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 modifier A (crmA) protein. Wild-type crmA inhibits caspases 1 and 8 and thereby protects cells from apoptosis triggered by ligation of CD95 or tumour necrosis factor (TNF) receptors, but it does not protect against death mediated by other caspases. By replacing the tetrapeptide pseudosubstrate region of crmA (LVAD) with tetrapeptides that are optimal substrates for the different families of caspases, or with the four residues from the cleavage site of the baculovirus protein p35 (DQMD), we have generated a family of caspase inhibitors that show altered ability to protect against cell death. Although DEVD is the optimal substrate for caspase 3, crmA DEVD was degraded rapidly and was a weaker inhibitor than crmA DQMD, which was not degraded. Unlike 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.
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The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria 3050, Australia.
Silke
J
J
Vaux
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DL
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D001483:Base Sequence; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D004789:Enzyme Activation; D005720:Gamma Rays; D015870:Gene Expression; D014644:Genetic Variation; D006801:Humans; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D015843:Serpins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection; D014764:Viral Proteins
D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; D009842:Oligopeptides; D015843:Serpins; D014764:Viral Proteins; D003907:Dexamethasone; C074641:interleukin-1beta-converting enzyme inhibitor; D020169:Caspases