Gene name - Relish
Cytological map position - 85C4--6
Function - transcription factor
Keywords - immune response
Symbol - Rel
FlyBase ID: FBgn0014018
Genetic map position - 3-
Classification - Ankyrin-repeat and NFkappaB domain protein
Cellular location - nuclear and cytoplasmic
|Recent literature||Sudmeier, L.J., Samudrala, S.S., Howard, S.P. and Ganetzky, B. (2015). Persistent activation of the innate immune response in adult Drosophila following radiation exposure during larval development. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 26333838
This study investigates the role of the innate immune system in response to radiation exposure. It was shown that the innate immune response and NF-ĸB target gene expression is activated in the adult Drosophila brain following radiation exposure during larval development and that this response is sustained in adult flies weeks after radiation exposure. Preliminary data suggest that innate immunity is radioprotective during Drosophila development. Together these data suggest that activation of the innate immune response may be beneficial initially for survival following radiation exposure but result in long-term deleterious consequences, with chronic inflammation leading to impaired neuronal function and viability at later stages. This work lays the foundation for future studies of how the innate immune response is triggered by radiation exposure and its role in mediating the biological responses to radiation.
|Ji, Y., Thomas, C., Tulin, N., Lodhi, N., Boamah, E., Kolenko, V. and Tulin, A. V. (2016). Charon mediates immune deficiency-driven PARP-1-dependent immune responses in Drosophila. J Immunol [Epub ahead of print]. PubMed ID: 27527593
Regulation of NF-κB nuclear translocation and stability is central to mounting an effective innate immune response. This article describes a novel molecular mechanism controlling NF-κB-dependent innate immune response. A previously unknown protein, termed as Charon, functions as a regulator of antibacterial and antifungal immune defense in Drosophila. Charon is an ankyrin repeat-containing protein that mediates poly(ADP-ribose) polymerase-1 (PARP-1)-dependent transcriptional responses downstream of the innate immune pathway. The results demonstrate that Charon interacts with the NF-κB ortholog Relish inside perinuclear particles and delivers active Relish to PARP-1-bearing promoters, thus triggering NF-κB/PARP-1-dependent transcription of antimicrobial peptides. Ablating the expression of Charon prevents Relish from targeting promoters of antimicrobial genes and effectively suppresses the innate immune transcriptional response. Taken together, these results implicate Charon as an essential mediator of PARP-1-dependent transcription in the innate immune pathway. Thus, these results are the first to describe the molecular mechanism regulating translocation of the NF-κB subunit from cytoplasm to chromatin.
|Morris, O., Liu, X., Domingues, C., Runchel, C., Chai, A., Basith, S., Tenev, T., Chen, H., Choi, S., Pennetta, G., Buchon, N. and Meier, P. (2016). Signal integration by the IkappaB protein Pickle shapes Drosophila innate host defense. Cell Host Microbe 20: 283-295. PubMed ID: 27631699
Pattern recognition receptors are activated following infection and trigger transcriptional programs important for host defense. Tight regulation of NF-κB activation is critical to avoid detrimental and misbalanced responses. This study describes Pickle (CG5118), a Drosophila nuclear IκB that integrates signaling inputs from both the Imd and Toll pathways by skewing the transcriptional output of the NF-κB dimer repertoire. Pickle interacts with the NF-kappaB protein Relish and the histone deacetylase dHDAC1, selectively repressing Relish homodimers while leaving other NF-κB dimer combinations unscathed. Pickle's ability to selectively inhibit Relish homodimer activity contributes to proper host immunity and organismal health. Although loss of pickle results in hyper-induction of Relish target genes and improved host resistance to pathogenic bacteria in the short term, chronic inactivation of pickle causes loss of immune tolerance and shortened lifespan. Pickle therefore allows balanced immune responses that protect from pathogenic microbes while permitting the establishment of beneficial commensal host-microbe relationships.
