AMPs in downstream tissues requires Imd. Consequently, Imd acts downstream of NO to induce AMPs in larvae. These findings argue that tissue-to-tissue signaling plays a role in a natural infection model in larvae and that CanA1 participates in this signaling (Dijkers, 2007).

PGRP-SC2 promotes gut immune homeostasis to limit commensal dysbiosis and extend lifespan

Interactions between commensals and the host impact the metabolic and immune status of metazoans. Their deregulation is associated with age-related pathologies like chronic inflammation and cancer, especially in barrier epithelia. Maintaining a healthy commensal population by preserving innate immune homeostasis in such epithelia thus promises to promote health and longevity. This study shows that, in the aging intestine of Drosophila, chronic activation of the transcription factor Foxo reduces expression of peptidoglycan recognition protein SC2 (PGRP-SC2), a negative regulator of IMD/Relish innate immune signaling, and homolog of the anti-inflammatory molecules PGLYRP1-4. This repression causes deregulation of Rel/NFκB activity, resulting in commensal dysbiosis, stem cell hyperproliferation, and epithelial dysplasia. Restoring PGRP-SC2 expression in enterocytes of the intestinal epithelium, in turn, prevents dysbiosis, promotes tissue homeostasis, and extends lifespan. These results highlight the importance of commensal control for lifespan of metazoans and identify SC-class PGRPs as longevity-promoting factors (Jasper, 2014).

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).

Peptidoglycan sensing by octopaminergic neurons modulates Drosophila oviposition

As infectious diseases pose a threat to host integrity, eukaryotes have evolved mechanisms to eliminate pathogens. In addition to develop strategies reducing infection, animals can engage in behaviours that lower the impact of the infection. The molecular mechanisms by which microbes impact host behaviour are not well understood. This study demonstrated that bacterial infection of Drosophila females reduces oviposition and that bacterial cell wall peptidoglycan, the component that activates Drosophila antibacterial response, is also the elicitor of this behavioral change. Peptidoglycan regulates egg laying rate by activating PGRP-LC -> NF-κB (Relish) signaling pathway in octopaminergic neurons and that, a dedicated peptidoglycan degrading enzyme acts in these neurons to buffer this behavioural response. This study shows that a unique ligand and signaling cascade are used in immune cells to mount an immune response and in neurons to control fly behavior following infection. This may represent a case of behavioural immunity (Kurz, 2017).

In addition to activate direct antimicrobial strategies, eukaryotes have developed behavioral mechanisms that facilitate the avoidance of pathogens or lower the impact of the infection. These phenotypes grouped under the term 'behavioral immunity' or 'sickness behavior' refer to a suite of neuronal mechanisms that allow organisms to detect the potential presence of disease-causing agents and to engage in behaviors which prevent contact with the invaders or reduce the consequences of the infection. Although such microbe-induced behavioral changes have been reported in Lepidoptera and Orthoptera, deciphering the molecular mechanisms involved is experimentally challenging in these insects. Indeed, such an analysis requires a model organism with genetic tools allowing the manipulation of actors and regulators of both the immune and neuronal systems. Recent reports, mainly in Drosophila, start to unravel some aspects of these peculiar host-microbe interactions. Stensmyr et al. demonstrated that Drosophila avoid food contaminated by pathogenic bacteria by using an olfactory pathway exquisitely tuned to a single microbial odor, Geosmin (Stensmyr, 2012). Produced by harmful microorganisms, Geosmin is detected by specific Drosophila olfactory sensory neurons which then transfer the message to higher brain centers. Activation of this olfactory circuit ultimately induces an avoidance response, and suppresses egg-laying and feeding behaviors, thereby reducing the infection risk of both the adult flies and their offspring. Drosophila not only modify their behavior to avoid contamination by microbes or parasites, but also once they have been contaminated in order to reduce the impact of infection. For instance, direct exposure to bacteria impacts sleep patterns and induces hygienic grooming. In addition, Drosophila plastically increases the production of recombinant offspring in response to parasite infection. Although certainly involving a neuro-immunological integration, these microbe-induced behavioral changes are rarely understood at the molecular level, namely with no information on the nature of the elicitor and on the cellular and molecular machineries that link bacteria detection to behavioral changes. Moreover, canonical immune signaling pathways were never reported as being involved in those processes (Kurz, 2017).

The data demonstrate that bacteria derived cell wall peptidoglycan (PGN) entry into the fly body cavity has, at least, two physiological consequences. In addition to activate innate immune response in fat body cells, it also blocks mature egg delivery in oviduct and hence reduces egg laying of infected females. It was further demonstrated that this bacterially induced behavioral change is due to an NF-κB pathway-dependent modulation in octopaminergic neurons. Evidence is presented that both responses, that are potentially detrimental if not down-regulated, are fine-tuned by distinct and specific PGN degrading enzymes. It is proposed that by regulating the level of internal PGN, flies adapt their egg-laying behavior to environmental conditions. In standard environmental conditions, PGRP-LB ensures that low level of PGN does not affect egg laying. However, whenever PGN concentration reaches a certain threshold, which either reflects an infection status or the presence of a highly contaminated food supply, NF-κB pathway activation in neurons is blocking egg release. As PGN of ingested bacteria is capable of reaching the internal fluid and triggering dedicated signaling cascades, one could imagine that such a mechanism prevents flies from laing their eggs in highly contaminated food, in which their development and that of the hatching larvae could be impaired by microbes. In this context, PGRP-LB mediated PGN scavenging is crucial since a non-regulated behavioral immune response would lead to a severe drop in the amount of progeny which may not be in keeping with the real threat. Another possibility could be that a reduced egg production will favor immune effector production. Indeed, it is often considered that the energy cost of an acute innate immune response needs can be balanced by a decreased offspring production. Blocking the energy-consuming egg production in infected flies could be a way for them to mobilize resources required for full activation of innate immune defences. A similar depression of oviposition has recently been documented in females flies exposed to parasitoid wasps who lay their eggs in Drosophila larvae. However, while visual perception of wasps by female flies induces a long-term decline in oviposition associated with an early stage-specific oocyte apoptosis, PGN effects are transient and rather lead to a late stage oocyte accumulation suggesting that although the final outcome is the same, the mechanisms differ (Kurz, 2017).

The data from this study indicate that PGN sensing acts on egg-laying behavior via neuronal modulation. NF-κB pathway signaling in octopaminergic neurons was identified as the actor of this PGN-dependent oviposition reduction. It would be informative to test whether bacterial infection is also affecting other octopamine-mediated behaviors such as reward in olfactory or visual learning, male-male courtship, male aggressive behavior. This would require to further characterize the nature of the octopamine neurons whose activation is modulated by infection and to consider that the phenotypes defined as being part of the sickness behaviours might be orchestrated directly by the immune system following the perception of microbes. Indeed, a PGRP-LBPD reporter line not only labels cells in the reproductive tract but also in thoraco-abdominal ganglia and in the brain with projections to proboscis, wings and legs. Likewise, octopaminergic neurons have been shown to innervate numerous areas in the brain and in the thoraco-abdominal ganglion and to project to various reproductive structures such as ovaries, oviducts and uterus, further work will be needed to exactly pinpoint the identity of the affected octopaminergic neurons, their targets and their effect on fly behavior. In addition, the question remains as to how NF-κB activation can modulate octopaminergic neurons activity. Among the possibilities is the modulation of octopamine neuron excitability, the regulation of octopamine production or its secretion. Knowing the NF-κB protein itself is required for this behavioral response and that increasing the amount of available octopamine via overexpression of the TβH enzyme rescues the oviposition drop, it is expected that IMD pathway activation in neurons will have transcriptional consequences. However, other hypotheses might be considered since Dorsal, a member of the other Drosophila NF-κB signaling cascade Toll, has been shown to function post-transcriptionally together with IκB and IRAK at the post-synaptic membrane to specify glutamate receptor density. It should also be noticed that PGRP-LC has recently been shown to control presynaptic homeostatic plasticity in mouse (Harris, 2015). One of the future challenges will be to understand how NF-κB activation is reducing octopaminergic signals (Kurz, 2017).

This study shows that Drosophila uses an unique bacteria associated molecular pattern to activate different processes related to host defence, namely the production of antimicrobial peptides and the modulation of oviposition behavior. Interestingly, it appears that in order to fine-tune these responses, different isoforms of the same PGN scavenging enzyme, PGRP-LB, are required. While the secreted PGRP-LBPC isoform certainly acts non cell-autonomously to dampen immune activation by circulating PGN, a putatively intracellular isoform PGRP-LBPD controls the effect of PGN on oviposition. Even more remarkable, this response is not transmitted via PGRP-LC but rather by the intracytoplasmic PGRP-LE receptor. Previous work has shown that PGRP-LE is also regulating response to bacteria in some part of the gut. Thus, it will be important to understand how PGN is trafficking within and through cells, and how PGRP-LBPD modulates PGRP-LE-dependent IMD pathway activation and whether it is also required to modulate other PGN/PGRP-LE-dependent responses (Kurz, 2017).

In essence, the results demonstrate that PGN, when ingested or introduced into the body cavity, not only activates antibacterial immune response but also influences neuronally controlled behaviors in flies. Importantly, the sickness behavior deciphered in this study does not appear to be a side effect of an energetically expensive immune response, but rather the result of a specific regulation. An orchestration of different processes required for the immune response was also exemplified by a recent report linking metabolism and immunity. Although not dissected to the molecular level, previous studies in mammals have suggested that similar interactions between PGN and neuronally controlled activities. For instance, PGN derived muropeptide MDP has been shown to display powerful somnogenic effect when injected into rabbit braint. It has also been shown that PGN produced by symbiotic microbiota may 'leak' into the bloodstream and reach organs distant to the gut, such as the bones. Finally, recent findings show that bacterial cell wall peptidoglycan traverses the murine placenta and reach the developing fetal brain where it triggers a TLR2-dependent fetal neuroproliferative response. A future challenge will be to test whether an NF-κB-dependent response to PGN is also taking place in mammalian neurons and directly influences the animal behavior (Kurz, 2017).

Targets of Activity

Since co-expression of Relish with Dif or Dorsal can enhance the expression of Drosomycin and defensin, it has been speculated that the proteins can interact directly with the target genes to regulate their expression. Among the immunity genes, the cecropin and diptericin promoters have been the most thoroughly analyzed. Previously, it has been shown that Dif can bind to the kappaB site in the cecropin promoter, whereas Dorsal can bind to a similar site in the diptericin promoter. The binding probably leads to activated transcription of cecropin and diptericin. Furthermore, Dif and Dorsal may preferentially activate cecropin and diptericin, respectively, based on DNA binding and transfection studies. Meanwhile, the results suggest the possibility that the heterodimers formed by these Rel proteins bind to kappaB sites in the promoters of drosomycin and defensin. Based on the conserved sequence of insect kappaB motif, similar kappaB sites have been identified in the promoters of both drosomycin and defensin. It was then tested whether Rel proteins expressed in the stable cell lines can interact directly with these kappaB sites using EMSA. The parental S2* cell extract exhibits very little DNA binding activity. The cell extract that expresses FLAG-tagged Relish has strong binding activity toward the oligonucleotide containing the drosomycin kappaB site. This activity is supershifted by anti-FLAG antibody, showing Relish is the protein factor that binds directly to the kappaB site. Compared with Relish, Dif and Dorsal have relatively lower binding affinity. When Relish is co-expressed with Dif or Dorsal, new complexes are formed that have faster mobility, probably due to the formation of Dif/Relish or Dorsal/Relish heterodimer, which has lower molecular weight. The result indicates that Relish enhances the binding of Dif and Dorsal to the kappaB site. Moreover, the corresponding bands are supershifted by anti-FLAG antibody, demonstrating that FLAG-tagged Relish complexes with Dif and Dorsal in binding to DNA (Han, 1999).

To further confirm that Rel proteins bind to the kappaB sites within the oligonucleotides, competition EMSA was carried out. Whereas 20-fold excess amount of the same wild type oligonucleotide effectively blocks the binding of the Rel proteins to the 32P-labeled wild type oligonucleotide, the oligonucleotide with mutated kappaB motif does not affect the binding, suggesting that the Rel proteins bind specifically to the kappaB motif of drosomycin (Han, 1999).

The binding activity of Rel proteins to the kappaB site of defensin promoter shows similar pattern to that of drosomycin promoter, albeit with lower affinity. Interestingly, this is coincident with lower level of defensin expression in S2* cells and in adult fly. Whether or not the affinity of the Rel proteins toward the target promoter determines the inducibility requires further investigation. When the cellular extracts that contain combinations of Rel proteins were examined, Relish was found to enhance the binding activity of both Dif and Dorsal. The corresponding bands could also be supershifted using the anti-FLAG antibody, showing that the complex indeed contains Relish. The result therefore demonstrates that Rel proteins can interact directly with the defensin promoter (Han, 1999).

The Cecropin A1 gene promoter is known to have a functional kappaB-site, and is thus a potential target for Relish regulation. The effect of Relish overexpression was tested on a CecA1-lacZ reporter gene construct after cotransfection of the mbn-2 blood cell line. In addition to a full-length Relish cDNA, the effect of a truncated 'Rel-only' construct that lacks the IkappaB-like domain was tested. Mammalian p50 is produced by the proteolytic degradation of the ankyrin domain of p105. The Rel-only contruct was designed to be similar to a p105 fragment that produces a stable p50 when transfected into mammalian cells. Overexpression of the full-length Relish gene stimulates expression from the CecA1-lacZ fusion reporter 3-fold over the maximally lipopolysaccharide-induced control, while the Rel-only construct increases expression as much as 10-fold. Relish can thus stimulate cecropin transcription, directly or indirectly, and the sequences present in the Rel-only construct are sufficient for this effect. The lesser effect seen with the full-length construct may be due to the presence of an inhibitory IkappaB-like domain. Alternatively, this construct may be less efficiently translated, since the cDNA clone contains a short open reading frame 5' of the start site that is likely to reduce the level of protein expression. Similar false starts are found at the 5' end of Dif cDNA clones, and these have been suggested to possibly serve a translational regulation function (Dushay, 1996).

The role of ubiquitination in Drosophila innate immunity: Signaling upstream of Relish

Infection of Drosophila by Gram-negative bacteria triggers a signal transduction pathway (the IMD pathway) culminating in the expression of genes encoding antimicrobial peptides. A key component in this pathway is a Drosophila IkappaB kinase (DmIKK) complex, which stimulates the cleavage and activation of the NF-kappaB transcription factor Relish. Activation of the DmIKK complex requires the MAP3K dTAK1, but the mechanism of dTAK1 activation is not understood. In human cells, the activation of TAK1 and IKK requires the human ubiquitin-conjugating enzymes Ubc13 and UEV1a. This study demonstrates that the Drosophila homologs of Ubc13, (Bendless) and UEV1a, are similarly required for the activation of dTAK1 and the DmIKK complex. Surprisingly, the Drosophila caspase DREDD and its partner dFADD are required for the activation of DmIKK and JNK, in addition to their role in Relish cleavage. These studies reveal an evolutionarily conserved role of ubiquitination in IKK activation, and provide new insights into the hierarchy of signaling components in the Drosophila antibacterial immunity pathway (Zhou, 2005).

A cell culture system was established to study the IMD and Toll signaling pathways in S2 cells. The IMD pathway is activated by treating the cells with Gram-negative peptidoglycan (which is present in crude preparations of lipopolysaccharides), whereas activation of the Toll pathway is achieved by treating the cells with the Spätzle ligand. Active Spätzle is produced from a cell line stably transfected with a plasmid containing the copper-inducible metallothionein promoter driving the expression of active Spätzle C-106. When these cells are treated with copper, active SPZ is secreted into the medium, and this conditioned media can be used to activate naïve cells. Using the RNAi-mediated gene inactivation method, it was found that SPZ-induced Drosomycin gene activation in S2 cells requires the Drosophila Rel proteins Dif and Dorsal, as well as Toll, dMyD88, Tube, Pelle, and Slimb, as expected. In sharp contrast, these dsRNAs do not block the expression of antimicrobial peptide genes induced by peptidoglycan. Instead, RNAi studies demonstrate that peptidoglycan-induced gene expression requires all known components of the IMD pathway. Therefore this RNAi approach was used to determine the roles of candidate signaling components in the IMD and Toll signaling pathways (Zhou, 2005).

