Genetic and Biochemical Analyses of the Necessity for Caspase Activation by the CED4-Domain Proteins, APAF-1 and DARK
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Activation of caspase proteases by Ced4 domain proteins is a critical step in the induction of programmed cell death, or apoptosis. Understanding of the genetic and biochemical regulation of the mammalian Ced-4 gene, Apaf-1, may be crucial to the understanding of autoimmune diseases, neurodegenerative disorders and cancer progression. Located in chromosomal band 12q22, Apaf-1 is in a locus frequently deleted in Male Germ Cell Tumors (GCT's). Though not homozygously inactivated in these tumors, Apaf-1 mediated caspase activation is impaired in GCT cell lines and may be a frequent event in other cancer types. Analysis of human genomic DNA facilitated the discovery of the homologous gene DARK, the Drosophila Apaf-1 Related Killer. Hypomorphic alleles of DARK cause developmental disruption, including wing defects, body wall defects, supernumerary bristles, male sterility and an enlarged nervous system. Mutation of DARK potently suppresses the apoptotic function of the genes reaper, grim, and hid. Recombinant Grim protein was shown to antagonize IAP-mediated caspase inhibition in vitro. Peptides corresponding to the conserved Amino-termini of the reaper, grim and hid genes could compete for a binding site also used by the mammalian anti-IAP protein SMAC to block IAP-caspase interaction. Despite these peptides' failure to allow for reconstitution of caspase activation in vitro, genetic inactivation of IAP's leads to significant activation of caspases in vivo. DARK plays a critical role in caspase activation in vivo, and mutations of DARK suppress several genetic measures of cell death due to IAP inactivation. These studies show DARK to be an important apoptosis gene in the fly and necessary for caspase amplification and apoptotic initiation in certain cell death pathways. Further understanding of the regulation of cell death in the genetically tractable Drosophila model may help shed light on the regulation of apoptosis in human cells and disease states as well.