InteractiveFly: GeneBrief

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


REGULATION

Transcriptional Regulation

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).

Protein Interactions

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).

Two roles for the Drosophila IKK complex in the activation of Relish and the induction of antimicrobial peptide genes

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).

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

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


DEVELOPMENTAL BIOLOGY

Embryonic

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).

Multiple apoptotic caspase cascades are required in nonapoptotic roles for Drosophila spermatid individualization

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).

Apoptosis controls the speed of looping morphogenesis in Drosophila male terminalia

In metazoan development, the precise mechanisms that regulate the completion of morphogenesis according to a developmental timetable remain elusive. The Drosophila male terminalia is an asymmetric looping organ; the internal genitalia (spermiduct) loops dextrally around the hindgut. Mutants for apoptotic signaling have an orientation defect of their male terminalia, indicating that apoptosis contributes to the looping morphogenesis. However, the physiological roles of apoptosis in the looping morphogenesis of male terminalia have been unclear. This study shows the role of apoptosis in the organogenesis of male terminalia using time-lapse imaging. In normal flies, genitalia rotation accelerates as development proceeds, and completes a full 360° rotation. This acceleration is impaired when the activity of caspases or JNK or PVF/PVR signaling is reduced. Acceleration is induced by two distinct subcompartments of the A8 segment that form a ring shape and surrounds the male genitalia: the inner ring rotates with the genitalia and the outer ring rotates later, functioning as a 'moving walkway' to accelerate the inner ring rotation. A quantitative analysis combining the use of a FRET-based indicator for caspase activation with single-cell tracking shows that the timing and degree of apoptosis correlates with the movement of the outer ring, and upregulation of the apoptotic signal increases the speed of genital rotation. Therefore, apoptosis coordinates the outer ring movement that drives the acceleration of genitalia rotation, thereby enabling the complete morphogenesis of male genitalia within a limited developmental time frame (Kuranaga, 2011; full text of article).


EFFECTS OF MUTATION

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).


EVOLUTIONARY HOMOLOGS

Other Drosophila Caspases

In comparison with other caspase family members, Drosophila caspase-1 is more homologous to CPP-32 and MCH-2alpha than to interleukin-1 beta-converting enzyme (ICE). It shares 37% sequence identity with both CPP-32 and MCH-2alpha, 29% identity with NEDD-2 (ICH-1), 28% homology with CED-2 and 25% homology with human ICE. This sequence similarity suggests that DCP-1 may be a member of the ced3-CPP-32 subfamily of caspases (Song, 1997).

A second Drosophila Caspase has been isolated and termed drICE. drICE is distinct from Death Caspase-1 and exhibits highest homology with the mammalian caspases, Mch2 and CPP32ß. drICE also contains a region in its putative small subunit that corresponds to the P4-specificity loop of CPP32ß. Overexpression of drICE sensitizes Drosophila cells to apoptotic stimuli; expression of an N-terminally truncated form of drICE rapidly induces apoptosis in Drosophila cells. Induction of apoptosis by reaper overexpression or by cycloheximide or etoposide treatment of Drosophila cells results in proteolytic processing of drICE. drICE is a cysteine protease that cleaves baculovirus p35 and Drosophila lamin DmO in vitro. drICE is expressed at all stages of Drosophila development at which programmed cell death can be induced. Levels are highest from 2-6 hours of embryogenesis, lower from 6-12 hours, and still lower after 12 hours of development. These results strongly argue that drICE is an apoptotic caspase that acts downstream of reaper (Fraser, 1997a).

The role of drICE was examined in in vitro apoptosis of the D. melanogaster cell line S2. Cytoplasmic lysates, made from S2 cells undergoing apoptosis induced by either reaper expression or cycloheximide treatment, contain a caspase activity with DEVD specificity that can cleave p35, lamin DmO, drICE and DCP-1 in vitro. This caspase activity can trigger chromatin condensation in isolated nuclei. Immunodepletion of drICE from lysates is sufficient to remove most measurable in vitro apoptotic activity; re-addition of exogenous drICE to such immunodepleted lysates restores apoptotic activity. It is concluded that, at least in S2 cells, drICE can be the sole caspase effector of apoptosis (Fraser 1997b).

Human CLARP, a caspase-like apoptosis-regulatory protein, contains two amino-terminal death effector domains fused to a carboxyl-terminal caspase-like domain. The structure and amino acid sequence of CLARP resembles those of caspase-8, caspase-10, and DCP2 (Caspase 2), a Drosophila melanogaster protein identified in this study. However, unlike caspase-8, two important residues predicted to be involved in catalysis are lost in the caspase-like domain of CLARP. Analysis with fluorogenic substrates for caspase activity confirms that CLARP is catalytically inactive. CLARP interacts with caspase-8 but not with FADD/MORT-1, an upstream death effector domain-containing protein of the Fas and tumor necrosis factor receptor 1 signaling pathways. Expression of CLARP induces apoptosis, which is blocked by the viral caspase inhibitor p35, dominant negative mutant caspase-8, and the synthetic caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk). Moreover, CLARP augments the killing ability of caspase-8 and FADD/MORT-1 in mammalian cells. The human clarp gene maps to 2q33. Thus, CLARP represents a regulator of the upstream caspase-8, which may play a role in apoptosis during tissue development and homeostasis (Inohara, 1997).