Previous studies have shown that activation of the mammalian IKK complex requires a ubiquitination step. In particular, TRAF6-mediated IKK activation was shown to require a dimeric ubiquitin-conjugating enzyme composed of the Ubc13 and UEV1a proteins. Bendless and dUEV1a are the Drosophila homologs of Ubc13 and UEV1a, respectively. Bendless and dUEV1a, like their mammalian counterparts, associate with each other in vivo. To investigate whether Bendless and dUEV1a are required for antibacterial gene expression in response to peptidoglycan, the RNAi-mediated gene inactivation method was used. S2 cells were transfected with various dsRNAs. After 48 h, cells were first treated with 20-hydroxy-ecdysone for 24 h to enhance their competence to induce antimicrobial genes in response to immune challenge, and then treated with peptidoglycan or SPZ to activate the IMD or Toll signaling pathways, respectively. Total RNA was isolated from these cells and subjected to Northern blotting analysis using cDNA fragments corresponding to Diptericin or Drosomycin as probes, to examine the activation of the IMD and Toll pathways, respectively. Both Bendless and dUEV1a are required for maximal levels of antibacterial peptide gene expression in response to peptidoglycan treatment. In fact, when both Bendless and dUEV1a are both targeted by RNAi, Diptericin induction is reduced to near background levels. (The partial effect of Bendless or dUEV1a RNAi alone is likely because of the fact that RNAi often does not generate a complete null phenotype.) By contrast, the induction of Drosomycin by Toll activation is unaffected by the RNAi-mediated knock-down of Bendless and dUEV1a. Note that the same cells were stimulated with either peptidoglycan or SPZ in. As a control, S2 cells treated with DmIKKγ or Toll dsRNA showed significantly reduced peptidoglycan-induced Diptericin or SPZ-induced Drosomycin gene expression, respectively. As a control, mRNA and/or protein levels of targeted genes were examined by RT-PCR and/or Western blotting to confirm the effectiveness of RNAi (Zhou, 2005).

To provide further evidence that Bendless is involved in the IMD pathway, a dominant negative mutant Bendless was used to determine whether peptidoglycan-induced antibacterial gene activation can be blocked. Stable S2 cell lines were generated that express either wild-type or C87A Bendless under the control of the metallothionein promoter. The cysteine to alanine mutation at position 87 creates a dominant-negative mutant because this residue, located in the catalytic pocket in ubiquitin-conjugating enzymes, is crucial for the catalytic activity of E2s. These cells were then treated with various combinations of peptidoglycan and copper, and Northern blotting was employed to examine the expression of antibacterial genes including Attacin, Cecropin and Diptericin. Overexpression of wild-type Bendless has no effect on peptidoglycan-induced antibacterial gene activation, since similar levels of antibacterial peptide gene expression were detected in cells treated with or without copper. In contrast, overexpression of Bendless C87A leads to a significant reduction in peptidoglycan-activated expression of antibacterial peptide genes (Zhou, 2005). Bendless flies have been identified which carry a proline to serine substitution at position 97 within the strictly conserved active site region of E2s. In order to determine whether bendless flies are defective in response to Gram-negative bacterial infection, both wild-type and bendless flies were subjected to E. coli infection and Diptericin gene activation was examined by Northern blotting. bendless flies display significantly weaker Diptericin gene activation compared with wild-type flies. These results indicate that the Bendless-dUEV1a E2 complex is required for signaling by the IMD pathway (Zhou, 2005).

Experiments were carried out to determine whether Bendless and dUEV1a are required for peptidoglycan-induced activation of the DmIKK complex. Previous studies have shown that peptidoglycan treatment induces the kinase activity of the endogenous DmIKK complex in S2 cells. Cells were transfected with dsRNAs corresponding to various mRNAs. After 48 h, these cells were treated with peptidoglycan for 15 min, and the endogenous DmIKK complex was immunoprecipitated and subjected to in vitro kinase assays using recombinant Relish protein as substrate. Bendless or dUEV1a dsRNA treatment leads to a significant decrease in peptidoglycan-induced DmIKK kinase activity, suggesting that Bendless and dUEV1a are required for peptidoglycan-induced DmIKK activation. As a control, DmIKKγ dsRNA treatment completely abolished peptidoglycan-induced DmIKK activation. It is concluded that the ubiquitin-conjugating enzymes Bendless and dUEV1a are specifically involved in the Drosophila IMD pathway, and they play a role upstream of the DmIKK complex (Zhou, 2005).

Overexpression of IMD in Drosophila results in the activation of antibacterial genes in the absence of bacterial infection. IMD overexpression, under control of the copper-inducible metallothionein promoter, can also strongly activate expression of the Diptericin gene in S2 cells. This stable cell line therefore provides a useful tool to perform an epistatic analysis to determine the position of Bendless/dUEV1a complex relative to IMD in the Drosophila antibacterial signaling pathway. The IMD stable cells were first transfected with dsRNAs derived from various genes and then stimulated with copper or peptidoglycan, and IMD- and peptidoglycan-induced Diptericin gene activation was examined. Overexpression of IMD, via the addition of copper, leads to strong activation of the Diptericin gene. In fact, copper-induced IMD expression is as potent as peptidoglycan in driving diptericin expression. Cells transfected with LacZ dsRNA show a similar Diptericin expression profile compared with cells that were mock-treated. Consistent with the observation that the DmIKK complex functions downstream of IMD, cells transfected with DmIKKγ dsRNA are severely defective in both IMD- and peptidoglycan-induced Diptericin expression. Furthermore, cells treated with dsRNAs derived from Bendless and dUEV1a genes display a significant reduction in both peptidoglycan- and IMD-mediated Diptericin gene activation. These results indicate that the ubiquitin conjugating enzymes Bendless and dUEV1a function downstream of IMD in this signaling pathway (Zhou, 2005).

Thus, by differentially activating the IMD and Toll signaling pathways in Drosophila S2 cells, this study shows that Bendless (dUbc13) and dUEV1a are required for the Drosophila IMD signaling pathway. Using RNAi to target Bendless and/or dUEV1a significantly reduces the levels of peptidoglycan-induced antibacterial peptide gene expression and activation of the Drosophila IKK complex. This mechanism of IKK activation is highly conserved; in mammals Ubc13 and UEV1a are required for TNFalpha-, IL-1β-, and TCR-mediated IKK and NF-kappaB activation (Zhou, 2005).

This ubiquitin-dependent kinase activation does not involve proteasome-mediated degradation. Proteasome inhibitors do not block IKK activation, in flies or humans. Moreover, ubiquitination without degradation has been shown to activate the human IKK complex, and a similar Drosophila activity has been identified, The primary sequence of the mammalian Ubc13/UEV1a and those of the Drosophila Bendless/dUEV1a are highly conserved (90% similarity for Ubc13, 79% for UEV1a). The crystal structures of the yeast and human Ubc13/UEV1a (Mms2) have shown clearly that this E2 complex can make only K63-linked polyubiquitin chains. Thus, it is likely that the Drosophila Bendless/dUEV1a E2 catalyzes the formation of K63-linked ubiquitin chains (Zhou, 2005).

In a cell-free system, human TRAF6 was shown to be an E3 ligase that auto-ubiquitinates in conjunction with Ubc13/UEV1a. This results in the activation of TRAF6 and, in turn, the activation of TAK1. Activated TAK1 phosphorylates key serine residues in the activation loop of IKKβ, resulting in the activation of IKKβ. It is suspected that similar mechanisms are involved in the Drosophila IMD pathway. This study demonstrates that the Drosophila TAK1 homolog functions downstream of Bendless and dUEV1a. Furthermore, a Drosophila homolog of TAB2/TAB3 is also required for the IMD pathway. Interestingly, the C-terminal zinc finger domain of TAB2, which is conserved in the Drosophila protein, has recently been shown to bind specifically to K63-polyubiquitin chains. Strikingly, it has found that a galere/dTAB2 mutant, which is defective in the IMD pathway, carries a mutation in this zinc finger domain (Zhou, 2005).

The Drosophila E3 ligase, analogous to human TRAF2 or TRAF6, which functions with Bendless and dUEV1a in the activation of dTAK1 and DmIKK remains to be identified. In Drosophila, the dTRAF2 protein is the closest homolog of mammalian TRAF6, and it is the only Drosophila TRAF protein that contains the RING domain, typical of E3 ligases. However, RNAi knockdown and dominant-negative studies suggest that dTRAF2 is not involved in either the IMD or the Toll signaling pathways in S2 cells. In fact this gene is expressed at undetectably low levels in S2 cells. In one previous study dTRAF2 was reported to interact physically and functionally with Pelle, a key signaling component in the Toll signaling pathway that controls the antifungal immune response. However, these studies were based on overexpression experiments and in vitro binding assays, which might not reflect the physiological role of dTRAF2. In contrast, a recent publication demonstrated that dTRAF2 mutants are not fully able to induce antimicrobial peptide genes following E. coli infection. However, these studies did not clearly determine whether dTRAF2 is involved in the Toll or IMD pathways. The data suggest that dTRAF2 is not a critical component of the IMD pathway in S2 cells. Further studies, in cells and in flies, are necessary to elucidate the role of dTRAF2 in Drosophila immunity (Zhou, 2005).

The possibility is considered that other Drosophila RING-containing proteins might be involved in Drosophila immunity. However, RNAi knockdown studies with 10 different RING domain-encoding genes failed to block either the Toll or IMD pathways. Finally, since the structure of the RING domain of Rad5 has been successfully modeled to fit into the structure of the dimeric ubiquitin-conjugating enzyme complex Ubc13/UEV1a, it was reasoned that the potential ubiquitin ligase involved in the IMD pathway might physically interact with Bendless and dUEV1a. Therefore yeast two-hybrid screens were performed using Bendless and dUEV1a as baits in an effort to identify their protein interaction partners. A Drosophila RING protein, CG14435, was identified in such screens. CG14435 interacts robustly with both Bendless and dUEV1a in yeast two-hybrid assays. Furthermore, the CG14435-Bendless and CG14435-dUEV1a interaction was confirmed by co-immunoprecipitation of overexpressed proteins in S2 cells. However, RNAi-based studies suggest that CG14435 is not involved in the Drosophila innate immunity signaling pathways. It has been shown that bendless flies display defective synaptic connectivity and abnormal morphology within the visual system, suggesting Bendless functions in a variety of developmental processes. In addition, the Saccharomyces cerevisiae homologs of Bendless and dUEV1a have been implicated in DNA damage repair. Therefore it is possible that CG14435 is involved in some cellular processes other than immunity which require Bendless and dUEV1a. Further studies are necessary to elucidate the physiological role of CG14435 and to identify the ubiquitin ligase activity required for ubiquitination-dependent DmIKK activation (Zhou, 2005).

As in the TRAF6 pathway, dTRAF2 and/or other E3 ligases that function with Bendless and dUEV1a in the IMD pathway may be the target(s) of K63 polyubiquitination. Another possible target of Bendless/dUEV1a-mediated ubiquitination is the Drosophila IKKγ subunit (also known as NEMO in mammals) of the IKK complex. In mammals, it has recently been shown that NEMO is K63 polyubiquitinated by the Ubc13/UEV1a complex in response to Bcl10 expression or T-cell activation. Other possible targets of ubiquitination by Bendless and dUEV1a in the IMD pathway include the Drosophila TAB2 homolog and IMD. Recently, it was shown that the two mammalian homologs of TAB2 and TAB3 were ubiquitinated or associated with other ubiquitinated proteins. In addition, the mammalian RIP1, which is homologous to IMD protein especially in its death domain, has recently been shown to be K63 polyubiquitinated and associated with TAB2 in a TNFalpha-dependent manner. In any case, K63 polyubiquitin chains likely function to recruit the Drosophila TAK1/TAB2 complex, via the TAB2 K63 polyubiquitin binding domain, to either (or both) the upstream activators, such as IMD, and/or the downstream target of dTAK1 kinase activity, the Drosophila IKK complex (Zhou, 2005).

As expected, the epistatic analyses presented in this study demonstrate that IMD functions upstream of all other components in the pathway except the receptor PGRP-LC, and is required for IKK activation. Moreover, Bendless and dUEV1a function downstream of IMD and upstream of dTAK1, as predicted from the model for Ubc13 and UEV1a in mammals. dTAK1 is required for activation of the Drosophila IKK complex and likely functions as the IKK kinase (Zhou, 2005).

Although it is established that dFADD and DREDD are required for the IMD pathway, previous experiments have suggested that they function downstream of the DmIKK complex. For example, DREDD overexpression in flies leads to Diptericin gene expression in the absence of Gram-negative bacterial infection, and DREDD-mediated Diptericin gene activation requires neither the DmIKK complex nor dFADD. Also, recent studies have shown that DREDD interacts with Relish, and that a caspase-cleavage site within Relish is required for peptidoglycan-induced Relish activation. Therefore, it was speculated that DREDD functions downstream of the DmIKK complex by directly cleaving DmIKK-phosphorylated Relish. This possibility is consistent with the observation that DREDD and dFADD are required for dTAK1Delta-mediated Diptericin gene activation. Surprisingly, dFADD and DREDD are required for peptidoglycan-induced DmIKK activation, arguing that DREDD and dFADD function upstream in the pathway. In addition, DREDD is also required for peptidoglycan-induced JNK activation, but neither DREDD nor dFADD are required for dTAK1- or dTAK1Delta-mediated DmIKK activation, suggesting that dFADD and DREDD act at a step upstream of dTAK1 in response to peptidoglycan. Based on these observations, it is proposed that dFADD and DREDD play dual roles in the Drosophila antibacterial signaling pathway. On the one hand, dFADD transduces signals from IMD to DREDD, resulting in DREDD activation and enabling Relish cleavage; in contrast, dFADD and DREDD contribute to peptidoglycan-induced DmIKK activation through a mechanism that remains to be elucidated. DREDD may function similarly to human Caspase-8, which is a DED-containing apical caspase similar to DREDD. Caspase-8 has recently been shown to be required for NF-kappaB activation in response TCR-signaling. This role of Caspase-8 requires the enzymatic activity of full-length protein Caspase-8 and is involved in recruiting the IKK complex to the upstream signaling complex of CARMA1, Bcl10, and MALT1. Interestingly MALT1 is also a caspase-like gene (sometimes referred to as a paracaspase), and it is thought to function as an E3-ligase accessory factor with Ubc13 and UEV1a in TCR-mediated NF-kappaB activation. DREDD may similarly function as E3-ligase accessory factors with Bendless and dUEV1a as the E2, in the IMD pathway (Zhou, 2005).

The following scheme is proposed for the Drosophila antibacterial signaling pathway. Peptidoglycan treatment or Gram-negative bacterial infection leads to the activation of the membrane-bound peptidoglycan-recognition protein, PGRP-LC. Activated PGRP-LC in turn transduces signal to IMD. IMD, in turn, interacts with dFADD and subsequently DREDD. It is proposed that IMD, dFADD and DREDD form a complex that contributes to dTAK1 activation, perhaps as part of an E3 ligase. This complex is likely to function in conjunction with Bendless/dUEV1a to activate dTAK1 and then DmIKK. Once activated, the Drosophila IKK complex phosphorylates Relish, which is subsequently cleaved. The N-terminal Relish cleavage product, an NF-kappaB transcription factor, then translocates to the nucleus where it activates antimicrobial peptide gene expression. In addition to their role in IKK activation, DREDD and dFADD are also proposed to function downstream in this pathway, in the signal-induced cleavage of phospho-Relish (Zhou, 2005).

IAP DIAP2, acting downstream of or in parallel to relish, functions in innate immunity, in a caspase independent role, and is essential to resist gram-negative bacterial infection

The founding member of the Inhibitor of Apoptosis (IAPs) protein family was originally identified as a cell death inhibitor. However, recent evidence suggest that IAPs are multifunctional signalling devices that influence diverse biological processes. In order to investigate the in vivo function of Drosophila IAP2 diap2 null alleles were generated. diap2 deficient animals develop normally and are fully viable suggesting that diap2 is dispensable for proper development. However, these animals are 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) signalling cascade, a NF-kappaB-dependent pathway that shares striking similarities with the one of mammalian tumour necrosis factor receptor 1 (TNFR1). diap2 mutant flies fail to activate NF-kappaB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumb to bacterial infection. Genetic epistasis analysis places diap2 downstream of or in parallel to imd, Dredd, Tak1 and Relish (see however the article by Huh, 2007, described below). Therefore, DIAP2 functions in host-immune response to Gram-negative bacteria. In contrast, it was found that the Drosophila Traf-family member Traf2 is dispensable to resist Gram-negative bacterial infection. Taken together, these genetic data identify DIAP2 as an essential component of the Imd signalling cascade protecting the organism from infiltrating microbes (Leulier, 2006).

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. 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, 2006).

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, 2006).

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, 2006).

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, 2006).

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, 2006).

Insect neuropeptide bursicon homodimers induce innate immune and stress genes during molting by activating the NF-κB transcription factor Relish

Bursicon is a heterodimer neuropeptide composed of two cystine knot proteins, bursicon α (burs α) and bursicon β (burs β), that elicits cuticle tanning (melanization and sclerotization) through the Drosophila leucine-rich repeats-containing G protein-coupled receptor 2 (DLGR2). Recent studies have show that both bursicon subunits also form homodimers. However, biological functions of the homodimers have remained unknown. This report shows in Drosophila that both bursicon homodimers induced expression of genes encoding antimicrobial peptides (AMPs), Diptericin (Dpt), Cecropin B (Cec B), Attacin A (Att A), Turandot B (Tot B), and Attacin B (Att B), but not Drosomycin (Drs) in neck-ligated adults following recombinant homodimer injection and in larvae fat body after incubation with recombinant homodimers. These AMP genes were also up-regulated in 24 h old unligated flies (when the endogenous bursicon level is low) after injection of recombinant homodimers. Up-regulation of AMP genes by the homodimers was accompanied by reduced bacterial populations in fly assay preparations. The induction of AMP expression is via activation of the NF-κB transcription factor Relish in the immune deficiency (Imd) pathway. The influence of bursicon homodimers on immune function does not appear to act through the heterodimer receptor DLGR2, i.e. novel receptors exist for the homodimers. These results reveal a mechanism of CNS-regulated prophylactic innate immunity during molting via induced expression of genes encoding AMPs and genes of the Turandot family. Turandot genes are also up-regulated by a broader range of extreme insults. From these data it is inferred that CNS-generated bursicon homodimers mediate innate prophylactic immunity to both stress and infection during the vulnerable molting cycle (An, 2012).

