The Drosophila Caspase DRONC Cleaves following Glutamate or Aspartate and Is Regulated by DIAP1, HID, and GRIM
2000; Elsevier BV; Volume: 275; Issue: 35 Linguagem: Inglês
10.1016/s0021-9258(19)61483-3
ISSN1083-351X
AutoresChristine J. Hawkins, Soon Ji Yoo, Erin P. Peterson, Susan L. Wang, Stephanie Y. Vernooy, Bruce A. Hay,
Tópico(s)Insect Resistance and Genetics
ResumoThe caspase family of cysteine proteases plays important roles in bringing about apoptotic cell death. All caspases studied to date cleave substrates COOH-terminal to an aspartate. Here we show that the Drosophila caspase DRONC cleaves COOH-terminal to glutamate as well as aspartate. DRONC autoprocesses itself following a glutamate residue, but processes a second caspase, drICE, following an aspartate. DRONC prefers tetrapeptide substrates in which aliphatic amino acids are present at the P2 position, and the P1 residue can be either aspartate or glutamate. Expression of a dominant negative form of DRONC blocks cell death induced by theDrosophila cell death activators reaper,hid, and grim, and DRONC overexpression in flies promotes cell death. Furthermore, the Drosophila cell death inhibitor DIAP1 inhibits DRONC activity in yeast, and DIAP1's ability to inhibit DRONC-dependent yeast cell death is suppressed by HID and GRIM. These observations suggest that DRONC acts to promote cell death. However, DRONC activity is not suppressed by the caspase inhibitor and cell death suppressor baculovirus p35. We discuss possible models for DRONC function as a cell death inhibitor. The caspase family of cysteine proteases plays important roles in bringing about apoptotic cell death. All caspases studied to date cleave substrates COOH-terminal to an aspartate. Here we show that the Drosophila caspase DRONC cleaves COOH-terminal to glutamate as well as aspartate. DRONC autoprocesses itself following a glutamate residue, but processes a second caspase, drICE, following an aspartate. DRONC prefers tetrapeptide substrates in which aliphatic amino acids are present at the P2 position, and the P1 residue can be either aspartate or glutamate. Expression of a dominant negative form of DRONC blocks cell death induced by theDrosophila cell death activators reaper,hid, and grim, and DRONC overexpression in flies promotes cell death. Furthermore, the Drosophila cell death inhibitor DIAP1 inhibits DRONC activity in yeast, and DIAP1's ability to inhibit DRONC-dependent yeast cell death is suppressed by HID and GRIM. These observations suggest that DRONC acts to promote cell death. However, DRONC activity is not suppressed by the caspase inhibitor and cell death suppressor baculovirus p35. We discuss possible models for DRONC function as a cell death inhibitor. polyacrylamide gel electrophoresis poly(ADP-ribosyl)transferase Programmed cell death, or apoptosis, is a process by which organisms remove unwanted or damaged cells during development and in the adult (reviewed in Ref. 1Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2403) Google Scholar). Central components of this process are a family of cysteine proteases known as caspases (2Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G.S. Thornberry N.A. Wong W.W. Yuan J. Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2139) Google Scholar). Caspases are translated as inactive precursors that are cleaved to generate proteolytically active enzymes. Caspase processing involves one or more cleavages COOH-terminal to the active site cysteine to produce large and small subunits. An NH2-terminal prodomain is also often removed. Studies of the crystal structures of caspases show that large and small subunits from two precursor molecules assemble to form an active heterotetramer (3Walker N.P.C. Talanian R.V. Brady K.D. Dang L.C. Bump N.J. Ferenz C.R. Franklin S. Ghayur T. Hackett M.C. Hammill L.D. et al.Cell. 1994; 78: 343-352Abstract Full Text PDF PubMed Scopus (527) Google Scholar, 4Wilson K.P. Black J.A.F. Thomson J.A. Kim E.E. Griffith J.P. Navia M.A. Murcko M.A. Chambers S.P. Aldape R.A. Raybuck S.A. Livingston D.J. Nature. 