RESUMEN
Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-ß2 (Kapß2) at 1:1 ratio. The nuclear magnetic resonances of Kapß2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapß2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Proteína C9orf72/química , Péptidos/química , beta Carioferinas/química , Sitios de Unión , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Clonación Molecular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Péptidos/genética , Péptidos/metabolismo , Transición de Fase , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMEN
Oligonucleotides represent powerful DNA-recognition tools, but the formation of undesirable "self-duplexes" becomes more probable with increasing DNA affinity. Herein, we have developed a modified nucleobase with "self-avoiding" properties. Facile methylation of guanine yields a cationic N7-methylguanine, which suppresses the formation of self-duplexes whilst improving DNA affinity through electrostatic interaction.
Asunto(s)
ADN/química , Guanina/química , Sitios de Unión , Cationes , Metilación , Ácidos Nucleicos de Péptidos/química , Electricidad EstáticaRESUMEN
Peptide nucleic acid (PNA) can form a stable duplex with DNA, and, accordingly, directly recognize double-stranded DNA through the formation of a double-duplex invasion complex, wherein a pair of complementary PNA strands form two PNA/DNA duplexes. Because invasion does not require prior denaturation of DNA, PNA holds great potential for in cellulo or in vivo applications. To broaden the applicability of PNA invasion, we developed a new conjugate of PNA with a ruthenium complex. This Ru-PNA conjugate exhibits higher DNA-binding affinity, which results in enhanced invasion efficiency, even under physiological conditions.
Asunto(s)
Complejos de Coordinación/química , ADN/química , Ácidos Nucleicos de Péptidos/química , Rutenio/química , Secuencia de Bases , Desnaturalización de Ácido Nucleico , Hibridación de Ácido NucleicoRESUMEN
This report describes two cases with obstructive jaundice caused by poorly differentiated gastric adenocarcinoma. Computed tomography scans showed circumferential stenosis in the hilar bile ducts. Endoscopic retrograde cholangiopancreatography showed dilatation of the bilateral hepatic ducts and stenosis of the common hepatic ducts from the bifurcation of the bilateral hepatic ducts. The first diagnoses were hilar cholangiocarcinoma and biliary drainage decreased serum bilirubin; however, both patients died of cancer within a short period of time. Autopsies revealed lymphatic vessel invasion and possible subepithelial invasion by gastric adenocarcinoma into the hilar bile ducts. A differential diagnosis should thus be required in suspected cases of hilar cholangiocarcinoma.