The data in this paper support the hypothesis that the CNS influences innate immunity via secretion of a neurohormone and thus expands the biological roles of bursicon beyond cuticle tanning and wing expansion. Several points are germane. First, the expression patterns of genes encoding burs alpha and burs β subunits and six AMPs in untreated pharate and newly-eclosed adults appear in strong inverse correlation. Second, injection of r-burs α/α or burs β/β homodimers to the 24 h-old files, which displayed low levels of bursicon transcripts and AMP genes, up-regulates AMP genes, demonstrating a role for bursicon homodimers in mediating AMP gene transcription in vivo. Third, bursicon acts in the novel homodimer configuration. Fourth, the bursicon homodimers induce expression of genes encoding AMPs via the activation of the NF-kappaB transcription factor Relish. And fifth, burs α/α or burs β/β homodimers do not appear to regulate their effects on the immune system via the established heterodimer receptor, DLGR2. Sequence analysis revealed that the burs α and β subunits have no similarity to bacterial cell wall proteins, which bind the peptidoglycan recognition protein (PGRP) to activate immune responses. Although PGRP binding to bursicon has not been experimentally ruled out, it is inferred that bursicon homodimers do not act through the PGRP. Hence, bursicon homodimers activate components in the Imd signaling pathway, downstream of PGRP, but upstream of Relish. Future studies will focus on the identification of the novel receptor(s) involved in the action of bursicon homodimers on the immune system. Despite the preponderance of work on mammalian immunity, exactly how the CNS controls inflammation and the immune response is not understood completely. Perhaps the Drosophila model with its abundant genetic repertoire will help solve this ancient problem(An, 2012).

Bursicon is a member of the cystine knot protein family, which includes vertebrate glycoprotein hormones, growth factors, mucins, and bone morphogenetic protein antagonists. All these hormones, including bursicon, share a common structural feature of a α-subunit and a β-subunit which form the physiologically operational heterodimers. Some, such as the placental chorionic gonadotropin, also form homodimers to execute a different physiological function. It has been shown that each of the bursicon subunits forms a homodimer, shown in vitro in Drosophila by Western blot and in vivo in several insect species including Drosophila by immunocytochemistry, but the function of the homodimers was, until now, unknown. This study identified one role for the bursicon homodimers as mediators of the prophylactic expression of genes encoding AMPs(An, 2012).

It is also noted that bursicon homodimers induce expression of several turandot and Tep1 genes, markers for the JAK/STAT pathway. Since the transcriptional regulation of the JAK/STAT pathway requires inputs from the Imd pathway, up-regulation of turandot and Tep1 genes could result from up-regulation of the Imd pathway. The biological significance of these gene products extends beyond anti-microbial actions to the generalized responses to extreme stressors (Ekengren, 2001a). While other stress-responsive proteins, such as heat shock proteins, act within cells, the Turandot proteins are secreted into the hemolymph following a variety of stress experiences (Ekengren, 2001b). Like developmental and reproductive events in many animals, molting produces actual and potential stresses in insects, including increased energy demands (producing reactive oxygen species), water loss, ion imbalances, injury and infection. It is concluded that the expression of general stress-responsive genes could be an important adaption during the highly susceptible time of the molting cycle (An, 2012).

Protein Interactions

Degradation of Relish

The Rel/NF-kappaB transcription factor Relish plays a key role in the humoral immune response in Drosophila. Activation of this innate immune response is preceded by rapid proteolytic cleavage of Relish into two parts. The proteolytic cleavage of Relish depends on Death related ced-3/Nedd2-like protein (Dredd). An N-terminal fragment, containing the DNA-binding Rel homology domain, translocates to the nucleus where it binds to the promoter of the Cecropin A1 gene and probably to the promoters of other antimicrobial peptide genes. The C-terminal IkappaB-like fragment remains in the cytoplasm. This endoproteolytic cleavage does not involve the proteasome, requires the DREDD caspase, and is different from previously described mechanisms for Rel factor activation (Stöven, 2000).

The inducible production of antimicrobial peptides is a major immune response in Drosophila. The genes encoding these peptides are activated by NF-κB transcription factors that are controlled by two independent signaling cascades: the Toll pathway that regulates the NF-κB homologs, Dorsal and DIF; and the IMD pathway that regulates the compound NF-κB-like protein, Relish. Although numerous components of each pathway that are required to induce antimicrobial gene expression have been identified, less is known about the mechanisms that either repress antimicrobial genes in the absence of infection or that downregulate these genes after infection. In a screen for factors that negatively regulate the IMD pathway, two partial loss-of-function mutations were isolated in the SkpA gene that constitutively induce the antibacterial peptide gene, Diptericin, a target of the IMD pathway. These mutations do not affect the systemic expression of the antifungal peptide gene, Drosomycin, a target of the Toll pathway. SkpA encodes a homolog of the yeast and human Skp1 proteins. Skp1 proteins function as subunits of SCF-E3 ubiquitin ligases that target substrates to the 26S proteasome, and mutations affecting either the Drosophila SCF components, Slimb and Cullin1, or the proteasome also induce Diptericin expression. In cultured cells, inhibition of SkpA and Slimb via RNAi increases levels of both the full-length Relish protein and the processed Rel-homology domain. It is concluded that in contrast to other NF-κB activation pathways, the Drosophila IMD pathway is repressed by the ubiquitin-proteasome system. A possible target of this proteolytic activity is the Relish transcription factor, suggesting a mechanism for NF-κB downregulation in Drosophila (Khush, 2002).

In wild-type flies Diptericin is tightly controlled by the IMD pathway. Therefore, to identify genes that normally function to repress the IMD pathway, 2,000 yellow, white (y,w) F1 male progeny from male flies mutagenized with ethyl methanesulfonate were screened for constitutive expression of a Green Fluorescent Protein (GFP) reporter gene under the control of the Diptericin promoter. Two males, J6 and G49, expressed Diptericin-GFP, and this gene was constitutively expressed in larvae and adults in homozygous lines derived from these males. Although flies carrying the J6 and G49 mutations are viable and fertile at 25°C, G49 is pupal lethal at 29°C, indicating temperature-sensitive phenotypes associated with this mutation (Khush, 2002).

Using recombination mapping, the J6 and G49 mutations were shown to be tightly linked to the y locus on the proximal tip of the X chromosome. To further localize the two mutations, deletions were used to determine that J6 falls in the area defined by the overlap of Df(1)74k24.1, Df(1)svr, and Df(1)su(s)83, placing it in cytological region 1B10 near the Dredd gene. Two lethal P-element insertions in the Bloomington stock center collection, l(1)G0389 and l(1)G0109, which map near this region, were shown to not complement the constitutive Diptericin expression in the J6 and G49 lines. By sequencing DNA flanking the P elements in the two insertion lines, both elements were ascertained to lie within 200 bp of each other in the 5' untranslated region of the SkpA gene. To confirm that J6 and G49 are mutations in SkpA, a wild-type SkpA transgene on the second chromosome was shown to suppress the constitutive Diptericin expression phenotype in G49 flies. The J6 and G49 lines were shown to each contain a point mutation in the SkpA gene that generates a single amino acid change in the SkpA protein: J6, renamed SkpAJ6, converts threonine 98 to an isoleucine, and G49, renamed SkpAG49, replaces glutamic acid 101 with a lysine. These alleles are hypomorphic mutations of SkpA since the P-element insertions are pupal lethal at 25°C. SkpAG49 is pupal lethal at 29°C, and homozygous SkpAG49 adults transferred to 29°C express Diptericin at similar levels as flies heterozygous for SkpAG49 and either the P-element insertions or deletions that remove SkpA. At 29°C, therefore, SkpAG49 behaves like a null mutation, which probably reflects the significant change from the negatively charged glutamic acid to the positively charged lysine in this allele (Khush, 2002).

The SkpA gene encodes a protein that is highly similar to Skp1 proteins in humans and yeast. Skp1 proteins are components of SCF ubiquitin ligases that target substrates to the proteasome, and crystal structures of human Skp1 complexed with the F-box protein Skp2 and the cullin protin Cul1 have been solved. SkpAJ6 and SkpAG49 both affect a conserved region of SkpA that corresponds to helix 5 of Skp1; helix 5 forms part of the core interface between Skp1, the F-box region of Skp2, and the amino-terminal domain of Cul1, with some amino acids in this helix making direct contact with residues in Skp2 and Cul1. This suggests that the SkpAJ6 and SkpAG49 mutations disrupt interactions between SkpA and the F-box protein and cullin components of an SCF complex. Protein interaction studies indicate that SkpA functions with the F-box protein Slimb and the Cullin-like protein Cullin1 (Cul1) in a Drosophila SCF complex. In support of this model, slimb1 and dcul1l(2)02074 mutant larvae, as well as larvae carrying the DTS5 mutation, a dominant-negative mutation that affects the β6 subunit of the 26S proteasome, were shown to express Diptericin at levels comparable to those in the SkpA mutants. To further test the DTS5 phenotype, the UAS-Gal4 system was used to overexpress a UAS-DTS5 transgene in larval fat bodies: DTS5 overexpression induces Diptericin to levels that are comparable to those generated by bacterial infection with Erwinia carotovora carotovora 15 (Ecc15). Flies heterozygous for mutations at both the SkpA and slimb loci were generated: these flies constitutively express Diptericin, indicating a synergistic interaction between SkpA and slimb. The constitutive Diptericin expression in the slimb1, dcul1l(2)02074, and DTS5 mutants and the interaction between SkpA and slimb together suggest that an SCFSkpA/Cul1/Slimb ubiquitin ligase represses Diptericin expression by targeting a regulatory factor for degradation by the 26S proteasome (Khush, 2002).

To determine if the constitutive Diptericin expression in the SCF complex mutants is mediated through the IMD pathway, Diptericin levels were examined in larvae homozygous for mutations in either SkpA, or slimb and various genes of the IMD pathway: SkpAJ6;imd1 and SkpAG49;dtak11 double mutants display constitutive Diptericin expression, although Diptericin levels are slightly reduced in the SkpAG49;dtak11 larvae. Mutations in DmIkkγ, DmIkkβ, and Relish, however, completely block Diptericin expression in the SkpAJ6 background, and a Dredd mutation completely blocks Diptericin expression in the slimb1 background. The constitutive Diptericin expression observed in SkpA and slimb mutants, therefore, does not require IMD and dTak1, but it is dependent on the DmIKK complex, Dredd, and Relish. These results imply that, in wild-type flies, the SCFSkpA/Cul1/Slimb negatively regulates the IMD pathway by targeting one of these factors, or an additional unidentified component of the IMD pathway, for degradation by the proteasome. In contrast to fat body cells, the IMD pathway is the primary regulator of all antimicrobial genes, including Drosomycin, in surface epithelial tissues. A Drosomycin-GFP transgene is constitutively expressed in tracheal cells but not in fat body cells of slimb1 mutant larvae; this expression pattern further demonstrates that the IMD pathway, but not the Toll pathway, is constitutively activated when the SCFSkpA/Cul1/Slimb complex is compromised (Khush, 2002).

Although the genetic results do not allow differentiation between the DmIKK complex, Dredd, Relish, or other unidentified downstream components of the IMD pathway as targets of the ubiquitin-proteasome pathway, the mammalian Relish homolog, P105, is regulated by an SCF complex that contains the Slimb homolog β-TrCP/E3RSIκB. Consequently, RNA-mediated interference (RNAi), an effective technique for specifically inhibiting targeted proteins, was used in cultured Drosophila S2 cells to test for interactions between the SCFSkpA/Cul1/Slimb complex and Relish. Initially, SkpA and Slimb activity were blocked in S2 cells via RNAi; then, transient expression of a full-length Relish protein, modified by an N-terminal FLAG tag, was induced in the same S2 cells and the effects of the SkpA and slimb RNAi treatments on FLAG-Relish protein stability was monitored using Western blots and anti-FLAG antibodies (Khush, 2002).

Reducing Slimb activity, in the absence of LPS stimulation, visibly increases steady-state levels of both full-length Relish and the active N-terminal Rel-homology domain; levels of both polypeptides are further increased by inhibiting Slimb and SkpA simultaneously. This effect is specific since RNAi of the SkpA homologs, SkpB and SkpD, does not increase Relish levels. Dredd RNAi does increase Relish levels at day 1, but this is probably because Dredd inhibition blocks Relish processing. Previous studies show that Relish processing in S2 cells is induced by lipopolysaccharide (LPS) and requires Dredd activity. As expected, therefore, RNAi of Dredd blocks LPS-induced Relish processing. Simultaneous RNAi of SkpA and Slimb in the presence of LPS, however, results in higher steady-state levels of the Rel-homology domain up to 4 days after Relish induction. Higher levels of the Rel-homology domain after SkpA and Slimb RNAi could be caused by increased processing of full-length Relish. However, because full-length Relish levels also mount, the explanation is favored that Rel-homology domain turnover is reduced. Although the Slimb and SkpA RNAi treatments appear to inhibit Relish turnover, Relish levels do eventually diminish. This suggests that RNAi efficiency decreases with time, possibly due to degradation of the transfected double-stranded RNA. These RNAi experiments indicate that the constitutive antimicrobial gene expression in SkpA and slimb mutant flies is caused by higher Relish levels, and they suggest that the SCFSkpA/Cul1/Slimb complex represses the IMD pathway by promoting the degradation of both full-length and processed Relish proteins (Khush, 2002).

If the constitutive antimicrobial gene expression in flies carrying mutations that affect the SCFSkpA/Cul1/Slimb complex or proteasome is due to higher Relish levels, this would imply some level of steady-state Relish activation. Low levels of the Rel-homology domain have been reported in nuclear extracts from unstimulated S2 cells, and these low levels indicate that Relish is constitutively processed. Increasing Relish levels in larvae and adults via the Gal4-UAS system is sufficient to induce low levels of Diptericin expression. These results indicate that Relish is constitutively processed and activated to some level, supporting the hypothesis that Relish activity, in the absence of infection, is countered by ubiquitination and degradation (Khush, 2002).

A Drosophila IkappaB kinase complex required for Relish cleavage and antibacterial immunity

A Drosophila IkappaB kinase complex containing DmIKKß (immune response deficient 5) and DmIKKγ (Kenny), homologs of the human IKKß and IKKγ proteins, has been identified. This complex is required for the signal-dependent cleavage of Relish, a member of the Rel family of transcriptional activator proteins, and for the activation of antibacterial immune response genes. In addition, the activated DmIKK complex, as well as recombinant DmIKKß, can phosphorylate Relish in vitro. Thus, it is proposed that the Drosophila IkappaB kinase complex functions, at least in part, by inducing the proteolytic cleavage of Relish. The N terminus of Relish then translocates to the nucleus and activates the transcription of antibacterial immune response genes. Remarkably, this Drosophila IkappaB kinase complex is not required for the activation of the Rel proteins Dif and Dorsal through the Toll signaling pathway, which is essential for antifungal immunity and dorsoventral patterning during early development. Thus, a yet to be identified IkappaB kinase complex must be required for Rel protein activation via the Toll signaling pathway (Silverman, 2000).

To identify the signaling components required for the Drosophila immune response a reverse genetic approach, taking advantage of the Drosophila Genome Project, was undertaken. A cDNA sequence with homology to the kinase domain of the human IKK genes was identified in the BDGP EST database. Examination of the amino acid sequence of the encoded protein, which has been designated DmIKKß (Drosophila melanogaster IKKß), displays significant similarity to the N-terminal region of hIKKalpha and hIKKß but is more similar to hIKKß. The amino acid sequence of the C terminus of DmIKKß is only weakly related to the corresponding regions of IKKalpha and IKKß. A predicted coiled-coil can be detected in a region corresponding to the predicted leucine zipper coiled-coil of hIKKalpha and hIKKß. The DmIKKß gene maps to chromosomal location 89B as determined with the BDGP P1 filter array (Silverman, 2000).

The mammalian IKKalpha and IKKß proteins are found in a high molecular weight IkappaB kinase complex that includes the structural component IKKγ or NEMO. To investigate the possibility that DmIKKß is also a component of a similar kinase complex, a yeast two-hybrid screen was performed using DmIKKß as bait. A Drosophila larval cDNA library was screened, and a total of 85 independent positive clones were analyzed. All of these clones were found to contain overlapping inserts from a cDNA that encodes a Drosophila protein with homology to hIKKγ primarily in its C terminus. This gene, referred to as DmIKKγ hereafter, maps to chromosomal location 60E as determined with the BDGP P1 filter array. Secondary structure predictions of the protein encoded by DmIKKγ suggest the existence of several coiled-coil regions. The overall amino acid sequence homology between DmIKKγ and hIKKγ suggests a putative structural and functional relationship between the two proteins, especially in their C-terminal halves. DmIKKß and DmIKKγ interact to form a complex both in vivo and in vitro (Silverman, 2000).

