Characterization of the DrosophilaCaspase, DAMM*

Caspases are main effectors of apoptosis in metazoans. Genome analysis indicates that there are seven caspases in Drosophila, six of which have been previously characterized. Here we describe the cloning and characterization of the last Drosophila caspase, DAMM. Similar to mammalian effector caspases, DAMM lacks a long prodomain. We show that the DAMM precursor, along with the caspases DRONC and DECAY, is partially processed in cells undergoing apoptosis. Recombinant DAMM produced inEscherichia coli shows significant catalytic activity on a pentapeptide caspase substrate. Low levels of damm mRNA are ubiquitously expressed in Drosophila embryos during early stages of development. Relatively high levels ofdamm mRNA are detected in larval salivary glands and midgut, and in adult egg chambers. Ectopic expression of DAMM in cultured cells induces apoptosis, and similarly, transgenic overexpression of DAMM, but not of a catalytically inactive DAMM mutant, in Drosophila results in a rough eye phenotype. We demonstrate that expression of the catalytically inactive DAMM mutant protein significantly suppresses the rough eye phenotype due to the overexpression of HID, suggesting that DAMM may be required in a hid-mediated cell death pathway.

Programmed cell death in metazoans is mediated by caspases, a family of cysteine proteases, which cleave their substrates following an Asp residue (1)(2)(3)(4). A number of caspases have been described in both vertebrates and invertebrates. To date 14 caspases have been cloned in mammals (1)(2)(3)(4). Gene targeting studies in the mouse suggest that some caspases play a signal specific and spatially restricted role in apoptosis, whereas others seem mainly involved in the processing and activation of proinflammatory cytokines (reviewed in Ref. 5). Four caspases exist in the nematode Caenorhabditis elegans, but only one, CED-3, is essential for all developmentally programmed cell death, and the functions of the remaining three are not known (6). In Drosophila melanogaster six caspases, named DCP-1, DREDD/DCP-2, DRICE, DRONC, DECAY, and STRICA have been cloned so far (7)(8)(9)(10)(11)(12)(13). In addition to the already described caspases, analysis of the Drosophila genomic database predicts one more caspase, which we have termed DAMM 1 (GenBank TM accession number AF240763; death-associated molecule related to Mch2) (14). Among the Drosophila caspases, DREDD and DRONC contain long prodomains carrying death effector domains (DEDs) and a caspase recruitment domain (CARD), respectively, suggesting that these two caspases may act as upstream initiator caspases (reviewed in Ref. 14). STRICA also has a long prodomain, but it lacks any CARD or DED structures (13). On the other hand, DCP-1, DRICE, and DECAY lack long prodomains and are thus similar to downstream effector caspases in mammals. dcp-1 mutants are larval lethal and exhibit melanotic tumors (7). Additionally, DCP-1 is required for Drosophila oogenesis, as dcp-1 mutants show a defect in transfer of nurse cell cytoplasmic contents to developing oocytes (15). The transcript for dredd accumulates in embryonic cells undergoing programmed cell death and in nurse cells in the ovary at a time that coincides with nurse cell death (8). Heterozygosity at the dredd locus suppresses cell death induced by the ectopic expression by rpr, hid, and grim in transgenic models, indicating that DREDD concentration may be a rate-limiting step in this pathway. In addition to its function in apoptosis, DREDD also plays a key role in the innate immune response (16,17).
dronc mRNA is widely expressed during development and is up-regulated several-fold by ecdysone in larval salivary glands and midgut prior to histolysis of these tissues (11). Heterozygosity at the dronc locus or the expression of a catalytically inactive DRONC mutant suppress the eye phenotype caused by rpr, grim, and hid, consistent with the idea that DRONC functions in the RPR, GRIM, and HID pathway (18 -20). DRONC also interacts, both biochemically and genetically, with the CED-4/Apaf-1 fly homolog, Dark (20). Furthermore, loss of DRONC function in early Drosophila embryos because of dronc RNA ablation results in a decrease in apoptosis, indicating that DRONC is required for programmed cell death during embryogenesis (20). These results suggest that DRONC is a key upstream caspase in mediating developmentally programmed cell death in Drosophila.
The precise roles of DRICE, DECAY, and STRICA in programmed cell death in Drosophila have not been established. However, in vitro antibody depletion experiments suggest that DRICE is required for apoptotic activity in the S2 Drosophila cell line (21). Similar to dronc, decay expression is high in larval midgut and salivary glands, but decay expression is not regulated by ecdysone (12). Accumulation of dronc and decay mRNA in salivary glands and midgut may be required to sensitize these tissues for deletion by apoptosis during metamorphosis.