Cloning of Caspase-8/FLICE

To identify CAP3 and CAP4, components of the CD95 (Fas/APO-1) death-inducing signaling complex, nano-electrospray tandem mass spectrometry was used. This is a recently developed technique to sequence femtomole quantities of polyacrylamide gel-separated proteins. Interestingly, CAP4 encodes a novel 55 kDa protein, designated FLICE, which has homology to both FADD and the ICE/CED-3 family of cysteine proteases. FLICE binds to the death effector domain of FADD and upon overexpression induces apoptosis that is blocked by the ICE family inhibitors: CrmA and z-VAD-fmk. CAP3 was identified as the FLICE prodomain, which likely remains bound to the receptor after proteolytic activation. Taken together, these data provide unique biochemical evidence to link a death receptor physically to the proapoptotic proteases of the ICE/CED-3 family (Muzio, 1996).

Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartate specific cysteine proteases (ASCPs) is responsible for the apoptotic changes observed in mammalian cells undergoing programmed cell death. Two novel ASCPs have been cloned from human Jurkat T-lymphocytes. Like other ASCPs, the new proteases, named Mch4 and Mch5, are derived from single chain proenzymes. However, their putative active sites contain a QACQG pentapeptide instead of the QACRG present in all known ASCPs. Also, their N termini contain FADD-like death effector domains, suggesting possible interaction with FADD. Expression of Mch4 in Escherichia coli produces an active protease that, like other ASCPs, is potently inhibited by the tetrapeptide aldehyde DEVD-CHO. Interestingly, h Mch4 and the serine protease granzyme B both cleave recombinant proCPP32 and proMch3 at a conserved IXXD-S sequence to produce the large and small subunits of the active proteases. Granzyme B also cleaves proMch4 at a homologous IXXD-A processing sequence to produce mature Mch4. These observations suggest that CPP32 and Mch3 are targets of mature Mch4 protease in apoptotic cells. The presence of the FADD-like domains in Mch4 and Mch5 suggest a role for these proteases in the Fas-apoptotic pathway. In addition, these proteases could participate in the granzyme B apoptotic pathways (Fernandes-Alnemri, 1996).

Fas (APO-1/CD95) is a transmembrane receptor protein that induces apoptosis upon activation. In apoptosis triggered by Fas, a subset of cysteine proteases (designated caspases) is activated, playing a central role as effector molecules. Among these caspases, human caspase-8 (FLICE/MACH/Mch5) has been isolated and shown to be indispensable for Fas-mediated apoptotic signaling. The mouse homolog to human caspase-8 has been isolated from a BaF3 cell cDNA library. This molecule conserves the death effector domain (DED) and protease domain as detected in human caspase-8, and is capable of inducing apoptosis in KB and Rat-1 cells when overexpressed. Expression of caspase-8 is detected by Northern-blot analysis in the various tissues of adult mouse and in embryos at 9.5 days and 17.5 days of development. Further, a chromosomal gene for caspase-8 has been isolated from a mouse genomic library: the gene consists of eight exons and seven introns spanning about 26 kb in the coding region (Sakamaki, 1998).

A pivotal discovery established that Fas-associated death domain protein (FADD) interleukin-1beta-converting enzyme (FLICE)/MACH has a role in initiating the death pathway: this protease is recruited to the CD95 signaling complex by virtue of CD95's ability to bind the adapter molecule FADD. A new member of the caspase family has been cloned, a homolog of FLICE/MACH (Caspase 8), and Mch4. Since the overall architecture and function of this molecule is similar to that of FLICE, it has been designated FLICE2. Importantly, the carboxyl-terminal half of the small catalytic subunit that includes amino acids predicted to be involved in substrate binding is distinct. The pro-domain of FLICE2 encodes a functional death effector domain that binds to the corresponding domain in the adapter molecule FADD. Consistent with this finding, FLICE2 is recruited to both the CD95 and p55 tumor necrosis factor receptor signaling complexes in a FADD-dependent manner. A functional role for FLICE2 is suggested by the finding that an active site mutant of FLICE2 inhibits CD95 and tumor necrosis factor receptor-mediated apoptosis. FLICE2 is therefore involved in CD95 and p55 signal transduction (Vincenz, 1997).

Induction of apoptosis by the cell surface receptor CD95 (APO-1/Fas) has been shown to involve activation of a family of cysteine proteases (caspases). The caspase FLICE is part of the CD95 death-inducing signaling complex and is therefore the most upstream caspase in the CD95 apoptotic pathway. A total of eight different isoforms of FLICE (caspase-8/a-h) have been described. To determine which isoforms are expressed in different cells a panel of monoclonal antibodies was generated, directed against all functional domains of FLICE. Using these antibodies it has been shown that only two of the FLICE isoforms (caspase-8/a and caspase-8/b) are predominantly expressed in cells of different origin. Both isoforms are recruited to the CD95 death-inducing signaling complex and are activated upon CD95 stimulation with similar kinetics. Taken together, only two of the eight published caspase-8 isoforms could be detected in significant amounts at the protein level (Scaffidi, 1997).