| Tavignot, R., Chaduli, D., Djitte, F., Charroux,
B. and Royet, J. (2017). Inhibition
of a NF-κB/Diap1 pathway by PGRP-LF is required for proper apoptosis
during Drosophila development. PLoS Genet [Epub ahead
of print]. PubMed ID: 28085885
NF-κB pathways are key signaling cascades of the Drosophila innate immune response. One of them, the Immune Deficiency (IMD) pathway, is under a very tight negative control. Although molecular brakes exist at each step of this signaling module from ligand availability to transcriptional regulation, it remains unknown whether repressors act in the same cells or tissues and if not, what is rationale behind this spatial specificity. This study shows that the negative regulator of IMD pathway PGRP-LF is epressed in ectodermal derivatives. In the absence of any immune elicitor, PGRP-LF loss-of-function mutants display a constitutive NF-κB/IMD activation specifically in ectodermal tissues leading to genitalia and tergite malformations. In agreement with previous data showing that proper development of these structures requires induction of apoptosis, it was found that ectopic activation of NF-κB/IMD signaling leads to apoptosis inhibition in both genitalia and tergite primordia. NF-κB/IMD signaling antagonizes apoptosis by up-regulating expression of the anti-apoptotic protein Diap1. Altogether these results show that, in the complete absence of infection, the negative regulation of NF-κB/IMD pathway by PGRP-LF is crucial to ensure proper induction of apoptosis and consequently normal fly development. These results highlight that IMD pathway regulation is controlled independently in different tissues, probably reflecting the different roles of this signaling cascade in both developmental and immune processes.
|He, X., Yu, J., Wang, M., Cheng, Y., Han, Y., Yang, S., Shi, G., Sun, L., Fang, Y., Gong, S. T., Wang, Z., Fu, Y. X., Pan, L. and Tang, H. (2017). Bap180/Baf180 is required to maintain homeostasis of intestinal innate immune response in Drosophila and mice. Nat Microbiol 2: 17056. PubMed ID: 28418397
Immune homeostasis is a prerequisite to protective immunity against gastrointestinal infections. In Drosophila, immune deficiency (IMD) signalling (tumour necrosis factor receptor/interleukin-1 receptor, TNFR/IL-1R in mammals) is indispensable for intestinal immunity against invading bacteria. However, how this local antimicrobial immune response contributes to inflammatory regulation remains poorly defined. This study shows that flies lacking intestinal Bap180 (a subunit of the ]SWI/SNF complex) are susceptible to infection as a result of hyper-inflammation rather than bacterial overload. Detailed analysis shows that Bap180 is induced by the IMD-Relish response to both enteropathogenic and commensal bacteria. Upregulated Bap180 can feed back to restrain overreactive IMD signalling, as well as to repress the expression of the pro-inflammatory gene eiger (TNF), a critical step to prevent excessive tissue damage and elongate the lifespan of flies, under pathological and physiological conditions, respectively. Furthermore, intestinal targeting of Baf180 renders mice susceptible to a more aggressive infectious colitis caused by Citrobacter rodentium. Together, Bap180 and Baf180 serve as a conserved transcriptional repressor that is critical for the maintenance of innate immune homeostasis in the intestines.