The presence of multiple caspases in Drosophila indicates that apoptotic pathways in insects are likely to be of similar complexity to those in mammals. To fully understand the role of various caspases in cell physiology, it is important to analyze all caspases in a given model organism. In this paper, we describe the initial characterization of DAMM, the last Drosophila caspase.

EXPERIMENTAL PROCEDURES
Cloning of damm cDNA-damm was identified using a TBLASTN search of the Berkeley Drosophila Genome Project database, as a region of genomic DNA sequence displaying significant homology to mammalian caspases. From this region of homology, damm specific primers were designed and were used in conjunction with library-specific vector primers to amplify the full-length open-reading frame of damm from a Drosophila larval cDNA library. The 5Ј-end of the damm open-reading frame was confirmed using 5Ј-rapid amplification of cDNA ends (RACE) (Life Technologies). The cDNA sequence of damm has been deposited in the GenBank TM database under accession number AF240763. Multiple sequence alignments and construction of phylogenetic trees were carried out using Bionavigator software packages ClustalW and Protpars at the Australian National Genome Information Services server.
In Vitro Caspase Cleavage Assays-Full-length damm in pcDNA3 was used as a template for the production of [ 35 S]methionine-labeled protein using a coupled transcription/translation kit (Promega). 5 l of translated product was incubated with recombinant enzyme lysates for 3 h at 37°C, electrophoresed on 15% SDS-polyacrylamide gels, transferred to polyvinylidene membrane (Dupont) and exposed to x-ray film.
Cell death assays in insect cells were carried out using Schneider L2 (SL2) cells as previously described (28,29). SL2 cells were maintained and transfected using Cellfectin (Life Technology) as described (8). For death assays, 1.5 ϫ 10 6 SL2 cells were cotransfected with either 2 g of vector or 1 g of pRM-damm together with 1 g of respective pRmHa.3 inhibitor constructs, pRM-diap1HA, pRM-diap2HA, or pRM-p35HA and 0.5 g of pCASPERhs-␤gal reporter. All death assays were performed in duplicate. 24 h post-transfection, cells were induced to express pCASPERhs-␤gal by three cycles of heat shock at 37°C for 30 min followed by 27°C for 30 min. Following heat shock, one sample of each duplicate transfection was induced to express the respective pRmHa.3 constructs by the addition of CuSO 4 at a final concentration of 0.7 mM. Cells were fixed and stained with X-gal 48 h post-CuSO 4 induction as previously described (29). Cell survival was calculated as the percent of transfected (␤-galactosidase-positive) cells in the CuSO 4 -treated population relative to the percent of transfected cells in the untreated population. All calculations were normalized against the 100% survival value of vector transfected cells. The results, shown as average percentages Ϯ S.E.M., were derived from three independent experiments. To check copper-induced protein expression after 48 h of CuSO 4 treatment, cells were lysed in SDS-PAGE buffer, and lysates were subjected to immunoblotting using a rat monoclonal ␣HA antibody (Roche Molecular Biochemicals) or a mouse monoclonal ␣FLAG antibody (Sigma Chemical Co.).
Caspase Processing in SL2 Transfectants-SL2 cells were transfected with pRM-dronc, pRM-dammFLAG, or pRM-decayFLAG as described above. 24 h following transfection, cells were induced to express respective constructs by the addition of CuSO 4 at a final concentration of 0.7 mM. 24 h following copper induction, transfectants were treated with cycloheximide at a final concentration of 25 g/ml for 8, 16, or 24 h. Expression and processing of DRONC, DAMM, and DECAY proteins was analyzed by SDS-PAGE electrophoresis of lysates from transfected cells and immunoblotting using either ␣Dronc (20) or the ␣FLAG antibody.
Northern and In Situ mRNA Analysis-Total RNA from various developmental stages of Drosophila or adult flies was prepared using RNAzol B according to the manufacturer's (Tel-Test Inc.) protocol. Approximately 20 g of total RNA was electrophoresed on a 2.2 M formaldehyde gel and transferred to Biodyne A nylon membrane (Pall). The blot was hybridized to a 32 P-labeled damm cDNA coding region probe and exposed to Kodak XAR-5 film. For in situ RNA analysis, antisense and sense digoxigenin-labeled riboprobes were prepared using the appropriate RNA polymerase from a linearized damm cDNA clone. Digoxigenin labeling was performed according to the manufac- turer's instructions (Roche Molecular Biochemicals). In situ hybridization to Drosophila embryos and larval tissues were essentially as described (12,29,30).
