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Published on: March 5, 2018
cIAP1 cooperatively inhibits procaspase-3 activation by the caspase-9 apoptosome
Stephen P Burke1, Lucinda Smith, Jeffrey B Smith
1Department of Pharmacology and Toxicology, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Alabama 35294-0019, USA.
Abstract:
Although early studies of inhibitor of apoptosis proteins (IAPs) suggested that cIAP1 directly binds and inhibits caspases similarly to X-linked IAP (XIAP), a recent one found that micromolar concentrations of cIAP1 only weakly inhibit caspase-3, -7, or -9. Here, we show that cIAP1 specifically and cooperatively blocks the cytochrome c-dependent apoptosome in vitro. Hence, cIAP1 prevented the activation of procaspase-3 but had no effect on the processing of procaspase-9 or the activity of prior activated caspase-3. Like cIAP1, XIAP had no effect on procaspase-9 processing and was a more potent inhibitor of procaspase-3 activation than of already activated caspase-3 activity. Inhibition of procaspase-3 activation depended on BIR2 and BIR3 of cIAP1 and was independent of BIR1, RING, CARD, and UBA domains. Smac prevented cIAP1 from inhibiting procaspase-3 activation and reversed the inhibition by prior addition of cIAP1. A procaspase-9 mutant (D315A) that cannot produce the p12 subunit was resistant to inhibition by cIAP1. Therefore, the N-terminal Ala-Thr-Pro-Phe motif of the p12 subunit of the caspase-9 apoptosome facilitates apoptosome blockade. Consequently, cIAP1 cooperatively interacts with oligomerized processed caspase-9 in the apoptosome and blocks procaspase-3 activation.
Insights
Cellular inhibitor of apoptosis protein 1 (cIAP1) blocks the apoptosome by binding processed caspase-9. This interaction prevents procaspase-3 activation, revealing a specific mechanism for apoptosis regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Inhibitor of Apoptosis Proteins (IAPs) play crucial roles in regulating programmed cell death.
- Previous studies suggested direct caspase inhibition by cIAP1, but recent findings indicated weak inhibitory effects.
- The precise mechanism of cIAP1's interaction with caspases and the apoptosome remains incompletely understood.
Purpose of the Study:
- To elucidate the specific mechanism by which cIAP1 inhibits apoptosis.
- To determine the interaction of cIAP1 with key components of the apoptosome.
- To identify the domains of cIAP1 responsible for its inhibitory activity.
Main Methods:
- In vitro biochemical assays using purified proteins.
- Analysis of caspase processing and activity.
- Site-directed mutagenesis of procaspase-9.
- Investigation of protein-protein interactions using cIAP1 domains.
Main Results:
- cIAP1 specifically blocks the cytochrome c-dependent apoptosome, not free caspases.
- cIAP1 inhibits procaspase-3 activation but not procaspase-9 processing or activated caspase-3.
- Inhibition requires BIR2 and BIR3 domains of cIAP1 and is reversed by Smac.
- A procaspase-9 mutant resistant to inhibition highlights the role of the p12 subunit's N-terminal motif.
Conclusions:
- cIAP1 acts as an apoptosome blocker by interacting with oligomerized, processed caspase-9.
- The N-terminal Ala-Thr-Pro-Phe motif of the caspase-9 p12 subunit is critical for this interaction.
- This mechanism provides a novel understanding of IAP function in apoptosis regulation.
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