|Maki, K., Shibata, T. and Kawabata, S. I. (2017). Transglutaminase-catalyzed incorporation of polyamines masks the DNA-binding region of the transcription factor Relish. J Biol Chem [Epub ahead of print]. PubMed ID: 28258224
In Drosophila, the immune deficiency (IMD) pathway-dependent signal is finally transmitted through proteolytic conversion of the nuclear factor-κB-like transcription factor Relish to the active N-terminal fragment Relish-N to induce its translocation from the cytosol into the nucleus for the expression of IMD-controlled genes. Previous studies have demonstrated that transglutaminase (TG) suppresses the IMD pathway by polymerizing Relish-N to inhibit its nuclear translocation. A synthetic amine, such as monodansylcadaverine (DCA) or biotin-labeled pentylamine, ingested by flies is TG-dependently incorporated into Relish-N, causing the nuclear translocation of modified Relish-N in gut epithelial cells. DCA-incorporated Gln residues were located in the Rel homology domain, the DNA-binding region of Relish-N. TG-catalyzed DCA incorporation inhibits the binding of Relish-N to the Rel-responsive element of the κB sequence. TG localizes not only in the cytosol but also in the nucleus. Natural polyamines, including spermidine and spermine, competitively inhibited TG-dependent DCA incorporation into Relish-N. Relish-N is also modified by spermine, and the transcription of cecropin A1 and diptericin genes controlled by the IMD pathway is reduced. These findings suggest that intracellular TG regulates the transcriptional activity of Relish-N through the incorporation of polyamines into Relish-N as well as through protein-protein crosslinking of Relish-N.
|Goto, A., Okado, K., Martins, N., Cai, H., Barbier, V., Lamiable, O., Troxler, L., Santiago, E., Kuhn, L., Paik, D., Silverman, N., Holleufer, A., Hartmann, R., Liu, J., Peng, T., Hoffmann, J. A., Meignin, C., Daeffler, L. and Imler, J. L. (2018). The kinase IKKbeta regulates a STING- and NF-kappaB-dependent antiviral response pathway in Drosophila. Immunity 49(2): 225-234.e224. PubMed ID: 30119996
Antiviral immunity in Drosophila involves RNA interference and poorly characterized inducible responses. This study showed that two components of the IMD pathway, the kinase dIKKbeta and the transcription factor Relish, were required to control infection by two picorna-like viruses. A set of genes was identified that was induced by viral infection and regulated by dIKKbeta and Relish, which included an ortholog of STING. This study showed that dSTING participated in the control of infection by picorna-like viruses, acting upstream of dIKKbeta to regulate expression of Nazo, an antiviral factor. These data reveal an antiviral function for STING in an animal model devoid of interferons and suggest an evolutionarily ancient role for this molecule in antiviral immunity.
|Li, Y. X., Sibon, O. C. M. and Dijkers, P. F. (2018). Inhibition of NF-kappaB in astrocytes is sufficient to delay neurodegeneration induced by proteotoxicity in neurons. J Neuroinflammation 15(1): 261. PubMed ID: 30205834
This study examined responses in astrocytes induced by expression of disease-associated, aggregation-prone proteins in other cells. A role was examined for intracellular astrocytic responses in a Drosophila model for Spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease), a disease caused by expansion of the polyglutamine (polyQ) stretch in the ATXN3 gene. In this Drosophila SCA3 model, eye-specific expression of a biologically relevant portion of the ATXN3 gene, containing expanded polyQ repeats (SCA3(polyQ78)) was expressed. Eye-specific expression of SCA3(polyQ78) resulted in the presence of astrocytes in the eye, suggesting putative involvement of astrocytes in SCA3. In a candidate RNAi screen, genes in astrocytes were identified that can enhance or suppress SCA3(polyQ78)-induced eye degeneration. Relish, a conserved NF-kappaB transcription factor, was identified as an enhancer of degeneration. Activity of Relish was upregulated in the SCA3 model. Relish can exert its effect via Relish-specific AMPs, since downregulation of these AMPs attenuated degeneration. Relish signaling was examined in astrocytes on neurodegeneration. Selective inhibition of Relish expression specifically in astrocytes extended lifespan of flies that expressed SCA3(polyQ78) exclusively in neurons. Inhibition of Relish signaling in astrocytes also extended lifespan in a Drosophila model for Alzheimer's disease. These data demonstrate that astrocytes respond to proteotoxic stress in neurons, and that these astrocytic responses are important contributors to neurodegeneration. The data provide direct evidence for cell-non-autonomous contributions of astrocytes to neurodegeneration, with possible implications for therapeutic interventions in multiple neurodegenerative diseases.
