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Characterization of the BH3 protein Bmf in Gallus gallus: identification of a novel chicken-specific isoform
Francisco Gómez-Esquer1, Ma Angustias Palomar, Inés Rivas
1Departamento de Ciencias de la Salud III, Universidad Rey Juan Carlos, Avenida de Atenas S/N, Alcorcón, Madrid, Spain.
Abstract:
Bmf is a proapoptotic member of the BH3-only subgroup of Bcl-2 family proteins, which is associated to myosin V motors by binding to the dynein light chain 2 (DLC2). It acts as a sentinel detecting intracellular damages on the main cytoskeletal structures. The cloning and characterization of the chicken (Gallus gallus) Bmf cDNA and splicing variant is described in this report. The Bmf cDNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The chicken Bmf cDNA encodes a protein of 193 amino acids, showing homology to mammalian Bmf proteins. A splicing variant of the chicken Bmf (Bmf(S), short isoform of Bmf) coding a protein of 118 amino acids was also identified. This is the first Bmf isoform identified so far which lacks the DLC2-binding domain although retaining the BH3 domain. Both chicken Bmf isoforms induced apoptosis 24 h after transfection in MCF7 and HeLa cell lines, but chicken Bmf(S) exhibits a higher proapoptotic activity. In addition, mRNA expression analysis showed that chicken Bmf transcription is ubiquitous in all embryo developmental stages, suggesting a role for this protein in the control of the development process.
Insights
Researchers cloned and characterized chicken Bmf, a proapoptotic protein. A novel short isoform, Bmf(S), shows higher apoptotic activity and may play a role in embryonic development.
Area of Science:
- Molecular Biology
- Cell Biology
- Developmental Biology
Background:
- Bmf is a proapoptotic protein in the BH3-only subgroup of Bcl-2 family proteins.
- Bmf interacts with myosin V motors via dynein light chain 2 (DLC2), acting as a sentinel for cytoskeletal damage.
Purpose of the Study:
- To clone and characterize the chicken (Gallus gallus) Bmf cDNA and identify its splicing variants.
- To investigate the proapoptotic activity and expression patterns of chicken Bmf isoforms.
Main Methods:
- Chicken Bmf cDNA was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR).
- In silico analysis and sequencing were used to identify splicing variants.
- Apoptosis assays were performed in MCF7 and HeLa cell lines after transfection.
- mRNA expression analysis was conducted across different embryonic developmental stages.
Main Results:
- Chicken Bmf cDNA encodes a 193-amino acid protein homologous to mammalian Bmf.
- A novel short isoform, Bmf(S) (118 amino acids), was identified, lacking the DLC2-binding domain but retaining the BH3 domain.
- Both chicken Bmf isoforms induced apoptosis post-transfection, with Bmf(S) exhibiting higher activity.
- Chicken Bmf mRNA expression is ubiquitous throughout embryonic development.
Conclusions:
- The study describes the cloning and characterization of chicken Bmf and its novel short isoform, Bmf(S).
- Chicken Bmf(S) demonstrates potent proapoptotic activity and lacks the DLC2-binding domain.
- Ubiquitous expression suggests a role for chicken Bmf in embryonic development control.
