Characterization of the DrosophilaCaspase, DAMM
2001; Elsevier BV; Volume: 276; Issue: 27 Linguagem: Inglês
10.1074/jbc.m009444200
ISSN1083-351X
AutoresNatasha L. Harvey, Tasman Daish, Kathryn Mills, Loretta Dorstyn, Leonie M. Quinn, Stuart H. Read, Helena E. Richardson, Sharad Kumar,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoCaspases 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.AF240763 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.AF240763 death-associated molecule related to Mch2 caspase recruitment domain death effector domain Drosophila inhibitor of apoptosis hemagglutinin 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid polymerase chain reaction rapid amplification of cDNA ends amino-trifluoromethylcoumarin amino-methylcoumarin Programmed cell death in metazoans is mediated by caspases, a family of cysteine proteases, which cleave their substrates following an Asp residue (1Nicholson D.W. Cell Death Diff. 1999; 6: 1551-1570Crossref Scopus (1297) Google Scholar, 2Kumar S. Cell Death Diff. 1999; 6: 1060-1066Crossref PubMed Scopus (183) Google Scholar, 3Cryns V. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1161) Google Scholar, 4Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). A number of caspases have been described in both vertebrates and invertebrates. To date 14 caspases have been cloned in mammals (1Nicholson D.W. Cell Death Diff. 1999; 6: 1551-1570Crossref Scopus (1297) Google Scholar, 2Kumar S. Cell Death Diff. 1999; 6: 1060-1066Crossref PubMed Scopus (183) Google Scholar, 3Cryns V. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1161) Google Scholar, 4Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). 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. 5Zheng T.S. Hunot S. Kuida K. Flavell R.A. Cell Death Diff. 1999; 6: 1043-1053Crossref PubMed Scopus (251) Google Scholar). 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 (6Shaham S. J. Biol. Chem. 1998; 273: 35109-35117Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). InDrosophila melanogaster six caspases, named DCP-1, DREDD/DCP-2, DRICE, DRONC, DECAY, and STRICA have been cloned so far (7Song Z. McCall K. Steller H. Science. 1997; 275: 536-540Crossref PubMed Scopus (249) Google Scholar, 8Chen P. Rodriguez A. Erskine R. Thach T. Abrams J.M. Dev. Biol. 1998; 201: 202-216Crossref PubMed Scopus (182) Google Scholar, 9Inohara N. Koseki T. Hu Y. Chen S. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10717-10722Crossref PubMed Scopus (278) Google Scholar, 10Fraser A.G. Evan G.I. EMBO J. 1997; 16: 2805-2813Crossref PubMed Scopus (171) Google Scholar, 11Dorstyn L. Colussi P. Quinn L.M. Richardson H. Kumar S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4307-4312Crossref PubMed Scopus (237) Google Scholar, 12Dorstyn L. Read S.H. Quinn L.M. Richardson H. Kumar S. J. Biol. Chem. 1999; 274: 30778-30783Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 13Doumanis J. Quinn L.M. Richardson H. Kumar S. Cell Death Diff. 2001; 8: 387-394Crossref PubMed Scopus (67) Google Scholar). In addition to the already described caspases, analysis of theDrosophila genomic database predicts one more caspase, which we have termed DAMM1(GenBankTM accession number AF240763; death-associated molecule related toMch2) (14Kumar S Doumanis J. Cell Death Diff. 2000; 7: 1039-1044Crossref PubMed Scopus (129) Google Scholar). 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. 14Kumar S Doumanis J. Cell Death Diff. 2000; 7: 1039-1044Crossref PubMed Scopus (129) Google Scholar). STRICA also has a long prodomain, but it lacks any CARD or DED structures (13Doumanis J. Quinn L.M. Richardson H. Kumar S. Cell Death Diff. 2001; 8: 387-394Crossref PubMed Scopus (67) Google Scholar). 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 (7Song Z. McCall K. Steller H. Science. 1997; 275: 536-540Crossref PubMed Scopus (249) Google Scholar). Additionally, DCP-1 is required for Drosophila oogenesis, as dcp-1mutants show a defect in transfer of nurse cell cytoplasmic contents to developing oocytes (15McCall K. Steller H. Science. 1998; 279: 230-234Crossref PubMed Scopus (143) Google Scholar). The transcript for dreddaccumulates in embryonic cells undergoing programmed cell death and in nurse cells in the ovary at a time that coincides with nurse cell death (8Chen P. Rodriguez A. Erskine R. Thach T. Abrams J.M. Dev. Biol. 1998; 201: 202-216Crossref PubMed Scopus (182) Google Scholar). 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 (16Elrod-Erickson M. Misra S. Schneider D. Curr. Biol. 2000; 10: 781-784Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 17Leulier F. Rodriguez A. Khush R.S. Abrams J.M. Lemaitre B. EMBO Rep. 2000; 1: 353-358Crossref PubMed Scopus (312) Google Scholar). 