1994; 370: 270-275Crossref PubMed Scopus (754) Google Scholar, 5Rotonda J. Nicholson D.W. Fazil K.M. Gallant M. Gareau Y. Labelle M. Peterson E.P. Rasper D.M. Ruel R. Vaillancourt J.P. Thornberry N.A. Becker J.W. Nat. Struct. Biol. 1996; 3: 619-625Crossref PubMed Scopus (401) Google Scholar, 6Mittl P.R. Di Marco S. Krebs J.F. Bai X. Karanewsky D.S. Priestle J.P. Tomaselli K.J. Grutter M.G. J. Biol. Chem. 1997; 272: 6539-6547Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Caspases described to date all cleave their substrates following aspartate residues (7Sleath P.R. Hendrickson R.C. Kronheim S.R. March C.J. Black R.A. J. Biol. Chem. 1990; 265: 14526-14528Abstract Full Text PDF PubMed Google Scholar, 8Howard A.D. Kostura M.J. Thornberry N. Ding G.J. Limjuco G. Weidner J. Salley J.P. Hogquist K.A. Chaplin D.D. Mumford R.A. Schmidt J.A. Tocci M.J. J. Immunol. 1991; 147: 2964-2969PubMed Google Scholar, 9Thornberry N.A. Bull H.G. Calaycay J.R. Chapman K.T. Howard A.D. Kostura M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. et al.Nature. 1992; 356: 768-774Crossref PubMed Scopus (2199) Google Scholar, 10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1844) Google Scholar, 11Talanian R.V. Quinlan C. Trautz S. Hackett M.C. Mankovich J.A. Banach D. Ghayur T. Brady K.D. Wong W.W. J. Biol. Chem. 1997; 272: 9677-9682Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar). Importantly, the sites at which caspase zymogens are cleaved to generate active tetramers often correspond to consensus caspase target sites. This has suggested that caspases can function in a cascade in which initiator caspases, activated by upstream death signals, cleave and activate a set of executioner caspases that carry out proteolytic cleavages of cellular proteins (12Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1936) Google Scholar, 13Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4124) Google Scholar). Seven caspases, DCP-1 (14Song Z. McCall K. Steller H. Science. 1997; 275: 536-540Crossref PubMed Scopus (251) Google Scholar), drICE (15Fraser A.G. Evan G.I. EMBO J. 1997; 16: 2805-2813Crossref PubMed Scopus (171) Google Scholar), DCP-2/DREDD (16Inohara 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, 17Chen P. Rodriguez A. Erskine R. Thach T. Abrams J.M. Dev. Biol. 1998; 201: 202-216Crossref PubMed Scopus (182) Google Scholar), DRONC (18Dorstyn L. Colussi P.A. Quinn L.M. Richardson H. Kumar S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4307-4312Crossref PubMed Scopus (239) Google Scholar), DECAY (19Dorstyn 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 two caspases predicted on the basis of genomic sequence (20Rubin G.M. Yandell M.D. Wortman J.R. Miklos G.L.G. Nelson C.R. Hariharan I.K. et al.Science. 2000; 287: 2204-2215Crossref PubMed Scopus (1368) Google Scholar) have been identified in Drosophila. Evidence that caspases are important for cell death in Drosophilacomes from several sets of observations. Expression of the baculovirus caspase inhibitor p35 or the Drosophila caspase inhibitor DIAP1 blocks cell death in the fly in a number of different contexts (reviewed in Ref. 21Bergmann A. Agapite J. Steller H. Oncogene. 1998; 17: 3215-3223Crossref PubMed Scopus (109) Google Scholar), including normally occurring cell death and death induced by overexpression of the cell death activatorsreaper (rpr), head involution defective (hid), and grim (22Hay B.A. Wolff T. Rubin G.M. Development. 1994; 120: 2121-2129Crossref PubMed Google Scholar, 23Grether M.E. Abrams J.M. Agapite J. White K. Steller H. Genes Dev. 1995; 9: 1694-1708Crossref PubMed Scopus (592) Google Scholar, 24Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (643) Google Scholar, 25White K. Tahaoglu E. Steller H. Science. 