The death proteases caspase 8 (FLICE) and caspase 10 (Mch4/FLICE2) are recruited to the CD-95 and tumor necrosis factor receptor-1 signaling complexes. This pivotal discovery suggested a mechanism used by these cytotoxic receptors to initiate apoptosis. The cloning and characterization of I-FLICE is described, a novel inhibitor of tumor necrosis factor receptor-1- and CD-95-induced apoptosis. The overall architecture of I-FLICE is strikingly similar to that of FLICE and Mch4/FLICE2. However, I-FLICE lacks both a catalytic active site and residues that form the substrate binding pocket, in keeping with its dominant negative inhibitory function. I-FLICE is the first example of a catalytically inert caspase that can inhibit apoptosis (Hu, 1997).

Caspase-8/FLICE interaction with FADD and recruitment to the CD95 signaling complex

Engagement of CD95 or tumor necrosis factor 1 receptor (TNFR-1) by ligand or agonist antibodies is capable of activating the cell death program, the effector arm of which is composed of mammalian interleukin-1beta converting enzyme (ICE)-like cysteine proteases (designated caspases) that are related to the Caenorhabditis elegans death gene, CED-3. Caspases, unlike other mammalian cysteine proteases, cleave their substrates following aspartate residues. Furthermore, proteases belonging to this family exist as zymogens, which in turn require cleavage at internal aspartate residues to generate the two-subunit active enzyme. As such, family members are capable of activating each other. Remarkably, both CD95 and TNFR-1 death receptors initiate apoptosis by recruiting a novel ICE/CED-3 family member, designated FLICE/MACH, to the receptor signaling complex. Therefore, FLICE/MACH represents the apical triggering protease in the cascade. Consistent with this, recombinant FLICE is capable of proteolytically activating downstream caspases. Furthermore, CrmA, a pox virus-encoded serpin that inhibits Fas and tumor necrosis factor-induced cell death attenuates the ability of FLICE to activate downstream caspases (Muzio, 1997).

Viruses have evolved many distinct strategies to avoid the host's apoptotic response. A new family of viral inhibitors (v-FLIPs) is described that interferes with apoptosis signaled through death receptors and which are present in several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as in the tumorigenic human molluscipoxvirus. v-FLIPs contain two death-effector domains that interact with the adaptor protein FADD: this inhibits the recruitment and activation of the protease FLICE by the CD95 death receptor. Cells expressing v-FLIPs are protected against apoptosis induced by CD95 or by the related death receptors TRAMP and TRAIL-R. The herpesvirus saimiri FLIP is detected late during the lytic viral replication cycle, at a time when host cells are partially protected from CD95-ligand-mediated apoptosis. Protection of virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to the persistence and oncogenicity of several FLIP-encoding viruses (Thome, 1997).

The binding of Fas ligand to Fas recruits caspase 8 to Fas via an adaptor, FADD/MORT1, and activates a caspase cascade leading to apoptosis. A human Jurkat-derived cell line (JB-6) is described that is deficient in caspase 8. This cell line is resistant to the apoptosis triggered by Fas engagement. However, the multimerization of Fas-associated protein with death domain, through the use of a dimerizing system, kills the JB-6 cells. This killing process is not accompanied by the activation of caspases or DNA fragmentation. The dying cells show neither condensation nor fragmentation of cells and nuclei, but the cells and nuclei swell in a manner similar to that seen in necrosis. These results suggest that Fas-associated protein with death domain can kill the cells via two pathways, one mediated by caspases and another that does not involve them (Kawahara, 1998).

Fas is a cell-surface receptor molecule that relays apoptotic (cell death) signals into cells. When Fas is activated by the binding of its ligand, the proteolytic protein caspase-8 is recruited to a signaling complex known as DISC by binding to a Fas-associated adapter protein. A large new protein, FLASH, has now been identified as a result of the cloning of its complementary DNA. This protein contains a motif with oligomerizing activity whose sequence is similar to that of the Caenorhabditis elegans protein CED-4, and another domain (DRD domain) that interacts with a death-effector domain in caspase-8 or in the adapter protein. Stimulated Fas binds FLASH, so FLASH is probably a component of the DISC signaling complex. Transient expression of FLASH activates caspase-8, whereas overexpression of a truncated form of FLASH containing only one of its DRD or CED-4-like domains does not allow activation of caspase-8 and Fas-mediated apoptosis to occur. Overexpression of full-length FLASH blocks the anti-apoptotic effect of the adenovirus protein E1B19K. FLASH is therefore necessary for the activation of caspase-8 in Fas-mediated apoptosis (Imai, 1999).

Caspase-8/FLICE activation

The assembly of the CD-95 (Fas/Apo-1) receptor death-inducing signaling complex occurs in a hierarchical manner; the death domain of CD-95 binds to the corresponding domain in the adapter molecule Fas-associated death domain (FADD) Mort-1, which in turn recruits the zymogen form of the death protease caspase-8 (FLICE/Mach-1) by a homophilic interaction involving the death effector domains. Immediately after recruitment, the single polypeptide FLICE zymogen is proteolytically processed to the active dimeric species composed of large and small catalytic subunits. Since all caspases cleave their substrates after Asp residues and are themselves processed from the single-chain zymogen to the two-chain active enzyme by cleavage at internal Asp residues, it follows that an upstream caspase can process a downstream zymogen. However, since FLICE represents the most apical caspase in the Fas pathway, its mode of activation has been enigmatic. It is hypothesized that the FLICE zymogen possesses intrinsic enzymatic activity such that when approximated, it autoprocesses to the active protease. Support for this is provided by two sources of evidence: (1) the synthesis of chimeric Fpk3FLICE molecules that can be oligomerized in vivo by the synthetic cell-permeable dimerizer FK1012H2, and (2) cells transfected with Fpk3FLICE undergo apoptosis after exposure to FK1012H2, producing a nonprocessable zymogen form of FLICE that retains low but detectable protease activity (Muzio, 1998).

