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Characterization of Bax-sigma, a cell death-inducing isoform of Bax
E Schmitt1, C Paquet, M Beauchemin
1Research Centre of the University of Montreal Hospital Centre, Notre Dame Hospital, Montreal, Quebec, H2L 4M1, Canada.
Abstract:
The Ced-9/Bcl-like family of genes codes for proteins that have antiapoptotic and proapoptotic activity. Several Bax isoproteins have been detected by 2-D gel electrophoresis, and a novel human member, designated as Bax-sigma, has been identified and cloned from human cancer promyelocytic cells. Bax-sigma contains BH-3, BH-1, and BH-2 domains, putative alpha-5 and alpha-6 helices, and the carboxy-terminal hydrophobic transmembrane domain but lacks amino acids 159 to 171 compared to Bax-alpha. mRNA expression analysis by reverse transcription-polymerase chain reaction and RNase protection assays have revealed that Bax-sigma is expressed in a variety of human cancer cell lines and normal tissues. To investigate the potential role of Bax-sigma in apoptosis, first its effects were compared to those of Bax-alpha by transient expression in human B lymphoma Namalwa cells. Both Bax-sigma and Bax-alpha promoted apoptosis, as detected by DNA fragmentation and morphological analysis by electron microscopy. The apoptosis induced by Bax-sigma and Bax-alpha was correlated with their expression, cytochrome c release, and caspase activation. In a yeast two-hybrid system, Bax-sigma interacted with several Ced-9/Bcl family members but had no affinity for the human Egl-1 homologs Bik and Bad and the Ced-4 homolog Apaf-1. In human cells, Bax-sigma function was counteracted by Bcl-xL overexpression, and co-immunoprecipitation experiments indicated that Bax-sigma was associated with Bcl-xL. Furthermore, Bax-sigma overexpression increased cell death induced by various concentrations of genotoxic agents with the most pronounced effect occurring at low camptothecin and vinblastine dose levels. Our results suggest that Bax-sigma, a novel variant of Bax, encodes a protein with a proapoptotic effect and mode of action similar to those of Bax-alpha.
Insights
A novel Bax-sigma protein variant was identified and cloned. Bax-sigma promotes apoptosis, similar to Bax-alpha, by influencing cell death pathways and interacting with Bcl-2 family members.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- The Ced-9/Bcl-2 family regulates apoptosis through antiapoptotic and proapoptotic proteins.
- Bax isoproteins, including a novel variant Bax-sigma, have been identified.
Purpose of the Study:
- To identify and characterize a novel human Bax isoprotein, Bax-sigma.
- To investigate the proapoptotic activity and molecular interactions of Bax-sigma.
Main Methods:
- Cloning of Bax-sigma from human cancer cells.
- Transient expression in human B lymphoma cells to assess apoptosis induction.
- Yeast two-hybrid assays and co-immunoprecipitation to determine protein interactions.
- Analysis of mRNA expression in cancer cell lines and normal tissues.
Main Results:
- Bax-sigma was cloned and found to contain key Bcl-2 homology domains (BH-3, BH-1, BH-2).
- Bax-sigma, like Bax-alpha, induced apoptosis, evidenced by DNA fragmentation and cytochrome c release.
- Bax-sigma interacted with Bcl-2 family members but not with Bik, Bad, or Apaf-1.
- Bcl-xL counteracted Bax-sigma's function and associated with it in human cells.
- Bax-sigma overexpression enhanced cell death induced by genotoxic agents.
Conclusions:
- Bax-sigma is a novel proapoptotic protein variant of Bax.
- Bax-sigma shares a similar mode of action with Bax-alpha in promoting apoptosis.
- Bax-sigma's function is modulated by interactions with other Bcl-2 family members, particularly Bcl-xL.