Relish was identified in a screen for genes involved in the Drosophila immune system by using PCR differential display to identify genes induced in infected flies. Unlike the single domain proteins Dorsal and Cactus (respectively the classic NF-kappaB- and IkappaB-related proteins of Drosophila), Relish contains both a Rel homology domain (hence the name Relish) and an IkappaB-like domain with six ankyrin repeats. Thus Relish is a dual domain protein. In this respect Relish is similar to the compound mammalian NF-kappaB precursors p100 and p105, although no obvious similarity is seen outside the two conserved domains (Dushay, 1996).
Interesting features have been noted in the regions outside the conserved domains. Like RelB, Relish has an unusually long region N-terminal of the Rel homology domain. Furthermore, just downstream of the Rel homology domain there is a serine-rich stretch, corresponding to the position where p100 and p105 have a glycine-rich region that serves as a processing signal for the generation Rel domain containing p50. The serine-rich sequence in Relish may serve a similar function. Another serine-rich region is found in the N-terminal region of Relish. Finally, there are several potential target sites for phosphorylation by casein kinase II, including four in the spacer between the Rel and ankyrin domains, and five either in or near the PEST region. Casein kinase II has been implicated in the constitutive phosphorylation of the PEST region in IkappaB, and the signal-induced phosphorylation of the same protein is mediated by a kinase with similar target sites (Dushay, 1996).
There is experimental evidence for at least three independent signal transduction pathways in the activation of the immune response in Drosophila. One pathway, defined by the Toll gene, is involved in the defense against fungi. A constitutively active form of Toll can mediate superinduction of the Cecropin gene promoter in the transfected hemocyte line mbn-2. Toll is preferentially involved in the induction of antifungal components such as Drosomycin and metchnikowin. The Toll pathway also requires other dorsal group genes such as tube, pelle, cactus, and spatzle (spz), but not dorsal itself. A second pathway directs an antibacterial response. It is defined by the immune deficiency (imd) mutation, which affects the expression of diptericin and other bactericidal factors, but not of drosomycin. In agreement with this model, factors that have both antibacterial and antifungal activity, such as the cecropins and metchnikowin, appear to respond to both pathways. In addition to these two pathways, recent experiments with the Toll-like gene, 18-wheeler, indicate that a third pathway may exist. Mutations in 18-wheeler affect the induction of Attacin, but not Diptericin or Drosomycin (Hedengren, 1999 and references therein).
Although one or more Rel factors are likely to mediate signals from these pathways in the immune response, the specific role of the three known Rel factors in Drosophila is unclear. Dorsal is not required, because dorsal mutants have no phenotypic effect on the induction of the antimicrobial genes. However, recent data show that Dif is specifically involved in the induction of drosomycin and that Dorsal may play a redundant role in the same pathway. Using P element-mediated mutagenesis, a series of deletions in the Relish gene have been created. These mutations have a profound effect on the induction of the entire set of antimicrobial genes, and it is concluded that Relish is a key factor that controls the antibacterial as well as the antifungal defense in Drosophila. The most straightforward interpretation of the broad effect of the Relish mutants on the antibacterial and antifungal responses is that Relish is involved in all three immunity pathways of Drosophila (Hedengren, 1999)
Relish is a key factor in the induction of the humoral immune response in Drosophila, including antibacterial as well as antifungal factors. In striking contrast, there is a complete lack of effect of Relish on cellular immune reactions and hematopoiesis. These observations indicate that cellular and humoral immune responses are controlled by distinct and independent systems in Drosophila. Hematopoiesis, and perhaps the activation of cellular reactions, may instead be controlled by another Rel factor, Dorsal. Overexpression of this factor has been shown to induce lamellocyte differentiation and cause the formation of melanotic capsules, and similar effects have been observed with mutations that lead to the activation of the Toll pathway (Hedengren, 1999 and references therein).
