From the Hanson Centre for Cancer Research, Institute
of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia and the ¶ Peter MacCallum Cancer Institute, St.
Andrew's Place, Melbourne, Victoria 8006, Australia
Received for publication, October 17, 2000, and in revised form, April 13, 2001
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ABSTRACT |
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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 in
Escherichia 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 of damm 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 (1-4). A number of caspases have been described in both
vertebrates and invertebrates. To date 14 caspases have been cloned in
mammals (1-4). 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
(7-13). In addition to the already described caspases, analysis of the
Drosophila genomic database predicts one more caspase, which
we have termed DAMM1
(GenBankTM 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.
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 5'-end of the
damm open-reading frame was confirmed using 5'-rapid
amplification of cDNA ends (RACE) (Life Technologies). The cDNA
sequence of damm has been deposited in the
GenBankTM 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.
Plasmid Constructs--
damm product amplified from
the Drosophila larval cDNA library was purified and
cloned into pGEM®-T Easy (Promega). The 765-base pair
coding region of damm was amplified from
pGEM®-T Easy-damm using Pwo
polymerase (Roche Molecular Biochemicals) and was cloned directionally
into pcDNA3 (Invitrogen) using the following oligonucleotides:
Primer A, 5'-ATGTATCTGCCCGAAAGAAC and Primer B,
5'-GCTCTAGATCACTTGTCATCGTCGTCCTTGTAGTCAGTGTTTTTAGCATAATTTCC. Primer B contained an XbaI site (italics) and sequence
encoding a FLAG tag (underlined). The catalytic Cys156
residue of DAMM was mutated to a Gly residue by QuikChange mutagenesis (Stratagene) using pcDNA3-dammFLAG as a template.
dammFLAG and damm(C156G)FLAG were subcloned into
pRmHa.3 (22) and pGMR (23). Primer C,
5'-GGAATTCCATATGTATCTGCCCGAAAGAAC containing a
NdeI site (denoted in italics) and primer D,
5'-CGCGGATCCCGAGTGTTTTTAGCATAATTTCC containing a
BamHI site (denoted in italics) were used to amplify wild-type and catalytic Cys mutant damm for directional
cloning into the pET32b vector (Novagen). Apoptosis inhibitor cDNAs
diap1, diap2, and p35 were amplified
using Pwo polymerase (Roche Molecular Biochemicals) and the
following primers: DIAP1A,
5'-CGGAATTCATGGCATCTGTTGTAGCTGATC; DIAP1B,
5'-CGCGGATCCGCGTCATGCGTAGTCTGGCACGTCGTATGGGTAAGAAAAATATACGCGCATC; DIAP2A, 5'-CGGAATTCATGACGGAGCTGGGCATG; DIAP2B,
5'-CGCGGATCCGCGTCATGCGTAGTCTGGCACGTCGTATGGGTAATCGATTTGCTTAACTGC; P35A, 5'-CGGAATTCATGTGTGTAATTTTTCCG; P35B,
5'-CGCGGATCCGCGTCATGCGTAGTCTGGCACGTCGTATGGGTATTTAATCATGTCTAATATTAC. In each case the forward primers contained an EcoRI site (in
italics), whereas the reverse primers contained a BamHI site
(in italics) and sequence encoding an HA tag (underlined).
diap1HA, diap2HA, and p35HA were
cloned into pRmHa.3 for expression in insect cells and into pcDNA3
for expression in mammalian cells.
Recombinant Caspases and Caspase Assays--
Recombinant DAMM
protein was generated by transformation of E. coli BL21
(DE3) cells with DAMM-His6 constructs in pET32b. pET-damm and pET-dammC156G 6-h
cultures were subcultured 1 in 25 and grown at 22 °C for 3 h.
Cultures were induced with 1 mM
isopropyl-1-thio- In Vitro Caspase Cleavage Assays--
Full-length
damm in pcDNA3 was used as a template for the production
of [35S]methionine-labeled protein using a coupled
transcription/translation kit (Promega). 5 µl 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.
