RESUMEN
Here we investigate the function of zebrafish Bcl-2 family proteins and demonstrate important conservation of function across zebrafish and mammalian systems. We have isolated a zebrafish ortholog of mammalian BIM and show that it is the most toxic of the zebrafish BH3-only genes examined, sharing this characteristic with the mammalian BIM gene. The zebrafish bad gene shows a complete lack of embryonic lethality, but like mammalian BAD, its pro-apoptotic activity is regulated through phosphorylation of critical serines. We also found that the pattern of mitochondrial dysfunction observed by zebrafish BH3 domain peptides in a mammalian cytochrome c release assay recapitulates the pattern of embryonic lethality induced by the respective mRNA injections in vivo. In contrast to zebrafish Bim, Bid exhibited only weak binding to zebrafish Bcl-2 and moderate-to-weak overall lethality in zebrafish embryos and isolated mitochondria. Given that zebrafish Bcl-2 binds strongly to mammalian BID and BIM peptides and proteins, the protein identified as the zebrafish Bid ortholog has different properties than mammalian BID. Overall, our results demonstrate the high degree of functional conservation between zebrafish and mammalian Bcl-2 family proteins, thus validating the zebrafish as a model system to further dissect the molecular mechanisms that regulate apoptosis in future forward genetic and chemical modifier screens.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/fisiología , Proteínas de la Membrana/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas de Pez Cebra/fisiología , Secuencia de Aminoácidos , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Línea Celular , Sistema Nervioso Central/efectos de la radiación , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Mutación , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/química , Tolerancia a Radiación , Homología de Secuencia de Aminoácido , Serina/genética , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteína Letal Asociada a bcl/química , Proteína Letal Asociada a bcl/metabolismoRESUMEN
The zebrafish has emerged as a powerful genetic model of cancer, but has been limited by the use of stable transgenic approaches to induce disease. Here, a co-injection strategy is described that capitalizes on both the numbers of embryos that can be microinjected and the ability of transgenes to segregate together and exert synergistic effects in forming tumors. Using this mosaic transgenic approach, gene pathways involved in tumor initiation and radiation sensitivity have been identified.