It is interesting to compare the roles of different Rel factors in the cellular and humoral immune responses of Drosophila with the situation in mammals. A parallel can be drawn between the phenotype of the Relish mutants and the effects seen in knockout mice that carry a disruption of the p105 gene. Such mice have a normal complement of lymphocytes, but they are deficient in both specific and nonspecific responses to infection (Sha, 1995). In contrast, disruption of the RelA and RelB genes in mice gives more complex developmental phenotypes, including effects on hematopoiesis. Thus, although the picture is less clear in mice than in Drosophila, it appears to be a conserved feature that different Rel factors regulate immune responses and hematopoiesis (Hedengren, 1999 and references therein).
The relative importance of the humoral factors in Drosophila immunity is dramatically illustrated by the phenotype of the Relish mutants. A single bacterial cell is sufficient to kill these mutants and the resistance to fungal infections is also impaired, even though the cellular immune defense appears to be intact and parasites are efficiently eliminated. This extreme sensitivity to a bacterial infection is surprising, since phagocytosis of bacteria appears to be normal. One possible explanation is that the antibacterial effector functions of the phagocytic cells are impaired in the Relish mutants. Alternatively, and perhaps more likely, enterobacteria such as Enterobacter cloacae may be relatively resistant to the killing mechanisms of the hemocytes. Indeed, injected cells of Enterobacter cloacae or Escherichia coli can survive for a long time in wild-type Drosophila, even though bacteria injected later are eliminated efficiently by the induced immune response. The surviving bacteria probably reside within phagocytic cells, where they are protected from the humoral factors. Thus, the humoral immune response may be the only efficient defense against bacteria such as Enterobacter cloacae or Escherichia coli (Hedengren, 1999 and references therein).
In spite of the dramatic effects on the immune response, homozygous Relish mutants are fertile and give rise to normal offspring. This lack of Relish developmental effects is surprising, considering the fact that early embryos express a specific, maternally contributed form of Relish that lacks the N-terminal domain (Dushay, 1996). The existence of a specific embryonic form suggests that Relish, like dorsal, could play a role in embryogenesis. However, the function of Relish in the early embryo, if any, must be redundant or else it is involved in a more subtle process that is not required for survival (Hedengren, 1999).
Two alternative models are suggested for the role of Relish in the three genetically defined induction pathways of immune responses in Drosophila. The most straightforward interpretation of the broad effect of the Relish mutants on the antibacterial and antifungal responses is that Relish is involved in all three pathways. The case is most clear for the imd pathway, which is of particular importance for the induction of Diptericin and other genes of the antibacterial defense. Since Diptericin induction has an absolute requirement for a functional Relish gene, it has been concluded that Relish is likely to be part of this pathway. Furthermore, Relish could also be involved in the Toll and 18-wheeler pathways, since the inducibility of both Drosomycin and Attacin is significantly reduced in Relish mutants. However, additional factors have to be invoked in order to explain how different pathways can specifically induce different genes. For the Toll pathway, this specificity could be provided by Dif and/or dorsal since these factors have been shown to participate in the Toll-dependent induction of Drosomycin, perhaps in the form of a Relish-Dif (or Relish-Dorsal) heterodimer. In this case, a homo- or a hetero-dimer of Dif and/or Dorsal could partially substitute for loss or Relish, explaining the residual expression of Drosomycin in the Relish mutants. Data supporting this model were very recently obtained by Han (1999). A synergistic effect on Drosomycin expression was found when Relish was overexpressed together with Dif. This indicates that the two factors may indeed cooperate, perhaps as a heterodimer, in the Toll pathway. For the other pathways, no candidates for such specificity factors have been found. The induction of Drosomycin and Attacin is not completely abolished in the Relish mutants, and for this reason at least one Relish-independent pathway must be included in the model (Hedengren, 1999 and references therein).
