Caspase 3 attenuates XIAP (X-linked inhibitor of apoptosis protein)-mediated inhibition of caspase 9.
During apoptosis, the initiator caspase 9 is activated at the apoptosome after which it activates the executioner caspases 3 and 7 by proteolysis. During this process, caspase 9 is cleaved by caspase 3 at Asp(330), and it is often inferred that this proteolytic event represents a feedback amplification loop to accelerate apoptosis. However, there is substantial evidence that proteolysis per se does not activate caspase 9, so an alternative mechanism for amplification must be considered. Cleavage at Asp(330) removes a short peptide motif that allows caspase 9 to interact with IAPs (inhibitors of apoptotic proteases), and this event may control the amplification process. We show that, under physiologically relevant conditions, caspase 3, but not caspase 7, can cleave caspase 9, and this does not result in the activation of caspase 9. An IAP antagonist disrupts the inhibitory interaction between XIAP (X-linked IAP) and caspase 9, thereby enhancing activity. We demonstrate that the N-terminal peptide of caspase 9 exposed upon cleavage at Asp330 cannot bind XIAP, whereas the peptide generated by autolytic cleavage of caspase 9 at Asp315 binds XIAP with substantial affinity. Consistent with this, we found that XIAP antagonists were only capable of promoting the activity of caspase 9 when it was cleaved at Asp315, suggesting that only this form is regulated by XIAP. Our results demonstrate that cleavage by caspase 3 does not activate caspase 9, but enhances apoptosis by alleviating XIAP inhibition of the apical caspase.
Denault
Jean-Bernard
JB
Program in Cell Death and Apoptosis Research, The Burnham Institute for Medical Research and the Graduate Program in Molecular Pathology, University of California San Diego, La Jolla, CA 92037, USA.
Eckelman
Brendan P
BP
Shin
Hwain
H
Pop
Cristina
C
Salvesen
Guy S
GS
D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism; D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology; D051017:Apoptosis Regulatory Proteins; D001224:Aspartic Acid / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism*; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism*; D061945:Caspase Inhibitors; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D051017:Apoptosis Regulatory Proteins; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D010455:Peptides; D011994:Recombinant Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D001224:Aspartic Acid; D053148:Caspase 3; D053453:Caspase 9