Transient Transfection--
NIH3T3 cells were maintained in
Dulbecco's modified Eagle's medium with 10% fetal calf serum. For
cell death assays, 2 × 105 cells were plated per
35-mm dish the day before transfection. 2 µg of plasmid DNA
comprising either 2 µg of empty vector or 1 µg of
pcDNA3-damm or pcDNA3-damm (C156G)
together with 1 µg of empty vector, pcDNA3-diap1Myc,
pcDNA3-p35HA, pRSV-bcl2,
pcDNA3-crmA, or pcDNA3-mihA, was
cotransfected with 0.5 µg of a
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 × 106 SL2 cells were
cotransfected with either 2 µg of vector or 1 µg of
pRM-damm together with 1 µg of respective pRmHa.3
inhibitor constructs, pRM-diap1HA, pRM-diap2HA,
or pRM-p35HA and 0.5 µg of pCASPERhs- 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 CuSO4 at a final concentration of 0.7 mM. 24 h following copper induction, transfectants
were treated with cycloheximide at a final concentration of 25 µg/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 DAMM Interaction Studies--
293T cells were transfected using
Fugene6. 1.5 µg of pcDNA3-dammFLAG was
cotransfected with either 1.5 µg of pcDNA3 vector or 1.5 µg of
pcDNA3-Mycdiap1, pcDNA3-diap2HA, or
pcDNA3-p35HA. As a positive control,
pcDNA3-droncFLAG was cotransfected with pcDNA3-Mycdiap1 (20). 24 h post-transfection, cell
lysates were prepared in lysis buffer (150 mM NaCl, 1%
Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl
pH 7.5, supplemented with a 1× protease inhibitor mixture
(CompleteTM, Roche Molecular Biochemicals). Lysates were
immunoprecipitated with 2 µg of either an isotype-matched negative
control antibody 3D3, or 2 µg of mouse monoclonal 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 µg 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 32P-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
manufacturer'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 dammC156G 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 GMR-dammC156G 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).
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
5'-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 ~29% 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 ~0.9-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 throughout 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 embryos (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 His6 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 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
DEVD-amc, but displayed substantial cleavage activity on VDVAD-amc
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, ~10% of cells had undergone
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 ~26 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 Expression of Mutant damm Inhibits hid-induced Cell Death in the
Fly Eye--
Catalytically inactive mutants of some
caspases have been shown to act as dominant negative
molecules. For example, catalytically inactive mutant of DRONC 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-dammC156G 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-dammC156G showed a significant improvement
of this ablated eye phenotype, and the eyes appeared larger and more
structured than in GMR-hid/+ 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-dammC156G (Fig.
8J). These results suggest that DAMM may be required for HID, but not REAPER-mediated cell death.
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.
In mammals, initiator caspases carrying specific protein-protein
interaction domains are believed to be autoactivated by a proximity-induced model (1-4). 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 P5
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
(45-48). 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside and grown for a
further 4 h. Cells were pelleted and lysed by sonication in assay
buffer (25 mM HEPES, pH 7.0, 1 mM EDTA, 10% sucrose, 0.1% CHAPS, 5 mM dithiothreitol). Cleared
E. coli lysates from cells expressing DAMM were assayed for
protein concentration, and an equal amount of total protein from each
lysate was incubated with 100 µM fluorogenic peptide
substrates and assayed for caspase activity as described previously
(24, 25). Recombinant DRONC, DRICE, DCP-1, DREDD, and DECAY were
prepared as described previously (12, 26). Caspase substrates
VEID-amino-methylcoumarin (-amc), DEVD-amino-trifluoromethylcoumarin
(-afc), and YVAD-afc were from Bachem. VDVAD-amc was purchased from
California Peptide Research Inc. LEHD-amc and IETD-amc were from Alexis
Biochemicals. Expression of His6-tagged caspases in
E. coli lysates was determined by immunoblotting using an
6×His antibody (Roche Molecular Biochemicals).
-galactosidase expression plasmid
(pEF-
gal) (27). All transfections in mammalian cells were carried
out using Fugene6 transfection reagent (Roche Molecular Biochemicals)
according to manufacturer's instructions. Cells were fixed and stained
with X-gal at 24 or 48 h post-transfection, and
-galactosidase-positive cells were scored for apoptotic morphology as previously described (27).
