Death related ced-3/Nedd2-like protein
Steroid hormones coordinate multiple cellular changes, yet the mechanisms by which these systemic signals are refined into stage- and tissue-specific responses remain poorly understood. The Drosophila gene Eip93F, more familiarly termed E93 determines the nature of a steroid-induced biological response. E93 mutants possess larval salivary glands that fail to undergo steroid-triggered programmed cell death, and E93 is expressed in cells immediately before the onset of death. E93 protein is bound to the sites of steroid-regulated and cell death genes on polytene chromosomes, and the expression of these genes is defective in E93 mutants. Furthermore, expression of E93 is sufficient to induce programmed cell death. It is proposed that the steroid induction of E93 determines a programmed cell death response during development (Lee, 2000).
The nuclear localization of E93
in larval salivary glands provided an opportunity to determine if E93
binds to the salivary gland polytene chromosomes and, if so, to
identify the sites bound by the protein. Salivary glands were
dissected 12-14 hr after puparium formation, fixed, squashed,
and photographed to acquire accurate cytology of the banding and
puffing patterns for mapping. The chromosomes were then stained with
affinity-purified E93 antibodies, and these patterns were compared
with the original set of photographs to allow accurate mapping of the
bound sites. E93 clearly binds to the polytene chromosomes in a
reproducible and site-specific manner and is consistently detected
at 65 chromosome sites, many of which contain ecdysone-regulated
genes or programmed cell death genes. Among these sites are the 74EF
and 75B early puffs, which contain the E74 and E75
ecdysone-inducible genes, as well as the 93F puff, which contains
E93. In addition, 1B, 21C, 59F, and 99B are bound by E93 and
contain the programmed cell death genes dredd, crq,
dcp-1, and drICE, respectively. The 2B5 early puff,
containing the BR-C ecdysone-inducible gene, and 75CD,
containing βFTZ-F1 and the programmed cell death genes
rpr, hid, and grim, were not bound by E93.
These data indicate that E93 may directly regulate the genes in bound
chromosome loci and may either encode a site-specific DNA binding
protein or a chromatin-associated protein that functions as a
transcriptional regulator (Lee, 2000).
Drosophila MyD88 is an adapter in the Toll signaling pathway that associates with both the Toll receptor and the downstream kinase Pelle. Expression of MyD88 in S2 cells strongly induces activity of a Drosomycin reporter gene, whereas a dominant-negative version of MyD88 potently inhibits Toll-mediated signaling. MyD88 associates with the death domain-containing adapter Drosophila Fas-associated death domain-containing protein (FADD), which in turn interacts with the apical caspase Dredd. This pathway links a cell surface receptor to an apical caspase in invertebrate cells and therefore suggests that the Toll-mediated pathway of caspase activation may be the evolutionary ancestor of the death receptor-mediated pathway for apoptosis induction in mammals (Horng, 2002).
A BLAST search of the Drosophila genome identified the sequence encoding MyD88, a Drosophila homolog of human MyD88. Similar to its human
homolog, Drosophila MyD88 contains an N-terminal death domain, an intermediate domain, and a TIR domain. However, unlike human MyD88, Drosophila MyD88 contains an additional 81 amino acids preceding the death domain and a 162-aa-long C-terminal region following the TIR domain (Horng, 2002).
Transfection of MyD88 into Drosophila S2 cells potently
induces a Drosomycin reporter gene but not an Attacin reporter gene. This preferential ability to induce an antifungal gene is similar to that of Toll 10b, a constitutively active form of Toll, and suggests that MyD88 may be a
component of the Toll-Tube-Pelle-Cactus-Dif signaling pathway. Previous
studies have demonstrated that Toll-mediated Drosomycin induction
requires the nuclear translocation of Dif. Dif is normally
retained in the cytoplasm by the IkappaB inhibitor Cactus and is released
only in response to signal-dependent degradation of Cactus. To test
whether MyD88-mediated Drosomycin induction also depends on
Cactus degradation, a Cactus mutant was constructed that contains mutations of
the conserved serine residues that, in mammalian IkappaB, are the targets
of signal-dependent phosphorylation. A Cactus mutant inhibits
Drosomycin induction by MyD88 and, as expected, by Toll. This result indicates that, similar to Toll, MyD88 regulates
Drosomycin induction through the Cactus-dependent pathway (Horng, 2002).
