RESUMEN
Intracellular accumulation of polyglutamine (polyQ)-expanded Huntingtin (Htt) protein is a hallmark of Huntington's disease (HD). This study evaluated whether activation of Sirt1 by the anti-cancer agent, ß-lapachone (ß-lap), induces autophagy in human neuroblastoma SH-SY5Y cells, thereby reducing intracellular levels of polyQ aggregates and their concomitant cytotoxicity. Treatment of cells with ß-lap markedly diminished the cytotoxicity induced by forced expression of Htt exon 1 containing a pathogenic polyQ stretch fused to green fluorescent protein (HttEx1(97Q)-GFP). ß-lap increased autophagy in SH-SY5Y cells, as evidenced by the increased formation of LC3-II and autolysosomes. Furthermore, ß-lap reduced HttEx1(97Q)-GFP aggregation, which was significantly prevented by co-incubation with 3-methyladenine, an inhibitor of autophagy. ß-lap increased Sirt1 activity, as shown by the increased deacetylation of the Sirt1 substrates, PARP-1 and Atg5, and the nuclear translocation of FOXO1. Both the induction of autophagy and attenuation of HttEx1(97Q)-GFP aggregation by ß-lap were significantly prevented by co-incubation with sirtinol, a general sirtuin inhibitor or by co-transfection with shRNA against Sirt1. The pro-autophagic actions of ß-lap were further investigated in a transgenic Caenorhabditis elegans (C. elegans) line that expressed Q67 fused to cyanine fluorescent protein (Q67). Notably, ß-lap reduced the number of Q67 puncta and restored Q67-induced defects in motility, which were largely prevented by pre-treatment with RNAi against sir-2.1, the C. elegans orthologue of Sirt1. Collectively, these data suggest that ß-lap induces autophagy through activation of Sirt1, which in turn leads to a reduction in polyQ aggregation and cellular toxicity. Thus, ß-lap provides a novel therapeutic opportunity for the treatment of HD.
Asunto(s)
Autofagia/efectos de los fármacos , Naftoquinonas/farmacología , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/patología , Péptidos/farmacología , Sirtuina 1/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/crecimiento & desarrollo , Animales Modificados Genéticamente/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteína Huntingtina , Técnicas para Inmunoenzimas , Inmunoprecipitación , Microscopía Fluorescente , Proteínas del Tejido Nervioso/genética , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Péptidos/química , Péptidos/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Inhibidores de la Transcriptasa Inversa/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirtuina 1/antagonistas & inhibidores , Sirtuina 1/genéticaRESUMEN
BACKGROUND: Polyglutamine (polyQ)-induced protein aggregation is the hallmark of a group of neurodegenerative diseases, including Huntington's disease. We hypothesized that a protease that could cleave polyQ stretches would intervene in the initial events leading to pathogenesis in these diseases. To prove this concept, we aimed to generate a protease possessing substrate specificity for polyQ stretches. METHODOLOGY/PRINCIPAL FINDINGS: Hepatitis A virus (HAV) 3C protease (3CP) was subjected to engineering using a yeast-based method known as the Genetic Assay for Site-specific Proteolysis (GASP). Analysis of the substrate specificity revealed that 3CP can cleave substrates containing glutamine at positions P5, P4, P3, P1, P2', or P3', but not substrates containing glutamine at the P2 or P1' positions. To accommodate glutamine at P2 and P1', key residues comprising the active sites of the S2 or S1' pockets were separately randomized and screened. The resulting sets of variants were combined by shuffling and further subjected to two rounds of randomization and screening using a substrate containing glutamines from positions P5 through P3'. One of the selected variants (Var26) reduced the expression level and aggregation of a huntingtin exon1-GFP fusion protein containing a pathogenic polyQ stretch (HttEx1(97Q)-GFP) in the neuroblastoma cell line SH-SY5Y. Var26 also prevented cell death and caspase 3 activation induced by HttEx1(97Q)-GFP. These protective effects of Var26 were proteolytic activity-dependent. CONCLUSIONS/SIGNIFICANCE: These data provide a proof-of-concept that proteolytic cleavage of polyQ stretches could be an effective modality for the treatment of polyQ diseases.
Asunto(s)
Muerte Celular/efectos de los fármacos , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Neuronas/citología , Péptidos/metabolismo , Péptidos/toxicidad , Ingeniería de Proteínas , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteasas Virales 3C , Línea Celular Tumoral , Cisteína Endopeptidasas/química , Virus de la Hepatitis A/enzimología , Humanos , Neuronas/efectos de los fármacos , Proteolisis , Especificidad por Sustrato , Proteínas Virales/químicaRESUMEN
BACKGROUND: The nuclear inclusion a (NIa) protease of turnip mosaic virus (TuMV) is responsible for the processing of the viral polyprotein into functional proteins. NIa was previously shown to possess a relatively strict substrate specificity with a preference for Val-Xaa-His-Gln↓, with the scissile bond located after Gln. The presence of the same consensus sequence, Val(12)-His-His-Gln(15), near the presumptive α-secretase cleavage site of the amyloid-ß (Aß) peptide led us to hypothesize that NIa could possess activity against Aß. METHODOLOGY/PRINCIPAL FINDINGS: Western blotting results showed that oligomeric as well as monomeric forms of Aß can be degraded by NIa in vitro. The specific cleavage of Aß was further confirmed by mass spectrometry analysis. NIa was shown to exist predominantly in the cytoplasm as observed by immunofluorescence microscopy. The overexpression of NIa in B103 neuroblastoma cells resulted in a significant reduction in cell death caused by both intracellularly generated and exogenously added Aß. Moreover, lentiviral-mediated expression of NIa in APP(sw)/PS1 transgenic mice significantly reduced the levels of Aß and plaques in the brain. CONCLUSIONS/SIGNIFICANCE: These results indicate that the degradation of Aß in the cytoplasm could be a novel strategy to control the levels of Aß, plaque formation, and the associated cell death.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Brassica napus/virología , Endopeptidasas/química , Regulación Enzimológica de la Expresión Génica , Virus del Mosaico/enzimología , Proteínas Virales/química , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Animales , Línea Celular Tumoral , Supervivencia Celular , Citoplasma/metabolismo , Progresión de la Enfermedad , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Virus del Mosaico/genética , Neuronas/citología , Ratas , Fracciones Subcelulares/metabolismo , Especificidad por SustratoRESUMEN
By using an improved genetic screening system, variants of the HAV 3CP protease which exhibit altered P2 specificity were obtained. We randomly mutated the His145, Lys146, Lys147, and Leu155 residues that constitute the S2 pocket of 3CP and then isolated variants that preferred substrates with Gln over the original Thr at the P2 position using a yeast-based screening method. One of the isolated variants cleaved the Gln-containing peptide substrate more efficiently in vitro, proving the efficiency of our method in isolating engineered proteases with desired substrate selectivity.