A second model, which is also consistent with these findings, is suggested by the fact that there is a residual inducibility of Drosomycin in Toll mutants and of Attacin in 18-wheeler mutants. According to this model, Relish and Toll (the latter probably via Dif) act independently to induce Drosomycin, and similarly Relish and 18-wheeler form independent pathways for the induction of Attacin (Hedengren, 1999 and references therein).
It is interesting to observe the complexity of the different signal transduction pathways that converge to activate the immune responses in Drosophila. At least two of these pathways depend on receptors of the Toll family. Several other members of this family can be identified in the database of the Drosophila genome project, and it is possible that yet other signal pathways will be identified as the role of these receptors becomes clear. The availability of Relish and other mutants will help in understanding these different pathways and their role in immunity, not only in Drosophila but probably also in humans (Hedengren, 1999).
The Drosophila NF-kappaB transcription factor Relish is an essential regulator of antimicrobial peptide gene induction after gram-negative bacterial infection. Relish is a bipartite NF-kappaB precursor protein, with an N-terminal Rel homology domain and a C-terminal IkappaB-like domain, similar to mammalian p100 and p105. Unlike these mammalian homologs, Relish is endoproteolytically cleaved after infection, allowing the N-terminal NF-kappaB module to translocate to the nucleus. Signal-dependent activation of Relish, including cleavage, requires both the Drosophila IkappaB kinase [consisting of 2 subunits: a catalytic kinase subunit encoded by ird5 (IKKβ) and a regulatory subunit encoded by kenny (IKKγ)] and death-related ced-3/Nedd2-like protein (DREDD), the Drosophila caspase-8 like protease. This report shows that the IKK complex controls Relish by direct phosphorylation on serines 528 and 529. Surprisingly, these phosphorylation sites are not required for Relish cleavage, nuclear translocation, or DNA binding. Instead they are critical for recruitment of RNA polymerase II and antimicrobial peptide gene induction, whereas IKK functions noncatalytically to support Dredd-mediated cleavage of Relish (Ertürk-Hasdemir, 2009).
These data suggest a new model for the regulation of Relish activity. In this model, Relish is controlled by 2 distinct mechanisms, both of which signal downstream of the receptor Peptidoglycan recognition protein LC (PGRP-LC). One arm controls the cleavage of Relish and requires IMD, FADD, DREDD, and the IKK complex. The other arm controls Relish phosphorylation through TAK1 and the IKK complex. Robust induction of antimicrobial peptide expression requires that both mechanisms of control are fully active; Relish must be cleaved and phosphorylated (Ertürk-Hasdemir, 2009).
Phosphorylation of Relish is critical for signal-dependent transcriptional activation of target genes. By using mass spectrometry and in vitro kinase assays, serines 528 and 529 were identified as targets of IKKβ phosphorylation. These serines are phosphorylated rapidly, in cell lines and flies, after immune challenge. Mutation of these residues to alanine resulted in a protein that acted as dominant negative in cell culture, inhibiting the PGN-induced expression of antimicrobial peptide genes Diptericin and Attacin. A recent study reported that ectopic expression of REL-68 (the N-terminal portion of Relish) was not sufficient to drive the expression of the antimicrobial peptide genes Attacin and Cecropin (Wiklund, 2009). Because REL-68 is not expected to be phosphorylated, these results support the conclusion that phosphorylation is critical for Relish-mediated transcriptional activation of AMP genes. However, this article also reported that REL-68 was able to induce Diptericin, which appears to contradict the current findings. In these experiments, transgenic REL-68 is overexpressed, which may contribute to these confusing results (Ertürk-Hasdemir, 2009).
Further supporting the conclusion that Relish phosphorylation is critical, kinase-dead IKKβ transgenic rescue supported only very weak induction of AMP genes in flies. In these experiments, Relish is expressed at normal levels. Surprisingly, serines 528 and 529, and IKKβ catalytic activity itself, were not required for signal-dependent Relish cleavage. Serines 528 and 529 were also not essential for nuclear translocation or DNA binding. Instead, ChIP experiments show that these serines are required for the efficient recruitment of RNA Pol II to the Diptericin locus (Ertürk-Hasdemir, 2009).