To determine whether DmIKKß and DmIKKγ are involved in the activation of antibacterial genes, an LPS-inducible Drosophila cell line was engineered to express different versions of these genes. When S2* cells are treated with LPS, the expression of antibacterial peptide genes, such as Diptericin, Cecropin, and Attacin, are induced. Thus, S2* cells were stably transfected with plasmids that express wild-type or potentially dominant negative versions of DmIKKß and DmIKKγ under the control of the copper-inducible metallothionein promoter. Four cell lines were generated that express one of the following proteins: DmIKKß wild type, DmIKKß K50A, DmIKKγ wild type, or DmIKKγ 201-387. DmIKKß K50A was chosen because similar mutations in hIKKalpha or hIKKß, which change a conserved lysine in the ATP binding domain, create dominant negative proteins, whereas the DmIKKγ201-387 was designed because a similarly truncated hIKKγ acts as a dominant negative mutant in human cells. Full-length hIKKγ can also act as a dominant negative when expressed at high levels, presumably by titrating limiting components required for functional complex assembly. DmIKKß and DmIKKγ interact to form a complex both in vivo and in vitro (Silverman, 2000).

After the addition of copper, DmIKK proteins are rapidly produced at high levels as detected by immunoblotting. To determine whether these DmIKK proteins block LPS induction of antibacterial peptide expression in S2* cells, the stable cell lines were first treated with copper to induce expression of the DmIKKs and then stimulated with LPS. DmIKKß K50A blocks the LPS induction of the Diptericin, Cecropin, and Attacin genes. Similarly, either full-length or truncated versions of DmIKKγ are potent inhibitors of antibacterial peptide gene expression. These data clearly show that DmIKKß K50A, DmIKKγ full-length, or DmIKKγ201-387 potently inhibit Diptericin, Cecropin, and Attacin gene induction. By contrast, the expression of wild-type DmIKKß slightly decreases the level of expression of these genes. It is concluded that DmIKKß and DmIKKγ are required for the activation of antibacterial gene expression in response to LPS (Silverman, 2000).

Genetic studies have shown that the Drosophila Relish gene is essential for the antibacterial immune response. Bacterial infection or LPS treatment activates the endoproteolytic cleavage of Relish. Once cleaved, the N-terminal RHD of Relish translocates into the nucleus, where it activates the transcription of antibacterial genes. Considering that the overexpression of dominant negative DmIKKß or DmIKKγ blocks the induction of antibacterial peptide genes, it is likely that DmIKKß (and the DmIKK complex) functions in the signaling pathway leading to the cleavage of Relish. In order to test this possibility, the DmIKK overexpressing cell lines were employed to follow the fate of Relish protein cleavage. The stable cell lines and the parental cells were first treated with copper and then induced with LPS for 15 min. LPS treatment of the parental cells, or any of the stable lines that were not treated with copper, results in Relish cleavage. Full-length Relish (~110 kD) is cleaved to generate fragments of ~68 kD and ~49 kD, corresponding to the N- and C-terminal fragments, respectively. Expression of DmIKKß K50A, DmIKKγ, or DmIKKγ201-387 blocks the cleavage of Relish. Overexpression of wild-type DmIKKß causes a slight accumulation of full-length Relish (Silverman, 2000).

To obtain additional evidence that DmIKKß and DmIKKγ are required for LPS-induced antibacterial gene expression, the inhibitory effect of double-stranded RNA (dsRNA), also referred to as RNAi, was exploited. The presence of gene-specific dsRNA molecules in Drosophila or Caenorhabditis elegans embryos has been shown to destabilize the cognate mRNAs. dsRNA corresponding to the DmIKKγ or DmIKKß genes was synthesized in vitro and transfected into the LPS-inducible S2* cell line. Transfection of gene-specific dsRNA causes a significant reduction in the amount of the corresponding mRNA. For example, DmIKKγ dsRNA lowers DmIKKγ mRNA to nearly undetectable levels, while DmIKKß or LacZ dsRNA has no effect on DmIKKγ mRNA levels. DmIKKγ protein levels are also greatly reduced only in those cells transfected with DmIKKγ dsRNA; however, it is important to note that although DmIKKγ protein is reduced approximately 10-fold, it is still detectable after RNAi treatment. Importantly, dsRNA-mediated interference of either DmIKKß or DmIKKγ greatly inhibits the LPS-induced expression of antibacterial genes, such as Attacin, Cecropin, and Diptericin. The LPS-induced cleavage of Relish is also inhibited by either DmIKKß or DmIKKγ dsRNA. Full-length Relish protein persists after the induction with LPS only in those lanes transfected with DmIKK dsRNA. Although the inhibition of Relish cleavage seen with DmIKK RNAi is not as dramatic as that observed with the dominant negative DmIKKs, it is clear that full-length Relish is not as efficiently cleaved in those cells with reduced levels of DmIKKß or DmIKKγ. In both the dominant negative and the RNAi experiments, a stronger inhibition is always observed at the transcriptional level, as compared to that seen at the protein level. This suggests that small perturbations in the amount of the nuclear translocated Relish can have dramatic effects on the level of transcriptional activation. Small differences in the amounts of transcription factors have been shown to exhibit all-or-none effects on promoters that require the cooperative assembly of transcription enhancer complexes. dsRNA-mediated interference does not create a null allele. Thus, the reduced amount of Relish cleavage and antibacterial gene expression still observed in the RNAi-treated cells is most likely due to the remaining DmIKKs found in these cells. Both the dominant negative and RNAi experiments show that DmIKKß and DmIKKγ are required for LPS-induced Relish endoproteolytic cleavage and transcriptional activation of antibacterial peptide genes in vivo (Silverman, 2000).

The Toll signaling pathway, which leads to Cactus degradation and activation of Dif and Dorsal, is necessary for the antifungal immune response as well as early embryonic patterning. The DmIKKß/γ complex could be involved in the Toll signaling pathway in addition to its role in the antibacterial pathway. To test this possibility directly, the RNAi technique was used with a Drosophila cell line specifically engineered to assay the Toll signaling pathway. Schneider S2* cells were stably transfected with a torso-pelle fusion gene controlled by the metallothionein promoter, creating the S2*tpll cell line. Previous studies have demonstrated that fusing the transmembrane domain of torso to the pelle gene creates a constitutively active pelle kinase that can stimulate the Toll signaling pathway in fly embryos or in tissue culture cells. In the S2*tpll cell line, addition of copper leads to the activation of the Toll antifungal pathway. Drosomycin transcription is induced, but Diptericin transcription is unaffected. Transfection of dsRNA into the S2*tpll cells leads to specific loss of the corresponding mRNA and protein. DmIKKγ dsRNA causes a substantial reduction in the level of DmIKKγ protein. However, neither DmIKKß nor DmIKKγ dsRNA blocks the torso-pelle-mediated activation of Drosomycin transcription. These experiments strongly argue that DmIKKß and DmIKKγ are not required for the Toll-mediated antifungal pathway but are required for the LPS-induced antibacterial pathway (Silverman, 2000).

The finding that dominant negative DmIKKs or DmIKK RNAi can block Relish cleavage and Relish-dependent gene activation suggests that Relish may be a bona fide target of the DmIKK complex. To test this possibility, experiments were carried out to determine whether DmIKKß or the DmIKK complex can phosphorylate Relish protein in vitro. Flag-tagged Relish was immunoprecipitated with Flag antibodies from a Schneider cell line that expresses very high levels of the epitope-tagged protein. Immunoprecipitated Relish was then used as a substrate with recombinant DmIKKß in a kinase assay using [γ-32P]ATP. A band the size of Relish was labeled with 32P by the recombinant kinase. As a control, extracts from the parental Schneider cell line, which does not express Flag-Relish, were also used in a Flag immunoprecipitation. When these immunoprecipitates were used as substrates for DmIKKß, phosphorylation of Relish was not observed. To further demonstrate that the phosphorylated band is Relish, a similar experiment was performed using anti-Relish antibodies, instead of Flag antibodies, to precipitate Relish. Again, a band corresponding to Relish was phosphorylated in a DmIKKß-dependent manner. It is concluded that DmIKKß can directly phosphorylate Relish (Silverman, 2000).

To determine whether this DmIKKß phosphorylation of Relish is specific, the ability of other, related kinases to phosphorylate Relish was tested. For these experiments, the specificity of DmIKKß was compared to that of recombinant human IKKß and IKKepsilon. While DmIKKß readily phosphorylates Relish, phosphorylation was not observed with either hIKKß or hIKKepsilon. When these same recombinant human IKKs were used in a GST-IkappaBalpha kinase assay, they were both active. Thus, the ability to phosphorylate Relish is not shared between the Drosophila and human IkappaB kinases. Preliminary experiments have shown that the DmIKKß can phosphorylate the linker region between the RHD and the Ankyrin domain. Surprisingly, DmIKKß can also phosphorylate Cactus and IkappaBalpha, and this phosphorylation occurs within the N-terminal regulatory domain, which is required for signal-dependent degradation by the proteasome. Thus, the ability to specifically phosphorylate IkappaB proteins is shared by the Drosophila and human IkappaB kinases, whereas only the Drosophila kinase can phosphorylate Relish. The mammalian IkappaB kinase complex has been shown to phosphorylate p105; however, this phosphorylation leads to its degradation rather than its processing (Silverman, 2000).

To test the possibility that the activated DmIKK complex can phosphorylate Relish, an immunocomplex kinase assay was performed. First, the DmIKK complex was precipitated from an LPS-inducible Schneider cell line using an anti-DmIKKγ antibody. This antibody recognizes the endogenous DmIKKγ that is expressed in this cell line. Although the anti-DmIKKß antibodies are less sensitive (DmIKKß cannot be detected in crude extracts), the DmIKKß protein can be detected after immunoprecipitation with anti-DmIKKγ antibodies. Immunoprecipitation experiments were performed with extracts prepared from cells treated with LPS or left untreated; no differences were detected, with or without LPS, in the levels of DmIKKß or DmIKKγ expressed and precipitated. However, LPS causes a specific increase in the level of Relish kinase activity that was detected in the immunoprecipitated DmIKK complex. Thus, the DmIKK complex that is precipitated with anti-DmIKKγ antibodies contains an LPS-inducible Relish kinase activity. This observation supports the view that DmIKKß and DmIKKγ form (part of) an LPS-inducible kinase complex that phosphorylates Relish, activating its cleavage in response to LPS (Silverman, 2000).

Role of Drosophila IKKgamma in a Toll-independent antibacterial immune response

Loss-of-function mutants in the Drosophila homolog of the mammalian I-kappa B kinase (IKK) complex component IKK γ have been generated. Drosophila IKK γ is required for the Relish-dependent immune induction of the genes encoding antibacterial peptides and for resistance to infections by Escherichia coli. However, it is not required for the Toll-DIF-dependent antifungal host defense. The results indicate distinct control mechanisms of the Rel-like transactivators DIF and Relish in the Drosophila innate immune response and show that Drosophila Toll does not signal through a IKK γ-dependent signaling complex. Thus, in contrast to the vertebrate inflammatory response, IKK γ is required for the activation of only one immune signaling pathway in Drosophila (Rutschmann, 2000).

The antibacterial arm of the Drosophila innate immune response requires an IkappaB kinase

The ird5 gene was identified in a genetic screen for Drosophila immune response mutants. Mutations in ird5 prevent induction of six antibacterial peptide genes in response to infection but do not affect the induction of an antifungal peptide gene. Consistent with this finding, Escherichia coli survive 100 times better in ird5 adults than in wild-type animals. The ird5 gene encodes a Drosophila homolog of mammalian IkappaB kinases (IKKs). The ird5 phenotype and sequence suggest that the gene is specifically required for the activation of Relish, a Drosophila NF-kappaB family member (Lu, 2001).

Mutations in ird5 prevent induction of a diptericin-lacZ reporter gene in response to infection and also prevent transcriptional induction of the endogenous diptericin gene. E. coli-induced expression of all seven classes of antimicrobial peptide genes in ird5 mutant larvae were examined by RNA blot hybridization, including the genes encoding antibacterial (Diptericin, Cecropin A, Defensin, Attacin, Drosocin, and Metchnikowin) and antifungal (Drosomycin) peptides. In wild-type larvae, all the antimicrobial genes are strongly induced after bacterial challenge. In contrast, in larvae homozygous for either ird5 allele, there is no detectable induction of the diptericin, cecropin A, defensin, drosocin, or metchnikowin genes. The attacin gene is induced in the mutants to ~30% of normal levels, while drosomycin is induced to normal levels. The same effects on the induction of antimicrobial peptide genes are seen in ird51/Df and ird52/Df animals, suggesting that both alleles cause a complete loss of gene function (Lu, 2001).

Mutations in three other genes, imd, Relish, and Death related ced-3/Nedd2-like protein, have been shown to prevent normal induction of antibacterial peptide genes in adult Drosophila. The pattern of antimicrobial peptide gene induction in ird5 mutants was compared with that in imd and Relish mutants in both larvae and adults. Mutations in all three genes have very similar effects on antimicrobial gene induction in larvae: diptericin and cecropin A are not induced; attacin induction is reduced and drosomycin induction is normal. In adult animals, the antimicrobial gene expression phenotypes of ird5 and Relish mutants are very similar: diptericin induction is blocked, cecropin A and attacin induction is reduced, and drosomycin induction is normal. The antimicrobial gene expression phenotype of imd adults is slightly different, with some residual diptericin expression. Mutations in Dredd, a Drosophila caspase, prevent normal induction of diptericin and attacin and allow induction of drosomycin. These comparisons suggest that ird5, Dredd, Relish, and probably imd act in the same signaling pathway to control the induction of antibacterial peptide genes in response to infection (Lu, 2001).

To assess the importance of the ird5 gene in controlling the growth of invading bacteria, bacterial survival and growth were compared in wild-type, ird5, imd, and Relish animals. In wild-type larvae, most of the E. coli injected into the animal are killed by 6 h after infection. At this same time point, there are four to 15 times as many E. coli in ird5 mutant larvae as in wild-type animals. The effects of the ird5 mutations are more striking in experiments with adults: at 24 h after infection, there are 20-350 times as many bacteria per animal in ird5 mutants compared to wild type. The bacterial growth phenotype of ird5 mutants is similar to that seen in Relish mutant larvae and adults and somewhat stronger than that of imd mutants. This is consistent with the stronger effects of ird5 and Relish on the antimicrobial peptide genes: Mutations in either ird5 or Relish prevent normal induction of diptericin, cecropin, drosocin, attacin, and metchnikowin, while induction of metchnikowin is induced in imd mutants (Lu, 2001).

The mutations responsible for the failure to induce the diptericin-lacZ reporter gene for both ird5 alleles were mapped between the visible markers cu (86D1-4) and sr (90D2-F7) on the right arm of the third chromosome. The deficiency Df(3R)sbd45(89B4-10) fails to complement the immune response defect of either ird5 allele. Further deficiency-complementation tests and male recombination mapping narrowed the ird5 interval to 89B4-9, between pannier and Stubble. Two molecular-defined genes in this interval were considered as candidates responsible for the ird5 phenotype, Akt and a gene defined by an EST that is related to mammalian IkappaB kinases (IKKs). Mutant alleles of Akt cause recessive lethality, and ird5/Akt[l(3)89Bq] heterozygous animals are viable and showed normal induction of the diptericin-lacZ reporter gene, indicating that the ird5 phenotypes are not caused by mutations in Akt (Lu, 2001).

Mammalian IKKbeta is required for activation of NF-kappaB in response to inflammatory signals such as TNF-alpha and IL-1; the IKK homolog was therefore considered as a candidate gene for ird5. A full-length cDNA was cloned for the IKK homolog, termed here DmIkkbeta. The same gene has also identified molecularly as encoding a kinase activated by LPS in a Drosophila cell line (Kim, 2000; Medzhitov, 2000; Silverman, 2000). Based on genomic DNA sequence, DmIkkbeta is located between pannier and mini-spindles. Two size classes of transcripts, 2.7 and 4.2 kb, were detected from the DmIkkbeta gene; the cDNA corresponds to the 2.7-kb transcript. Both transcripts are expressed at higher levels after infection. The complete open reading frame of DmIkkbeta was sequenced from the ird51 and ird52 chromosomes. A single C-to-T nucleotide substitution was found in ird51 that would change a glutamine codon (CAA) at amino acid 266 of the open reading frame to a stop codon (TAA) within the conserved kinase domain. No sequence changes were identified in the open reading frame in ird52; however, neither DmIkkbeta transcript was detectable in ird52 homozygotes. This analysis indicates that both ird5 alleles are associated with mutations that should abolish DmIkkbeta activity (Lu, 2001).

The ird5 immune response phenotype shows striking specificity: all of the antibacterial peptide genes are strongly affected by the ird5 mutations, but the antifungal peptide gene drosomycin is induced normally in ird5 mutants. The specific immune response phenotype of ird5/DmIkkbeta in vivo contrasts with the global effects on antimicrobial peptide genes seen in cell lines when a dominant negative form of the same gene is expressed in cultured cells (Kim, 2000). The ird5/DmIkkbeta mutant phenotype implies that, in vivo, ird5 is not an essential component of the Toll pathway, which is required for the induction of drosomycin. The ird5/DmIkkbeta gene is therefore a component of an independent signaling pathway, which could be activated by another member of the Drosophila Toll-like receptor family (Lu, 2001).