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 (11Dorstyn L. Colussi P. Quinn L.M. Richardson H. Kumar S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4307-4312Crossref PubMed Scopus (237) Google Scholar). Heterozygosity at thedronc 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 (18Meier P. Silke J. Leevers S.J. Evan G.I. EMBO J. 2000; 19: 598-611Crossref PubMed Scopus (273) Google Scholar, 19Hawkins C.J. Yoo S.J. Peterson E.P. Wang S.L. Vernooy S.Y. Hay B.A. J. Biol. Chem. 2000; 275: 27084-27093Abstract Full Text Full Text PDF PubMed Google Scholar, 20Quinn L.M. Dorstyn L. Mills K. Colussi P.A. Chen P. Coombe M. Abrams J. Kumar S Richardson H. J. Biol. Chem. 2000; 275: 40416-40424Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). DRONC also interacts, both biochemically and genetically, with the CED-4/Apaf-1 fly homolog, Dark (20Quinn L.M. Dorstyn L. Mills K. Colussi P.A. Chen P. Coombe M. Abrams J. Kumar S Richardson H. J. Biol. Chem. 2000; 275: 40416-40424Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Furthermore, loss of DRONC function in earlyDrosophila embryos because of dronc RNA ablation results in a decrease in apoptosis, indicating that DRONC is required for programmed cell death during embryogenesis (20Quinn L.M. Dorstyn L. Mills K. Colussi P.A. Chen P. Coombe M. Abrams J. Kumar S Richardson H. J. Biol. Chem. 2000; 275: 40416-40424Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). 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 (21Fraser A.G. McCarthy N.J. Evan G.I. EMBO J. 1997; 16: 6192-6199Crossref PubMed Scopus (125) Google Scholar). Similar to dronc, decay expression is high in larval midgut and salivary glands, but decayexpression is not regulated by ecdysone (12Dorstyn L. Read S.H. Quinn L.M. Richardson H. Kumar S. J. Biol. Chem. 1999; 274: 30778-30783Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Accumulation ofdronc 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. 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 aDrosophila larval cDNA library. The 5′-end of thedamm 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. 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 Pwopolymerase (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 Cys156residue 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 (22Bunch T.A. Grinblat Y. Goldstein L.S. Nucleic Acids Res. 1988; 16: 1043-1061Crossref PubMed Scopus (367) Google Scholar) and pGMR (23Hay B.A. Wolff T. Rubin G.M. Development. 1994; 120: 2121-2129Crossref PubMed Google Scholar). Primer C, 5′-GGAATTCCATATGTATCTGCCCGAAAGAAC containing aNdeI site (denoted in italics) and primer D, 5′-CGCGGATCCCGAGTGTTTTTAGCATAATTTCC containing aBamHI 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 cDNAsdiap1, 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 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 mmisopropyl-1-thio-β-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). ClearedE. 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 (24Harvey N.L. Butt A.J. Kumar S. J. Biol. Chem. 1997; 272: 13134-13139Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 25Butt A.J. Harvey N.L. Parasivam G. Kumar S. J. Biol. Chem. 1998; 273: 6763-6768Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Recombinant DRONC, DRICE, DCP-1, DREDD, and DECAY were prepared as described previously (12Dorstyn L. Read S.H. Quinn L.M. Richardson H. Kumar S. J. Biol. Chem. 1999; 274: 30778-30783Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 26Kanuka H. Hisahara S. Sawamoto K. Shoji S. Okano H Miura M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 145-150Crossref PubMed Scopus (39) Google Scholar). 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 inE. coli lysates was determined by immunoblotting using an α6×His antibody (Roche Molecular Biochemicals). Full-lengthdamm 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. 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 β-galactosidase expression plasmid (pEF-βgal) (27Kumar S. Kinoshita M. Noda M. Copeland N.G. Jenkins N.A. Genes Dev. 1994; 8: 1613-1626Crossref PubMed Scopus (585) Google Scholar). 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 β-galactosidase-positive cells were scored for apoptotic morphology as previously described (27Kumar S. Kinoshita M. Noda M. Copeland N.G. Jenkins N.A. Genes Dev. 1994; 8: 1613-1626Crossref PubMed Scopus (585) Google Scholar). Cell death assays in insect cells were carried out using Schneider L2 (SL2) cells as previously described (28Chen P. Lee P. Otto L Abrams J.M. J. Biol. Chem. 1996; 271: 25735-25737Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 29Colussi P.A. Quinn L.M. Huang D.C.S. Coombe M. Read S.H. Richardson H. Kumar S. J. Cell Biol. 2000; 148: 703-710Crossref PubMed Scopus (136) Google Scholar). SL2 cells were maintained and transfected using Cellfectin (Life Technology) as described (8Chen P. Rodriguez A. Erskine R. Thach T. Abrams J.M. Dev. Biol. 1998; 201: 202-216Crossref PubMed Scopus (182) Google Scholar). 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-βgalreporter. All death assays were performed in duplicate. 24 h post-transfection, cells were induced to express pCASPERhs-βgal 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 (29Colussi P.A. Quinn L.M. Huang D.C.S. Coombe M. Read S.H. Richardson H. Kumar S. J. Cell Biol. 2000; 148: 703-710Crossref PubMed Scopus (136) Google Scholar). Cell survival was calculated as the percent of transfected (β-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 αHA antibody (Roche Molecular Biochemicals) or a mouse monoclonal αFLAG antibody (Sigma Chemical Co.). 