For further analyses, various deletion mutants of MyD88 were generated. Two of the deletion mutants, one
containing the TIR domain and the C-terminal domain (amino acids
237-537) and another containing the intermediate, TIR, and C-terminal
domains (amino acids 176-537), activate the Drosomycin reporter
weakly (10-fold) in comparison to full length MyD88, indicating that the intact protein is required for optimal activity. However, the fact that these truncation mutants can still induce signaling is surprising, since they lack the death domain that mediates interactions with downstream signaling components. Moreover, similar analyses of human MyD88 have shown that a combination of the death domain and the intermediate domain is sufficient to induce signaling activity comparable to that of the wild-type protein. An equivalent truncation of dMyD88 (amino acids 1-237) retains no residual activity despite being well expressed, suggesting that there
are some differences in domain function between human and Drosophila MyD88 proteins (Horng, 2002).
To determine whether MyD88 is a component of the Toll signaling
pathway, attempts were made to identify a deletion mutant that would have
dominant-negative activity. Therefore, three MyD88 deletion
mutants that do not activate the Drosomycin reporter were tested for
their ability to inhibit Toll-mediated Drosomycin induction. The strongest inhibitor
was the death domain- and middle domain-containing construct (amino
acids 1-237), which at low concentrations potently inhibits
Toll-mediated Drosomycin induction in a dose-dependent manner (Horng, 2002).
To order MyD88 in the pathway with respect to Pelle, MyD88 was tested
for its ability to be inhibited by PelleN, a dominant-negative form of
Pelle that consists of the N-terminal death domain-containing region of
Pelle. MyD88, like Toll, is strongly inhibited by
PelleN. MyD88, however, does not inhibit Pelle, demonstrating that, similar to the mammalian pathway, MyD88 functions upstream of Pelle (Horng, 2002).
To further establish MyD88 as a component of the Toll pathway, whether MyD88 interacts with Toll was tested by coimmunoprecipitation assays. The TIR domain-containing MyD88 construct is detected in
anti-Toll immunoprecipitates. Interestingly, when
cotransfected with Toll 10b, MyD88 reproducibly appears as two distinct
bands -- a slower migrating upper band that may correspond to
phosphorylated MyD88 construct and a faster migrating lower band. The
predominant form of MyD88 detected in immunoprecipitates is the faster
migrating species. MyD88 therefore associates with Toll, presumably
through TIR domains, and is a component of the active receptor complex (Horng, 2002).
Because human MyD88 associates with IRAK through death domains, a
likely immediate downstream target of MyD88 is the IRAK homolog
Pelle. Interaction between the death
domain-containing dMyD88 construct (amino acids 1-237) and Pelle was examined. MyD88 is detected in Pelle immunoprecipitates, indicating that MyD88 interacts with Pelle, presumably through their death domains (Horng, 2002).
These results therefore demonstrate that MyD88 is an adaptor in the Toll
signaling pathway downstream of the receptor and upstream of Pelle.
From genetic analyses, the adaptor protein Tube has also been
implicated to be downstream of Toll and upstream of Pelle in the Toll
signaling pathway. The death domain of Tube also interacts with Pelle. Because Tube and MyD88 also contain death domains that could potentially mediate their interaction, tests were performed for association between these two proteins in immunoprecipitation assays; Tube and MyD88 do indeed interact. Therefore, MyD88 and Tube both function as adaptors downstream of Toll, exist in the same active complex along with Pelle, and are probably both involved in the recruitment and/or activation of Pelle. Understanding functional differences between these two adapters will require further analysis (Horng, 2002).
To identify other potential downstream targets of MyD88,
a search of the Drosophila genome was performed for other sequences that encode death domain-containing proteins that may interact with MyD88. One such
sequence encodes a protein with a death domain as well as a death
effector domain and appears to be a homolog of mammalian FADD. This
cDNA has been identified and named FADD (Hu, 2000). Whether FADD can interact with MyD88 was tested. Lysates from S2 cells
transfected with MyD88 were incubated
with anti-Flag beads to immunoprecipitate FADD, and immunoprecipitates
were blotted with anti-V5 antibody to look for associated MyD88. A strong band corresponding to MyD88 was observed, indicating
that MyD88 can interact with FADD through death domains.
Overexpression of FADD in S2 cells, however, does not lead to
activation of either the Drosomycin or Attacin reporters (Horng, 2002).
