RESUMEN
Members of the Inhibitor of Apoptosis Protein (IAP) family block activation of the intrinsic cell death machinery by binding to and neutralizing the activity of pro-apoptotic caspases. In Drosophila melanogaster, the pro-apoptotic proteins Reaper (Rpr), Grim and Hid (head involution defective) all induce cell death by antagonizing the anti-apoptotic activity of Drosophila IAP1 (DIAP1), thereby liberating caspases. Here, we show that in vivo, the RING finger of DIAP1 is essential for the regulation of apoptosis induced by Rpr, Hid and Dronc. Furthermore, we show that the RING finger of DIAP1 promotes the ubiquitination of both itself and of Dronc. Disruption of the DIAP1 RING finger does not inhibit its binding to Rpr, Hid or Dronc, but completely abrogates ubiquitination of Dronc. Our data suggest that IAPs suppress apoptosis by binding to and targeting caspases for ubiquitination.
Asunto(s)
Apoptosis/fisiología , Caspasas/metabolismo , Proteínas de Drosophila/metabolismo , Ubiquitina/metabolismo , Animales , Células Cultivadas , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas Inhibidoras de la Apoptosis , Ligasas/metabolismo , Mutagénesis/fisiología , Neuropéptidos/metabolismo , Péptidos/metabolismo , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/enzimología , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Retina/citología , Retina/enzimología , Ubiquitina-Proteína LigasasAsunto(s)
Infecciones Bacterianas/microbiología , Micosis/microbiología , Animales , Bacterias/genética , Bacterias/aislamiento & purificación , Infecciones Bacterianas/inmunología , Fenómenos Fisiológicos Bacterianos , Políticas Editoriales , Hongos/genética , Hongos/aislamiento & purificación , Hongos/fisiología , Interacciones Huésped-Patógeno , Humanos , Micosis/inmunologíaRESUMEN
The Drosophila inhibitor of apoptosis protein DIAP1 ensures cell viability by directly inhibiting caspases. In cells destined to die this IAP-mediated inhibition of caspases is overcome by IAP-antagonists. Genetic evidence indicates that IAP-antagonists are non-equivalent and function synergistically to promote apoptosis. Here we provide biochemical evidence for the non-equivalent mode of action of Reaper, Grim, Hid and Jafrac2. We find that these IAP-antagonists display differential and selective binding to specific DIAP1 BIR domains. Consistently, we show that each DIAP1 BIR region associates with distinct caspases. The differential DIAP1 BIR interaction seen both between initiator and effector caspases and within IAP-antagonist family members suggests that different IAP-antagonists inhibit distinct caspases from interacting with DIAP1. Surprisingly, we also find that the caspase-binding residues of XIAP predicted to be strictly conserved in caspase-binding IAPs, are absent in DIAP1. In contrast to XIAP, residues C-terminal to the DIAP1 BIR1 domain are indispensable for caspase association. Our studies on DIAP1 and caspases expose significant differences between DIAP1 and XIAP suggesting that DIAP1 and XIAP inhibit caspases in different ways.