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Proapoptotic activity of Caenorhabditis elegans CED-4 protein in Drosophila: implicated mechanisms for caspase
H Kanuka1, S Hisahara, K Sawamoto
1Department of Neuroanatomy, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Abstract:
CED-4 protein plays an important role in the induction of programmed cell death in Caenorhabditis elegans through the activation of caspases. However, the precise mechanisms by which it activates caspases remain unknown. To investigate the conservation of CED-4 function in evolution, transgenic Drosophila lines that express CED-4 in the compound eye were generated. Ectopic expression of CED-4 in the eyes induced massive apoptotic cell death through caspase activation. An ATP-binding site (P-loop) mutation in CED-4 (K165R) causes a loss of function in its ability to activate Drosophila caspase, and an ATPase inhibitor blocks the CED-4-dependent caspase activity in Drosophila S2 cells. Immunoprecipitation analysis showed that both CED-4 and CED-4 (K165R) bind directly to Drosophila caspase drICE, and the overexpression of CED-4 (K165R) inhibits CED-4-, ecdysone-, or cycloheximide-dependent caspase activation in S2 cells. Furthermore, CED-4 (K165R) partially prevented cell death induced by CED-4 in Drosophila compound eyes. Thus, CED-4 function is evolutionarily conserved in Drosophila, and the molecular mechanisms by which CED-4 activates caspases might require ATP binding and direct interaction with the caspases.
Insights
The CED-4 protein
Area of Science:
- Cell biology
- Developmental biology
- Genetics
Background:
- Programmed cell death (apoptosis) is crucial for development and tissue homeostasis.
- CED-4 protein is a key regulator of apoptosis in Caenorhabditis elegans.
- The precise molecular mechanisms of CED-4-mediated caspase activation are not fully understood.
Purpose of the Study:
- To investigate the evolutionary conservation of CED-4 function in apoptosis.
- To elucidate the molecular mechanisms underlying CED-4's activation of caspases.
Main Methods:
- Generation of transgenic Drosophila expressing Caenorhabditis elegans CED-4 in compound eyes.
- Utilizing an ATP-binding site mutant (K165R) of CED-4.
- Employing Drosophila S2 cells for biochemical assays, including immunoprecipitation.
- Assessing caspase activation and cell death induction in Drosophila models.
Main Results:
- Ectopic CED-4 expression in Drosophila eyes induced massive apoptosis via caspase activation.
- A CED-4 (K165R) mutant lost the ability to activate Drosophila caspases and partially inhibited CED-4-induced cell death.
- ATP-binding site mutation and ATPase inhibitors blocked CED-4-dependent caspase activity.
- Both wild-type CED-4 and CED-4 (K165R) directly bind to the Drosophila caspase drICE.
Conclusions:
- CED-4's role in apoptosis is evolutionarily conserved from C. elegans to Drosophila.
- ATP binding and direct interaction with caspases are likely essential for CED-4's mechanism of caspase activation.