DAMM Transgenic Flies and Genetic Interactions-Wild-type damm or damm C156G mutant cDNA, tagged with FLAG was cloned into the pGMR vector and transgenic flies were generated and maintained as previously described (31). For testing the interaction of GMRdamm C156G with GMR-hid or GMR-rpr, crosses were carried out at 18°C. Progeny were scored by examining eye phenotypes using a light microscope, as previously described (31).

RESULTS
Identification of DAMM-While searching for new molecules with homology to various mammalian caspases using the TBLASTN program, we identified a genomic sequence contained in an entry (accession no. AC005466) in the Berkeley Drosophila Genome Project database that encoded a partial caspase-like molecule. We cloned the corresponding cDNA for this gene by a combination of PCR and 5Ј-RACE. The cDNA contained a predicted open-reading frame of 255 amino acid residues with a high degree of homology to mammalian caspases, particularly those related to the caspase-3 subfamily (Fig. 1). We named this new molecule DAMM, for death-associated molecule related to Mch2. A comparison of the damm cDNA sequence and the annotated Drosophila genome sequence reveals that the coding region for DAMM is contained in 5 exons (Fig. 1A). This differs from the predicted gene structure for damm in the flybase. DAMM shares ϳ29% amino acid sequence identity and 43% sequence similarity with caspase-6 (Mch-2) and 27% sequence identity and 46% sequence similarity with caspase-3. Of the Drosophila caspases, DAMM is most homologous to the newly identified caspase STRICA (Fig. 1C) (13), sharing 44% sequence identity and 60% sequence similarity. The degree of homology between these two caspases is particularly striking given that DAMM does not possess the long prodomain of STRICA. Among other Drosophila caspases, DAMM shares 28% sequence identity with DCP-1 and 26% identity with DECAY, DRICE, and DREDD. DAMM shares the least homology with DRONC among the Drosophila caspases with 23% sequence identity and 39% similarity.
damm mRNA Expression during Drosophila Development-In RNA blots, damm was present as an ϳ0.9-kilobase transcript in most developmental stages, larvae, pupae, and in the adult fly (Fig. 2). Relatively high levels of damm transcript were detected in early 3rd instar larvae and in the adult fly (Fig. 2). We further analyzed the expression pattern of damm during Drosophila development by in situ hybridization to embryos and larval tissues using a digoxigenin-labeled antisense mRNA probe (Fig. 3). damm is expressed at low levels through-out embryogenesis and shows no up-regulation at stage 11 (Fig.  3), when programmed cell death first becomes evident in Drosophila (32). In addition to data shown for stage 5 and later  (Fig. 3, A-F) damm mRNA was also detected in stage 1-4 syncitial embryos (not shown), suggesting that it is maternally deposited into the embryo, because zygotic expression does not begin before stage 5 (33). In stage 5 cellularized embyros, damm mRNA is ubiquitously expressed (Fig. 3A), but in later stages higher levels of damm transcript were evident in specific cells and tissues (Fig. 3, C-F). For example, specific cells in developing salivary gland (Fig. 3D) and hindgut (Fig.  3E) showed staining for damm transcript. No staining was seen when a damm sense probe was hybridized to embryos at various stages of development ( Fig. 3G and data not shown).
We also examined the expression of damm in third instar larval tissues and during oogenesis. Low levels of damm expression were observed in brain lobes (Fig. 3H), which contain apoptotic cells at this stage (34). A relatively high level of damm expression was observed in midgut (Fig. 3I) and salivary glands (Fig. 3J) from late third instar larvae, preceding the onset of apoptosis in these tissues, which occurs after pupariation (35). During oogenesis damm mRNA is detected in egg chambers of all stages and in the nurse cells ( Fig. 3L and data not shown). No staining was seen when a damm sense probe was hybridized to salivary glands (Fig. 3K), midgut (data not shown), or egg chambers at various stages of development ( Fig.  3M and data not shown). Several other Drosophila caspases, including DCP-1 (15), DRONC (11), DECAY (12), and STRICA (13) are also expressed in egg chambers; however, the function of these caspases, except for DCP-1, in oocyte and nurse cell death remains unclear.
