RESUMEN
Apaf1 is a key regulator of the mitochondrial intrinsic pathway of apoptosis, as it activates executioner caspases by forming the apoptotic machinery apoptosome. Its genetic regulation and its post-translational modification are crucial under the various conditions where apoptosis occurs. Here we describe Ku70/86, a mediator of non-homologous end-joining pathway of DNA repair, as a novel regulator of Apaf1 transcription. Through analysing different Apaf1 promoter mutants, we identified an element repressing the Apaf1 promoter. We demonstrated that Ku70/86 is a nuclear factor able to bind this repressing element and downregulating Apaf1 transcription. We also found that Ku70/86 interaction with Apaf1 promoter is dynamically modulated upon DNA damage. The effect of this binding is a downregulation of Apaf1 expression immediately following the damage to DNA; conversely, we observed Apaf1 upregulation and apoptosis activation when Ku70/86 unleashes the Apaf1-repressing element. Therefore, besides regulating DNA repair, our results suggest that Ku70/86 binds to the Apaf1 promoter and represses its activity. This may help to inhibit the apoptosome pathway of cell death and contribute to regulate cell survival.
Asunto(s)
Antígenos Nucleares/metabolismo , Factor Apoptótico 1 Activador de Proteasas/genética , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Animales , Antígenos Nucleares/química , Muerte Celular/efectos de los fármacos , Línea Celular , Daño del ADN/genética , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Proteínas de Unión al ADN/química , Etopósido/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Autoantígeno Ku , Ratones , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Proteínas Represoras/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transcripción Genética/efectos de los fármacosRESUMEN
Apoptosis-inducing factor (AIF) has important supportive as well as potentially lethal roles in neurons. Under normal physiological conditions, AIF is a vital redox-active mitochondrial enzyme, whereas in pathological situations, it translocates from mitochondria to the nuclei of injured neurons and mediates apoptotic chromatin condensation and cell death. In this study, we reveal the existence of a brain-specific isoform of AIF, AIF2, whose expression increases as neuronal precursor cells differentiate. AIF2 arises from the utilization of the alternative exon 2b, yet uses the same remaining 15 exons as the ubiquitous AIF1 isoform. AIF1 and AIF2 are similarly imported to mitochondria in which they anchor to the inner membrane facing the intermembrane space. However, the mitochondrial inner membrane sorting signal encoded in the exon 2b of AIF2 is more hydrophobic than that of AIF1, indicating a stronger membrane anchorage of AIF2 than AIF1. AIF2 is more difficult to be desorbed from mitochondria than AIF1 on exposure to non-ionic detergents or basic pH. Furthermore, AIF2 dimerizes with AIF1, thereby preventing its release from mitochondria. Conversely, it is conceivable that a neuron-specific AIF isoform, AIF2, may have been 'designed' to be retained in mitochondria and to minimize its potential neurotoxic activity.
Asunto(s)
Factor Inductor de la Apoptosis/metabolismo , Encéfalo/metabolismo , Mitocondrias/metabolismo , Secuencia de Aminoácidos , Animales , Factor Inductor de la Apoptosis/química , Factor Inductor de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Humanos , Ratones , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Neuronas/citología , Neuronas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alineación de SecuenciaRESUMEN
Fas-associated factor 1 (Faf1) has been described as a Fas-binding pro-apoptotic protein and as a component of the death-inducing signaling complex (DISC) in Fas-mediated apoptosis. Faf1 is able to potentiate Fas-induced apoptosis in several cell lines, although its specific functions are still not clear. Here we show that Faf1 is highly expressed in several areas of the developing telencephalon. Its expression pattern appears to be dynamic at different embryonic stages and to be progressively confined within limited territories. To decipher the specific role of Faf1 in developing brain, we used cDNA over-expression and mRNA down-regulation experiments to modulate Faf1 expression in telencephalic neural precursor cells, and we showed that in neural cell death Faf1 acts as a Fas-independent apoptotic enhancer. Moreover, we found that Faf1 protein level is down-regulated during apoptosis in a caspase- and Apaf1-dependent manner.