Structure-function analysis of an evolutionary conserved protein, DAP3, which mediates TNF-alpha- and Fas-induced cell death

A novel approach to the isolation of positive mediators of programmed cell death, based on random inactivation of genes by expression of anti sense RN As, was employed to identify mediators of interferon-gamma-induced apoptosi s, One of the several genes identified is DAP3, which codes for a 46 kDa pr otein with a potential nucleotide-binding motif, Structure-function studies of the protein indicate that the intact full-length protein is required fo r its ability to induce apoptosis when overexpressed, The N-terminal 230 am ino acids, on the other hand, act in a dominant-negative fashion. Both of t hese functions are dependent on the integrity of the nucleotide binding mot if, Expression of anti-sense DAP3 RNA and of the dominant interfering form of DAP3 both protected cells from apoptosis induced by activation of Fas an d tumor necrosis factor alpha (TNF-alpha) receptors, Thus, DAP3 is implicat ed as a positive mediator of these death-inducing stimuli. It functions dow nstream of the receptor signaling complex and its death promoting effects d epend on caspase activity. In the nematode Caenorhabditis elegans, a potent ial homolog of DAP3 showing 35% identity and 64% similarity to the human pr otein was isolated. Overexpression of the nematode DAP3 cDNA in mammalian c ells induced cell death, indicating that the protein is conserved at the fu nctional level as well as the structural level.