RESUMEN
Aggresomes are associated with many neurodegenerative disorders, including Parkinson's disease, and polyglutamine disorders such as Huntington's disease. These inclusions commonly contain ubiquitylated proteins. The stage at which these proteins are ubiquitylated remains unclear. A malfunction of the ubiquitin/proteasome system (UPS) may be associated with their formation. Conversely, it may reflect an unsuccessful attempt by the cell to remove them. Previously, we demonstrated that overexpression of Parkin, a ubiquitin-protein ligase associated with autosomal recessive juvenile Parkinsonism, generates aggresome-like inclusions in UPS compromised cells. Mutations in the de-ubiquitylating enzyme, UCH-L1, cause a rare form of Parkinsonism. We now demonstrate that overexpression of UCH-L1 also forms ribbon-like aggresomes in response to proteasomal inhibition. Disease-associated mutations, which affect enzymatic activities, significantly increased the number of inclusions. UCH-L1 aggresomes co-localized with ubiquitylated proteins, HSP70, gamma-tubulin and, to a lesser extent, the 20S proteasome and the chaperone BiP. Similar to Parkin inclusions, we found UCH-L1 aggresomes to be surrounded by a tubulin rather than a vimentin cage-like structure. Furthermore, UCH-L1 aggregates with Parkin and alpha-synuclein in some, but not all inclusions, suggesting the heterogeneous nature of these inclusion bodies. This study provides additional evidence that aggregation-prone proteins are likely to recruit UPS components in an attempt to clear proteins from failing proteasomes. Furthermore, UCH-L1 accumulation is likely to play a pathological role in inclusion formation in Parkinson's disease.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Cuerpos de Inclusión/metabolismo , Complejos Multienzimáticos/metabolismo , Neuronas/metabolismo , Enfermedad de Parkinson/genética , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Animales , Células COS , Proteínas Portadoras/metabolismo , Línea Celular , Chlorocebus aethiops , Chaperón BiP del Retículo Endoplásmico , Inhibidores Enzimáticos/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Cuerpos de Inclusión/química , Cuerpos de Inclusión/patología , Cuerpos de Lewy/metabolismo , Cuerpos de Lewy/patología , Sustancias Macromoleculares , Chaperonas Moleculares/metabolismo , Complejos Multienzimáticos/antagonistas & inhibidores , Mutagénesis Sitio-Dirigida , Mutación , Proteínas del Tejido Nervioso/metabolismo , Neuronas/patología , Enfermedad de Parkinson/metabolismo , Pruebas de Precipitina , Complejo de la Endopetidasa Proteasomal , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad , Sinucleínas , Transfección , Tubulina (Proteína)/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , alfa-SinucleínaRESUMEN
Human homologue of Drosophila ariadne (HHARI) is a RING-IBR-RING domain protein identified through its ability to bind the human ubiquitin-conjugating enzyme, UbcH7. We now demonstrate that HHARI also interacts with the eukaryotic mRNA cap binding protein, translation initiation factor 4E homologous protein (4EHP), via the N-terminal RING1 finger of HHARI. HHARI, 4EHP and UbcH7 do not form a stable heterotrimeric complex as 4EHP cannot immunoprecipitate UbcH7 even in the presence of HHARI. Overexpression of 4EHP and HHARI in mammalian cells leads to polyubiquitylation of 4EHP. By contrast, HHARI does not promote its own autoubiquitylation. Thus, by promoting the ubiquitin-mediated degradation of 4EHP, HHARI may have a role in both protein degradation and protein translation.
Asunto(s)
Proteínas Portadoras/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Ubiquitina/metabolismo , Proteínas Portadoras/química , Línea Celular , Humanos , Pruebas de Precipitina , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Homología de Secuencia de Aminoácido , Transfección , Técnicas del Sistema de Dos Híbridos , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
Association between protein inclusions and neurodegenerative diseases, including Parkinson's and Alzheimer's diseases, and polyglutamine disorders, has been widely documented. Although ubiquitin is conjugated to many of these aggregated proteins, the 26S proteasome does not efficiently degrade them. Mutations in the ubiquitin-protein ligase Parkin are associated with autosomal recessive juvenile Parkinsonism. Although Parkin-positive inclusions are not detected in brains of autosomal recessive juvenile Parkinsonism patients, Parkin is found in Lewy bodies in sporadic disease. This suggests that loss of Parkin ligase activity via mutation, or sequestration to Lewy bodies, is a contributory factor to sporadic disease onset. We now demonstrate that decreased proteasomal activity causes formation of large, noncytotoxic inclusions within the cytoplasm of both neuronal and nonneuronal cells overexpressing Parkin. This is not a general phenomenon as there is an absence of similar inclusions when HHARI, a structural homolog of Parkin, is overexpressed. The inclusions colocalize with ubiquitin and with proteasomes. Furthermore, Parkin inclusions colocalize with gamma-tubulin, acetylated alpha-tubulin, and cause redistribution of vimentin, suggesting aggresome-like properties. Our data imply that lower proteasomal activity, previously observed in brain tissue of Parkinson's disease patients, leads to Parkin accumulation and a concomitant reduction in ligase activity, thereby promoting Lewy body formation.