1996; 271: 805-807Crossref PubMed Scopus (337) Google Scholar, 26Chen P. Nordstrom W. Gish B. Abrams J.M. Genes Dev. 1996; 10: 1773-1782Crossref PubMed Scopus (362) Google Scholar). Dominant negative forms of DCP-2/DREDD (27Rodriguez A. Oliver H. Zou H. Chen P. Wang X. Abrams J.M. Nat. Cell Biol. 1999; 1: 272-279Crossref PubMed Scopus (293) Google Scholar) and DRONC (28Meier P. Silke J. Leevers S.J. Evan G.I. EMBO J. 2000; 19: 598-611Crossref PubMed Scopus (275) Google Scholar) (this work) inhibitrpr-, hid-, andgrim-dependent cell death. Mutations indcp-1 (29McCall K. Steller H. Science. 1998; 279: 230-234Crossref PubMed Scopus (143) Google Scholar), the Drosophila homolog of the caspase-activating adaptor Apaf-1 (27Rodriguez A. Oliver H. Zou H. Chen P. Wang X. Abrams J.M. Nat. Cell Biol. 1999; 1: 272-279Crossref PubMed Scopus (293) Google Scholar, 30Kanuka H. Sawamoto K. Inohara N. Matsuno K. Okano H. Miura M. Mol. Cell. 1999; 4: 757-769Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 31Zhou L. Song Z. Tittel J. Steller H. Mol. Cell. 1999; 4: 745-755Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), or heterozygosity for deficiencies that remove the dcp-2/dredd (17Chen P. Rodriguez A. Erskine R. Thach T. Abrams J.M. Dev. Biol. 1998; 201: 202-216Crossref PubMed Scopus (182) Google Scholar) ordronc loci (28Meier P. Silke J. Leevers S.J. Evan G.I. EMBO J. 2000; 19: 598-611Crossref PubMed Scopus (275) Google Scholar), suppress apoptosis in specific contexts. Also, immunodepletion of drICE prevents apoptotic events in cell extracts (32Fraser A.G. McCarthy N.J. Evan G.I. EMBO J. 1997; 16: 6192-6199Crossref PubMed Scopus (125) Google Scholar). Finally, mutants that eliminate the function of aDrosophila caspase inhibitor, DIAP1, result in massive cell death (33Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 34Goyal L. McCall K. Agapite J. Hartwieg E. Steller H. EMBO J. 2000; 19: 589-597Crossref PubMed Scopus (379) Google Scholar, 35Lisi S. Mazzon L. White W. Genetics. 2000; 154: 669-678PubMed Google Scholar), which is associated with an increase in caspase activity (33Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). How do the Drosophila caspases function to bring about cell death in the fly? DCP-1, drICE, DECAY, and one caspase predicted by genomic sequence (daydream; GenBank™ accession numberAF281077) have short prodomains characteristic of executioner caspases. In contrast, DCP-2/DREDD and DRONC have large NH2-terminal prodomains with homology to mammalian death effector and caspase recruitment domains, respectively. A second caspase predicted by genomic sequence (dream; GenBank™ accession number AF275814) has a long prodomain that lacks homology with any known death regulators. Death effector and caspase recruitment domains in caspases are thought to mediate their recruitment to death signal-dependent complexes in which activation occurs in response to oligomerization (reviewed in Ref. 36Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6151) Google Scholar). Thus DCP-2/DREDD and DRONC may act as initiator caspases in apoptotic signaling. In most caspases the catalytic site cysteine (C) is present in the pentapeptide sequence QAC(R/Q/G)(G/E), in which the QAC motif is invariant. DRONC is unique among caspases in that the sequence surrounding the active site is PFCRG (Fig. 1 A) (18Dorstyn L. Colussi P.A. Quinn L.M. Richardson H. Kumar S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4307-4312Crossref PubMed Scopus (239) Google Scholar). Caspase crystal structures indicate that the glutamine at the first position of the canonical caspase pentapeptide QACRG is part of the substrate binding pocket. The fact that DRONC has a proline at this position suggests that it has a novel substrate specificity. Several Caenorhabditis elegans caspases, CSP-1a and CSP-2a, with pentapeptide sequences that differ in the first two pentapeptide positions, SACRG and VCCRG, respectively, have also been described (37Shaham S. J. Biol. Chem. 1998; 273: 35109-35117Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Here we show that, unique among caspases characterized to date, DRONC cleaves tetrapeptide and protein