Mammalian IKKalpha and IKKbeta phosphorylate serine residues in the N-terminal domain of IkappaB; these serine residues target IkappaB for degradation, thereby allowing the nuclear localization and activation of NF-kappaB. The ird5/DmIkkbeta sequence suggests that the protein encoded by this gene phosphorylates an IkappaB-like protein. There are two known Drosophila IkappaB-like proteins that could act as inhibitor proteins in the immune response: Cactus, and the C-terminal ankyrin repeat domain of Relish. In cactus mutants, drosomycin is expressed constitutively, but the antibacterial peptide genes are not, which indicates that Cactus is not involved in the pathways that regulate the antibacterial peptide genes. Furthermore, ird5/DmIkkbeta homozygous mutant females are fertile, demonstrating that this gene is not required for degradation of Cactus during dorsal-ventral patterning in the embryo (Lu, 2001).

The ird5/DmIkkbeta phenotype is similar to the phenotype of Relish mutants. For both genes, homozygous mutant flies are viable and fertile, indicating that the two genes are not essential for development. Mutations in either Relish or ird5/DmIkkbeta completely prevent induction of diptericin and cecropin but allow some induction of attacin and drosomycin. Mutations in either gene produce comparable effects on bacterial growth. These results argue that ird5/DmIkkbeta and Relish act in the same signaling pathway and suggest that Ird5/DmIkkbeta activates Relish-containing dimers. Relish activation requires proteolytic cleavage of Relish protein into an N-terminal Rel domain that translocates to the nucleus and a C-terminal ankyrin repeat domain that remains in the cytoplasm. Recent biochemical experiments have shown that DmIkkbeta can phosphorylate Relish protein (Silverman, 2000), which is consistent with the model that phosphorylation of Relish by DmIkkbeta leads to targeted proteolysis and activation of Relish (Lu, 2001).

Although ird5/DmIkkbeta is expressed maternally, ird5 mutant females are fertile, demonstrating that the gene is not required for embryonic dorsal-ventral patterning. However, a small fraction of embryos (~0.5%) produced by homozygous ird51 or ird51/ird52 females show a weakly dorsalized phenotype, suggesting that ird5/DmIkkbeta does have a minor role in the maternal pathway that activates Dorsal. It is suggested that there is another kinase in the early embryo that is primarily responsible for phosphorylation and degradation of Cactus. The normal induction of drosomycin in ird5/DmIkkbeta mutants suggests that there will also be another kinase activated by the Toll pathway in the immune response -- perhaps the same kinase that acts downstream of Toll to activate Dorsal in the embryo. The genome sequence indicates that there is one additional IkappaB kinase gene in Drosophila. Future experiments will test whether this gene plays a role in embryonic patterning and the antifungal immune response (Lu, 2001).

The data suggest that different Drosophila Rel dimers are activated by homologous but distinct signaling pathways. Given the similarities of innate immune response pathways in Drosophila and mammals, it is likely that similar pathway-specific signaling components will mediate the activities of the members of the mammalian Rel proteins (Lu, 2001).

Interaction of Relish with Dif and Dorsal

NF-kappaB/Rel proteins function as homodimers or heterodimers, which recognize specific DNA sequences within target promoters. The activity of different Drosophila Rel-related proteins in modulating Drosophila immunity genes was examined by expressing the Rel proteins in stably transfected cell lines. How different combinations of these transcriptional regulators control the activity of various immunity genes was also examined. The results show that Rel proteins are directly involved in regulating the Drosophila antimicrobial response. Furthermore, the drosomycin and defensin expression is best induced by the Relish/Dif and the Relish/Dorsal heterodimers, respectively, whereas the attacin activity can be efficiently up-regulated by the Relish homodimer and heterodimers. These results illustrate how the formation of Rel protein dimers differentially regulate target gene expression (Han, 1999).

The parental S2 cell line used in this series of experiments can be induced by lipopolysaccharide (LPS) to express members of a subset of antimicrobial genes and these are therefore designated as S2* cells. In these cells, attacin, cecropin, and diptericin are inducible, but drosomycin induction is not detectable. Comparing with the induction levels of attacin and cecropin, defensin is less inducible. The differential expression of the antimicrobial genes is consistent with a multiple pathway model in which either LPS activates multiple receptors leading to the induction of individual immunity genes or a single LPS receptor can activate an intracellular pathway that branches at a certain point to regulate different immunity genes. The S2* cells may lack some, but not all, of the regulatory molecules that act downstream of the branch point, resulting in a partial inducibility by LPS (Han, 1999).

Although different pathways have been proposed to regulate various immunity gene expression, the utilization of specific Rel proteins in each pathway is not known even though most of these genes can be regulated by NF-kappaB-binding elements. To correlate the function of Rel proteins with the regulation of immune response, the mRNA expression of Rel protein genes in S2* cells was examined after LPS challenge. The mRNA for all three Rel protein genes is detectable in S2* cells, albeit at different levels. relish mRNA has high basal expression level, and the expression can be further up-regulated by 4-fold after 1 h of LPS treatment. Dif and dorsal mRNA levels are low and less responsive to LPS treatment. The different expression levels of Dif, dorsal, and relish mRNA may account for the inducibility of only a subset of immunity genes and suggest that Relish can function as a primary transcription factor in controlling attacin and cecropin expression (Han, 1999).

Stable cell lines that overexpress different Rel proteins, including Dif, Dorsal, and Relish, were established and used to analyze immunity gene regulation. S2* cells were transfected with different combination of plasmids that contained the Rel protein coding sequences under the control of metallothionein promoter. Stably transfected cells were selected. The transfected constructs were examined using FLAG or hexahistidine (His) epitope. Western blot results show that all three proteins are expressed, individually or in combination, to significant levels. The FLAG-tagged Relish expression is relatively uniform in cells designed to express Relish. Expression of His-tagged Dif and Dorsal shows some variation. Despite the variation, the interaction of different Rel proteins seems to be a more important determinant in gene regulation as demonstrated by examining immunity gene induction (Han, 1999).

The expression of anti-microbial genes was examined in the presence of exogenous Rel proteins prior to or after LPS treatment. After 18 h of incubation with CuSO4 to induce the Rel proteins, the cells were challenged with LPS, and RNA samples were analyzed by Northern blot. The expression of Dif or Dorsal alone prior to LPS incubation causes a modest up-regulation of drosomycin gene expression. Dif and Dorsal together have a similar modest effect on drosomycin induction. 1-6 h of LPS treatment further up-regulates drosomycin expression. This Dif or Dorsal dependent up-regulation is specific, because Relish itself does not cause S2* cells to express drosomycin (Han, 1999).

A functional interaction between Relish and Dif or Relish and Dorsal was detected by co-expressing two Rel proteins in the S2* cells. When Relish is co-expressed with Dif, drosomycin induction is enhanced significantly. This increased expression is observed before LPS challenge. Treatment of the cells with LPS further increases drosomycin expression by 2-fold. Even though there is a significant amount of Relish RNA in the parental S2* cell, Dif functions better with more exogenous Relish expression. It is possible that Dif and Relish form an unstable heterodimer, and the formation of the heterodimer requires higher concentrations of Dif and Relish than that produced by the endogenous genes. The result nonetheless suggests that the formation of Dif/Relish dimer is the rate-limiting step for full activation of drosomycin. Although the Relish and Dorsal combination also increases the expression and the inducibility of drosomycin, the effect of Dif/Relish on drosomycin expression is 4-fold higher than that of Dorsal/Relish. It is possible that the promoter of drosomycin has higher affinity toward Dif/Relish heterodimer. Alternatively, additional protein factors are involved in activating drosomycin expression, and these proteins coordinate better with Dif/Relish heterodimer. In summary, these results demonstrated that Relish functionally interacts with Dif and Dorsal to control drosomycin expression and that drosomycin expression is most efficiently activated by Dif/Relish heterodimer (Han, 1999).

The induction of defensin prefers the Relish/Dorsal heterodimer over other combinations, which is different from that of drosomycin. Surprisingly, the induction of the defensin gene is down-regulated by the expression of Dif or Dorsal alone. The Dif/Dorsal combination shows similar repressive effect. These results suggest that the Dif or Dorsal homodimers bind to the defensin promoter in a nonproductive conformation, thereby blocking the activation by an endogenous factor such as Relish. In contrast, Expression of Relish in combination with Dorsal and Dif restores the inducibility of defensin upon LPS treatment. Unlike the induction of drosomycin by Dif/Relish and Dorsal/Relish, defensin is not expressed prior to LPS treatment even in the presence of the heterodimers. One interpretation is that an additional protein factor, activated by LPS, coordinates with Rel proteins to activate defensin expression. The results, therefore, show that although the expression of Dif or Dorsal alone represses defensin expression, the co-expression of Dorsal and Relish best enhances defensin gene induction, and the co-expression of Dif and Relish can also mediate the induction (Han, 1999).

Dif, when expressed alone, is sufficient to activate cecropin and diptericin expression before LPS treatment. The result is consistent with the previous reports that Dif can activate cecropin and diptericin expression in transfection assays. However, the co-expression of Dif or Dorsal, in combination with Relish, does not show additional effect on the expression levels of cecropin and diptericin induced by LPS. Taken together, most combinations of Rel proteins can mediate cecropin and diptericin induction, and Dif homodimer is an efficient activator for these two genes (Han, 1999).

The Relish protein contains both Rel domain and IkappaB domain. It is likely that the IkappaB domain of Relish can complex with the Rel domain (RelD) to prevent the activation of Relish when expressed as a full-length protein. To study the function of Relish without the interference of the inhibitory domain, RelD (from amino acid 4 to 600) was expressed in a stable cell line. When RelD is expressed in S2* cells, attacin and cecropin are already activated prior to LPS treatment. In contrast, full-length Relish protein expression does not significantly activate attacin expression prior to LPS treatment. Furthermore, RelD does not affect drosomycin expression. In the presence of LPS, the expression of attacin and cecropin in the RelD-expressing cells is further elevated by only 2-fold. This suggests that RelD, in the absence of other Rel factors, can activate transcription of specific target genes. The result is in agreement with the observation that the ability of the parental S2* cells to up-regulate Relish by LPS is coincidental with the expression of attacin and cecropin. It may be that the formation of the processed Relish (RelD-like product) is the rate-limiting step, which is regulated by LPS. Therefore, the result supports the notion that Relish can function as the primary transcription factor in controlling attacin and cecropin gene induction. Because RelD alone can function to activate attacin and cecropin but not drosomycin expression, it further supports the idea that different combinations of Rel proteins have preferred target genes in vivo (Han, 1999).

Based on the functional interaction of Drosophila Rel proteins, it was expected that part of the protein function is achieved by the formation of heterodimers. To analyze further the interaction of Relish with Dif or Dorsal, co-immunoprecipitation experiments were performed. Anti-FLAG antibody was used to pull down FLAG-tagged Relish from extracts of cells that express Relish. The reaction is specific because the antibody does not recognize Dif or Dorsal protein, which contains the HIS epitope. From the samples that had Relish co-expressed with Dif or Dorsal, the His-tagged Dif and Dorsal can be co-immunoprecipitated as examined by Western blot. Quantitative analysis showed that 1-2% of total Dif or Dorsal can be co-immunoprecipitated along with Relish. It is possible that higher percentage of heterodimers are formed in the cells but escape detection. Furthermore, such amounts of heterodimer formation may be sufficient to account for the biological activities observed when assaying for the immunity gene expression. Nevertheless, the results demonstrate the specific interactions between Relish and Dif or Relish and Dorsal (Han, 1999).

Cactus-independent nuclear translocation of Relish

Insects can effectively and rapidly clear microbial infections by a variety of innate immune responses including the production of antimicrobial peptides. Induction of these antimicrobial peptides in Drosophila has been well established to involve NF-kappaB elements. Evidence is presented for a molecular mechanism of Lipopolysaccharide (LPS)-induced signaling involving Drosophila NF-kappaB, Relish, in Drosophila S2 cells. LPS induces a rapid processing event within the Relish protein releasing the C-terminal ankyrin-repeats from the N-terminal Rel homology domain (RHD). Examination of the cellular localization of Relish reveals that the timing of this processing coincides with the nuclear translocation of the RHD and the retention of the ankyrin-repeats within the cytoplasm. Both the processing and the nuclear translocation immediately precede the expression of antibacterial peptide genes cecropin A1, attacin, and diptericin. Over-expression of the RHD but not full-length Relish results in an increase in the promoter activity of the cecropin A1 gene in the absence of LPS. Furthermore, the LPS-induced expression of these antibacterial peptides is greatly reduced when RELISH expression is depleted via RNA-mediated interference. In addition, loss of Cactus expression via RNAi has revealed that Relish activation and nuclear translocation is not dependent on the presence of Cactus. Taken together, these results suggest that this signaling mechanism involving the processing of RELISH followed by nuclear translocation of the RHD is central to the induction of at least part of the antimicrobial response in Drosophila, and is largely independent of Cactus regulation (Cornwell, 2001).

Drosophila Tak1 functions upstream of Relish

In mammals, TAK1, a MAPKKK kinase, is implicated in multiple signaling processes, including the regulation of NF-kappaB activity via the IL1-R/TLR pathways. TAK1 function has been studied primarily in cultured cells, and its in vivo function is not fully understood. Null mutations have been isolated in the Drosophila Tak1 gene that encodes Tak1, a homolog of TAK1. Tak1 mutant flies are viable and fertile, but they do not produce antibacterial peptides and are highly susceptible to Gram-negative bacterial infection. This phenotype is similar to the phenotypes generated by mutations in components of the Drosophila Imd pathway. Genetic studies also indicate that Tak1 functions downstream of the Imd protein and upstream of the IKK complex in the Imd pathway that controls the Rel/NF-kappaB like transactivator Relish. In addition, epistatic analysis places the caspase, Dredd, downstream of the IKK complex, which supports the idea that Relish is processed and activated by a caspase activity. This genetic demonstration of Tak1's role in the regulation of Drosophila antimicrobial peptide gene expression suggests an evolutionary conserved role for TAK1 in the activation of Rel/NF-kappaB-mediated host defense reactions (Vidal, 2001).

The Toll signaling pathway, which was first identified as a regulator of embryonic dorsal-ventral patterning, is one regulator of antimicrobial peptide gene expression in Drosophila. Upon infection, the Spaetzle (Spz) protein is cleaved to generate a ligand for the Toll transmembrane receptor protein; Toll binding by Spz stimulates the degradation of the IkappaB homolog, Cactus, and the nuclear translocation of the Rel proteins Dorsal and Dorsal-like immunity factor (Dif). A second pathway regulating antimicrobial peptide gene expression in flies was initially identified by a mutation in the immune deficiency (imd) gene that results in susceptibility to Gram-negative bacterial infection and an impairment of antibacterial peptide gene expression. imd encodes a homolog of the mammalian Receptor Interacting Protein (RIP) (P. Georgel, in preparation). The imd gene encodes a death domain-containing protein. In mammals, RIP appears to function in an adaptor complex associated with the tumor necrosis factor (TNF) receptor, and genetic placement of imd suggests that IMD has a conserved function in flies as part of a receptor-adaptor complex that responds to Gram-negative bacterial infection. Molecular studies have isolated four additional factors that appear to define the Imd pathway: Relish, a third Drosophila Rel protein; two members of a Drosophila IkappaB kinase (IKK) complex, that is, the kinase DmIKKß and a structural component DmIKKγ and Dredd, a caspase. Relish is a homolog of the mammalian P100 and P105 compound Rel proteins that contain both Rel domains and inhibitory ankyrin domains. A receptor for the Imd pathway has not been identified, but infection triggers Relish cleavage and nuclear translocation of the Rel domain (Stöven et al. 2000). Relish cleavage requires DmIKKß activity, indicating that, like the mammalian IKK complex that functions in the IL1-R and TNF-R pathways, the Drosophila IKK complex regulates Rel protein activity. However, and in contrast to P100 and P105 processing, Relish cleavage is not blocked by proteasome inhibitors but does require a functional Dredd gene, suggesting that Dredd may cleave Relish directly after infection (Stöven, 2000). Like imd, mutations in DmIKKß, DmIKKγ, Dredd, and Relish affect antibacterial peptide gene expression after infection and induce susceptibility to Gram-negative bacterial infections. However, mutations in these genes do not induce susceptibility to fungal infections, demonstrating that the immune responses regulated by the Imd pathway are required to resist Gram-negative bacterial but not fungal infections (Vidal, 2001 and references therein).

Some significant conclusions of recent studies on the regulation of Drosophila antimicrobial peptide gene expression are that the Toll and Imd pathways do not share any components and that each pathway regulates specific Rel proteins. The only evidence of interactions between the two pathways is the observation that both pathways are required to fully induce some of the antimicrobial peptide genes, suggesting that these genes respond to combinations of Rel proteins controlled by the two pathways. The influence of each pathway on the expression of each antimicrobial peptide gene is apparent in flies carrying mutations that affect either the Toll or the Imd pathway: Drosomycin is mainly controlled by the Toll pathway; Diptericin and Drosocin can be fully activated by the Imd pathway; and full Metchnikowin, Defensin, Cecropin A and Attacin activation requires both pathways (Vital, 2001 and references therein).