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 CuSO4 at a
final concentration of 0.7 mM. Cells were fixed and stained
with X-gal 48 h post-CuSO4 induction as previously
described (29). Cell survival was calculated as the percent of
transfected (
-galactosidase-positive) cells in the
CuSO4-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 ± S.E.M., were
derived from three independent experiments. To check copper-induced
protein expression after 48 h of CuSO4 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.).
Dronc (20) or the
FLAG antibody.
FLAG antibody,
the rat monoclonal
HA antibody, or a mouse monoclonal
Myc
antibody (Roche Molecular Biochemicals). Immunoprecipitated proteins
were separated by SDS-PAGE and analyzed by immunoblotting using the
above mentioned antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (50K):
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Fig. 1.
DAMM sequence and its relationship to
Drosophila caspases. A, genomic
organization of the damm gene. The non-coding regions of
exons 1 and 5 are shown as open boxes. The 5' boundary of
exon 1 and 3' boundary of exon 5 have not been characterized.
B, an amino acid sequence alignment of the seven
Drosophila caspases. The deduced amino acid sequence of DAMM
consists of 255 amino acid residues. Alignments were obtained using
ClustalW program. Residues conserved in at least six caspases are shown
in black boxes. Similar residues in at least five caspases
or those identical in four caspases are shown in gray boxes.
C, phylogenetic relationship between various
Drosophila and mammalian caspases.
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Fig. 2.
Expression of damm mRNA
during Drosophila development. Approximately 20 µg of total RNA isolated from various developmental stages and from
adult flies was analyzed by Northern blotting using a probe
encompassing nucleotides 1-690 of the damm open-reading
frame. damm transcript is present as a single, 0.9-kilobase
band. The lower panel depicts the corresponding ribosomal
RNA bands in each sample as visualized by ethidium bromide staining of
the gel prior to transfer to membrane.
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Fig. 3.
In situ mRNA analysis of
damm expression during Drosophila
development. damm mRNA was detected by
in situ hybridization with a digoxigenin-labeled antisense
mRNA probe. A, a stage 5 embryo showing a uniform level
of damm expression. B, a stage 10/11 embryo.
C, a stage 14 embryo showing damm expression in
specific tissues. CNS, central nervous system;
SG, salivary gland; HG, hindgut. D and
E, the higher magnification of the panels shown in
C. Arrowheads indicate examples of specific cells
showing damm expression. F, a stage 17 embryo.
SG, salivary gland; HG, hindgut; MG,
midgut. G, a stage 14 embryo hybridized with a control
damm sense probe showing no staining. All embryos are
oriented with anterior to the left. H, the brain
lobes (BL) from third instar larvae showing low
damm expression. VG, ventral ganglion.
I, a third instar midgut (MG) displaying slightly
higher damm expression than seen in brain lobes.
GC, gastric caeca; PV, proventriculus.
J, a third instar salivary gland showing a high level of
damm expression. K, a third instar salivary gland
hybridized with a control damm sense probe showing no
staining. Panels J and K are oriented with the
salivary gland duct toward the top left hand corner.
L, a stage 10B adult egg chamber showing high level
damm expression in both the nurse cells (NC) and
the oocyte (OC). M, a stage 10B adult egg chamber
hybridized to control damm sense probe exhibiting no
staining. Panels L and M are oriented with the
oocyte to the right.
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Fig. 4.
Enzymatic activity of recombinant DAMM on
fluorogenic peptide substrates. A, recombinant DAMM was
expressed in E. coli as His6-tagged protein, and
protein expression detected by immunoblotting of bacterial lysates
using an His6 tag antibody. Note that following
induction with isopropyl-1-thio-
-D-galactopyranoside,
both DAMM and DAMM C156G mutant proteins were expressed at similar
levels. All lanes contain ~10 µg of E. coli lysate.
B, equivalent amount of E. coli lysates
containing recombinant DAMM or DAMM C156G were incubated with various
fluorogenic caspase substrates at 37 °C for 30 min and release of
-amc and -afc was monitored by a fluorimeter. Data (±S.E.M.) are
derived from two separate experiments performed in triplicate.
View larger version (31K):
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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 ± 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
CuSO4, 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 CuSO4-treated
samples as compared with untreated samples. Values were normalized
against vector alone-transfected samples (100%). The data shown were
derived from three independent experiments. C,
expression of DAMM and inhibitor proteins 48 h following
CuSO4 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.