Many forms of apoptosis, including that caused by the death receptor CD95/Fas/APO-1, depend on the activation of caspases, which are proteases that cleave specific intracellular proteins to cause orderly cellular disintegration. The requirements for activating these crucial enzymatic mediators of death are not well understood. Using molecular chimeras with either CD8 or Tac, it was found that oligomerization at the cell membrane powerfully induces caspase-8 autoactivation and apoptosis. Death induction is abrogated by the z-VAD-fmk, z-IETD-fmk, or p35 enzyme inhibitors or by a mutation in the active site cysteine but is surprisingly unaffected by death inhibitor Bcl-2 (Drosophila homolog: death executioner Bcl-2 homologue). Amino acid substitutions that prevent the proteolytic separation of the caspase from its membrane-associated domain completely block apoptosis. Thus, oligomerization at the membrane is sufficient for caspase-8 autoactivation, but apoptosis could involve a death signal conveyed by the proteolytic release of the enzyme into the cytoplasm (Martin, 1998).

Cytotoxic T lymphocytes induce apoptosis in target cells through the CD95(APO-1/Fas) and the perforin/granzyme B (GrB) pathway. The exact substrate of GrB in vivo is still unknown, but to induce apoptosis GrB requires the activity of caspases in target cells. In HeLa target cells induction of apoptosis through the perforin/GrB pathway results in minor direct cleavage of CPP32 (caspase-3) by GrB. Most caspase-3 cleavage results from activation of an upstream caspase. Moreover, target cells derived from caspase-3(-/-) mice display GrB-induced poly(ADP-ribose) polymerase (PARP) cleavage with only partially reduced efficiency, as compared to wild-type target cells. This indicates that other PARP-cleaving caspases can be activated during perforin/GrB-induced cell death. In contrast to caspase-3, FLICE (caspase-8) is directly cleaved by GrB in HeLa cells. It is therefore concluded that FLICE not only plays a central role in CD95(APO-1/Fas)-induced apoptosis but can also be directly activated during perforin/GrB-induced apoptosis (Medema,1998).

The exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homolog, Apaf-1 (Drosophila homolog: Apaf-1-related-killer), thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and three others (caspases-1, -4, and -5) fail to be activated under the same conditions. In vitro association assays confirm that caspase-9 selectively binds to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 do not. Depletion of caspase-9 from cell extracts abrogates cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enables an ordering of the sequence of caspase activation events downstream of caspase-9 and reveals the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9 (Slee, 1999).

Cytotoxic T lymphocytes induce apoptosis in target cells through the CD95(APO-1/Fas) and the perforin/granzyme B (GrB) pathway. The exact substrate of GrB in vivo is still unknown, but to induce apoptosis GrB requires the activity of caspases in target cells. In HeLa target cells induction of apoptosis through the perforin/GrB pathway results in minor direct cleavage of CPP32 (caspase-3) by GrB. Most caspase-3 cleavage results from activation of an upstream caspase. Moreover, target cells derived from caspase-3(-/-) mice display GrB-induced poly(ADP-ribose) polymerase (PARP) cleavage with only partially reduced efficiency, when compared to wild-type target cells. This indicates that other PARP-cleaving caspases can be activated during perforin/GrB-induced cell death. In contrast to caspase-3, FLICE (caspase-8) is directly cleaved by GrB in HeLa cells. It is therefore concluded that FLICE not only plays a central role in CD95(APO-1/Fas)-induced apoptosis but that it can also be directly activated during perforin/GrB-induced apoptosis (Medema, 1997).

A phosphoprotein protein with a death effector domain has been characterized that has a novel bifunctional role in programmed cell death. The 15-kDa phosphoprotein enriched in astrocytes (PEA-15) inhibits Fas-mediated apoptosis and increases tumor necrosis factor receptor-1 (TNF-R1)-mediated apoptosis in the same cell type in a ligand-dependent manner. Phosphorylation appears to play a role in its differential effects, since point mutations at one or both phosphorylation consensus sites within PEA-15 destroy its effect on Fas-mediated, but not TNF-R1-mediated, apoptosis. Furthermore, the differential effect is evident at the level of caspase-8 activity, which is inhibited via Fas activation, but increased via TNF-R1 activation upon PEA-15 expression. These results show that PEA-15 provides a potential mechanism during development for distinguishing between diverse extracellular death-inducing signals that culminate either in apoptosis or in survival (Estelles, 1999).

PEA-15 mRNA is abundant as early as embryonic day 12. Changes in the phosphorylation state of PEA-15 in response to hormones and neurotransmitters suggest that phosphorylation may be important to the function of this protein. Although PEA-15 has a death effector domain (DED) domain, the physiological role of PEA-15 and the significance of its phosphorylation state remain unclear. PEA-15 is unlike other DED-containing proteins described thus far in that its expression provides a means by which a cell can respond differently to two extracellular apoptotic stimuli. Such proteins are likely to serve an important role during embryonic and adult development (Estelles, 1999).