Mammalian FADD is recruited to the tumor necrosis factor
receptor complex through homophilic death domain interactions with the
adapter TNFR-associated death domain-containing protein (TRADD).
In turn, FADD recruits procaspase-8 through homophilic death effector
domain associations. It is speculated that Drosophila FADD may likewise recruit a Drosophila caspase to the Toll receptor complex. A
potential candidate caspase is Dredd, an apical caspase with a long
prodomain shown to be essential for induction of antibacterial genes. Indeed, analysis of immunoprecipitated lysates from cells
cotransfected with Drosophila FADD, and either full length Dredd or the death
effector domain of Dredd showed strong association of Dredd with FADD. A second study (Hu, 2000) has also shown interaction of dFADD with
Dredd (Horng, 2002).
Thus Drosophila MyD88 is an adapter in
the Toll signaling pathway. MyD88 associates with both Toll and Pelle
and functions upstream of Pelle. Tube is known from genetic studies to
be an adapter in the Toll pathway that functions upstream of Pelle.
Why Toll should signal through MyD88 and Tube, two receptor-proximal
adapters with seemingly similar functions, is not yet clear. MyD88 associates with the receptor Toll as well as the downstream
adapter FADD, which in turn interacts with the apical caspase Dredd.
Because caspases are essential executioners of the apoptotic
machinery in organisms from nematodes to mammals, and because Dredd has
been shown to be involved in apoptosis during
Drosophila development, it is possible that Toll-1 or
some of the other eight Tolls that exist in Drosophila may
induce apoptosis (or another Dredd-dependent pathway) through
the MyD88/dFADD/Dredd pathway in a cell-type specific and/or
developmental stage-specific manner. The pathway comprised of Toll,
MyD88, dFADD, and Dredd would be the first description of a pathway in
invertebrates that links a cell surface receptor to an apical caspase.
Such a pathway, if it exists, would enable extracellular stimuli,
perhaps ligands secreted by other cells during development or
pathogen-derived products during infection, to instruct invertebrate
cells to undergo cell death. In addition, the
Toll/MyD88/dFADD/Dredd pathway is remarkably similar to that activated by the receptors of the tumor necrosis factor
receptor (TNFR) superfamily in mammals, in which FADD-mediated recruitment of caspase-8 leads to induction of apoptosis. Since
the Drosophila genome does not encode any cell surface
receptors homologous to TNFRs, it appears that the
Toll/MyD88/dFADD/Dredd pathway is the evolutionary ancestor of
the mammalian death receptor pathways. This possibility is further
supported by the recent finding that human TLR2 can induce
apoptosis through the MyD88/FADD/Caspase-8 pathway (Horng, 2002).
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 with th 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).
The earliest expression of DREDD mRNA in young embryos is detected prior to the onset of zygotic transcription, indicating that DREDD mRNA is maternally derived. Low ubiquitous levels of DREDD mRNA expression are also observed in the middle stages of embryogenesis (through stage 10/11). However, during stage 11 and beyond, a distinct accumulation of DREDD mRNA is observed in spatial and temporal patterns that are strikingly coincident with well-documented patterns of programmed cell death (PCD) in the embryo. Conspicuous expression of DREDD mRNA coincides with the initial appearance of dying cells (stage 11) in the subepidermis of the gnathal segments, the clypeolabrum, and the caudal tip of the retracting germ band. During stage 13, punctate DREDD accumulation occurs in dying cells distributed around the supraesophageal ganglia and in the dorsal ridge. During stage 16, when most if not all PCD is confined to the central nervous system, DREDD mRNA expression also occurs throughout the brain and ventral cord in patterns coincident with PCD in this tissue. Accumulation of DREDD mRNA in the ventral cord at this time occurs in cells that are positioned at the midline and examples of asymmetric staining are also evident. While mRNAs occurring in the ventral cord are not macrophage associated (circulating hemocytes do not enter the ventral nerve cord), hybridization signals in other tissue are clearly associated with phagocytic macrophages (Chen, 1998b).
