RESUMEN
Aggresomes are pericentrosomal cytoplasmic structures into which aggregated, ubiquitinated, misfolded proteins are sequestered. Misfolded proteins accumulate in aggresomes when the capacity of the intracellular protein degradation machinery is exceeded. Previously, we demonstrated that an intact microtubule cytoskeleton is required for the aggresome formation [Johnston et al., 1998: J. Cell Biol. 143:1883-1898]. In this study, we have investigated the involvement of microtubules (MT) and MT motors in this process. Induction of aggresomes containing misfolded DeltaF508 CFTR is accompanied by a redistribution of the retrograde motor cytoplasmic dynein that colocalizes with aggresomal markers. Coexpression of the p50 (dynamitin) subunit of the dynein/dynactin complex prevents the formation of aggresomes, even in the presence of proteasome inhibitors. Using in vitro microtubule binding assays in conjunction with immunogold electron microscopy, our data demonstrate that misfolded DeltaF508 CFTR associate with microtubules. We conclude that cytoplasmic dynein/dynactin is responsible for the directed transport of misfolded protein into aggresomes. The implications of these findings with respect to the pathogenesis of neurodegenerative disease are discussed.
Asunto(s)
Citoplasma/metabolismo , Células Eucariotas/metabolismo , Cuerpos de Inclusión/metabolismo , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Orgánulos/metabolismo , Pliegue de Proteína , Antígenos/metabolismo , Antígenos/ultraestructura , Células Cultivadas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Citoplasma/ultraestructura , Complejo Dinactina , Dineínas/metabolismo , Dineínas/ultraestructura , Células Eucariotas/ultraestructura , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes , Humanos , Inmunohistoquímica , Cuerpos de Inclusión/ultraestructura , Proteínas Luminiscentes , Microscopía Electrónica , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/ultraestructura , Microtúbulos/ultraestructura , Proteínas Motoras Moleculares/ultraestructura , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/fisiopatología , Orgánulos/ultraestructura , Transporte de Proteínas/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The inherited retinal degenerations are typified by retinitis pigmentosa (RP), a heterogeneous group of inherited disorders that causes the destruction of photoreceptor cells, the retinal pigmented epithelium, and choroid. This group of blinding conditions affects over 1.5 million persons worldwide. Approximately 30-40% of human autosomal dominant (AD) RP is caused by dominantly inherited missense mutations in the rhodopsin gene. Here we show that P23H, the most frequent RP mutation in American patients, renders rhodopsin extremely prone to form high molecular weight oligomeric species in the cytoplasm of transfected cells. Aggregated P23H accumulates in aggresomes, which are pericentriolar inclusion bodies that require an intact microtubule cytoskeleton to form. Using fluorescence resonance energy transfer (FRET), we observe that P23H aggregates in the cytoplasm even at extremely low expression levels. Our data show that the P23H mutation destabilizes the protein and targets it for degradation by the ubiquitin proteasome system. P23H is stabilized by proteasome inhibitors and by co-expression of a dominant negative form of ubiquitin. We show that expression of P23H, but not wild-type rhodopsin, results in a generalized impairment of the ubiquitin proteasome system, suggesting a mechanism for photoreceptor degeneration that links RP to a broad class of neurodegenerative diseases.