Inhibition of apoptosis and clonogenic survival of cells expressing crmA variants: optimal caspase substrates are not necessarily optimal inhibitors

To study the role of various caspases during apoptosis, we have designed a series of caspase inhibitors based on the cowpox virus cytokine response mo difier A (crmA) protein. Wild-type crmA inhibits caspases 1 and 8 and there by protects cells from apoptosis triggered by ligation of CD95 or tumour ne crosis factor (TNF) receptors, but it does not protect against death mediat ed by other caspases. By replacing the tetrapeptide pseudosubstrate region of crmA (LVAD) with tetrapeptides that are optimal substrates for the diffe rent families of caspases, or with the four residues from the cleavage site of the baculovirus protein p35 (DQMD), we have generated a family of caspa se inhibitors that show altered ability to protect against cell death. Alth ough DEVD is the optimal substrate for caspase 3, crmA DEVD was degraded ra pidly and was a weaker inhibitor than crmA DQMD, which was not degraded. Un like wild-type crmA and crmA DEVD, crmA DQMD was able to inhibit apoptosis caused by direct activation of caspase 3 and protected lymphoid cells from death induced by radiation and dexamethasone, Significantly, the protected cells were capable of sustained growth.