RESUMEN
The neuronal long isoform of Fas Apoptotic Inhibitory Molecule (FAIM-L) protects from death receptor (DR)-induced apoptosis, yet its mechanism of protection remains unknown. Here, we show that FAIM-L protects rat neuronal Type II cells from Fas-induced apoptosis. XIAP has previously emerged as a molecular discriminator that is upregulated in Type II and downregulated in Type I apoptotic signaling. We demonstrate that FAIM-L requires sustained endogenous levels of XIAP to protect Type II cells as well as murine cortical neurons from Fas-induced apoptosis. FAIM-L interacts with the BIR2 domain of XIAP through an IAP-binding motif, the mutation of which impairs the antiapoptotic function of FAIM-L. Finally, we report that FAIM-L inhibits XIAP auto-ubiquitinylation and maintains its stability, thus conferring protection from apoptosis. Our results bring new understanding of the regulation of endogenous XIAP by a DR antagonist, pointing out at FAIM-L as a promising therapeutic tool for protection from apoptosis in pathological situations where XIAP levels are decreased.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/farmacología , Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Fármacos Neuroprotectores , Ubiquitinación/efectos de los fármacos , Proteína Inhibidora de la Apoptosis Ligada a X/fisiología , Receptor fas/fisiología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Citocromos c/metabolismo , Femenino , Inmunoprecipitación , Proteínas Inhibidoras de la Apoptosis/genética , Lentivirus/genética , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Mutagénesis Sitio-Dirigida , Células PC12 , Plásmidos/genética , Unión Proteica , Conformación Proteica , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína X Asociada a bcl-2/metabolismo , Receptor fas/genéticaRESUMEN
TNFα can promote either cell survival or cell death. The activation of NF-κB plays a central role in cell survival while its inhibition makes TNFα-triggered cytotoxicity possible. Here, we report that the overexpression of a non-degradable mutant of the inhibitor of NF-κB (super-repressor (SR)-IκBα) sensitizes HeLa cells towards TNFα-induced apoptosis, involving caspases activation and cytocrome C release from the mitochondria. Interestingly, we describe that the specific knockdown of Bcl-xL, but not that of Bcl-2, Bcl-w or Mcl-1, renders cells sensitive to TNFα-induced apoptosis. This cytotoxic effect occurs without altering the activation of NF-κB. Then, the activation of the NF-κB pathway is not sufficient to protect Bcl-xL-downregulated cells from TNFα-induced cell death, meaning that TNFα is not able to promote cell survival in the absence of Bcl-xL. In addition, Bcl-xL silencing does not potentiate the cytotoxicity afforded by the cytokine in SR-IκBα-overexpressing cells. This indicates that TNFα-induced apoptosis in SR-IκBα-overexpressing cells relies on the protein levels of Bcl-xL. We have corroborated these findings using RD and DU-145 cells, which also become sensitive to TNFα-induced apoptosis after Bcl-xL knockdown despite that NF-κB remains activated. Altogether, our results point out that the impairment of the anti-apoptotic function of Bcl-xL should make cells sensitive towards external insults circumventing the TNFα-triggered NF-κB-mediated cytoprotective effect. Hence, the specific inhibition of Bcl-xL could be envisaged as a promising alternative strategy against NF-κB-dependent highly chemoresistant proliferative malignancies.
