ABSTRACT
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.
Subject(s)
Active Transport, Cell Nucleus/genetics , C9orf72 Protein/chemistry , Peptides/chemistry , beta Karyopherins/chemistry , Binding Sites , C9orf72 Protein/genetics , C9orf72 Protein/metabolism , Cloning, Molecular , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HeLa Cells , Humans , Models, Molecular , Nuclear Localization Signals/genetics , Nuclear Localization Signals/metabolism , Peptides/genetics , Peptides/metabolism , Phase Transition , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , RNA-Binding Protein FUS/genetics , RNA-Binding Protein FUS/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , beta Karyopherins/antagonists & inhibitors , beta Karyopherins/genetics , beta Karyopherins/metabolismABSTRACT
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.
Subject(s)
DNA/chemistry , Guanine/chemistry , Binding Sites , Cations , Methylation , Peptide Nucleic Acids/chemistry , Static ElectricityABSTRACT
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.
Subject(s)
Coordination Complexes/chemistry , DNA/chemistry , Peptide Nucleic Acids/chemistry , Ruthenium/chemistry , Base Sequence , Nucleic Acid Denaturation , Nucleic Acid HybridizationABSTRACT
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.