substrates COOH-terminal to glutamate as well as aspartate residues. A role for DRONC as a cell death caspase is suggested by the observations that expression of a dominant negative form of DRONC blocks cell death, that DRONC expression induces cell death, and that DRONC interacts with other Drosophila cell death regulators, including DIAP1, drICE, hid, andgrim. Interestingly, however, DRONC is not inhibited by baculovirus p35, which inhibits cell death in flies. The substrate specificity of recombinant DRONC was tested using positional scanning synthetic combinatorial libraries, as described previously (10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1844) Google Scholar, 38Rano T.A. Timkey T. Peterson E.P. Rotunda J. Nicholson D.W. Becker J.W. Chapman K.T. Thornberry N.A. Chem. Biol. 1997; 4: 149-155Abstract Full Text PDF PubMed Scopus (238) Google Scholar). The DRONC coding region was amplified by polymerase chain reaction and introduced into pET23(a) (Novagen) to produce pDRONC-His6. This plasmid was used to prepare active DRONC from Escherichia coli as described for DCP-131-His6 (39Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (140) Google Scholar). The subunits were resolved on a 15% SDS-PAGE1 gel and transferred to polyvinylidene difluoride (Millipore). The membrane was stained with Coomassie and the smaller band excised. Microsequencing was carried out at the Caltech Protein Microanalytical Laboratory under the direction of Gary M. Hathaway. Fluorogenic peptide cleavage assays were carried out as described (39Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (140) Google Scholar), using DRONC or DCP-1 at 0.2 μm or 0.2 nm final concentration as specified in the text. AFC-tetrapeptide substrates were purchased from Enzyme Systems (Livermore, CA), including the custom made Ac-TQTE-AFC and AC-TQTD-AFC. Bacterially produced DCP-1-His6 and drICE-His6 have been described (39Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (140) Google Scholar). The coding regions for DRONC, drICE, and DCP-1 were cloned from the yeast expression constructs into Bluescript KS+ (Stratagene). The coding regions of mutant versions of these proteins were generated using polymerase chain reaction and the QuickChange site-directed mutagenesis kit (Stratagene). Sequencing verified the existence of the desired mutation and the absence of other polymerase chain reaction-generated mutations. A transcription-coupled rabbit reticulocyte translation system (TNT, Promega) with Redivue [35S]methionine (Amersham Pharmacia Biotech) was used to generate 35S-labeled proteins. 2 μl of each 35S-labeled product was incubated with 2 μm of active caspase or buffer alone in caspase activity buffer (39Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (140) Google Scholar) in a total 10-μl volume. The products were resolved by 15% SDS-PAGE, the proteins transferred to Hybond ECL membrane (Amersham Pharmacia Biotech). Bio-Max MR-1 film (KODAK) was used to visualize the labeled fragments. The concentration of active DRONC was determined by active site titration using the active site inhibitor carbobenzoxy-VAD-fluoromethyl ketone (z-VAD-fmk), as described in Ref. 40Garcia-Calvo M. Peterson E.P. Rasper D.M. Vaillancourt J.P. Zamboni R. Nicholson D.W. Thornberry N.A. Cell Death Diff. 1999; 6: 362-369Crossref PubMed Scopus (194) Google Scholar. The activity of DRONC was measured in triplicate for each substrate using continuous fluorometric assays as described in Ref. 33Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar. Appropriate dilutions of enzyme were added to reaction mixtures containing substrate and caspase activity buffer (33Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar) in a total volume of 100 μl.k cat/K m values were calculated as described in Ref. 41Stennicke H.R. Salvesen G.S. Methods. 