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Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation
Y Hu1, M A Benedict, D Wu
1Departments of Pathology and Comprehensive Cancer Center, 1500 East Medical Center Drive, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Abstract:
Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promoted the processing and activation of caspase-9 in vivo. Bcl-XL, an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-XL was mediated through both its CED-4-like domain and the C-terminal domain containing WD-40 repeats. Expression of Bcl-XL inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.
Insights
Mammalian Apaf-1 interacts with caspase-9, promoting its activation. The anti-apoptotic protein Bcl-XL inhibits this process by binding Apaf-1, revealing a conserved mechanism of cell death regulation from nematodes to humans.
Area of Science:
- Cell Biology
- Molecular Biology
- Apoptosis Research
Background:
- The nematode CED-4 protein activates the CED-3 death protease, a process inhibited by CED-9.
- Understanding the mammalian counterparts and their regulatory mechanisms is crucial for cell death research.
Purpose of the Study:
- To investigate the interaction of mammalian CED-4 homolog, Apaf-1, with caspases.
- To elucidate the role of Bcl-XL in Apaf-1 and caspase-9 regulation.
- To determine the evolutionary conservation of these apoptotic pathways.
Main Methods:
- Co-immunoprecipitation assays to study protein interactions in mammalian cells.
- Expression studies to assess the functional impact of Apaf-1 and Bcl-XL.
- In vitro assays with purified proteins to confirm regulatory mechanisms.
Main Results:
- Mammalian Apaf-1 interacts with multiple caspases, including caspase-9, via its N-terminal CED-4-like domain.
- Apaf-1 enhances caspase-9 activity and promotes its in vivo processing and activation.
- Bcl-XL physically interacts with both Apaf-1 and caspase-9, inhibiting Apaf-1-mediated caspase-9 maturation.
Conclusions:
- Apaf-1 is a key mediator in caspase-9 activation.
- Bcl-XL acts upstream of caspase-9 maturation by interacting with Apaf-1.
- The regulatory pathway involving Apaf-1, caspase-9, and Bcl-XL is conserved across species, highlighting fundamental mechanisms of apoptosis.