None of the antimicrobial peptide genes are induced in imd;Toll double mutant flies, demonstrating that Imd and Toll are two essential pathways that regulate antimicrobial gene expression pathways. Despite an increased understanding of the regulation of antimicrobial peptide gene expression in flies, various intermediates in the Toll and Imd pathways remain uncharacterized: for example, neither the kinase that targets Cactus for degradation in the Toll pathway nor the receptor-adaptor complex that regulates the Imd pathway have been identified. Following the observations that null mutations affecting the Imd pathway are not required for viability, a search for additional members of the Imd pathway was initiated by screening for nonlethal mutations that induce susceptibility to Gram-negative bacterial infection in adult flies. Null mutations in the Drosophila transforming growth factor activated kinase 1 gene (Tak1) encoding the Drosophila homolog of the mammalian mitogen-activated protein kinase kinase kinase (MAPKKK) TAK1 induce high susceptibility to Gram-negative bacterial infection and block antibacterial peptide gene expression. These results indicate that Drosophila Tak1 codes for a new component of the Imd pathway (Vidal, 2001).

To identify Drosophila genes that mediate defense reactions to bacterial infection, ~2500 lines carrying ethyl methanesulfonate (EMS)-induced mutations on the X chromosome were tested for susceptibility to bacterial infection: male adult flies were pricked with a needle dipped into a pellet of the Gram-negative bacterial species Erwinia carotovora carotovora 15 (E. carotovora 15) and screened for mutants that failed to survive infection. Using this assay, nine recessive, homozygous viable mutations were isolated that render flies highly susceptible to E. carotovora 15 infection: Less than 10% of the mutated flies survived 48 h postinfection, whereas more than 90% of the wild-type flies survived. These nine mutations fall into two complementation groups: B118, which represents five of the mutations, and D10, corresponding to Tak1, which represents the other four mutations. The B118 group corresponds to the caspase encoding gene Dredd (Vidal, 2001),

To compare the D10 phenotype with the phenotypes generated by mutations in other genes that regulate Drosophila immune responses, the susceptibility of D10 and other mutant lines to infection by four microorganisms was assayed: flies were pricked with the Gram-negative bacteria Escherichia coli (E. coli), the Gram-positive bacteria Micrococcus luteus (M. luteus), or the fungus Aspergillus fumigatus, and flies were naturally infected with the entomopathogenic fungus Beauveria bassiana (B. bassiana). The D10 phenotype is similar to the imd and Relish phenotypes; flies carrying the D10, imd, and Relish mutations are susceptible to Gram-negative bacterial infection and resistant to Gram-positive bacterial and fungal infections, although D10 flies, like imd flies, exhibit slightly lower susceptibility to Gram-negative bacterial infection compared to Relish mutants. In contrast, mutations in the spz gene render flies susceptible to fungal infections, and only flies carrying mutations in both spz and imd are susceptible to Gram-positive bacterial infection. This survival analysis demonstrates that the D10 gene product, like Imd and Relish, is required to resist Gram-negative bacterial infection (Vidal, 2001).

The Toll pathway is required for the full induction of the antifungal peptide genes and a subset of the antibacterial peptide genes. Mutations that block the Toll pathway reduce the expression of these genes; conversely, mutations that block the Imd pathway reduce the expression of genes with antibacterial activity. To determine how the D10 mutation affects antimicrobial peptide gene expression, the levels of Diptericin, Cecropin A, Defensin, and Attacin, which encode antibacterial peptides, Drosomycin, which encodes an antifungal peptide, and Metchnikowin, which encodes a peptide with both antibacterial and antifungal activity, were monitored in flies homozygous for two D10 alleles. In addition, the D10 phenotype was compared with all of the previously identified mutations affecting the Imd pathway and with a spz mutation that blocks the Toll pathway (Vidal, 2001).

Pricking adult flies with a mixture of Gram-positive and Gram-negative bacteria activates the expression of all the antimicrobial peptide genes; in the D10 mutants, however, mixed Gram-negative/Gram-positive infections induce significant levels of only Drosomycin and Metchnikowin. Quantitative measurements of three independent RNA blot experiments show that in D10 flies, Drosomycin is induced to wild-type levels; Metchnikowin is induced to 70% of wild-type levels; Cecropin A, Defensin, and Attacin are induced to <25% of wild-type levels, and Diptericin is induced to <5% of wild-type levels. This pattern of antimicrobial peptide gene expression in the D10 mutants is similar to the patterns displayed in mutants of the Imd pathway, although the D10 mutations, like imd, have slightly weaker effects on antimicrobial peptide gene expression compared to the Dredd, DmIKKß, DmIKKγ, and Relish mutations. This weaker phenotype in D10 and imd flies correlates well with their lower susceptibility to E. coli infection (Vidal, 2001).

Because of its chromosomal location and the functions of its mammalian homologs, Tak1 was chosen as a candidate gene mutated in D10 flies. Several different experiments to determine whether the D10 alleles correspond to mutations in Tak1. Tested f wasirst, the ability of a 15 Kb genomic fragment that contains Tak1 to rescue the immune response deficiency in D10 flies. D10 adults carrying the Tak1 transgene (P[Tak1+]) both express Diptericin and resist Gram-negative bacterial infection at levels comparable to wild-type flies, demonstrating that this genomic fragment rescues the D10 phenotypes. Overexpression of the Tak1 cDNA using the UAS/GAL4 system partially rescues Diptericin expression in the D101 mutant. The Tak1 genomic coding sequence was tested. The four D10 alleles all contain mutations within the Tak1 kinase domain. This led to the renaming of the D10 alleles Tak11 to Tak14: Tak11 and Tak14 were generated by missense mutations in conserved residues, Tak12 was generated by a point mutation that creates a stop codon, and Tak13 contains a deletion of 31 base pairs that also results in a premature stop codon. All four Tak1 alleles inhibit Diptericin induction by Gram-negative bacterial infection to the same degree, and this inhibition is not enhanced in flies heterozygous for each allele and a deficiency spanning Tak1. In addition, flies homozygous for the four alleles are equally susceptible to Gram-negative bacterial infection. The apparent null phenotype manifested by the four Tak1 alleles indicates that the Tak1 kinase domain is essential for Tak1 function in the Imd pathway. This observation is supported by results from experiments with a kinase dead form of Tak1, Tak1-K46R, which acts as a dominant negative inhibitor of Tak1. Tak1-K46R expression driven by the UAS/GAL4 system blocks Diptericin expression after mixed bacterial infection, confirming that Tak1 is required for the Drosophila antibacterial immune response. The results of the rescue experiments, sequencing data, and the dominant negative Tak1 mutant phenotypes together demonstrate that D10 encodes the MAPKKK Tak1 (Vidal, 2001).

The loss-of-function Tak1 mutations display immune response phenotypes that are very similar to the phenotypes generated by mutations in imd, DmIKKß, DmIKKγ, Dredd, and Relish, suggesting that these genes function together in the Imd pathway. Previous studies indicated that DmIKKγ and DmIKKß directly regulate Relish activity (Silverman, 2000); however, the positions of Imd, Tak1, and Dredd in the Imd pathway were not determined. To avail of a genetic approach for ordering the Imd pathway, both Dredd and Tak1 were overexpressed via the UAS/GAL4 system. In lines carrying a heat shock (hs)-GAL4 driver and either the Dredd or Tak1 cDNAs under the control of a UAS promoter, heat shock induces Diptericin expression to about 15%-20% of the level observed in adults 6 h after bacterial infection. This result indicates that the overexpression of these two genes is sufficient to activate the antibacterial pathway in the absence of infection. By using this UAS/GAL4 system to overexpress Tak1 and Dredd in various mutant backgrounds, the epistatic relationships were tested between Tak1 and Dredd, and the other genes of the Imd pathway. Mutations in DmIKKß and DmIKKγ, but not imd, block Diptericin induction by Tak1 overexpression, indicating that Tak1 functions downstream of Imd and upstream of the IKK complex. However, the Diptericin expression induced by Dredd overexpression is not affected by mutations in imd or DmIKKγ. For genetic reasons, Northern blot analysis could not be used to test the effect of mutations in Relish and DmIKKß, which are on the third chromosome, on the UAS-Dredd-induced Diptericin expression. Therefore, the UAS-Dredd transgene was overexpressed through a female, adult fat body driver (yolk-GAL4) and Diptericin expression was monitored with a Diptericin-LacZ reporter gene. LacZ titration assays have demonstrated that ß-galactosidase activity is induced in lines overexpressing UAS-Dredd in the absence of infection. The Dredd-mediated Diptericin-LacZ induction is strongly reduced in Relish but not in DmIKKß mutants, confirming the Northern blot results showing that Dredd functions downstream of the IKK complex and demonstrating that Dredd regulates Diptericin expression through Relish (Vidal, 2001).

Natural fungal infections highlight the ability of Drosophila to discriminate between pathogens and activate specific immune response pathways that lead to adapted immune responses. Although only the Imd pathway is required to resist Gram-negative bacterial infection, the Toll pathway is still activated to some degree in flies pricked with Gram-negative bacteria. This suggests that injury (and associated contamination) contributes to a nonspecific immune response. E. carotovora 15 naturally infects the Drosophila larval gut and triggers both the local and systemic expression of antimicrobial peptide genes. In contrast to infection by pricking, natural E. carotovora 15 infection induces Diptericin expression more strongly than Drosomycin expression. To compare the contribution of the Toll and Imd pathways to antimicrobial peptide induction after natural E. carotovora 15 infection, Diptericin and Drosomycin expression were quantified in larvae from various mutant lines after natural E. carotovora 15 infections. The Diptericin expression induced by E. carotovora 15 natural infection is entirely dependent on the genes of the Imd pathway, confirming that Diptericin is exclusively regulated by the Imd pathway. Interestingly, the level of Diptericin induction is higher in spz mutants than in wild-type larvae. In addition, Drosomycin induction after E. carotovora 15 natural infection of larvae is also dependent on the Imd pathway: mutations blocking the Imd pathway have stronger effects than the spz mutation on Drosomycin expression induced by natural infection. These data contrast somewhat with previous observations that the imd mutation does not block Drosomycin induction by E. carotovora 15 infection. However, the earlier analysis of Drosomycin induction by E. carotovora 15 was based on the qualitative analysis of a Drosomycin-lacZ reporter gene; it is thought that that the quantitative analysis of Drosomycin expression by Northern blot is a more accurate determination of Drosomycin expression patterns. In conclusion, the current results indicate that Drosomycin gene induction after natural Gram-negative bacterial infection is largely mediated by the Imd pathway (Vidal, 2001).

Natural infections by E. carotovora 15 also trigger the local expression of antimicrobial peptide genes in various epithelial tissues, and both Diptericin expression in the anterior midgut and Drosomycin expression in the trachea are dependent on the imd gene. By assaying the expression of Diptericin-lacZ and Drosomycin-GFP reporter genes in naturally infected Tak1 and Dredd mutant larvae, it has been shown that both genes are required for Diptericin and Drosomycin expression in epithelial tissues after natural E. carotovora. These data confirm the predominant role of the Imd pathway in antimicrobial peptide regulation after natural E. carotovora 15 infection and suggest that Gram-negative bacterial recognition in flies preferentially activates the Imd pathway (Vidal, 2001).

Drosophila Traf2 functions upstream of Relish

Two Drosophila tumor necrosis factor receptor-associated factors (TRAFs), Traf1 and Traf2, are proposed to have similar functions with their mammalian counterparts as a signal mediator of cell surface receptors. However, as yet their in vivo functions and related signaling pathways are not fully understood. Traf1 is shown to be an in vivo regulator of c-Jun N-terminal kinase (JNK) pathway in Drosophila. Ectopic expression of Traf1 in the developing eye induces apoptosis, thereby causing a rough-eye phenotype. Further genetic interaction analyses reveal that the apoptosis in the eye imaginal disc and the abnormal eye morphogenesis induced by Traf1 are dependent on JNK and its upstream kinases, Hep and TGF-ß activated kinase 1. In support of these results, the Traf1-null mutant shows a remarkable reduction in JNK activity with an impaired development of imaginal discs< and a defective formation of photosensory neuron arrays. In contrast, Traf2 was demonstrated as an upstream activator of nuclear factor-kappaB (NF-kappaB). Ectopic expression of Traf2 induces nuclear translocation of two Drosophila NF-kappaBs, Dif and Relish, consequently activating the transcription of the antimicrobial peptide genes diptericin, diptericin-like protein, and drosomycin. Consistently, the null mutant of Traf2 shows immune deficiencies in which NF-kappaB nuclear translocation and antimicrobial gene transcription against microbial infection were severely impaired. Collectively, these findings demonstrate that Traf1 and Traf2 play pivotal roles in Drosophila development and innate immunity by differentially regulating the JNK- and the NF-kappaB-dependent signaling pathway, respectively (Cha, 2003).

Microbial infection studies have demonstrated the ability of Drosophila to detect pathogens and activate specific signaling pathways, Toll or Imd pathways, which lead to adapted immune responses. In recent years, several families of antimicrobial peptides and their coding genes have been successfully identified: cecropins, attacins, diptericin, defensin, drosomycin, drosocin, and diptericin-like protein (dptlp). Understanding the molecular mechanisms underlying how microbial infection induces expression of these antimicrobial peptides has been the main question to answer in this field. Meanwhile, Traf2 have been identified as a downstream adaptor for Toll receptor (Shen, 2001) and Toll activation leads to immune responses. Therefore, it was suspected that DTRAFs would be involved in this defense mechanism (Cha, 2003).

Three representative antimicrobial genes, diptericin, dptlp, and drosomycin, were chosen as probes to determine the activity of the antimicrobial defense system. To examine whether Drosophila Traf1 and Traf2 have the ability to induce the transcription of diptericin, dptlp, and drosomycin, Traf1 or Traf2 was ectopically expressed in third-instar larvae by using hs-GAL4 driver, and the expression levels of diptericin, dptlp, and drosomycin were monitored by Northern blot analyses (Cha, 2003).

The transcription of diptericin, dptlp, and drosomycin was increased by ectopic expression of Traf2 in the absence of microbial infection. However, the expression levels of diptericin, dptlp, and drosomycin were not altered by Traf1 overexpression. In addition, Traf2-induced expression of diptericin and dptlp is completely inhibited in a relish (rel, Drosophila NF-kappaB)-null mutant background, whereas drosomycin expression is partially inhibited by the same mutation. The partial inhibition of the drosomycin expression by rel mutation suggests that the involvement of another Drosophila NF-kappaB, such as Dif, in antimicrobial response gene transcription. These results strongly suggest that Traf2, but not Traf1, functions downstream of microbial sensory receptors, Toll or Imd, and upstream of the NF-kappaBs to regulate Drosophila immune responses (Cha, 2003).

To further confirm the results, transgenic fly lines that have a GFP or a LacZ reporter gene fused to the drosomycin or the diptericin promoter, respectively, were used, allowing observation of the reporter gene activity, which reflects the drosomycin or diptericin gene expression level. The drosomycin-GFP reporter activity is dramatically increased in the microbe-infected larva compared to the uninfected control. As expected, Traf2 overexpression alone in the absence of microbial infection strongly induces drosomycin-GFP reporter gene activity. Further dissection analyses show that drosomycin-GFP and diptericin-LacZ reporter activities are highly induced in the fat body, which is a representative target tissue for immune responses in Drosophila. However, Traf1 overexpression fails to induce the reporter activities in both whole larvae and their fat bodies, further confirming the noninvolvement of Traf1 in the immune responses of Drosophila (Cha, 2003).

In order to confirm that the Traf2-induced immune responses are mediated by Dif and Relish, which are Drosophila NF-kappaBs specifically activated by Toll and Imd pathways, respectively, the subcellular localization of Dif and Relish was determened by using their specific antibodies (Cha, 2003).

Dif and Relish are dispersed in the cytoplasm of fat body cells in the absence of microbial infection. In contrast to this, either the microbial infection or overexpression of Traf2 fully induces the nuclear translocation of both Dif and Relish, demonstrating that both Dif and Relish participate in the Traf2-mediated immune responses. However, the subcellular localization of Dif and Relish is not altered by Traf1 induction, further confirming that Traf1 is not involved in the NF-kappaB signaling pathway. These data clearly demonstrated that Traf2, but not Traf1, has the capability to induce transcriptional activation of immune response genes by specifically activating NF-kappaBs (Cha, 2003).

The Traf2-null mutant, Traf2ex1, was generated by P-element excision method. RT-PCR analysis shows that the homozygous Traf2ex1 mutant fails to produce Traf2 mRNA. Intriguingly, the mutant flies manage to develop into adults and show no morphological defects. To determine whether the Traf2ex1 mutant shows a deficiency in immune responses, the transcriptional induction level of diptericin and drosomycin was examined after microbial infection. The null mutation of Traf2 drastically disrupts the transcriptional induction of diptericin and drosomycin when compared to the wild-type control. However, Traf1-null mutation (Traf1ex1) has no effect on the induction of diptericin and drosomycin gene expression after microbial infection. The nuclear translocation of Dif and Relish was examined in the Traf2-null mutant. Consistent with Northern blot analysis, the nuclear translocation of Dif and Relish induced by microbial infections is impaired in the Traf2ex1 mutant. These results support the position that Traf2, but not Traf1, is critical for the NF-kappaB-mediated Drosophila innate immune responses (Cha, 2003).