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 αDronc (20Quinn L.M. Dorstyn L. Mills K. Colussi P.A. Chen P. Coombe M. Abrams J. Kumar S Richardson H. J. Biol. Chem. 2000; 275: 40416-40424Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) or the αFLAG antibody. 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 (20Quinn L.M. Dorstyn L. Mills K. Colussi P.A. Chen P. Coombe M. Abrams J. Kumar S Richardson H. J. Biol. Chem. 2000; 275: 40416-40424Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). 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 (Complete™, Roche Molecular Biochemicals). Lysates were immunoprecipitated with 2 μg of either an isotype-matched negative control antibody 3D3, or 2 μg of mouse monoclonal αFLAG antibody, the rat monoclonal αHA antibody, or a mouse monoclonal αMyc antibody (Roche Molecular Biochemicals). Immunoprecipitated proteins were separated by SDS-PAGE and analyzed by immunoblotting using the above mentioned antibodies. 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 a32P-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 dammcDNA 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 (12Dorstyn L. Read S.H. Quinn L.M. Richardson H. Kumar S. J. Biol. Chem. 1999; 274: 30778-30783Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 29Colussi P.A. Quinn L.M. Huang D.C.S. Coombe M. Read S.H. Richardson H. Kumar S. J. Cell Biol. 2000; 148: 703-710Crossref PubMed Scopus (136) Google Scholar, 30Lehner C.F. O'Farrell P.H. Cell. 1989; 56: 957-968Abstract Full Text PDF PubMed Scopus (284) Google Scholar). Wild-typedamm or dammC156G mutant cDNA, tagged with FLAG was cloned into the pGMR vector and transgenic flies were generated and maintained as previously described (31Richardson H. O'Keefe L.V. Marty T. Saint R. Development. 1995; 121: 3371-3379PubMed Google Scholar). For testing the interaction of GMR-dammC156G withGMR-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 (31Richardson H. O'Keefe L.V. Marty T. Saint R. Development. 1995; 121: 3371-3379PubMed Google Scholar). 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, fordeath-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 fordamm 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 theDrosophila caspases, DAMM is most homologous to the newly identified caspase STRICA (Fig. 1C) (13Doumanis J. Quinn L.M. Richardson H. Kumar S. Cell Death Diff. 2001; 8: 387-394Crossref PubMed Scopus (67) Google Scholar), 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 otherDrosophila 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. 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 ofdamm 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 inDrosophila (32Abrams J.M. White K. Fessler L.I. Steller H. Development. 1993; 117: 29-43Crossref PubMed Google Scholar). 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 (33Edgar B.A. Schubiger G. Cell. 1986; 44: 871-877Abstract Full Text PDF PubMed Scopus (272) Google Scholar). In stage 5 cellularized embyros, damm mRNA is ubiquitously expressed (Fig.3A), but in later stages higher levels of dammtranscript 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 adamm sense probe was hybridized to embryos at various stages of development (Fig. 3G and data not shown).Figure 3In situmRNA analysis ofdamm expression during Drosophiladevelopment.damm mRNA was detected byin 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 andE, the higher magnification of the panels shown inC. 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 controldamm sense probe showing no staining. All embryos are oriented with anterior to the left. H, the brain lobes (BL) from third instar larvae showing lowdamm 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 ofdamm 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 leveldamm 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.View Large Image Figure ViewerDownload (PPT) We also examined the expression of damm in third instar larval tissues and during oogenesis. Low levels of dammexpression were observed in brain lobes (Fig. 3H), which contain apoptotic cells at this stage (34Wolff T. Ready D.F. Development. 1991; 113: 825-839PubMed Google Scholar). A relatively high level ofdamm 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 (35Jiang C. Baehrecke E.H. Thummel C.S. Development. 1997; 124: 4673-4683Crossref PubMed Google Scholar). 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 adamm 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 (15McCall K. Steller H. Science. 1998; 279: 230-234Crossref PubMed Scopus (143) Google Scholar), DRONC (11Dorstyn L. Colussi P. Quinn L.M. Richardson H. Kumar S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4307-4312Crossref PubMed Scopus (237) Google Scholar), DECAY (12Dorstyn L. Read S.H. Quinn L.M. Richardson H. Kumar S. J. Biol. Chem. 1999; 274: 30778-30783Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), and STRICA (13Doumanis J. Quinn L.M. Richardson H. Kumar S. Cell Death Diff. 2001; 8: 387-394Crossref PubMed Scopus (67) Google Scholar) are also expressed in egg chambers; however, the function of these caspases, except for DCP-1, in oocyte and nurse cell death remains unclear. 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 displ
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