Spermatozoa are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Apoptosis is mediated by caspase family proteases. Many apoptotic cell deaths in Drosophila utilize the REAPER/HID/GRIM family proapoptotic proteins. These proteins promote cell death, at least in part, by disrupting interactions between the caspase inhibitor DIAP1 and the apical caspase DRONC, which is continually activated in many viable cells through interactions with ARK, the Drosophila homolog of the mammalian death-activating adaptor APAF-1. This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC. This study demonstrates that ARK- and HID-dependent activation of DRONC occurs at sites of spermatid individualization and that all three proteins are required for this process. dFADD, the Drosophila homolog of mammalian FADD, an adaptor that mediates recruitment of apical caspases to ligand-bound death receptors, and its target caspase DREDD are also required. A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization. These results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process (Huh, 2004; full text of article).
Ectopic death of retinal cells results from ectopic expression of rpr and grim in eye discs. Reduction of the level of Dredd in Drosophila eyes reduces the level of ectopic cell death. Heterozygosity at the Dredd locus suppresses apoptosis in transgenic
models of reaper- and grim-induced cell killing, demonstrating that levels of Dredd product can modulate signaling
triggered by these death activators (Chen, 1998b).
The Drosophila innate immune system discriminates between pathogens and responds by inducing the expression of specific antimicrobial peptide-encoding genes through distinct signaling cascades. Fungal infection activates NF-kappaB-like transcription factors via the Toll pathway, which also regulates innate
immune responses in mammals. The pathways that mediate antibacterial defenses, however, are less defined. Loss-of-function mutations are reported in the
caspase encoding gene dredd, which block the expression of all genes that code for peptides with antibacterial activity. These mutations also render flies highly susceptible to infection by Gram-negative bacteria. These results demonstrate that Dredd regulates antibacterial peptide gene expression, and it is proposed that Dredd, Immune Deficiency and the P105-like rel protein Relish define a pathway that is required to resist Gram-negative bacterial infections (Leulier, 2000).
To identify genes that control Drosophila antibacterial immune responses, a screen was carried out for mutations on the X chromosome that affect the expression of the antibacterial peptide gene diptericin after bacterial infection. Among 2500 EMS mutagenized lines, five viable, recessive mutations (named B118, F64, L23, D55, D44) were isolated of a gene that is required for the expression of a diptericin-GFP reporter gene in larvae after bacterial infection. In addition, Northern blot analysis shows that adults homozygous for each of the five alleles do not express the diptericin gene after bacterial injection. The B118 allele was mapped to cytological region 1B9-1B13 on the proximal tip of the X chromosome and a small deficiency, Df(1)R194, was identified that does not complement B118. Deficiency Df(1)R194 spans four previously identified genes: rpL36, l(1)1Bi, dredd and su(s). Several results demonstrate that B118 is a mutation in dredd: (1) B118 is allelic to a viable P element insertion (EP-1412) inserted 50 bp upstream of dredd coding sequences; (2) the two genes flanking dredd, su(s) and l(1)1Bi complement B118; (3) a small deficiency, Df(1)dreddD3, which was generated by imprecise P element excision, and which removes dredd and affects the 5' upstream sequences of su(s), blocks diptericin expression after bacterial infection, and (4) a P element insertion, P[dredd+], containing 7.6 kb of genomic DNA, including dredd but not su(s) and l(1)1Bi , fully restores diptericin expression in B118 flies. All five dredd EMS mutations block diptericin expression after infection to the same degree as Df(1)dreddD3, indicating that they are probably null alleles. The P element insertion in line EP-1412 generates a strong hypomorphic dredd mutation since a small amount of diptericin expression is detectable after infection (Leulier, 2000).
dredd encodes an apical caspase and is an effector of the apoptosis activators reaper, grim and hid. One or more dredd transcripts are specifically enriched in cells programmed to die and dredd overexpression induces apoptosis in SL2 cells. In mammals, the closest dredd homologs are caspases 8 and 10, which mediate apoptosis induced by members of the tumor necrosis factor receptor family. Caspases are produced as inactive zymogens termed pro-caspases; when activated, mature caspases catalyze the proteolytic cleavage of death substrates that are associated with apoptosis. The isolation of mutations in dredd that block diptericin expression after infection demonstrate that Dredd also regulates immune responses. In addition, a dredd-lacZ reporter gene is constitutively expressed in all adult and larval tissues including the fat body, the major immuno-responsive tissue in insects. Infection does not, however, appear to increase dredd expression levels (Leulier, 2000).
