RESUMEN
It is well established that during the execution of the apoptotic cascade, activated caspase 3 releases sterol regulatory element-binding proteins (SREBP) from the membrane of the endoplasmic reticulum in a proteolytic reaction that is distinct from their normal sterol-dependent activation. However, it is not known whether these transcription factors are capable of activating sterol-responsive genes under such conditions. The construction of SRE expression vectors has permitted characterization of the apoptotic activation of SREBP. Cell lines stably expressing the plasma membrane marker CD32, or GFP, under the control of the SRE promoter were shown to modulate SRE gene expression on the basis of the levels of available sterols. However, during the induction of apoptosis, expression of CD32 and GFP was highly induced, even in the presence of ample sterols. Apoptotic induction of sterol-regulated genes was due to activation of caspase 3 and was impervious to treatment with sphingomyelinase, indicating that activation of SRE genes during apoptosis is sterol independent. Further characterization of this apoptotic response indicated that sterol-regulated genes are activated at an early stage in the apoptotic cascade, preceding the externalization of phosphatidylserine on the plasma membrane of apoptotic cells. These results suggest that activation of sterol-responsive genes early during apoptosis may play a role in the proper execution of this program.
Asunto(s)
Apoptosis , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Elementos de Respuesta/genética , Esteroles/farmacología , Factores de Transcripción/metabolismo , Apoptosis/efectos de los fármacos , Camptotecina/farmacología , Caspasa 3 , Caspasas/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Genes Reporteros/genética , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Fosfatidilserinas/metabolismo , Receptores de IgG/inmunología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Proteína 2 de Unión a Elementos Reguladores de EsterolesRESUMEN
Niemann-Pick type C (NPC) disease is a severe cell lipidosis characterized by the accumulation of unesterified cholesterol in the endosomal/lysosomal system. Recently the primary disease-causing gene, NPC1, was identified, but few clues regarding its potential function(s) could be derived from its predicted amino acid sequence. Therefore, efforts were directed at characterizing the subcellular location of the NPC1 protein. Initial studies with a FLAG-tagged NPC1 cDNA demonstrated that NPC1 is a glycoprotein that associates with the membranes of a population of cytoplasmic vesicles. Immunofluorescence microscopy using anti-NPC1 polyclonal antibodies confirmed this analysis. Double-label immunofluorescence microscopy and subcellular fractionation studies indicated that NPC1 associates predominantly with late endosomes (Rab9 GTPase-positive vesicles) and, to a lesser extent, with lysosomes and the trans-Golgi network. When cholesterol egress from lysosomes was blocked by treatment of cells with U18666A, the NPC1 location shifted from late endosomes to the trans-Golgi network and lysosomes. Subcellular fractionation of liver homogenates from U18666A-treated mice confirmed these observations. These data suggest that U18666A may inhibit the retrograde transport of NPC1 from lysosomes to late endosomes for subsequent transfer to the trans-Golgi network.
Asunto(s)
Proteínas Portadoras , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Lisosomas/metabolismo , Proteínas/metabolismo , Androstenos/farmacología , Animales , Anticolesterolemiantes/farmacología , Transporte Biológico/efectos de los fármacos , Células COS , Línea Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/metabolismo , Microscopía Fluorescente , Proteína Niemann-Pick C1 , Oligopéptidos , Péptidos/genética , Proteínas/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.