Death related ced-3/Nedd2-like protein: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

Gene name - Death related ced-3/Nedd2-like protein

Synonyms - Ced-3-like/Nedd2-like protein

Cytological map position - 1B13--1B13

Function - protease

Keywords - apoptosis, oogenesis

Symbol - Dredd

FlyBase ID:FBgn0020381

Genetic map position - 1-

Classification - Ced-3-like/Nedd2-like protein

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

Ced-3 is the product of a gene that is necessary for programmed cell death (PCD) in the nematode C. elegans. Using the sequence of Ced-3 in a Blast search, the Drosophila gene Dredd was identified, and found to be coded for by sequences at the 3' end of l(1)1Bi (accession No. U20542). Dredd (the name stands for "Death related ced-3/Nedd2-like") protein is a Drosophila member of the caspase gene family; it encodes a 128 kD nucleolar protein. To date, the mammalian caspase transcripts described are, under normal conditions, ubiquitously distributed in many, if not all, cell types. Similarly, constitutive embryonic expression has been reported for the two other Drosophila caspases, Dcp-1 and drICE (Fraser, 1997a and Song, 1997). In contrast to this, pronounced elevation of Dredd transcripts occurs in normal development and this unique regulation is tightly linked to apoptotic signaling by Reaper, Grim and Head involution defective (Hid). Expression of Reaper, Grim, and Hid triggers processing of Dredd protein precursors by means of a mechanism that is insensitive to, and upstream of, known caspase inhibitors (Chen, 1998b).

An example of stage specific expression of dredd is the expressing of dredd associated with PCD during oogenesis. During oogenesis, nurse cells synthesize essential cytoplasmic materials and transport these to the developing oocyte. Once this phase is accomplished (stage 12), the nurse cells degenerate, exhibiting apoptotic characteristics that include cellular condensation, DNA fragmentation, and changes in cytochrome c. An attractive feature of this system is that a stereotypical sequence of morphological changes permits the identification of doomed cells prior to any overt signs of apoptosis. For this reason, expression of DREDD mRNA was examined in developing egg chambers. DREDD mRNA first appears at stage 10 in both nurse cells and the developing oocute, suggesting that at least some DREDD mRNAS are maternally supplied to the egg. In later-stage egg chambers (stages 12-13), DREDD mRNA persists within nurse cells and accumulates to very high levels at a time coincident with nurse cell death (Chen, 1998b).

To discover whether expression of apoptosis activators reaper, grim and hid triggers the accumulation of DREDD mRNA, the three apoptosis activators were ectopically expressed in mesoderm, and the expression of DREDD mRNA examined. Expression of the apoptosis activators triggers excessive apoptosis in mesoderm. During stage 13 and beyond, DREDD mRNA is not widely expressed in the developing musculature in wild-type flies. However, when misexpression of each of the death activators is directed to these tissues, prominent levels of ectopic DREDD mRNA are detected. Expression of grim in the ectoderm also results in DREDD mRNA accumulation. DREDD mRNA accumulation has also been examined in embryos homozygous for crumbs (crb). In crb mutants, reaper is ectopically expressed in the disorganized epidermis. As anticipated, ectopic accumulation of DREDD mRNA is found scattered throughout the ectoderm in crb embryos, coincident with widespread patterns of rpr expression. Perhaps the most compelling evidence for a direct role for Dredd in apoptosis comes from an examination of accumulation of DREDD mRNA in embryos carrying a homozygous deletion of the entire reaper region (mutated for rpr, hid, and grim). No apoptosis occurs in these deletion mutants. The selective accumulation of DREDD mRNA fails to occur in these mutants. This is the first report of a molecular activity that is completely blocked by the absence of H99-associated signaling (Chen, 1998b).