Upon activation by liver injury, hepatic stellate cells produce excessive fibrous tissue leading to cirrhosis. The hepatotoxin CCl4 induces activation of RSK (see Drosophila RSK), phosphorylation of C/EBPß on Thr217, and proliferation of stellate cells in normal mice, but causes apoptosis of these cells in C/EBPß-/- or C/EBPß-Ala217 (a dominant-negative nonphosphorylatable mutant) transgenic mice. Both C/EBPß-PThr217 and the phosphorylation mimic C/EBPß-Glu217, but not C/EBPß-Ala217, associate with procaspases 1 and 8 in vivo and in vitro and inhibit their activation. These data suggest that C/EBPß phosphorylation on Thr217 creates a functional XEXD caspase substrate/inhibitor box (K-Phospho-T217VD) that is mimicked by C/EBPß-Glu217 (KE217VD). C/EBPß-/- and C/EBPß-Ala217 stellate cells are rescued from apoptosis by the cell permeant KE217VD tetrapeptide or C/EBPß-Glu217. It is concluded that C/EBPß phosphorylation by RSK creates a functional XEXD caspase inhibitory box critical for cell survival (Buck, 2001).

Caspase-8/FLICE targets and downstream signaling

The apoptotic signal triggered by ligation of members of the death receptor family is promoted by sequential activation of caspase zymogens. In a purified system, the initiator caspases-8 and -10 directly process the executioner pro-caspase-3. These rates of activation are of sufficient magnitude to indicate direct processing in vivo. Differentially processed forms of caspase-3 that accumulate during caspase-3 activation have similar rates of activation, activities, and specificities. The pattern and rate of caspase-8 induced activation of pro-caspase-3 in cytosolic extracts is the same as in a purified system. Moreover, immunodepletion of pro-caspase-9, a putative intermediary in the pathway to activation, is without consequence. Taken together these data demonstrate that the initiator caspase-8 can directly activate pro-caspase-3 without the requirement for an accelerator. The in vitro data thus help to deconvolute previous in vivo transfection studies that have debated the role of a direct versus indirect transmission of the apoptotic signal generated by ligation of death receptors (Stennicke, 1998).

A cytosolic protein has been purified that induces cytochrome c release from mitochondria in response to caspase-8, the apical caspase activated by cell surface death receptors such as Fas and TNF. Peptide mass fingerprinting identified this protein as Bid, a BH3 domain-containing protein known to interact with both Bcl2 and Bax. Caspase-8 cleaves Bid, and the COOH-terminal part translocates to mitochondria where it triggers cytochrome c release. Immunodepletion of Bid from cell extracts eliminates the cytochrome c releasing activity. The cytochrome c releasing activity of Bid is antagonized by Bcl2. A mutation at the BH3 domain diminishes its cytochrome c releasing activity. Bid, therefore, relays an apoptotic signal from the cell surface to mitochondria (Luo, 1998).

BID, a BH3 domain-containing proapoptotic Bcl2 family member, is a specific proximal substrate of Casp8 in the Fas apoptotic signaling pathway. While full-length BID is localized in cytosol, truncated BID (tBID) translocates to mitochondria and thus transduces apoptotic signals from cytoplasmic membrane to mitochondria. tBID induces first the clustering of mitochondria around the nuclei and release of cytochrome c independent of caspase activity, and then the loss of mitochondrial membrane potential, cell shrinkage, and nuclear condensation in a caspase-dependent fashion. Coexpression of BclxL inhibits all the apoptotic changes induced by tBID. These results indicate that BID is a mediator of mitochondrial damage induced by Casp8 (Li, 1998).

Signaling through the CD95/Fas/APO-1 death receptor plays a critical role in the homeostasis of the immune system. RICK, a novel protein kinase that regulates CD95-mediated apoptosis has been identified and characterized. RICK is composed of an N-terminal serine-threonine kinase catalytic domain and a C-terminal region containing a caspase-recruitment domain. RICK physically interacts with CLARP, a caspase-like molecule known to bind to Fas-associated protein with death domain (FADD) and caspase-8. Expression of RICK promotes the activation of caspase-8 and potentiates apoptosis induced by Fas ligand, FADD, CLARP, and caspase-8. Deletion mutant analysis reveals that both the kinase domain and caspase-recruitment domain are required for RICK to promote apoptosis. A RICK mutant (in which the lysine of the putative ATP-binding site at position 38 is replaced by a methionine) functions as an inhibitor of CD95-mediated apoptosis. Thus, RICK represents a novel kinase that may regulate apoptosis induced by the CD95/Fas receptor pathway (Inohara, 1998).

Genetic studies of the nematode C. elegans have identified several important components of the cell death pathway, most notably CED-3, CED-4, and CED-9. CED-4 directly interacts with the Bcl-2 homolog CED-9 (or the mammalian Bcl-2 family member Bcl-xL) and the caspase CED-3 (or the mammalian caspases ICE and FLICE). This trimolecular complex of CED-4, CED-3, and CED-9 is functional in that CED-9 inhibits CED-4 from activating CED-3 and thereby inhibits apoptosis in heterologous systems. The E1B 19,000-molecular weight protein (E1B 19K) is a potent apoptosis inhibitor and the adenovirus homolog of Bcl-2-related apoptosis inhibitors. Since E1B 19K and Bcl-xL have functional similarity, it was hypothesized that E1B 19K interacts with CED-4 and regulates CED-4-dependent caspase activation. Binding analysis indicates that E1B 19K interacts with CED-4 in a Saccharomyces cerevisiae two-hybrid assay, in vitro, and in mammalian cell lysates. The subcellular localization pattern of CED-4 is dramatically changed by E1B 19K, supporting the theory of a functional interaction between CED-4 and E1B 19K. Whereas expression of CED-4 alone could not induce cell death, coexpression of CED-4 and FLICE augments cell death induction by FLICE, which is blocked by expression of E1B 19K. Even though E1B 19K does not prevent FLICE-induced apoptosis, it does inhibit CED-4-dependent, FLICE-mediated apoptosis, which suggests that CED-4 is required for E1B 19K to block FLICE activation. Thus, E1B 19K functions through interacting with CED-4, and presumably a mammalian homolog of CED-4, to inhibit caspase activation and apoptosis (Han, 1998).