These ChIP assays used the 8WG16 mAb, which preferentially recognizes unphosphorylated CTD repeats of the largest subunit of RNA Pol II. The unphosphorylated CTD is associated with the preinitiating RNA Pol II complex recruited to promoters. Thus, these results argue that phosphorylation of Relish on serines 528 and 529 is required for efficient recruitment of RNA Pol II to the Diptericin and Diptericin-B promoters. An alternate possibility, suggested by recent findings on gene regulation in Drosophila, is that phosphorylated Relish could stimulate elongation from paused RNA Pol II. However, a genome-wide analysis of promoters containing stalled RNA Pol II has found that many Drosophila antimicrobial peptide genes are not sites of paused RNA Pol II. The use of 8WG16 in the experiments presented in this study, further argues that phosphorylation of serines 528 and 529 does not modulate RNA Pol II pausing but instead regulates polymerase recruitment to the preinitiation complex at the Diptericin locus. The exact mechanism by which phosphorylation of serines 528 and 529 affect RNA Pol II recruitment remains to be elucidated. It may involve interaction with coactivators, such as components of the mediator complex, or it may involve the recently discovered IMD component Akirin (Goto, 2008), which is argued to function in the nucleus, downstream of Relish (Ertürk-Hasdemir, 2009).
This report also provides further supporting evidence that DREDD may be the caspase that directly cleaves Relish. This study shows that overexpression of DREDD is sufficient to cause Relish cleavage. Relish cleavage required catalytically active DREDD and expression of another apical caspase, the caspase-9 like DRONC, did not generate cleaved Relish. Interestingly, DREDD-mediated Relish cleavage did not lead to Relish phosphorylation and was not sufficient to drive Diptericin expression. Furthermore, immunopurified DREDD, but not drICE, cleaved Relish in vitro, albeit not very efficiently. The poor efficiency of Relish cleavage, in vitro, may be due to the highly oligomeric state of purified Relish and/or the low activity of DREDD, which has proven to be very difficult to produce in an active form. It was also found that a biotinylated peptide with the Relish cleavage site bound active DREDD; although strong evidence for a direct interaction, this assay is not particularly specific. Together, these data strongly suggest that DREDD directly cleaves Relish, but it cannot yet be concluded with certainty that other proteases, such as an effector caspase, are not involved (Ertürk-Hasdemir, 2009).
In addition to DREDD, Relish cleavage also requires both IKK subunits. However, Relish cleavage does not require catalytically active IKKβ. Delaney (2006) showed that TAK1 is not required for Relish cleavage. Because TAK1 is required for the immune-induced activation of the IKK kinase, this result is consistent with the data indicating that IKK catalytic activity is not involved in Relish cleavage. Instead, IKK complex may function as a scaffold or adaptor, but not as a kinase, in controlling the cleavage of Relish (Ertürk-Hasdemir, 2009).
Taken together, these data demonstrate that Relish is regulated by 2 distinct mechanisms. Relish is probably cleaved by DREDD and phosphorylated by the IKK complex. These 2 regulatory mechanisms appear to be independent, because phosphorylation can occur without cleavage, and vice versa, although they are both triggered by PGN stimulation of the receptor PGRP-LC. Surprisingly, the IKK complex also plays a role in the cleavage of Relish, but not through its kinase activity. Instead, IKK-mediated phosphorylation of Relish on serines 528 and 529, within its N-terminal transcription factor module, is necessary for transcriptional activation of target genes (Ertürk-Hasdemir, 2009).
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).
