RESUMEN
NPGPx is a member of the glutathione peroxidase (GPx) family; however, it lacks GPx enzymatic activity due to the absence of a critical selenocysteine residue, rendering its function an enigma. Here, we show that NPGPx is a newly identified stress sensor that transmits oxidative stress signals by forming the disulfide bond between its Cys57 and Cys86 residues. This oxidized form of NPGPx binds to glucose-regulated protein (GRP)78 and forms covalent bonding intermediates between Cys86 of NPGPx and Cys41/Cys420 of GRP78. Subsequently, the formation of the disulfide bond between Cys41 and Cys420 of GRP78 enhances its chaperone activity. NPGPx-deficient cells display increased reactive oxygen species, accumulated misfolded proteins, and impaired GRP78 chaperone activity. Complete loss of NPGPx in animals causes systemic oxidative stress, increases carcinogenesis, and shortens life span. These results suggest that NPGPx is essential for releasing excessive ER stress by enhancing GRP78 chaperone activity to maintain physiological homeostasis.
Asunto(s)
Proteínas Portadoras/metabolismo , Estrés del Retículo Endoplásmico , Proteínas de Choque Térmico/metabolismo , Estrés Oxidativo , Peroxidasas/metabolismo , Deficiencias en la Proteostasis/enzimología , Transducción de Señal , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Cisteína , Daño del ADN , Disulfuros/metabolismo , Relación Dosis-Respuesta a Droga , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Fibroblastos/enzimología , Fibroblastos/patología , Glutatión Peroxidasa , Proteínas de Choque Térmico/genética , Homeostasis , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Mutación , Oxidantes/farmacología , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Peroxidasas/genética , Unión Proteica , Pliegue de Proteína , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/patología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Tiempo , TransfecciónRESUMEN
Glucagon is important for regulating lipid metabolism in part through its inhibition of fatty acid synthesis in adipocytes. Acetyl-CoA carboxylase 1 (ACC1) is the rate-limiting enzyme for fatty acid synthesis. Glucagon has been proposed to activate cAMP-dependent protein kinase A (PKA), which phosphorylates ACC1 to attenuate the lipogenic activity of ACC1. Because AMP-activated protein kinase (AMPK) also inhibits fatty acid synthesis by phosphorylation of ACC1, we examined the involvement of AMPK and its upstream kinase in the glucagon-elicited signaling in adipocytes in vitro and in vivo. LC-MS-MS analysis suggested that ACC1 was phosphorylated only at Ser(79), an AMPK-specific site, in glucagon-treated adipocytes. Pharmacological inhibitors and siRNA knockdown of AMPK or PKA in adipocytes demonstrate that glucagon regulates ACC1 and ACC2 activity through AMPK but not PKA. By using Ca(2+)/calmodulin-dependent protein kinase kinase-ß knockout (CaMKKß(-/-)) mice and cultured adipocytes, we further show that glucagon activates the CaMKKß/AMPK/ACC cascade. Additionally, fasting increases the phosphorylation of AMPK and ACC in CaMKKß(+/+) but not CaMKKß(-/-) mice. These results indicate that CaMKKß/AMPK signaling is an important molecular component in regulating lipid metabolism in adipocytes responding to glucagon and could be a therapeutic target for the dysregulation of energy storage.