RESUMEN
Histone H2A monoubiquitination (H2Aub1) by the PRC1 subunit RING1B entails a positive feedback loop, mediated by the RING1B-interacting protein RYBP. We uncover that human RYBP-PRC1 binds unmodified nucleosomes via RING1B but H2Aub1-modified nucleosomes via RYBP. RYBP interactions with both ubiquitin and the nucleosome acidic patch create the high binding affinity that favors RYBP- over RING1B-directed PRC1 binding to H2Aub1-modified nucleosomes; this enables RING1B to monoubiquitinate H2A in neighboring unmodified nucleosomes.
Asunto(s)
Histonas , Nucleosomas , Complejo Represivo Polycomb 1 , Proteínas Represoras , Ubiquitinación , Humanos , Proteínas de Ciclo Celular , Histonas/metabolismo , Histonas/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Modelos Moleculares , Nucleosomas/metabolismo , Nucleosomas/química , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 1/química , Unión Proteica , Proteínas Represoras/metabolismo , Proteínas Represoras/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/químicaRESUMEN
The biological effects of flavonoids on mammal cells are diverse, ranging from scavenging free radicals and anti-cancer activity to anti-influenza activity. Despite appreciable effort to understand the anti-influenza activity of flavonoids, there is no clear consensus about their precise mode-of-action at a cellular level. Here, we report the development and validation of a screening assay based on AlphaScreen technology and illustrate its application for determination of the inhibitory potency of a large set of polyols against PA N-terminal domain (PA-Nter) of influenza RNA-dependent RNA polymerase featuring endonuclease activity. The most potent inhibitors we identified were luteolin with an IC50 of 72 ± 2 nM and its 8-C-glucoside orientin with an IC50 of 43 ± 2 nM. Submicromolar inhibitors were also evaluated by an in vitro endonuclease activity assay using single-stranded DNA, and the results were in full agreement with data from the competitive AlphaScreen assay. Using X-ray crystallography, we analyzed structures of the PA-Nter in complex with luteolin at 2.0 Å resolution and quambalarine B at 2.5 Å resolution, which clearly revealed the binding pose of these polyols coordinated to two manganese ions in the endonuclease active site. Using two distinct assays along with the structural work, we have presumably identified and characterized the molecular mode-of-action of flavonoids in influenza-infected cells.
Asunto(s)
Antivirales/química , Endonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Flavonoides/química , Virus de la Influenza A/enzimología , Proteínas Virales/antagonistas & inhibidores , Antivirales/metabolismo , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Endonucleasas/química , Endonucleasas/metabolismo , Pruebas de Enzimas/métodos , Inhibidores Enzimáticos/metabolismo , Flavonoides/metabolismo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Unión Proteica , Dominios Proteicos , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Relación Estructura-Actividad , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Neuraminidase is the main target for current influenza drugs. Reduced susceptibility to oseltamivir, the most widely prescribed neuraminidase inhibitor, has been repeatedly reported. The resistance substitutions I223V and S247N, alone or in combination with the major oseltamivir-resistance mutation H275Y, have been observed in 2009 pandemic H1N1 viruses. We overexpressed and purified the ectodomain of wild-type neuraminidase from the A/California/07/2009 (H1N1) influenza virus, as well as variants containing H275Y, I223V, and S247N single mutations and H275Y/I223V and H275Y/S247N double mutations. We performed enzymological and thermodynamic analyses and structurally examined the resistance mechanism. Our results reveal that the I223V or S247N substitution alone confers only a moderate reduction in oseltamivir affinity. In contrast, the major oseltamivir resistance mutation H275Y causes a significant decrease in the enzyme's ability to bind this drug. Combination of H275Y with an I223V or S247N mutation results in extreme impairment of oseltamivir's inhibition potency. Our structural analyses revealed that the H275Y substitution has a major effect on the oseltamivir binding pose within the active site while the influence of other studied mutations is much less prominent. Our crystal structures also helped explain the augmenting effect on resistance of combining H275Y with both substitutions.