Inhibitor specificity of recombinant and endogenous caspase-9.
Apoptosis triggered through the intrinsic pathway by radiation and anti-neoplastic drugs is initiated by the activation of caspase-9. To elucidate control mechanisms in this pathway we used a range of synthetic and natural reagents. The inhibitory potency of acetyl-Asp-Glu-Val-Asp-aldehyde ('Ac-DEVD-CHO'), benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone ('Z-VAD-FMK') and the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis protein ('XIAP') against recombinant caspase-9 were predictive of the efficacy of these compounds in a cell-free system. However, the viral proteins CrmA and p35, although potent inhibitors of recombinant caspase-9, had almost no ability to block caspase-9 in this system. These findings were also mirrored in cell expression studies. We hypothesize that the viral inhibitors CrmA and p35 are excluded from reacting productively with the natural form of active caspase-9 in vivo, making the potency of inhibitors highly context-dependent. This is supported by survival data from a mouse model of apoptosis driven by Sindbis virus expressing either p35 or a catalytic mutant of caspase-9. These results consolidate previous findings that CrmA is a potent inhibitor of caspase-9 in vitro, yet fails to block caspase-9-mediated cell death.
Ryan
Ciara A
CA
Program for Apoptosis and Cell Death Research, The Burnham Institute, 10901 North, Torrey Pines Road, La Jolla, CA 92037, USA.
Stennicke
Henning R
HR
Nava
Victor E
VE
Burch
Jennifer B
JB
Hardwick
J Marie
JM
Salvesen
Guy S
GS
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D053453:Caspase 9; D061945:Caspase Inhibitors; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D006801:Humans; D007668:Kidney; D007700:Kinetics; D009842:Oligopeptides / Q000494:pharmacology; D011506:Proteins / Q000502:physiology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D013379:Substrate Specificity; D014960:X Chromosome; D051636:X-Linked Inhibitor of Apoptosis Protein; D016335:Zinc Fingers
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D011506:Proteins; D011994:Recombinant Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C506433:CASP9 protein, human; C506434:Casp9 protein, mouse; D053453:Caspase 9