The Bcl-2 family member Bcl-xL has often been correlated with apoptosis resistance. In peripheral human T cells, resistance to CD95-mediated apoptosis is characterized by a lack of caspase-8 recruitment to the CD95 death-inducing signaling complex (DISC) and by increased expression of Bcl-xL. This raises the possibility that Bcl-xL directly prevents caspase-8 activation by the DISC. To test this hypothesis a cell line in which CD95 signaling is inhibited by overexpression of Bcl-xL was used. In these MCF7-Fas-bcl-xL cells Bcl-xL has no effect on the recruitment of caspase-8 to the DISC. It does not affect the activity of the DISC nor the generation of the caspase-8 active subunits p18 and p10. In contrast, cleavage of a typical substrate for caspase-3-like proteases, poly(ADP-ribose) polymerase, is inhibited in comparison with the control-transfected CD95-sensitive MCF7-Fas cells. To test whether Bcl-xL inhibits active caspase-8 subunits in the cytoplasm, a number of immunoprecipitation experiments were performed. Using monoclonal antibodies directed against different domains of caspase-8, anti-Bcl-xL antibodies, or fusion proteins of glutathione S-transferase with different domains of caspase-8, no evidence for a direct or indirect physical interaction between caspase-8 and Bcl-xL was found. Moreover, overexpression of Bcl-xL does not inhibit the activity of the caspase-8 active subunits p18/p10. Therefore, in this cell line that has become resistant to CD95-induced apoptosis due to overexpression of Bcl-xL, Bcl-xL acts independently and downstream of caspase-8 (Medema, 1998).

Two cell types have been identified, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 is activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells, cleavage of both caspases is delayed for approximately 60 min. However, both type I and type II cells show similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (DeltaPsim). Upon CD95 triggering, all mitochondrial apoptogenic activities are blocked by Bcl-2 or Bcl-xL overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-xL blocks caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis is accompanied by activation of large amounts of caspase-8 by the death-inducing signaling complex (DISC), whereas in type II cells DISC formation is strongly reduced and activation of caspase-8 and caspase-3 occurs following the loss of DeltaPsim. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-xL, converts these cells into true type I cells in which apoptosis is no longer inhibited by Bcl-xL. In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells) (Scaffidi, 1998).

Apoptosis often involves the release of cytochrome c from mitochondria, leading to caspase activation. However, in apoptosis mediated by CD95 (Fas/APO-1), caspase-8 (FLICE/MACH/Mch5) is immediately activated and, in principle, could process other caspases directly. To investigate whether caspase-8 could also act through mitochondria, active caspase-8 was added to a Xenopus cell-free system requiring these organelles. Caspase-8 rapidly promotes the apoptotic program, culminating in fragmentation of chromatin and the nuclear membrane. In extracts devoid of mitochondria, caspase-8 produces DNA degradation, but leaves nuclear membranes intact. Thus, mitochondria are required for complete engagement of the apoptotic machinery. In the absence of mitochondria, high concentrations of caspase-8 are required to activate downstream caspases. However, when mitochondria are present, the effects of low concentrations of caspase-8 are vastly amplified through cytochrome c-dependent caspase activation. Caspase-8 promotes cytochrome c release indirectly, by cleaving at least one cytosolic substrate. Bcl-2 blocks apoptosis only at the lowest caspase-8 concentrations, potentially explaining why CD95-induced apoptosis can often evade inhibition by Bcl-2 (Kuwana, 1998).

Although the molecular mechanisms of TNF signaling have been largely elucidated, the principle that regulates the balance of life and death is still unknown. The death domain kinase RIP, a key component of the TNF signaling complex, is cleaved by Caspase-8 in TNF-induced apoptosis. The cleavage site maps to the aspartic acid at position 324 of RIP. The cleavage of RIP results in the blockage of TNF-induced NFkappa-B activation. RIPc, one of the cleavage products, enhances interaction between TRADD and FADD/MORT1 and increases cell sensitivity to TNF. Most importantly, the Caspase-8 resistant RIP mutants protect cells against TNF-induced apopotosis. These results suggest that cleavage of RIP is an important process in TNF-induced apoptosis. Furthermore, RIP cleavage is also detected in other death receptor-mediated apoptosis. Therefore, this study provides a potential mechanism to convert cells from life to death in death receptor-mediated apoptosis (Lin, 1999).