Caspase Activity of DAMM-To investigate the caspase activity of DAMM, we expressed full-length wild-type and C156G mutant DAMM fused to His 6 in E. coli. Lysates prepared from cultures induced with IPTG were tested for DAMM expression using an antibody against the 6xHis tag. Full-length DAMM

FIG. 5. Effects of DAMM protein expression in cultured cells.
A, effect of DAMM expression in transfected mammalian cells. Various expression constructs were cotransfected with pEF-␤gal into NIH3T3 cells by lipofection. At 48 h post-transfection, cells were fixed, stained with X-gal, and blue cells were observed for apoptotic morphology. Bars represent apoptotic cells as percent of total ␤-galactosidase-positive cells Ϯ S.E.M. At least 300 blue cells were scored for each dish. The data shown were derived from three independent experiments. B, effect of DAMM expression in Drosophila SL2 cells. SL2 cells were cotransfected with various caspase and inhibitor constructs in conjunction with pCASPERhs-lacZ. At 24 h post-transfection, cells were treated with heat shock, followed by CuSO 4 , to induce expression of the ␤-galactosidase and caspase/inhibitor constructs, respectively. 48 h later, cell loss because of apoptosis was calculated by counting the number of residual ␤-galactosidase positive cells. Bars represent the percent of ␤-galactosidase-positive cells in CuSO 4 -treated samples as compared with untreated samples. Values were normalized against vector alonetransfected samples (100%). The data shown were derived from three independent experiments. C, expression of DAMM and inhibitor proteins 48 h following CuSO 4 treatment. Protein expression of respective constructs was analyzed by Western blotting using antibodies to DAMM FLAG or Diap1HA, Diap2HA, P35HA. The data shown in C are derived from a single experiment. Data from multiple similar experiments indicated that all proteins were expressed at roughly equal levels, and all proteins had similar stability in transfected cells. Therefore, the variations in the intensity of bands in C are likely to be caused by the loading variations, rather than protein stability. was clearly detectable in E. coli lysates, however, processed fragments were not visible ( Fig. 4A; data not shown). The E. coli lysates containing DAMM were incubated with a variety of fluorogenic tetrapeptide caspase substrates to analyze substrate preference. DAMM did not show any activity on DEVDamc, but displayed substantial cleavage activity on VDVADamc compared with a control lysate expressing catalytically inactive DAMM C156G mutant (Fig. 4B). Because DAMM is more similar to caspase-6 than to any other known mammalian caspase, we tested whether DAMM can cleave the optimal caspase-6 tetrapeptide substrate, VEID-amc, and in vitro translated Nedd4 protein, which we have previously shown to be cleaved by caspase-6 (36). DAMM displayed no activity on either substrate (Fig. 4B and data not shown). Interestingly, DAMM showed small but significant cleavage activity on LEHD-amc and YVAD-afc, optimal substrates for caspase-4/ caspase-5, and caspase-1, respectively, and to a much lesser extent on IETD-amc, a caspase-9 substrate (Fig. 4).
Ectopic Expression of DAMM Induces Apoptosis in Cultured Cells-Many caspases, when overexpressed in cultured cells, induce apoptosis to some degree. We therefore analyzed whether DAMM is able to induce apoptosis in transfected cells. In NIH3T3 cells, at 48 h following transfection, around 10% of cells transfected with the wild-type damm construct showed apoptotic morphology when compared with cells transfected with the empty vector or an expression construct carrying the C156G mutant DAMM (Fig. 5A). A similar level of apoptosis has been observed when DECAY is ectopically expressed in NIH3T3 cells (12). We and others have previously demonstrated that caspases lacking a long prodomain are not as efficient at inducing apoptosis because of the fact that they may rely on the activity of upstream caspases for their initial activation (37,38). DAMM induced apoptosis in NIH3T3 cells was partially inhibited by the Drosophila inhibitor of apoptosis protein Diap1 as well as the baculovirus apoptosis inhibitor P35 and other inhibitors Bcl-2, CrmA, and MIHA. damm overexpression also induced a small degree of apoptosis in Drosophila SL2 cells (Fig. 5B). In these cells, damm was transfected under the control of a copper inducible promoter. 48 h following the induction of damm expression, ϳ10% of cells had under-gone apoptosis, as noted by their loss. Damm-induced apoptosis in these cells was inhibited by the Drosophila inhibitors Diap1 and Diap2 as well as by P35 (Fig. 5B). The expression of respective proteins was confirmed by Western blotting of lysates from transfected cells (Fig. 5C).