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Fig. 6.
DRONC, DAMM, and DECAY are processed in SL2
transfectants in response to cycloheximide treatment. SL2 cells
were transfected with pRM-dronc, pRM-dammFLAG, or
pRMdecay-FLAG and were induced to express respective
constructs 24 h post-transfection by the addition of
CuSO4 as described under "Experimental Procedures." At
the indicated times post-treatment with 25 µg/ml cycloheximide, cells
were harvested and analyzed by SDS-PAGE and Western blotting using
DRONC or
FLAG antibodies.
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Fig. 7.
DAMM interaction studies in transfected
cells. Immunoprecipitation analysis of DAMM and inhibitors in
transfected 293T cells was performed as described under "Experimental
Procedures." The antibodies used in each immunoprecipitation are
denoted as F, FLAG; M,
MYC; and
H,
HA. In each panel, the position of the IgG
heavy (IgGH) and light (IgGL) chains are
indicated. In panel C, asterisks indicate the
position of the IgG(H) chain in
HA and
-Myc blots. In these
samples, the IgG(H) band runs just below the indicated DRONC and Diap1
bands, with the exception of the far right lane. In this
instance, only the Diap1 band is visible, because the rat IgG(H) band
for
HA is not recognized by the mouse
Myc antibody (see
first panel, vector-transfected control IP). In
B the slower migrating band in the right panel,
indicated by the
symbol, represents a nonspecific cross-reacting
protein.
-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.
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Fig. 8.
GMR-damm flies exhibit a rough eye
phenotype and the GMR-hid eye phenotype is suppressed by
catalytically inactive DAMM. Photographs of wild-type and
transgenic GMR-damm fly eyes and analysis of DAMM-FLAG
transgene expression in larval eye discs. 3rd instar larval eye disc
from wild type (A) and GMR-damm H (B)
flies immunostained with an anti-FLAG antibody. Eye discs are
orientated with posterior upwards. Arrowheads in
A and B indicate morphogenetic furrow. The
GMR enhancer drives expression of DAMM-FLAG in cells
posterior to the morphogenetic furrow, which show staining with the
anti-FLAG antibody. Examples of positive-staining cells are indicated
by arrows. C-J represent light microscopic
pictures of the wild-type and various transgenic fly eyes. Eyes are
oriented with posterior to the left. C, wild type
(Canton-S); D, GMR-damm H; E,
GMR-damm F. Note that GMR-damm line F
(E) shows a rough eye phenotype compared with wild-type
(C), whereas GMR-damm line H (D) does
not. F, GMR- damm
C163G/+; G,
GMR-hid/+; H, GMR-hid/+ GMR-damm
C163G/+; I, GMR-rpr/+;
J, GMR-rpr/+ GMR-damm
C163G/+. Note that GMR- damm
C163G suppresses the GMR-hid but not the
GMR-rpr ablated eye phenotype.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Bruce Hay, Tatsushi Igaki, and the Bloomington Stock Center for reagents and Paul Colussi for helpful discussions.
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FOOTNOTES |
---|
* This work was supported in part by funds from the Wellcome Trust, Anti-Cancer Foundation of South Australia, and the National Health and Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF240763.
§ Supported by an Anti-Cancer Foundation Research Associateship.
Wellcome Trust Senior Fellow in Medical Science.
** To whom correspondence should be addressed: Hanson Center for Cancer Research, IMVS, P.O. Box 14, Rundle Mall, Adelaide, SA 5000, Australia. Tel.: 61-8-8222-3738; Fax: 61-8-8222-3139; E-mail: sharad.kumar@imvs.sa.gov.au.
Published, JBC Papers in Press, May 3, 2001, DOI 10.1074/jbc.M009444200
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ABBREVIATIONS |
---|
The abbreviations used are: DAMM, death-associated molecule related to Mch2; CARD, caspase recruitment domain; DED, death effector domain; Diap, Drosophila inhibitor of apoptosis; HA, hemagglutinin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; afc, amino-trifluoromethylcoumarin; amc, amino-methylcoumarin.
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REFERENCES |
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