Caspase-8/FLICE interaction with CED-3/Apaf-1

Previous genetic studies of the nematode C. elegans have identified three important components of cell death machinery. CED-3 and CED-4 (Drosophila homolog: Apaf-1-related-killer) function to kill cells, whereas CED-9 protects cells from death. Both CED-9 and its mammalian homolog Bcl-xL (a member of the Bcl-2 family of cell death regulators) interact with and inhibit the function of CED-4. In addition, CED-4 can simultaneously interact with CED-3 and its mammalian counterparts interleukin-1beta-converting enzyme (ICE) and FLICE. Thus, CED-4 plays a central role in the cell death pathway, biochemically linking CED-9 and the Bcl-2 family to CED-3 and the ICE family of pro-apoptotic cysteine proteases (Chinnaiyan, 1997).

Miscellaneous Caspase-8/FLICE interacting proteins

The CD95 signaling pathway comprises proteins that contain one or two death effector domains (DED), such as FADD/Mort1 or caspase-8. A novel 37 kDa protein, DEDD, is described that contains an N-terminal DED. DEDD is highly conserved between human and mouse (98. 7% identity) and is ubiquitously expressed. Overexpression of DEDD in 293T cells induces weak apoptosis, mainly through its DED by which it interacts with FADD and caspase-8. Endogenous DEDD is found in the cytoplasm and translocates into the nucleus upon stimulation of CD95. Immunocytological studies reveal that overexpressed DEDD directly translocates into the nucleus, where it co-localizes in the nucleolus with UBF, a basal factor required for RNA polymerase I transcription. Consistent with its nuclear localization, DEDD contains two nuclear localization signals and the C-terminal part shares sequence homology with histones. Recombinant DEDD binds to both DNA and reconstituted mononucleosomes and inhibits transcription in a reconstituted in vitro system. The results suggest that DEDD is a final target of a chain of events by which the CD95-induced apoptotic signal is transferred into the nucleolus to shut off cellular biosynthetic activities (Stegh, 1998).

Adenovirus type 5 encodes a 14.7-kDa protein that protects infected cells from tumor necrosis factor-induced cytolysis by an unknown mechanism. Infection of cells with an adenovirus vector expressing Fas ligand induces rapid apoptosis that is blocked by coinfection with a virus expressing the 14.7-kDa protein. Moreover, FasL promotes the rapid activation of DEVD-specific caspases, and caspase activation is blocked by coinfection with Adenovirus coding for the 14.7-kDa protein. Cell death induced by the overexpression of Fas ligand, Fas-associated death domain-containing protein (FADD)/MORT1, or FADD-like interleukin-1beta-converting enzyme (FLICE)/caspase-8 in a virus-free system is efficiently blocked by 14.7K expression. Moreover, 14.7K is shown to interact with FLICE. These results support the idea that FLICE is a cellular target for the 14.7-kDa protein (Chen, 1998a).

BH3-only proapoptotic proteins of the Bcl-2 family such as Bad, Bid, Bim, or Bik transduce death stimuli from the cell surface to the central death machinery. Following apoptosis stimulation, these molecules translocate from the cytosol to mitochondria where they bind to membrane-based Bcl-2 family members. Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells. In type II cells, such as Jurkat cells or hepatocytes, death-inducing signaling complex (DISC) formation is strongly reduced compared to type I cells in which activation of large amounts of caspase 8 by the DISC enables direct activation of downstream caspases leading to irreversible cell damage. In type II cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerates Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delays apoptosis. When phosphorylated, Bid is insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis (Desagher, 2001).

Caspase-8/FLICE deficiency

Fas (APO-1/CD95) is a member of the tumor necrosis factor receptor (TNF-R) family and induces apoptosis when crosslinked with either Fas ligand or agonistic antibody (Fas antibody). The Fas-Fas ligand system has an important role in the immune system, where it is involved in the downregulation of immune responses and the deletion of peripheral autoreactive T lymphocytes. The intracellular domain of Fas interacts with several proteins including FADD (MORT-1), DAXX, RIP, FAF-1, FAP-1 and Sentrin. The adaptor protein FADD can, in turn, interact with the cysteine protease caspase-8 (FLICE/MACH/Mch5). In a genetic screen for essential components of the Fas-mediated apoptotic cascade, a Jurkat T lymphocyte cell line deficient in caspase-8 has been isolated that was completely resistant to Fas-induced apoptosis. Complementation of this cell line with wild-type caspase-8 restores Fas-mediated apoptosis. Fas activation of multiple caspases and of the stress kinase p38 and c-Jun NH2-terminial kinase (JNK) is completely blocked in the caspase-8-deficient cell line. Furthermore, the cell line is severely deficient in cell death induced by TNF-alpha and is partially deficient in cell death induced by ultraviolet irradiation, adriamycin and etoposide. This study provides the first genetic evidence that caspase-8 occupies an essential and apical position in the Fas signaling pathway and suggests that caspase-8 may participate broadly in multiple apoptotic pathways (Juo, 1998).