DAMM, DRONC, and DECAY Are Processed during Apoptosis of SL2 Cells-To analyze whether DAMM can be processed by various Drosophila caspases, in vitro translated DAMM was incubated with lysates expressing DAMM, DRONC, DECAY, DCP-1, or DRICE. In a similar manner to DECAY (12), no significant DAMM processing was evident by any of the caspases tested (data not shown). These results suggest that DAMM may require a caspase/protease, other than those tested here, for processing and activation.
We further analyzed the processing of DAMM in Drosophila SL2 cells undergoing apoptosis in response to cycloheximide treatment. In this study, we also analyzed the processing of DRONC, a key initiator caspase in the Drosophila cell death pathway (20), and DECAY, which, like DAMM, does not show any processing in vitro (12). We show that all three caspases are processed in SL2 transfectants in response to cycloheximide treatment (Fig. 6). As expected of an upstream initiator caspase, DRONC processing occurred early and by 8 h after cycloheximide treatment, DRONC precursor was completely cleaved to smaller fragments. The processing of DAMM and DECAY became apparent following that of DRONC, with processed products visible 16 h after the addition of cycloheximide to culture medium. Whereas a processed product of ϳ26 kDa was seen in both DAMM and DECAY transfectants, a 10 kDa product, possibly corresponding to the small subunit of DECAY, was visible only in DECAY-transfected cells. It is possible that processed DAMM subunits have a very short half-life as is the case with many mammalian caspases where mature subunits are often difficult to visualize by immunoblotting. Conversely, DAMM may not undergo full processing into two subunits for its activation.
DAMM Does Not Directly Associate With Diap1, Diap2, or P35-We and others have shown that Diap1 is able to inhibit several Drosophila caspases, including DRONC, DRICE, and DCP-1 (18 -20, 39 -41). To determine whether inhibition of DAMM-mediated cell death by Diap1 is because of a direct interaction between the two proteins, 293T cells were cotransfected with damm and diap1, and immunoprecipitation analysis was performed. As shown in Fig. 7, DAMM did not coprecipitate with Diap1, whereas in a control experiment DRONC was immunoprecipitated in a complex with Diap1, as has been previously shown (20). In a manner similar to DAMM, DRICE precursor does not bind Diap1 (41), whereas DRONC can directly interact with Diap1 in a CARD-dependent manner (18). DAMM also failed to interact with Diap2, which does not interact with any of the Drosophila caspases tested (41), except STRICA (13). The baculovirus protein P35, that is thought to inhibit caspases by binding to active caspases (42), did not coprecipitate with DAMM. This may be because of the fact that no significant processing of DAMM was seen in transiently tranfected cells.
High Level damm Expression Induces a Rough Eye Phenotype-To investigate the effects of ectopic damm expression in vivo, we generated transgenic flies that express FLAG-tagged damm under the control of the eye-specific GMR promoter (Fig.  8). Among the various lines examined, lines with a single insert, such as line H, showed a normal eye phenotype (Fig.  8D), whereas one line with multiple GMR-damm inserts (line F) showed a roughened eye phenotype (Fig. 8E). Although all damm lines expressed DAMM-FLAG protein (e.g. Fig. 8B), low levels of DAMM protein expression did not appear to have any effect on eye phenotype, suggesting that the rough eye phenotype in line F is because of higher levels of DAMM expression. Interestingly, the GMR-damm F phenotype was significantly enhanced as compared with controls, by ␥-irradiation (data not shown), suggesting that ectopic expression of damm sensitizes cells to apoptosis. All transgenic lines expressing the catalytically inactive C156G mutant DAMM protein showed normal eye phenotype (Fig. 8F), suggesting that the rough eye phenotype in GMR-damm F was dependent upon the caspase activity of DAMM. has been shown to suppress cell death mediated by the Drosophila death activators REAPER and HID in the transgenic fly eye model (18,19). To test whether DAMM plays a role in HID and REAPER-mediated cell death pathways, we crossed GMR-damm C156G flies with GMR-hid and GMR-rpr flies. As expected, flies containing one copy of GMR-hid or GMR-rpr showed a severely ablated eye phenotype (Fig. 8, G and I). Flies containing one copy each of GMR-hid and GMR-damm C156G showed a significant improvement of this ablated eye phenotype, and the eyes appeared larger and more structured than in GMR-hid/ϩ flies (Fig. 8H). Conversely, no significant suppression of the GMR-rpr ablated eye phenotype was seen in flies carrying single copies of GMR-rpr and GMR-damm C156G (Fig.  8J). These results suggest that DAMM may be required for HID, but not REAPER-mediated cell death. DISCUSSION We have described here the characterization of a new Drosophila caspase, DAMM. Low levels of damm transcript are widely expressed during Drosophila embryogenesis. Higher expression of damm mRNA in larval salivary glands and midgut suggests a possible role for DAMM in the programmed deletion of these larval tissues during larval/pupal metamorphosis. Additionally, high expression of damm mRNA in egg chambers suggests a possible role for DAMM in nurse cell death. Because high levels of damm transcript are also found in adult animals, DAMM may additionally be involved in regulating normal cell turnover in the adult.

