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Cloning of a novel human caspase-9 splice variant containing only the CARD domain
Pingzhang Wang1, Taiping Shi, Dalong Ma
1Chinese National Human Genome Center, Beijing. #3-707 North YongChang Road BDA, Beijing 100176, PR China. sicau2000@yahoo.com.cn
Insights
A novel splice variant, Caspase-9 gamma, inhibits apoptosis by interfering with key protein interactions. This finding suggests Caspase-9 gamma acts as an endogenous inhibitor, impacting cell death pathways.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Caspase-9 is crucial for the intrinsic apoptotic pathway.
- Two splice variants, caspase-9-alpha and -beta, are known.
- The role of additional splice variants in apoptosis regulation is under investigation.
Purpose of the Study:
- To clone and characterize a novel splice variant of caspase-9, designated Casp9-gamma.
- To investigate the functional role of Casp9-gamma in apoptosis and NF-kappaB activation.
Main Methods:
- Cloning and characterization of the Casp9-gamma splice variant.
- Overexpression studies in mammalian cells to assess apoptosis.
- Analysis of procaspase-3 cleavage in the presence of apoptotic stimuli (Bax, staurosporine).
- Investigation of NF-kappaB activation in transfected 293T and ND7 cells.
Main Results:
- Casp9-gamma is a novel splice variant resulting from an alternative 3' splice site, leading to a preterminated open reading frame encoding only the CARD domain.
- Casp9-gamma does not induce apoptosis upon overexpression.
- Casp9-gamma inhibits procaspase-3 cleavage induced by Bax or staurosporine, suggesting an anti-apoptotic function.
- Casp9-gamma does not enhance NF-kappaB activation in 293T cells, contrasting with previous findings in ND7 cells.
Conclusions:
- Casp9-gamma functions as an endogenous inhibitor of apoptosis, potentially by blocking Apaf-1/procaspase-9 CARD-CARD interactions.
- The observed inhibition of apoptosis by Casp9-gamma highlights its regulatory role in the intrinsic apoptotic pathway.
- Procaspase-9-mediated NF-kappaB activation appears to be cell type-specific, indicating complex regulatory mechanisms.
Abstract:
Caspase-9 plays a key role in the intrinsic apoptotic pathway and currently two splice variants (caspase-9-alpha and -beta) have been identified. The present study cloned and characterized a novel caspase-9 splice variant, hereby designated Casp9-gamma. Casp9-gamma is generated from an additional alternative 3' splice site in the fourth exon of caspase-9, resulting in a 58-nucleotide fragment insertion compared with the full-length caspase-9-alpha. The fragment introduces an in-frame stop codon, and the resulting open reading frame (ORF) is preterminated. The Casp9-gamma comprises the deduced 154 amino acid residues containing only the caspase recruitment domain (CARD) and does not contain the large and small subunits. The Casp9-gamma does not promote apoptosis when overexpressed in mammalian cells. Moreover, it inhibits the cleavage of procaspase-3 mediated by proapoptotic member Bax or apoptosis inductor staurosporine. Therefore, Casp9-gamma may function as an endogenous apoptotic inhibitor by interfering with the CARD-CARD interaction between Apaf-1 (apoptotic protease activating factor-1) and procaspase-9. In addition, Casp9-gamma does not enhance NF-kappaB activation in transfected 293T cells, conflicting with previous evidence that the isolated CARD of caspase-9 activates NF-kappaB in ND7 cells. This suggests that the procaspase-9-mediated NF-kappaB activation in response to cellular stresses is cell type-specific through an unidentified mechanism.