The five dredd alleles all contain point mutations that affect different regions of the dredd protein. Alleles B118, D55 and F64 generate either premature stop codons or frameshift changes in the Dredd prodomain. D44 has a missense mutation in sequences encoding the first death effector domain (DED), a region thought to mediate protein-protein interactions. In the protein encoded by allele L23, a tryptophan (W) in the caspase domain is replaced by an arginine (R) residue. The strong phenotype of alleles D44 and L23 indicates that Dredd domains affected in these alleles are essential for Dredd function in immunity (Leulier, 2000).
The isolation of dredd mutations that block diptericin expression enabled the characterization of dredd's role in mediating Drosophila antimicrobial host defense as well as dredd's relationship to other genes that function in this response. Pricking adult flies with a mixture of Gram-positive and Gram-negative bacteria activates the expression of all the genes that encode antimicrobial peptides in Drosophila. In the dreddB118 mutant, however, mixed Gram-positive/Gram-negative infections only induce the expression of the antifungal gene drosomycin and the gene coding for Metchnikowin, which has both antifungal and antibacterial activity; diptericin, cecropin A, attacin A and defensin are expressed at <5% of wild-type levels and metchnikowin is expressed at 50% of the wild-type level. Antimicrobial gene expression is similarly affected in flies homozygous for relE20, a strong or null mutant allele of relish and imd, although most of the antibacterial genes are expressed at slightly higher levels in imd flies. By contrast, a mutation in the spz gene, which blocks Toll activation, reduces drosomycin induction by mixed Gram-negative/Gram-positive bacterial infection and reduces the induction of some of the antibacterial genes (defensin, attacin, cecropin A). These data demonstrate that mutations in dredd are phenotypically similar to mutations in imd and relish, and that these three genes regulate all Drosophila antibacterial peptide gene expression (Leulier, 2000).
To define further the roles of imd, dredd and relish in activating metchnikowin and drosomycin after different types of bacterial infection, metchnikowin and drosomycin expression were quantified in different mutant backgrounds 6 h after infection with either Gram-negative Escherichia coli or Gram-positive Micrococcus luteus bacteria. The dreddB118 and relE20 mutations strongly reduce metchnikowin and drosomycin induction by Gram-negative bacterial infections, while the imd mutation has a weak effect; by contrast, metchnikowin and drosomycin are expressed at close to wild-type levels in the imd, dreddB118 and relE20 mutants after Gram-positive bacterial infection. It is concluded, therefore, that dredd and relish play a greater role in inducing metchnikowin and drosomycin after Gram-negative bacterial infection than after Gram-positive bacterial infection (Leulier, 2000).
The observation that drosomycin and metchnikowin expression is almost completely abolished in imd;Toll double mutants suggests that Gram-positive bacterial infection triggers the expression of metchnikowin and drosomycin via the Toll pathway. In agreement, this analysis shows that mutations in spz affect drosomycin gene expression more strongly after Gram-positive than after Gram-negative bacterial infection, and that the constitutive activation of the Toll pathway in the Tl10b mutant leads to drosomycin expression in the absence of dredd activity. metchnikowin, however, is still expressed to a high level in spz mutants after Gram-positive bacterial infection, indicating that metchnikowin induction by Gram-positive bacterial infection may also be mediated in part by the Imd pathway (Leulier, 2000).
The susceptibility to microbial infection observed in dredd, imd, relish, spz and imd;spz mutants is correlated with the expression pattern of antimicrobial genes in these mutants. dreddB118, relE20 and imd;spzrm7 adults are highly susceptible to bacterial infection by Gram-negative bacteria, and imd adults are slightly less susceptible. These survival results confirm that the activation of defense responses to Gram-negative bacterial infection require imd, dredd and relish. Only the imd;spzrm7 double mutants, however, are highly susceptible to bacterial infection by Gram-positive bacteria, indicating that resistance to Gram-positive bacteria is regulated by both the Toll and Imd pathways. Finally, only spzrm7 and imd;spzrm7 mutants are highly sensitive to natural infection by the entomopathogenic fungus Beauveria bassiana or injection of Aspergillus fumigatus spores, confirming that responses to fungi are largely activated by the Toll pathway (Leulier, 2000).
The dredd immune phenotype is similar to the relish and imd phenotypes; it is predicted that the Imd, Dredd and Relish proteins function in a common signaling pathway that regulates antibacterial peptide gene expression. Based on the respective activites of Dredd as a caspase and Relish as a transcriptional transactivator, it is also hypothesized that Dredd functions upstream of Relish in the control of antimicrobial gene expression. This hypothesis is supported by the observation that Dredd is required for Relish activation via endoproteolytic cleavage. It is believed that the weaker effects of the imd mutation on antibacterial gene expression place the imd gene product at an early stage of the antibacterial cascade where multiple responses, some of which bypass imd, trigger the activation of the pathway. Alternatively, the imd mutation may represent a hypomorphic allele (Leulier, 2000).