All three Relish transcripts contain both Rel and ankyrin domains, since hybridization of Northern blots with probes from both domains show identical results. To further characterize the maternal transcript, five cDNA clones were isolated from an ovarian library. These clones are identical to those from the adult and mbn-2 cell libraries except for various degrees of truncation at the 5' end. The longest clone has an insert of 2.7 kb and thus must be near full-length. 5'-rapid amplification of cDNA end products from 0- to 2-h-old embryos show the same sequence and terminate near the 5' end of this clone. It is concluded that the maternal transcript differs from the other two Relish transcripts at the 5' end only, and that if there is a unique 5' exon it must be very short. The maternal transcript is too short to encode the N-terminal part of the open reading frame, and has an alternative translation start site (Dushay, 1996).
Relish maps near the end of the 85C region on the right arm of the third chromosome. This is a well-characterized region of the genome, covered by contiguous genomic walks around the oskar and pumilio genes, as well as by genomic clones around neuralized. Cosmid clones were obtained from the relevant interval and they were probed with the Relish cDNA clone 5.3 (Dushay, 1996). In this way, Relish could be mapped to a cosmid, cos 1698, between pumilio and neuralized. 10.8 kb around the Relish gene were sequenced and several cDNA clones corresponding to the transcripts of this region were isolated. The genomic organization in this region turns out to be complex, with overlapping transcripts from at least four different genes. Twenty cDNA clones from the Relish gene have been described (Dushay, 1996). They are all collinear, and there is no evidence for alternative splicing of the Relish gene or for the use of alternative polyadenylation sites. Nevertheless, at least three different Relish transcripts of 2.7, 3.1, and 3.4 kb can be detected on Northern blots (Dushay, 1996). The 2.7 kb transcript is expressed in females and in early embryos, presumably as a maternally contributed transcript. This conclusion is supported by the observation that Relish is expressed in nurse cells in the ovary, as detected by in situ hybridization (Hedengren, 1999).
The 3.1 kb transcript is very strongly induced in infected flies, whereas the 3.4 kb transcript is constitutively present. 5' RACE has been used to sequence and accurately map the 5' ends of these transcripts, as well as of a fourth, 3.5 kb transcript of low abundance. The latter transcript can also occasionally be detected as a faint band on Northern blots. These data show that the different Relish transcripts originate from one minor and three major transcription start sites that are arranged in tandem. The entire Relish transcription unit is situated inside an intron of the previously described Nmdmc gene (Price, 1993), which encodes a putative NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (Hedengren, 1999).
This is a bifunctional enzyme involved in tetrahydrofolate metabolism, with no obvious relationship to immune responses. From the Nmdmc gene, four different cDNA clones have been isolated, all of them representing the same splice form (transcript A) with a first exon 2.4 kb upstream of Relish, spliced to two exons downstream of the Relish gene. The first exon of this transcript differs from that of the previously published Nmdmc transcript (transcript B; Price, 1993) for which the first exon could be mapped to a position 1.2 kb upstream of Relish. Thus, the Nmdmc gene has two alternative splice forms, both of them spanning the region that contains the four Relish transcripts. The protein products encoded by the two transcripts are essentially identical. In transcript B, the first exon is noncoding, whereas transcript A encodes an additional six N-terminal amino acid residues (Hedengren, 1999).
Relish contains both a Rel homology domain and an IkappaB-like domain with six ankyrin repeats. A phylogenetic reconstruction of the possible relationship between the different Rel homology domains shows that Relish may have branched off from other Rel proteins at a very early stage. The sequences of the ankyrin repeats are also quite different from those of other IkappaB-like proteins, and they are about equally close to the ankyrin repeats in the Notch, ankyrin and IkappaB families. As in other Rel proteins, a putative nuclear localization sequence is found at the C-terminal end of the Rel homology domain, and the ankyrin repeats are followed by an acidic, PEST-like sequence. PEST sequences are rich in proline, glutamic acid, serine and threonine residues, and they have been implicated in protein turnover (Dushay, 1996).
date revised: 5 November 2000
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