In the mammalian system, when interleukin-1 (IL-1) receptor, a Toll-like receptor, is stimulated by binding of its ligand, IL-1 receptor associated kinase (IRAK) is recruited to the IL-1 receptor complex and phosphorylated. Consequently, the receptor associated IRAK (Drosophila homolog: Pelle) binds to TRAF6, which evokes a strong activation of the NF-kappaB signaling pathway. The importance of TRAF6 in the activation of this pathway has been confirmed by various experiments. For example, overexpression of TRAF6 can lead to NF-kappaB activation, and a dominant-negative mutant of TRAF6 inhibits IL-1-induced NF-kappaB activation (Cha, 2003).

Between the two Drosophila homologs of mammalian TRAFs, the TRAF domain of Traf2 is most closely related to that of mammalian TRAF6. Based on this structural similarity, there have been reports that Traf2 contributes to dorsal activation and immune responses by activating NF-kappaB in a cell culture system (Kopp, 1999; Shen, 2001). Thus, in agreement with these results, it has been demonstrated that Traf2 can activate Drosophila NF-kappaBs and their downstream target genes diptericin, dptlp and drosomycin. Also, it has been suggested that Traf1 is involved in the NF-kappaB-mediated immune response (Zapata, 2000). However, in the present study, Traf1 does not induce NF-kappaB activation and the consequent NF-kappaB-dependent immune responses in vivo. These data suggest that Traf2 is a highly specific signal mediator activating the NF-kappaB signaling pathway (Cha, 2003).

Although overexpression of Traf2 is sufficient to activate the NF-kappaB signaling pathway and induce innate immune responses, the Traf2-null mutation could not completely block the processes. This suggests the presence of other signaling pathway(s) that bypass Traf2 to transmit the exogenous microbial signals to NF-kappaBs. Further studies with the Traf2-null mutant are required to elucidate the unknown signaling mechanism (Cha, 2003).

Targeting of TAK1 by the NF-kappaB protein Relish regulates the JNK-mediated immune response in Drosophila

The molecular circuitry underlying innate immunity is constructed of multiple, evolutionarily conserved signaling modules with distinct regulatory targets. The MAP kinases and the IKK-NF-kappaB molecules play important roles in the initiation of immune effector responses. The Drosophila NF-kappaB protein Relish plays a crucial role in limiting the duration of JNK activation and output in response to Gram-negative infections. Relish activation is linked to proteasomal degradation of TAK1, the upstream MAP kinase kinase kinase required for JNK activation. Degradation of TAK1 leads to a rapid termination of JNK signaling, resulting in a transient JNK-dependent response that precedes the sustained induction of Relish-dependent innate immune loci. Because the IKK-NF-kappaB module also negatively regulates JNK activation in mammals, thereby controlling inflammation-induced apoptosis, the regulatory cross-talk between the JNK and NF-kappaB pathways appears to be broadly conserved (Park, 2004).

The JNK and IKK/Relish branches of the Imd pathway mediate distinct gene induction responses in Drosophila innate immunity. After diverging downstream from TAK1, these two signaling cascades regulate two separate groups of target genes that are distinct in their induction kinetics and function. The IKK/Relish targets have been extensively characterized and most encode products whose role in innate immunity is relatively well established. In contrast, the JNK-regulated, LPS-responsive genes represent a largely uncharacterized set of loci whose function in innate immunity is not clear. These genes exhibit transient induction kinetics, reaching a maximum ~1 h after induction. It was found that the transient kinetics of the JNK target genes is controlled by the transient kinetics of the JNK module of the Imd pathway and that the IKK/Relish branch plays an active role in turning off JNK activity. Hence, the two seemingly independent branches of the Imd pathway are wired in such a way as to coordinate the temporal order of individual responses (Park, 2004).

The evidence indicates that Relish-mediated JNK inhibition involves proteasomal degradation of TAK1, the MAPKKK responsible for JNK activation in response to LPS. Treatment with proteasomal inhibitors or RNAi against a component of the proteasome complex results in sustained JNK activation during the LPS response. Furthermore, in cells expressing constitutively active Relish, the stability of TAK1 is greatly decreased. Based on these findings, it is suggested that certain targets of Relish that are induced during immune responses facilitate destruction of TAK1 and switch off the JNK cascade. The fact that cycloheximide and actinomycin D also block the down-regulation of JNK activity indicates the involvement of targets of Relish rather than Relish itself. The Relish target involved in this cross-talk likely increases the susceptibility of TAK1 to proteasomal degradation by direct targeting of TAK1 or by antagonizing factors responsible for TAK1 stabilization (Park, 2004).

In considering this model, it should be noted that TAK1 is critically required for activating both IKK and JNK. Elimination of TAK1 during the LPS response thus turns off both of the downstream signaling cascades. Yet IKK/Relish target genes do not show a transient expression pattern, whereas JNK targets do. One possible explanation for this discrepancy lies in the nature of JNK and Relish activation. JNK is activated through its phosphorylation, a modification that is highly reversible. Thus, termination of the input that contributes to JNK phosphorylation is sufficient to result in its rapid inactivation, especially when one of the JNK targets encode a JNK phosphatase, Puckered. Relish, in contrast, is activated through proteolysis, an irreversible modification. Once activated Relish enters the nucleus, it may remain bound to its target genes for some time even after termination of the upstream signal (Park, 2004).

Interestingly, the antagonism between NF-kappaB and JNK signaling is evolutionarily conserved. In mice, inactivation of either IKKß or NF-kappaB RelA (p65) in cells leads to a sustained JNK activation in response to TNFalpha. The sustained JNK activation by TNFalpha has been associated with TNFalpha-induced apoptosis. Several independent studies have proposed different molecules as mediators for the NF-kappaB inhibition of JNK signaling in the TNFalpha pathway including XIAP, GADD45ß, and reactive oxygen species. Nevertheless, the underlying mechanism remains largely unknown. As the Drosophila Imd pathway and the signaling pathway downstream of mammalian TNF receptor share many conserved features, a similar mechanism may govern the antagonistic relationship between IKK/NF-kappaB and JNK in both flies and mammals. Thus, the mechanistic insights gained from studies in Drosophila should be relevant when elucidating the mechanism that connects NF-kappaB to JNK signaling in mammals (Park, 2004).

The RING-finger scaffold protein Plenty of SH3s targets TAK1 to control immunity signalling in Drosophila: POSH is required for JNK activation and Relish induction

Imd-mediated innate immunity is activated in response to infection by Gram-negative bacteria and leads to the activation of Jun amino-terminal kinase (JNK) and Relish, a NF-kappaB transcription factor responsible for the expression of antimicrobial peptides. Plenty of SH3s (POSH) has been shown to function as a scaffold protein for JNK activation, leading to apoptosis in mammals. This study reports that POSH controls Imd-mediated immunity signalling in Drosophila. In POSH-deficient flies, JNK activation and Relish induction were delayed and sustained, which indicated that POSH is required for properly timed activation and termination of the cascade. The RING finger of POSH, possessing ubiquitin-ligase activity, is essential for termination of JNK activation. POSH binds to and degrades TAK1, a crucial activator of both the JNK and the Relish signalling pathways. These results establish a novel role for POSH in the Drosophila immune system (Tsuda, 2005).

Among the components of the Imd pathway, TAK1 plays a crucial role as an activator of both the JNK and the Relish pathways (Vidal 2001; Silverman, 2003; Park, 2004). Flies that are deficient in TAK1 do not produce antibacterial peptides and are therefore highly susceptible to Gram-negative bacterial infection (Vidal, 2001). In Drosophila S2 cells, TAK1 has been shown to be required for the peptidoglycan-induced activation of IkappaB kinase (IKK) and JNK (Leulier, 2003; Silverman, 2003; Kaneko, 2004). TAK1 is also important for limiting the duration of JNK activation, thereby resulting in a transient JNK-dependent response that precedes the sustained induction of the Relish-dependent genes (Tsuda, 2005).

Although it has been shown that proteasomal degradation of TAK1 leads to the rapid termination of JNK signalling (Park, 2004), the degradation mechanism has yet to be entirely clarified, and a ubiquitin ligase that promotes TAK1 degradation has not been reported. This study identifies Plenty of SH3s (POSH) as a crucial component that controls the termination of immunity signalling in Drosophila. POSH, which was initially isolated as a Rac1-interacting protein, consists of a RING-finger domain and four SH3 domains, and has been shown to function as a scaffold protein for JNK signalling components leading to apoptosis in mammals (Tapon, 1998; Xu, 2003). In Drosophila, overexpression of POSH in imaginal discs activates JNK, which results in various defects in adult morphology (Seong, 2001). Based on flies that are deficient in POSH, evidence was provided that POSH is required for the properly timed activation and termination of Imd-mediated immune responses, and that the RING finger of POSH, which shows ubiquitin-ligase activity, is essential for this regulation (Tsuda, 2005).

To assess the role of POSH in vivo, a null allele of POSH was created by the excision of a flanking P-element in EP(2)1026. A mutant was obtained bearing a deletion that included the start codon and extended 1,282 nucleotide bases into the coding region of POSH. Northern blot analysis indicated that POSH transcripts were not detectable in POSH74 homozygous animals. Most POSH74 homozygous individuals survived embryogenesis, and the JNK cascade operating during dorsal closure remained intact. Furthermore, POSH74 showed no genetic interaction with the JNK pathway components in embryos. Taken together, these results indicate that not every instance of JNK signalling requires POSH as a scaffold. Although mutants showed no obvious developmental defects, adult flies did not live as long as control flies, and older cultures often contained dead larvae and pupae that were heavily melanized. In addition, the viability of POSH-deficient flies after injection with Escherichia coli was significantly lower than that of wild type. A possible cause for such phenotypes is defective immune function, and therefore a role for POSH in the immune response was sought (Tsuda, 2005).

Whether the TAK1-mediated immune response was compromised by the POSH74 mutation was tested. Infection with E. coli activates TAK1, which consequently phosphorylates JNK and induces the expression of Relish/NF-kappaB, resulting in the subsequent induction of a battery of genes encoding antimicrobial peptides, such as Diptericin. Northern blot analysis indicated that the induction of relish and diptericin (dpt) messenger RNAs after E. coli infection was significantly delayed in POSH74 flies compared with that in controls. Interestingly, it was also observed that expression of relish was sustained for a longer period after infection in POSH74 flies (Tsuda, 2005).

To investigate the effects of the POSH mutation more quantitatively, real-time PCR analysis of immune response genes was performed. Consistent with Northern blot analysis, the induction of dpt was delayed, and relish induction was sustained, with a maximum expression at 2 h after infection. The expression pattern of attacin A (attA), which is dependent on Relish, was also analyzed. Induction of attA was almost normal, but unlike in the control flies, its expression level continued to increase up to 4 h after infection. The maximum level of expression was twice that of control flies, consistent with Relish activity being elevated in POSH mutant flies. The expression profile of puckered (puc), a target gene of the JNK pathway, was also analyzed. Induction of puc was slightly delayed, with the highest level of expression at 1 h after infection, and expression was sustained for a longer period in POSH74 flies compared with that in controls. These results indicate that POSH is required for properly timed activation and rapid termination of both JNK- and Relish-mediated immune response pathways (Tsuda, 2005).

TAK1 has been shown to be required for the maintenance of relish mRNA expression, and its proteasomal degradation is responsible for the rapid termination of Relish and JNK signalling (Park, 2004). POSH contains a CH4C4-type RING-finger domain near its N terminus. It has been shown that the RING-finger domain of mammalian POSH possesses E3 ubiquitin-ligase activity and regulates the level of POSH protein through the proteasomal pathway (Xu, 2003). Drosophila POSH also had E3 ubiquitin-ligase activity; its auto-ubiquitination has been demonstrated. To assess the functional importance of the RING domain, POSHmR was constructed, bearing point mutations in the RING domain, and POSHDeltaR lacking the RING-finger domain altogether. Neither of these proteins was able to auto-ubiquitinate after expression in cultured Drosophila cells (S2). It is conceivable, therefore, that POSH is responsible for the feedback regulation of the immune response through TAK1 ubiquitination and its subsequent degradation. Observations indicate that the RING-finger function of POSH is essential for the degradation of TAK1 (Tsuda, 2005).

To characterize further the role of POSH in the regulation of JNK signalling, immunocompetent S2 cells were used in an in vitro model system used to study the immune response triggered by peptidoglycan in a commercial lipopolysaccharide preparation. Consistent with puc expression results in POSH mutant flies, the reduction of POSH by RNA interference (RNAi) significantly decreased the level of JNK activation, but it was sustained for a longer period than in control cells. In contrast, overexpression of POSH induced JNK activation at a high level, but then inactivated it rapidly. When POSHmR was transfected into S2 cells, phosphorylated JNK persisted for a longer period than in control cells. The effects of POSH overexpression and the role of the RING-finger function were examined in vivo using armadillo-GAL4 (arm-GAL4) as a ubiquitous expression driver. Following bacterial infection, the expression level of puc, a target of JNK signalling, rapidly declined in flies overexpressing POSH (arm>POSH). In contrast, puc expression was sustained in flies overexpressing POSHmR (arm>POSHmR), which suggests that POSHmR has a dominant-negative effect on JNK signalling. Together, these results indicate that POSH affects both activation and inactivation of JNK signalling induced by microbial infection, and that the RING-finger domain of POSH is essential for the negative feedback regulation of JNK signalling. The profile of puc expression in POSH74 flies is remarkably similar to that seen in relish flies, which supports the idea that POSH mediates Relish-dependent inactivation of JNK in the immune system (Tsuda, 2005).

These results show that POSH modulates the TAK1-mediated innate immune response. Following microbial infection, POSH facilitates rapid JNK activation and the induction of relish/NF-kappaB expression, both of which are mediated by TAK1 (Silverman, 2003). Conversely, POSH is also required for the rapid termination of both JNK activation and relish transcription through the E3 ubiquitin-ligase activity of the RING-finger domain. The results indicate that POSH is involved in a mechanism of negative feedback regulation of JNK and NF-kappaB signalling. The Drosophila TAK1-mediated innate immune response is similar to the tumour necrosis factor (TNF) signalling cascade in mammals. Imd protein contains a death domain with homology to that of mammalian TNF-receptor-interacting protein (RIP). Most of the pathway components (such as FADD, TAK1) and Drosophila homologues of IKKγ and IKKbeta are conserved in mammalian TNF signalling. The activation of Relish involves its phosphorylation and cleavage by DREDD, the Drosophila homologue of caspase 8. Eiger, a Drosophila homologue of the TNF-alpha superfamily ligand, has recently been identified. Overexpression of Eiger induces cell death by activating the JNK signalling pathway, as in mammals. Although the Eiger signalling pathway has not been fully elucidated in Drosophila, it has been clearly shown that TAK1 is essential to transduce the cell-death signal. It is possible that POSH is involved in Eiger signalling and modulates TAK-mediated JNK signalling as in the immune system. It will be of interest to determine whether POSH functions in mammalian TNF signalling (Tsuda, 2005).

Inhibitor kappaB-like proteins from a polydnavirus inhibit NF-kappaB activation and suppress the insect immune response

Complex signaling pathways regulate the innate immune system of insects, with NF-kappaB transcription factors playing a central role in the activation of antimicrobial peptides and other immune genes. Although numerous studies have characterized the immune responses of insects to pathogens, comparatively little is known about the counter-strategies pathogens have evolved to circumvent host defenses. Among the most potent immunosuppressive pathogens of insects are polydnaviruses that are symbiotically associated with parasitoid wasps. This study reports that the Microplitis demolitor bracovirus encodes a family of genes with homology to inhibitor kappaB (IkappaB) proteins from insects and mammals. Functional analysis of two of these genes, H4 and N5, were conducted in Drosophila S2 cells. Recombinant H4 and N5 greatly reduce the expression of drosomycin and attacin reporter constructs, which are under NF-kappaB regulation through the Toll and Imd pathways. Coimmunoprecipitation experiments indicated that H4 and N5 bind to the Rel proteins Dif and Relish, and N5 also weakly binds to Dorsal. H4 and N5 also inhibit binding of Dif and Relish to kappaB sites in the promoters of the drosomycin and cecropin A1 genes. Collectively, these results indicate that H4 and N5 function as IkappaBs and, circumstantially, suggest that other IkappaB-like gene family members are involved in the suppression of the insect immune system (Thoetkiattikul, 2005).

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

Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeat domains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. 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. 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 and the caspases Drice and Strica. Overexpression of DIAP2 can also inhibit Rpr- and Hid-dependent apoptosis, developmental apoptosis in the eye, as well as apoptosis associated with decreased levels of diap1. In addition, RNAi-mediated knockdown of DIAP2 in the S2 cell line has been reported to result in increased susceptibility to stress-induced apoptosis. 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, and second group reported organismal lethality in response to ubiquitous diap2 dsRNA expression. 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. 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 and to result in pupal lethality when expressed ubiquitously. 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. 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).