Expression of Mutant damm Inhibits hid-induced Cell Death in the Fly
In mammals, initiator caspases carrying specific proteinprotein interaction domains are believed to be autoactivated by a proximity-induced model (1)(2)(3)(4). The downstream effector caspases on the other hand require processing by initiator caspases. Because DAMM lacks a long prodomain, we analyzed whether it could be processed by known Drosophila caspases in vitro. However, as these caspases were unable to process DAMM, and because DAMM expression in E. coli does not result in cleavage of the DAMM precursor, it is possible that DAMM either does not require proteolytic processing, or its full activation requires an as yet uncharacterized protease. In contrast to DAMM and DECAY, DRICE and DCP-1 are efficiently processed in vitro by the putative initiator fly caspase DRONC (18,19). It is possible that the recently discovered prodomain containing caspase, STRICA (13), may be involved in processing DAMM and DECAY. Interestingly, we observed processing of DAMM, along with DRONC and DECAY, in cycloheximide treated SL2 cells. However, the processing of DAMM resulted in a slightly smaller product than the DAMM precursor, which may be generated by the removal of a small amino-terminal peptide. This observation suggests that DAMM precursor may not be cleaved into two subunits. Most mammalian caspase precursors have some intrinsic caspase activity, and recent data suggest that processing is not always a prerequisite for caspase activation (43). It is thus possible that mechanisms, other than full processing, may be involved in the activation of DAMM.
Among several commercially available caspase substrates examined, recombinant DAMM produced in E. coli showed highest activity on pentapeptide VDVAD-amc. As there was little cleavage of a similar tetrapeptide substrate DEVD-afc, it appears that DAMM, like caspase-2 and DRONC (11,19,44), prefers a residue in the P 5 position of the substrate. DAMM also showed significant activity on LEHD-amc and YVAD-afc substrates. However, activity of DAMM on these substrates was 4 -5-fold lower when compared with its activity on VDVAD-amc, suggesting that among the peptides tested, VDVAD is the optimal substrate for DAMM. Interestingly, despite its overall similarity to caspase-6, DAMM had no significant activity on caspase-6 substrate VEID-amc.
In our coprecipitation experiments, DAMM did not interact with Diap1 and Diap2. Diap1 is known to interact with a number of Drosophila caspases including DRONC, DRICE, STRICA, and DCP-1 (13,18,19,41), whereas Diap2 has only been shown to interact with STRICA (13). The exact mechanisms by which insect IAPs interact with and inhibit caspases are not known, but recent data with mammalian XIAP show that it interacts with the processed forms of both effector caspases, such as caspase-3, -7, and the initiator caspase, caspase-9 (45)(46)(47)(48). By analogy, Diap1 is also likely to associate with and inhibit processed forms of caspases, although it has also been shown to interact with the prodomain region of DRONC (18). We believe that the lack of interaction between Diap1 and DAMM may be because of the fact that DAMM is not significantly processed in our transient overexpression experiments.
The strongest evidence for a role for DAMM in fly cell death comes from our genetic studies showing that the expression of a catalytically inactive DAMM mutant protein significantly suppresses the eye phenotype because of ectopic expression of HID. Interestingly, cell death because of the overexpression of REAPER (Fig. 8) or DRONC (data not shown) was not inhibited by the catalytically inactive DAMM, suggesting that DAMM may function only in specific cell death pathway(s). Generation of a loss-of-function damm mutant and genetic-interaction analysis with the use of such a mutant would shed further light on the role of DAMM in programmed cell death in Drosophila.