1999; 17: 313-319Crossref PubMed Scopus (160) Google Scholar. Yeast expression plasmids for galactose inducible expression of DCP-1, DIAP1, DIAP2, and P35 have been previously described (39Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (140) Google Scholar). pCUP-DIAP1, for copper inducible expression of DIAP1, pGALL-RPR, pGALL-GRIM, and pGALL-HID have been previously described (33Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). The coding region of DRONC was amplified from a clone (LD12627) obtained from the BerkleyDrosophila Genome project, and cloned into pGALL-(LEU2) (39Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (140) Google Scholar) to produce pGALL-DRONC. A DRONC active site mutant, DRONCC318S, was generated by polymerase chain reaction using a mutagenic oligonucleotide encompassing the internalSacII site of DRONC, and cloned to replace the corresponding section of wild-type DRONC in pGALL-(LEU2), generating pGALL-DRONCC318S. A section of human PARP encoding amino acids 1–337, spanning the caspase cleavage site, was amplified from a HeLa cell Matchmaker library (CLONTECH) and cloned into pADH-(TRP1) (33Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar) in front of a Myc epitope tag (encoding MEQKLISEEDLAS), to generate pADH-mycPARP337. A fragment encoding p35 was excised from pEF-p35 (42Hawkins C.J. Uren A.G. Hacker G. Medcalf R.L. Vaux D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13786-13790Crossref PubMed Scopus (96) Google Scholar) and cloned into pADH-(TRP1) (33Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar) to give pADH-p35. W303α yeast were transformed as described previously (39Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (140) Google Scholar). For survival assays, transformants were grown in selective liquid medium to saturation, then pelleted, and resuspended in TE toA 600 = 0.02. Five-fold dilutions were prepared, and 2-μl drops of each solution were spotted onto selective plates with galactose and 0, 10, or 100 μm CuSO4 as specified. For Western blotting to analyze p35 and PARP cleavage, transformants were grown in glucose-containing selective medium to stationary phase, washed in TE and grown in YP medium with galactose for 8 h. Yeast were pelleted, lysed by 2 cycles of boiling, and vortexing with glass beads in cracking buffer (8 m urea, 5% SDS, 40 mm Tris, pH 6.8, 0.1 mm EDTA, 1% β-mercaptoethanol, 0.4 mg/ml bromphenol blue). Samples were subjected to 12% SDS-PAGE, transferred to Hybond ECL membrane (Amersham Pharmacia Biotech), and probed with antibodies recognizing the Myc epitope or p35. Washes were followed by incubation with a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:2000), followed by detection with ECL (Pierce). The coding regions for full-length wild type DRONC, DRONCC318S, GRIM, and HID were cloned into pGMR (22Hay B.A. Wolff T. Rubin G.M. Development. 1994; 120: 2121-2129Crossref PubMed Google Scholar), and introduced into theDrosophila germline using standard techniques (43Spradling A.C. Rubin G.M. Science. 1982; 218: 341-347Crossref PubMed Scopus (1169) Google Scholar), generating GMR-DRONC GMR-DRONCC318S, GMR-grim,and GMR-hid flies, respectively. GMR-rpr (24Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (643) Google Scholar) and GMR-p35 (22Hay B.A. Wolff T. Rubin G.M. Development. 1994; 120: 2121-2129Crossref PubMed Google Scholar) flies have been described previously. Fixation, embedding and sectioning of adult fly heads, and acridine orange staining were carried out as described in Ref. 44Wolff T. Ready D.F. Development. 