Fraser, Andrew; (1997) Cloning and characterisation of a novel Drosophila melanogaster caspase, drICE. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Caspases are cysteine proteases whose activity is required for the execution of cell death in all multicellular organisms. The morphology of Drosophila cells undergoing cell death is identical to that observed in other organisms, suggesting that Drosophila also have a caspase-containing cell death machinery; however, until recently no Drosophila caspases had been identified. Here I describe the first cloning and characterisation of a Drosophila caspase. I have used a degenerate PCR approach to clone a novel Drosophila melanogaster caspase, which I called drICE. drICE contains all the residues required for caspase activity. Expression of full-length drICE sensitises the S2 Drosophila cell-line to induction of apoptosis by etoposide and cycloheximide treatment; expression of an N-terminally truncated form of drICE, p30drICE, mimicking the proteolytic removal of the prodomain during caspase activation, induces apoptosis in S2 cells. This requires p30drICE caspase activity. drICE is proteolytically activated during S2 cell apoptosis as would be predicted if drICE is part of the Drosophila cell death machinery. drICE auto-processes when expressed in E. coli, and the resulting protein has DEVD-cleaving substrate specificity. The purified active enzyme has two subunits and cleaves lamin DmO and baculovirus p35 in vitro. Lysates of S2 cells undergoing apoptosis contain a caspase activity that can cleave p35, lamin DmO and drICE in vitro and can activate chromatin condensation in added HeLa nuclei. drICE is required for all these activities, suggesting that it has both a role as a necessary 'effector' caspase and as an upstream caspase: whether this is an 'amplifier' or an 'apical' role is unclear. The cloning of drICE described in this thesis should allow genetic screens to identify either upstream regulators or downstream targets of drICE; these in turn should illuminate caspase function in general.

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