Caspases were originally identified as effectors of apoptosis, but there is increasing evidence that caspases also function in other physiological processes. Recent studies suggest that the recruitment of the caspase-8 precursor to the TNF-R1 signaling complex either activates NF-kappaB through a Traf2-, RIP-, NIK- and IKK-dependent pathway or, after proteolytic processing of caspase-8, induces apoptosis. The data indicate that Dredd, a close homolog of caspase-8, may also have dual functions in NF-kappaB signaling and apoptosis in Drosophila. Further biochemical analysis is necessary to determine whether Dredd participates directly in Relish activation or functions further upstream (Leulier, 2000).
Deciphering the mechanisms that enable Drosophila to differentiate between pathogens and mount specific immune responses is essential for understanding innate immunity. Recent studies indicate that the Toll pathway is mainly activated in response to fungal and Gram-positive bacterial infection. Several observations suggest that imd, dredd and relish mediate most of the responses to Gram-negative bacterial infection: (1) these genes regulate the antimicrobial peptide genes that are most highly induced by Gram-negative bacterial infection; (2) dredd and relish control the induction of metchnikowin and drosomycin after Gram-negative bacterial infection, and (3) these three genes are required for resistance to Gram-negative bacterial infection. A model is proposed whereby antimicrobial gene expression in Drosophila adults is regulated by a balance of inputs from the Toll pathway and the Imd pathway, which includes Imd, Dredd and Relish, and that these two pathways are differentially activated by different classes of microorganisms. Identifying the receptors that discriminate between invading microbes and stimulate these pathways presents an exciting challenge in the study of innate immunity (Leulier, 2000).
Coexpression of an active-site C408A mutant of the fly apical caspase, Dredd [producing Dredd(C/A)], substantially attenuates cell killing triggered by Apaf-1-related-killer (Ark). In contrast, a comparable C211A mutation in the putative effector caspase drICE [producing drICE(C/A)] did not have similar effects even though it was prominently expressed. Therefore, Ark-mediated cell killing is generally not suppressed by the coexpression of mutant caspases, and the effect of Dredd(C/A) is specific. These data indicated that the Dredd mutant might exert a dominant-negative effect through a physical interaction with Ark. Whether Ark associates with Dredd was tested. A strong interaction between these proteins was detected when using either Ark(1-411) or the full-length protein. Similar tests with a comparable mutant form of drICE showed no evidence for an interaction between this caspase and Ark. These results do not address the question of whether a cofactor is necessary to regulate the Ark-Dredd interaction, since apoptotic SL2 cells may contain other proteins needed for their association. Nevertheless, Ark specifically interacts with the apical caspase Dredd but not with the effector caspase drICE. These data raise parallels to the binding observed between counterparts in the worm (CED-4 and CED-3) and in mammals (Apaf-1 and caspase-9) (Rodriguez, 1999).
The imd gene acts upstream of DmIKKgamma and the Caspase Dredd to activate the expression of antibacterial peptide genes. The dominant effect on antibacterial peptide genes of overexpression of imd driven by the hs-GAL4 driver (even in the absence of heat shock) serves to establish the epistatic relationships of imd with other genes in the pathway. In dredd and kenny mutant flies, expression of all antibacterial peptide genes in response to bacterial challenge, as well as survival to infection, is strongly impaired. The overexpression of imd is unable to confer challenge-independent expression of diptericin in dreddD55 and kenny1 mutant backgrounds. Furthermore, it does not restore immune inducibility of the diptericin gene in these mutants. It is noteworthy that drosomycin expression is not affected in these flies. These results demonstrate that imd acts upstream of both dredd and kenny to activate the antibacterial peptide genes during an immune response. In keeping with these results, transcription of the attacin-luciferase reporter gene induced by overexpression of imd in S2 cells is abolished by an ird5 (IKKß homolog) dominant-negative expression construct (Georgel, 2001).
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Death related ced-3/Nedd2-like protein:
Biological Overview
| Evolutionary Homologs
| Developmental Biology
| Effects of Mutation
date revised: 10 August 2010
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