1991; 113: 825-839PubMed Google Scholar. Some caspases autocatalytically cleave and activate themselves. To determine if and where DRONC cleaves itself we expressed and purified fromE. coli a COOH-terminal His6-tagged version of DRONC. The purified protein consisted of two major bands, presumably consisting of the processed large and small subunits (data not shown). As discussed below, this protein is active as a caspase. To determine the site of cleavage between the large and small subunits, Edman degradation amino-terminal sequencing was performed on the smaller band. The NH2-terminal sequence determined (Fig.1 A) occurs COOH-terminal to the sequence TQTE352, suggesting that DRONC cleaves itself following a glutamate rather than an aspartate. To test this hypothesis directly we mutated DRONC TQTE352 to TQTA352.35S-Labeled wild type DRONC and DRONC TQTAE352Awere generated by in vitro translation and incubated with bacterially produced DRONC or DCP-1. As shown in Fig. 1 B, DRONC cleaved itself to generate a product corresponding in size to the prodomain and large subunit. This band was not seen when DRONC TQTAE352A was the substrate, consistent with the hypothesis that DRONC processes itself following TQTE352. DCP-1 (Fig.1 B) and drICE (data not shown) cleaved DRONC at several sites. This cleavage was unaffected by the presence of the TQTA352 mutation, suggesting that these caspases cleaved elsewhere in DRONC, perhaps in the DRONC prodomain. To explore the possibility of cleavage within the DRONC prodomain we generated a form of DRONC, DRONCpD4A, in which the P1 aspartates of four potential caspase target sites within the prodomain, DEKD66, ESVD110, DESD113, and DIVD135, were changed to alanine. As shown in Fig.1 B, 35S-labeled in vitro translated DRONCpD4A was still processed by wild type DRONC, but not by DCP-1. Similar results were obtained with cleavage by drICE (data not shown). Thus DCP-1 and drICE can process DRONC within the prodomain, but not at the large-small subunit boundary. If this processing occurs in vivo it may serve as a point of regulation of DRONC function. Positional scanning synthetic combinatorial libraries (PS-SCL) have been a useful tool to determine cleavage site specificities of other caspases (10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1844) Google Scholar). The PS-SCL is composed of three separate sublibraries of 8,000 compounds each. In each sublibrary, one position is defined with one of 20 amino acids (excluding cysteine), while the remaining two positions contain a mixture of amino acids present in approximately equimolar concentrations. Analysis of the three sublibraries (20 samples each) affords a complete understanding of the amino acid preferences in the P2, P3, and P4 positions. We used this approach to characterize DRONC's preferences for given amino acids at each of these positions. The positional scanning synthetic combinatorial libraries available all contain aspartate at the P1 position. While DRONC cleaves itself after glutamate, it is also able to cleave protein substrates after aspartate (below). Thus we reasoned that the existing aspartate-based libraries would yield useful information about DRONCs cleavage specificity. As shown in Fig.2 A, DRONC shows a strong preference for Thr, Ile, or Val at the P2 position. A wider spectrum of amino acids was tolerated at the P3 and P4 positions. This analysis suggests that TATD constitutes an optimal DRONC P1 aspartate tetrapeptide cleavage site. The results of the PS-SCL analysis were supported by experiments in which DRONC activity was tested directly with a number of commonly used tetrapeptide activity substrates. DRONC showed highest levels of activity with the tetrapeptides VEID-AMC and IETD-AMC, and somewhat lower levels of activity with DEVD-AMC (Fig. 2 B). However, little if any activity was seen with WEHD-AMC or YVAD-AMC, which are predicted to be poor substrates. Fig. 2 B also showed that DRONC had higher levels of activity with the pentapeptide GIETD-AMC than with the tetrapeptide IETD-AMC. This, as well as other observations below, suggests that a P5 residue is important for optimal DRONC activity. To further characterize DRONCs cleavage preferences we carried out assays in which the cleavage activities of DRONC and DCP-1 were measured for two different peptide substrates: Ac-TQTE-AFC and Ac-DEVD-AFC (Fig. 2 C). Ac-TQTE-AFC is derived from the known DRONC autoprocessing site and is also predicted to correspond to a good DRONC cleavage site based on the results obtained from PS-SCL analysis. Ac-DEVD-AFC is a tetrapeptide