Local caspase activation interacts with Slit-Robo signaling to restrict axonal arborization

In addition to being critical for apoptosis, components of the apoptotic pathway, such as caspases, are involved in other physiological processes in many types of cells, including neurons. However, very little is known about their role in dynamic, nonphysically destructive processes, such as axonal arborization and synaptogenesis. This study shows that caspases are locally active in vivo at the branch points of young, dynamic retinal ganglion cell axonal arbors but not in the cell body or in stable mature arbors. Caspase activation, dependent on Caspase-3, Caspase-9, and p38 mitogen-activated protein kinase (MAPK), rapidly increased at branch points corresponding with branch tip addition. Time-lapse imaging revealed that knockdown of Caspase-3 and Caspase-9 led to more stable arbors and presynaptic sites. Genetic analysis showed that Caspase-3, Caspase-9, and p38 MAPK interacted with Slit1a-Robo2 signaling, suggesting that localized activation of caspases lie downstream of a ligand receptor system, acting as key promoters of axonal branch tip and synaptic dynamics to restrict arbor growth in vivo in the central nervous system (Campbell, 2013).

Regulation of Caspase-8/FLICE

Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. The mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases was compared in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-caspase-9. In contrast, these IAP family proteins do not prevent the caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibit active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-caspase-9 as a new target for IAP-mediated inhibition of apoptosis (Deveraux, 1998).

Tumor necrosis factor alpha (TNF-alpha) binding to the TNF receptor (TNFR) potentially initiates apoptosis and activates the transcription factor nuclear factor kappa B (NF-kappaB), which suppresses apoptosis by an unknown mechanism. The activation of NF-kappaB blocks the activation of caspase-8. TRAF1 (TNFR-associated factor 1), TRAF2, and the inhibitor-of-apoptosis (IAP) proteins c-IAP1 and c-IAP2 have been identified as gene targets of NF-kappaB transcriptional activity. In cells in which NF-kappaB is inactive, all of these proteins are required to fully suppress TNF-induced apoptosis, whereas c-IAP1 and c-IAP2 are sufficient to suppress etoposide-induced apoptosis. Thus, NF-kappaB activates a group of gene products that function cooperatively at the earliest checkpoint to suppress TNF-alpha-mediated apoptosis and that function more distally to suppress genotoxic agent-mediated apoptosis (Wang, 1998).

Cells of the monocyte/macrophage lineage play a central role in both innate and acquired immunity of the host. However, the acquisition of functional competence and the ability to respond to a variety of activating or modulating signals require maturation and differentiation of circulating monocytes and entails alterations in both the biochemical and phenotypic profiles of the cells. The process of activation also confers survival signals essential for the functional integrity of monocytes enabling the cells to remain viable in microenvironments of immune or inflammatory lesions that are rich in cytotoxic inflammatory mediators and reactive free-radical species. However, the molecular mechanisms of activation-induced survival signals in monocytes remain obscure. To define the mechanistic basis of activation-induced resistance to apoptosis in human monocytes at the molecular level, the modulation of expression profiles of genes associated with the cellular apoptotic pathways upon activation was evaluated and the following three results have been demonstrated: (1) activation results in selective resistance to apoptosis particularly to that induced by signaling via death receptors and DNA damage; (2) concurrent with activation, the most apical protease in the death receptor pathway, caspase-8/FLICE is rapidly down-regulated at the mRNA level representing a novel regulatory mechanism; and (3) activation of monocytes also leads to dramatic induction of the Bfl-1 gene, an anti apoptotic member of the Bcl-2 family. These findings thus provide a potential mechanistic basis for the activation-induced resistance to apoptosis in human monocytes (Perera, 1998).

Caspase-8/FLICE and Sphingomyelinase activation

Sphingomyelinase (SMase) activation and ceramide generation have emerged as an important signaling pathway transducing diverse biological effects of cytokine receptors like p55 tumor necrosis factor (TNF) receptor or Fas. The TNF-dependent activation of acid SMase (A-SMase) through the p55 TNF receptor-associated proteins TRADD and FADD is described. Overexpression of TRADD and FADD in 293 cells does not change the basal activity of A-SMase but enhances TNF-induced stimulation of A-SMase. Other TNF R55-associated proteins like TRAF2 and RIP, which have been reported to mediate TNF R55-mediated activation of nuclear factor kappaB, do not affect activation of A-SMase. Caspase inhibitors markedly reduce A-SMase activity, suggesting the involvement of an ICE-like protease in TRADD/FADD-mediated activation of A-SMase. Overexpression of caspase-8/a (FLICE/MACH) or caspase-10/b (FLICE2) does not change A-SMase activity, suggesting that TRADD/FADD-mediated activation of A-SMase involves a yet to be defined caspase-like protease distinct from caspase-8/a or -10/b (Schwandner, 1998).

Post-transcriptional control of executioner caspases by RNA-binding proteins

Regulation of caspases occurs at several levels, including transcription, proteolytic processing, inhibition of enzymatic function, and protein degradation. In contrast, little is known about the extent of post-transcriptional control of caspases. This study describes four conserved RNA-binding proteins (RBPs)-PUF-8, MEX-3, GLD-1, and CGH-1-that sequentially repress the CED-3 caspase in distinct regions of the Caenorhabditis elegans germline. GLD-1 represses ced-3 mRNA translation via two binding sites in its 3' untranslated region (UTR), thereby ensuring a dual control of unwanted cell death: at the level of p53/CEP-1 and at the executioner caspase level. Moreover, seven RBPs were identified that regulate human caspase-3 expression and/or activation, including human PUF-8, GLD-1, and CGH-1 homologs PUM1, QKI, and DDX6. Given the presence of unusually long executioner caspase 3' UTRs in many metazoans, translational control of executioner caspases by RBPs might be a strategy used widely across the animal kingdom to control apoptosis (Subasic, 2016).


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