Ku protein as the main cellular target of cell-surface-bound circulating DNA

• Published in: Expert opinion on biological therapy Vol. 12 Suppl 1; p. S35
• Main Authors: Cherepanova, Anna V, Bushuev, Alexander V, Duzhak, Tatyana G, Zaporozhchenko, Ivan A, Vlassov, Valentin V, Laktionov, Pavel P
• Format: Journal Article
• Language: English
• Published: England 01.06.2012
• Subjects: Base Sequence; Blotting, Western; Cell Membrane - metabolism; Cells, Cultured; Chromatography, Affinity; DNA - blood; DNA Helicases - isolation & purification; DNA Helicases - metabolism; DNA Primers; Electrophoresis, Polyacrylamide Gel; Electrophoretic Mobility Shift Assay; Humans; Ku Autoantigen; Oligodeoxyribonucleotides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 1744-7682
• DOI: 10.1517/14712598.2012.676035

An immunomodulatory activity of circulating DNA (cirDNA) is implemented via the interactions of cirDNA with the targets exposed on the cell membrane and/or intracellular targets. The goal of this work was to identify the cellular targets of immunoinhibiting cell-surface-bound cirDNA (csbDNA) using its oligodeoxyribonucleotide (ODN) analogs containing the nucleotide motifs frequently found in csbDNA and displaying the same effects. The binding of [(32)P]-labeled single- and double-stranded ODNs (ss- and ds-ODNs) with membrane-cytosolic (MC) extracts and living human umbilical vein endothelial cells (HUVEC) was studied by electromobility shift assay (EMSA). Complexes of biotinylated ODNs with target proteins were affinity isolated using streptavidin Sepharose with subsequent SDS-PAGE and identified by MALDI-TOF mass spectrometry. Both ss- and ds-ODNs form strong ODN-protein complexes with similar electrophoretic mobilities after incubation with the MC extracts of HUVEC either when added extracellularly or lipofected into cells. The ODN-binding proteins were identified as the DNA-binding components of DNA-dependent protein kinase (DNA-PK), namely, Ku70 and Ku80 proteins. Diverse cellular localizations and functions of the Ku proteins demand further clarification of Ku70/80 role as a mediator of the csbDNA immunoinhibiting effects.