substrate for caspases generally grouped together as effectors of apoptosis (group II caspases (10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1844) Google Scholar)). DRONCs activity is low in absolute terms compared with DCP-1. However, DRONC shows a clear cleavage preference for the Ac-TQTE-AFC substrate over Ac-DEVD-AFC (Fig. 2 C). As expected, DCP-1, which has a common variant of the standard caspase active site pentapeptide (QACQG), has a strong preference for the tetrapeptide substrate with a P1 aspartate, Ac-DEVD-AFC (Fig. 2 C) (45Song Z. Bergmann A. Nicholson D.W. Thornberry N.A. Peterson E.P. Steller H. Mol. Cell. Biol. 2000; 20: 2907-2914Crossref PubMed Scopus (79) Google Scholar). Despite the fact that DRONC shows relatively low levels of activity with tetrapeptide substrates containing a P1 aspartate, DRONC is efficiently inhibited by the broad range tripeptide caspase inhibitor carbobenzoxy-VAD-fluoromethyl ketone (z-VAD-fmk) (data not shown). We wanted to determine DRONC's P1 specificity with respect to aspartate and glutamate. To do this we synthesized a second tetrapeptide substrate, Ac-TQTD-AFC, that differs from Ac-TQTE-AFC only by the P1 residue. We used these substrates to measure DRONCs activity (k cat/K m), as described under “Experimental Procedures.” We calculated a (k cat/K m) of 2.73 for TQTE-AFC and a (k cat/K m) of 3.36 for TQTD-AFC. Thus DRONC shows only a slight preference for cleavage of tetrapeptide substrates with a P1 aspartate over those with a P1 glutamate. DRONC is, however, a particularly poor catalyst of tetrapeptide hydrolysis. The calculated (k cat/K m) values for DRONC are roughly 40–180-fold lower than those described for caspase-9, which itself is a very inefficient enzyme in isolation as compared with most other caspases (40Garcia-Calvo M. Peterson E.P. Rasper D.M. Vaillancourt J.P. Zamboni R. Nicholson D.W. Thornberry N.A. Cell Death Diff. 1999; 6: 362-369Crossref PubMed Scopus (194) Google Scholar). This may reflect the fact that DRONC has an intrinsically low turnover rate or that we have not identified optimalin vitro assay conditions. However, DRONC activity may also be regulated allosterically through interactions with theDrosophila homolog of Apaf-1 (variously known as Dapaf-1 (30Kanuka H. Sawamoto K. Inohara N. Matsuno K. Okano H. Miura M. Mol. Cell. 1999; 4: 757-769Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), HAC-1 (31Zhou L. Song Z. Tittel J. Steller H. Mol. Cell. 1999; 4: 745-755Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), and DARK (27Rodriguez A. Oliver H. Zou H. Chen P. Wang X. Abrams J.M. Nat. Cell Biol. 1999; 1: 272-279Crossref PubMed Scopus (293) Google Scholar)) in a manner similar to that of mammalian caspase-9 by Apaf-1 (46Rodriguez J. Lazebnik Y. Genes Dev. 1999; 13: 3179-3184Crossref PubMed Scopus (458) Google Scholar). Alternatively, since DRONC shows similar levels of activity to DCP-1 on the protein substrate drICE (below), optimal DRONC cleavage may require additional sequences surrounding the target site. If DRONC is an apical cell death caspase, likely substrates include other Drosophila caspases. drICE is a good candidate to be such a target since immunodepletion experiments show that drICE is required for rpr-dependent apoptotic events in cell extracts (32Fraser A.G. McCarthy N.J. Evan G.I. EMBO J. 1997; 16: 6192-6199Crossref PubMed Scopus (125) Google Scholar), and genetic interactions suggest that DRONC contributes to rpr-, hid-, andgrim-dependent cell death (see Ref. 28Meier P. Silke J. Leevers S.J. Evan G.I. EMBO J. 2000; 19: 598-611Crossref PubMed Scopus (275) Google Scholar and below). We generated 35S-labeled in vitrotranslated drICE and incubated it with bacterially produced DRONC or DCP-1. We found, consistent with the observations of others (28Meier P. Silke J. Leevers S.J. Evan G.I. EMBO J. 2000; 19: 598-611Crossref PubMed Scopus (275) Google Scholar, 45Song Z. Bergmann A. Nichol
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