Dredd has a long prodomain that contains significant sequence similarity to mammalian counterparts [caspase-8/FLICE/Mac5/MACH, and caspase-10(Mach4)]. This homology spans a region that is believed to promote homotypic interactions, which establish a regulatory interface between death signals and caspase function. In mammals, caspase-8 is the most upstream caspase activated by cell surface death receptors such as Fas and TNF. Dredd may serve a similar function related to the activation of other Drosophila caspases such as Dcp-1 and drICE. When apoptosis is independently triggered by expression of Rpr, Grim or Hid, processing of Dredd-gamma, -delta and -alpha (see below: Gene Structure) is readily observed. An N-terminal tagged version of Dredd was produced and tested for the production of the processed small subunit (p10) that represents the initial product of the caspase cleavage reaction. Using these N-terminal tagged versions of Dredd, intermediates were detected that anticipate the onset of apoptosis by at least two hours. Later, additional cleavage products were detected. Rpr- and Grim-induced apoptosis can be blocked by caspase peptide inhibitors, yet in the presence of inhibitors of apoptosis, initial processing intermediates of Dredd-gamma still appear. Therefore, proteolytic cleavage of Dredd is a direct consequence of signaling by death activators and is not inhibited by inhibitors of apoptosis, indicating that the processing of Dredd is a primary process and not a secondary consequence of apoptosis (Chen, 1998b).

Cells treated with caspase inhibitors accumulate Dredd proform and intermediates to levels that are notably higher than in the absence of inhibitors. This elevation might result from inhibition of downstream proteases responsible for further processing of intermediates. Thus despite the fact that classical caspase inhibitors and p35 completely prevent activator-induced apoptosis, the initial cleavage of Dredd is not prevented by these agents. These data raise the possibility that the initial step in the processing of Dredd may occur through proteolytic activities that are upstream of caspase action, suggesting that Dredd could function as an apical or initiator caspase for apoptosis in Drosophila. If signaling by Rpr, Grim and Hid engages Dredd at a direct level of activational processing, then these activators could propagate a feed-forward amplification loop of caspase activity. If such activity ultimately exceeds a threshold, the capacity of negative regulators, such as IAPs, may be overwhelmed and cell death may ensue (Chen, 1998b).

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

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

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

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

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

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

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

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

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

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


GENE STRUCTURE

Three alternate splice variants of Dredd have been identifed, two of which encode a relatively long prodomain and another that possibly utilizes an internal translation start truncating much of the prodomain. The Dredd-alpha and -gamma isoforms process the second intron at different splice donor sites and thus differ by six amino acids at the intron II position. However, this intron is retained in the beta variant and, as a result, the beta transcript includes a premature stop codon that separates the prodomain from the rest of the enzymatic open reading frame. In principle, the beta splice form could lead to an interrupted translation product producing only the prodomain; alternatively, translation starting at an internal AUC could produce a polypeptide including the large and small subunits but lacking much of the prodomain. Determinations of the translation initiation site suggests uniform utilization of a single 5' start, suggesting that the Dredd-alpha isoform is not formed. Uniformity of 3' ends of several alpha, beta and gamma cDNA clones suggests that most if not all the diversity associated with DREDD mRNAs arises from alternate processing of the second intron. PCR assays do not detect the gamma isoform, suggesting that this variant might be expressed at developmental stages that were not analyzed or that this isoform is expressed at levels below the limits of detection (Chen, 1998b).

Exons - 5


PROTEIN STRUCTURE

Amino Acids - 488

Structural Domains

Drosophila Dredd shares extensive homology with all members of the caspase gene family. Dredd includes essential residues required for catalysis and stabilization of the P1 Asp found to be absolutely conserved among all caspases thus far identified. In addition, the catalytic site for this enzyme (QACQE) is unique among the caspases, bearing a glutamic acid in a position typically occupied by a glycine. Gapped Blast analysis identifies marked similarity to caspase-8 (Mch5/FLICE/MACH) and caspase 10 (Mch4) throughout the entire protein, including significant sequence similarities within the prodomain. These similarities span regions of the prodomain in caspase-8 and-10 referred to as death effector domains, which are believed to mediate critical protein interactions required for activation of some "initiator" caspases (Chen, 1998b).


Death related ced-3/Nedd2-like protein: Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

date revised: 20 November 98

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