The Drosophila Caspase DRONC Cleaves following Glutamate or Aspartate and Is Regulated by DIAP1, HID, and GRIM

Abstract

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. 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