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Identification of an endogenous dominant-negative short isoform of caspase-9 that can regulate apoptosis
S M Srinivasula1, M Ahmad, Y Guo
1Center for Apoptosis Research and Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Abstract:
Alternatively spliced isoforms of certain apoptosis regulators, such as Bcl-x, Ced-4, and Ich-1, have been shown to play opposing roles in regulating apoptosis. Here, we describe the identification of an endogenous alternatively spliced isoform of caspase-9, named caspase-9b, which lacks the central large subunit caspase domain. Caspase-9b is detectable in many cell lines by PCR and at the mRNA and protein levels. Caspase-9b can interact with the caspase recruitment domain of Apaf-1, and like the active site mutant of caspase-9, it can inhibit multiple forms of apoptosis, including those triggered by oligomerization of death receptors. It can also block activation of caspase-9 and -3 by Apaf-1 in an in vitro cytochrome c-dependent caspase activation assay. These results suggest that caspase-9b functions as an endogenous apoptosis inhibitory molecule by interfering with the formation of a functional Apaf-1-caspase-9 complex.
Insights
Researchers identified caspase-9b, an apoptosis inhibitor. This alternatively spliced isoform of caspase-9 blocks apoptosis by preventing the formation of a functional Apaf-1-caspase-9 complex.
Area of Science:
- Molecular Biology
- Cell Biology
Background:
- Alternative splicing generates protein isoforms with diverse functions.
- Apoptosis regulators, like Bcl-x and Ich-1, can have opposing roles.
- Caspase-9 is a key mediator of apoptosis.
Purpose of the Study:
- To identify and characterize novel alternatively spliced isoforms of caspase-9.
- To investigate the role of any identified isoforms in apoptosis regulation.
Main Methods:
- Polymerase Chain Reaction (PCR) for detection.
- mRNA and protein level analysis.
- In vitro biochemical assays to assess protein interactions and enzymatic activity.
Main Results:
- Identified an endogenous alternatively spliced isoform of caspase-9, named caspase-9b.
- Caspase-9b lacks the central large subunit caspase domain.
- Caspase-9b interacts with Apaf-1 and inhibits apoptosis.
- Caspase-9b blocks Apaf-1-mediated activation of caspase-9 and caspase-3 in vitro.
Conclusions:
- Caspase-9b functions as an endogenous inhibitor of apoptosis.
- It interferes with the assembly of the Apaf-1-caspase-9 apoptosome complex.
- This discovery reveals a new regulatory mechanism in apoptosis control.