It has been proposed previously that DIAP2 functions in the IMD pathway downstream of Relish cleavage (Leulier, 2006) because no effect was seen 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. Previous studies 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 and references therein).

What do these observations tell us 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. 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. Binding of DIAP2 to Drice and Strica has been reported. 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. 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. 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).

Dorsal interacting protein 3 potentiates activation by Drosophila Rel homology domain proteins

Dorsal interacting protein 3 (Dip3) contains a MADF DNA-binding domain and a BESS protein interaction domain. The Dip3 BESS domain was previously shown to bind to the Dorsal (DL) Rel homology domain. This study shows that Dip3 also binds to the Relish Rel homology domain and enhances Rel family transcription factor function in both dorsoventral patterning and the immune response. While Dip3 is not essential, Dip3 mutations enhance the embryonic patterning defects that result from dorsal haplo-insufficiency, indicating that Dip3 may render dorsoventral patterning more robust. Dip3 is also required for optimal resistance to immune challenge since Dip3 mutant adults and larvae infected with bacteria have shortened lifetimes relative to infected wild-type flies. Furthermore, the mutant larvae exhibit significantly reduced expression of antimicrobial defense genes. Chromatin immunoprecipitation experiments in S2 cells indicate the presence of Dip3 at the promoters of these genes, and this binding requires the presence of Rel proteins at these promoters (Ratnaparkhi, 2009).

The Drosophila genome encodes three rel homology domain (RHD) containing proteins, Dorsal (Dl), Dorsal-related immunity factor (Dif), and Relish (Rel). The RHD, which is also found in the human NFκB family of transcriptional activators, mediates dimerization and sequence-specific DNA binding. Rel/NFκB family proteins in vertebrates and invertebrates play central roles in the innate immune response by triggering the expression of antimicrobial defense genes in response to signals transduced by Toll and the Immune deficiency (Imd) signal transduction pathways. In Drosophila, Dl also directs dorsoventral (D/V) patterning of the embryo. Specifically, the regulated nuclear localization of maternally expressed Dl in response to Toll signaling in the embryo leads to the formation of a ventral-to-dorsal nuclear concentration gradient of Dl and to the spatially restricted regulation of a large number of genes, including twist (twi), snail (sna), and rhomboid (rho), which are activated by Dl, and zerknullt and decapentaplegic, which are repressed by Dl. This serves to subdivide the embryo into multiple developmental domains along its D/V axis (Ratnaparkhi, 2009).

Unlike Dl, Dif and Rel are not required for D/V patterning. Instead, these two rel-family proteins function along with Dl in the innate immune response. Toll signaling in the immune system leads to the translocation of Dl and Dif to the nucleus and the consequent activation of a subset of anti-microbial defense genes, including drosomycin (drs) and Immune induced molecule 1. Dl and Dif are believed to have redundant roles in this process and thus either one alone is sufficient for the induction of drs. Activation of the Imd signal transduction pathway, leads to proteolytic cleavage of Rel. The N-terminal region of Rel, which contains the RHD, then translocates into the nucleus where it activates expression of anti-bacterial genes, such as diptericin (dipt), cecropin-A1 (cec-A), and attacin-A. Dl, Dif, and Rel homo- and hetero-dimerize to activate different subsets of the anti-microbial defense genes in response to signals from the Toll and Imd pathways (Ratnaparkhi, 2009).

Very little is known about the identity of factors that assist the RHD proteins in the activation of the anti-microbial defense genes. Proteins that modulate expression of these genes include transcription factors such as the GATA factor Serpent (Srp), Hox factors, Helicase89B, and an unknown protein that binds region 1 (R1), a regulatory module in cec-A and other anti-microbial defense genes. In addition, a recent screen identified several POU domain proteins as potential regulators of anti-microbial defense genes (Ratnaparkhi, 2009).

To date, about a dozen proteins that interact directly with Dl and modulate its regulatory functions have been identified by genetic and biochemical means. For example, an interaction between Dl and Twist (Twi) enhances the activation of Dl target genes, while an interaction between Dl and Groucho (Gro) is essential for Dl-mediated repression. A yeast two-hybrid screen to identify Dl interacting proteins yielded, in addition to the well characterized Dl-interactors Twi and Cactus, four novel Dl-interactors (Dip1, Dip2, Dip3, and Dip4/Ubc9). Conjugation of SUMO to Dl by Ubc9 was subsequently shown to result in more potent activation by Dl (Ratnaparkhi, 2009).

Dip3 belongs to a family of proteins that contain both MADF (for Myb/SANT-like in ADF) and BESS (for BEAF, Stonewall, SuVar(3)7-like) domains. While MADF-BESS domain proteins are found in both insects and vertebrates, only a few have been characterized and their functions are largely unknown. The Drosophila genome encodes 14 MADF-BESS domain factors. In addition to Dip3, these include Adf-1, which was initially found as an activator of Alcohol dehydrogenase, and Stonewall, which is required for oogenesis. The Dip3 MADF domain mediates sequence specific binding to DNA, while the Dip3 BESS domain mediates binding to a subset of TATA binding protein associated factors as well as to the Dl RHD and to Twi. In addition to functioning as an activator, Dip3 can function as a coactivator to stimulate synergistic activation by Dl and Twi in S2 cells (Ratnaparkhi, 2009).

This study shows that Dip3 assists RHD proteins during both embryonic development and the innate immune response. By stimulating the expression of antimicrobial defense genes, Dip3 improves survival of both larvae and adults following septic injury. The presence of Dip3 near the promoters of antimicrobial defense genes depends upon Rel family proteins suggesting that Dip3 functions as a coactivator at these promoters (Ratnaparkhi, 2009).

It has been shown that Dip3, which binds both Dl and Twi via its BESS domain, synergistically enhances the activation of a luciferase reporter with multiple Dl and Twi binding sites upstream of the promoter. In addition, Dip3 has been implicated as the 'mystery protein' which binds to sites adjacent to Dl and Twi binding sites in a subset of Dl target genes. Therefore the ability of Dip3 to enhance the expression of the Dl target promoters twi, sna, and rho in S2 cell transient transfection assays was examined. All three promoters require both Dl and Twi for full activity. Dip3 was found to synergize with Dl and Twi in the activation of the sna and twi promoters, but not in the activation of the rho promoter (Ratnaparkhi, 2009).

A polyclonal antibody against recombinant Dip3 was generated, and used to determine where and when Dip3 is present in the embryo. Maternally expressed Dip3 is observed in all nuclei as early as nuclear cycle 7. It was detected in subsequent nuclear cycles during formation of the Dl nuclear concentration gradient. In interphase embryonic as well as S2 cell nuclei, Dip3 localizes to nuclear speckles of unknown identity. During mitosis Dip3 is enriched on chromosomes. It associates with the centrosome proximal portion of the anaphase chromatids and the inside ring of the polar body rosette suggesting a predominant pericentromeric location at this stage of the cell cycle and hinting at a possible role of Dip3 in centromeric function. Confirming the specificity of the antibodies, the immunoreactivity is absent from Dip31 embryos in which the Dip3 transcriptional and translational start sites as well as a large segment of the Dip3 coding region have been deleted. Weak Dip3 expression is also detected in the fat body (Ratnaparkhi, 2009).

Homozygous Dip31 flies are viable and fertile, indicating that Dip3 cannot have an essential role in embryonic D/V pattern formation. However, a small proportion (7±4%) of the embryos fail to hatch and exhibit D/V patterning defects. Embryos produced by females transheterozygous for Dip31 and a deficiency that removes a portion of the second chromosome containing the Dip3 gene (Df(PC4) exhibit similar embryonic lethality (10%) and D/V patterning defects. Also, maternal overexpression of Dip3 using the Gal4-UAS system leads to 54±9 % embryonic lethality with cuticles of the dead embryos showing both anteroposterior and D/V patterning defects, indicating that Dip3 may have a role in embryonic pattern formation (Ratnaparkhi, 2009).

Consistent with a non-essential role for Dip3 in D/V patterning, a Dip3 mutation enhances the temperature sensitive dl haploinsufficieny phenotype. The degree of dorsalization is often quantified by categorizing embryos on a scale from D0 (completely dorsalized, lacking all dorsoventral pattern elements other than dorsal epidermis) to D3 (inviable, but with little or no apparent defect in the cuticular pattern). At 29°, about half the dead embryos produced by dl1/+ females exhibit detectable D/V patterning defects and the majority of these fall into the D2 category (moderately dorsalized, exhibiting mildly expanded ventral denticle belts and a twisted germ band). Removal of maternal Dip3 increases the proportion of dorsalized embryos to about 75% with most of the increase being due to an increase in the number of D2 embryos. The effect seems to be strictly maternal as the paternal genotype does not modulate the dl haploinsufficiency phenotype (Ratnaparkhi, 2009).

Dip3 is present in the fat body, the organ in which RHD factors activate antimicrobial defense genes in response to infection. Since Dip3 binds the Dl RHD, the role of Dip3 in the innate immune response was examined by assessing the sensitivity of Dip31 flies to bacterial and fungal infection. Wild-type and Dip31 adults and larvae were injected with gram positive bacteria (M. luteus), gram negative bacteria (E. coli), and fungi (B. brassiana). For comparison, flies were infected that contained mutations in known components of the Toll (spzrm7) and Imd (RelE20) pathways. Wild-type, RelE20, spzrm7, and Dip31 adults showed little lethality (<15%) 30 days after mock infection. However, the Dip31 adult flies exhibited 55% lethality one month after injection with a 1:1 mixture of M. luteus and E. coli, compared to 10% lethality after 30 days for wild-type flies and 98% after 30 days for RelE20 flies. In contrast, wild-type and Dip31 adults were equally sensitive to fungal infection, both showing 55-70% lethality after 30 days compared to 100% lethality after 22 days for RelE20 adults and 100% lethality after 7 days for spzrm7 adults. Similar results were seen in larvae in which Dip31, RelE20 and spzrm7 mutations resulted in reduced rates of eclosion following septic injury compared to wild-type. The effectiveness of the immune challenge was further verified by an experiment showing that septic injury leads to translocation of Dl into the nucleus (Ratnaparkhi, 2009).

To determine if the sensitivity of Dip31 flies to infection results from reduced induction of antimicrobial peptides, the expression of dipt, drs and cec-A was monitored as a function of time following septic injury. Relative to uninfected flies, the levels of expression of drs and dipt were reduced by the Dip31 mutation, especially at the 2 and 4 hr time points, while the levels of cec-A expression were not significantly altered. Thus, some, but not all, antimicrobial defense genes that are regulated by RHD family proteins exhibit dependence on Dip3. At the 4 hr time point, relative to infected, wild type flies, the spzrm7 mutation reduced drs expression to basal levels while the RelE20 mutation reduced dipt expression ten fold (Ratnaparkhi, 2009).

Dip3 was over expressed in the larvae using the Cg-Gal4 driver to examine the effect of increasing levels of Dip3 on the expression of antimicrobial defense genes in the fat body. Cec-A and drs levels were unaffected, while dipt levels increased two-fold in infected flies. Thus, both loss-of-function and over expression data are consistent with the conclusion that Dip3 makes the immune response more robust by elevating the expression of a subset of antimicrobial defense genes (Ratnaparkhi, 2009).

Radiolabeled Dip3 interacts with FLAG-tagged Dl and Rel immobilized on anti-FLAG beads. Similarly, immobilized FLAG-Dip3 binds Dl (Bhaskar, 2002) and Rel (Residues 1-600). Dip3 binds to DNA via its MADF domain and to the RHD via its BESS domain, and can thus function either as an activator or as a coactivator (Bhaskar, 2002). To determine if Dip3 is present at the promoters of antimicrobial defense genes, ChIP assays were carried out in S2 cells transfected with FLAG-Dip3. FLAG antibody was used to immunoprecipitate Dip3 crosslinked to chromatin. Compared both to mock-transfected cells and to the transcribed region of a ribosomal protein-encoding gene (rp49), Dip3 was highly enriched at the drs, dipt and cecA promoters. As expected, dsRNA directed against Dip3 eliminated the ChIP signal verifying antibody specificity. The association of Dip3 with the promoters of the anti-microbial defense genes depended on Rel family proteins, since knockdown of these proteins by dsRNAi significantly reduced association of Dip3 with the promoters. Similar results were observed with an anti-GFP antibody and cells expressing a Dip3-GFP fusion protein (Ratnaparkhi, 2009).

These results suggest that Dip3 may synergize with RHD proteins in multiple developmental contexts possibly through contact with the Dl rel homology domain. Dip3 is expressed maternally and present in cleavage stage nuclei at the time that Dl is functioning to pattern the D/V axis. Furthermore, Dip3 can potentiate Dl-mediated activation of the twist and snail promoters in S2 cells. These observations suggest that Dip3 might have a role in D/V patterning. Consistent with this possibility, it was found that removal of maternal Dip3 results in occasional D/V patterning defects and significantly enhances the dl haploinsufficiency phenotype suggesting the Dip3 renders D/V patterning more robust perhaps by assisting in Dl-mediated activation (Ratnaparkhi, 2009).

An important aspect of the immune response is activation in the fat body of genes encoding antimicrobial peptides by the Rel family transcription factors Dl, Dif, and Rel. This study found that synergistic killing of flies by a mixture of E.coli and M. luteus is enhanced in Dip31 flies. This suggests roles for Dip3 in the Imd and/or Toll pathways, which mediate the response to microbial infection. In accord with this idea, it was found that activation of the Imd pathway target dipt and the Toll pathway target drs are compromised in Dip3 mutant larvae (Ratnaparkhi, 2009).

To determine if the role of Dip3 at antimicrobial defense gene promoters is direct, ChIP assays were carried out demonstrating that this factor associates directly with the drs, dipt, and cec-A promoters in S2 cells. Since Dip3 contains a DNA binding domain, it is possible that it binds to these promoters through a direct interaction with DNA. However, with one exception in the drs promoter, these promoters lack matches for the consensus Dip3 binding sites. Thus, Dip3 may be acting as a coactivator at these promoters consistent with its ability to bind the rel homology domain. In support of this idea, it was found that simultaneous knockdown of all three rel family proteins significantly reduced recruitment of Dip3 to the promoters (Ratnaparkhi, 2009).

The mechanism of Dip3 co-activation remains unclear. The finding that the Dip3 BESS domain binds TAFs (Bhaskar, 2002) suggests a role for Dip3 in the recruitment of the basal machinery. In addition, the MADF domain is closely related to the SANT domain, which binds histone tails and may have a role in interpreting the histone code. While analysis of RHD targets suggests roles for Dip3 in activation, Dip3 also associates with pericentromeric heterochromatin during mitosis, consistent with a possible role in silencing. Other heterochromatic proteins including a suppressor of position effect variegation (Su(Var)3-7) also contain BESS domains. However, the loss of Dip3 does not appear to modify position effect variegation (Ratnaparkhi, 2009).

In flies, additional roles for RHD-mediated activation have been demonstrated in haematopoesis, neural fate specification, and glutamate receptor expression. Antimicrobial defense genes are also expressed constitutively in barrier epithelia and in the male and female reproductive tracts. It will be interesting to determine if Dip3 is involved in rel protein-dependent and independent gene activation in some or all of these tissues. One tissue in which Dip3 appears to have clear rel-independent functions is in the developing compound eye, where Dip3 overexpression results in conversion of eye to antenna, while Dip3 loss-of-function leads to mispatterning of the retina (Ratnaparkhi, 2009 and references therein).

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. DAP-type PGN binding causes these receptors to multimerize or cluster, triggering signal transduction. IMD signaling culminates in activation of the NF-κB precursor Relish and transcriptional induction of AMP genes (Paquette, 2010 and references therein).

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 have been shown to be required for immune-induced activation of the IKK complex. 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. 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 it has been shown, 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 and references therein).

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 the 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).

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).

Akirin specifies NF-kappaB selectivity of Drosophila innate immune response via chromatin remodeling

The network of NF-kappaB-dependent transcription that activates both pro- and anti-inflammatory genes in mammals is still unclear. As NF-kappaB factors are evolutionarily conserved, Drosophila was used to understand this network. The NF-kappaB transcription factor Relish activates effector gene expression following Gram-negative bacterial immune challenge. This study shows, using a genome-wide approach, that the conserved nuclear protein Akirin is a NF-kappaB co-factor required for the activation of a subset of Relish-dependent genes correlating with the presence of H3K4ac epigenetic marks. A large-scale unbiased proteomic analysis revealed that Akirin orchestrates NF-kappaB transcriptional selectivity through the recruitment of the Osa-containing-SWI/SNF-like Brahma complex (BAP). Immune challenge in Drosophila shows that Akirin is required for the transcription of a subset of effector genes, but dispensable for the transcription of genes that are negative regulators of the innate immune response. Therefore, Akirins act as molecular selectors specifying the choice between subsets of NF-kappaB target genes. The discovery of this mechanism, conserved in mammals, paves the way for the establishment of more specific and less toxic anti-inflammatory drugs targeting pro-inflammatory genes (Bonnay, 2014).

Relish : Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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