NR AUDZ
AU Strom,A.; Diecke,S.; Hunsmann,G.; Stuke,A.W.
TI Identification of prion protein binding proteins by combined use of far-Western immunoblotting, two dimensional gel electrophoresis and mass spectrometry
QU Proteomics 2006 Jan; 6(1): 26-34
PT journal article
AB The cellular prion protein (PrPc), a highly conserved glycoprotein predominantly expressed by neuronal cells, can convert into an abnormal isoform (PrPsc) and provoke a transmissible spongiform encephalopathy. In spite of many studies, the physiological function of PrPc remains unknown. Recent findings suggest that PrPc is a multifunctional protein participating in several cellular processes. Using recombinant human PrP as a probe, we performed far-Western immunoblotting (protein overlay assay) to detect cellular PrPc interactors. Brain extracts of wild-type and PrP knockout mice were screened by far-Western immunoblotting for PrP-specific interactions. Subsequently, putative ligands were isolated by 2-DE and identified by MALDI-TOF MS, enabling identification of heterogeneous nuclear ribonucleoprotein A2/B1 and aldolase C as novel interaction partners of PrPc. These data provide the first evidence of a molecule indicating a mechanism for the predicted involvement of PrPc in nucleic acid metabolisms. In summary, we have shown the successful combination of 2-DE with far-Western immunoblotting and MALDI-TOF MS for identification of new cellular binding partners of a known protein. Especially the application of this technique to investigate other neurodegenerative diseases is promising.
MH Animals; Blotting, Far-Western/*methods; Brain/metabolism; Cytosol/metabolism; Electrophoresis, Gel, Two-Dimensional/*methods; Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism; Immunoprecipitation; Membrane Proteins/*metabolism; Mice; Nerve Tissue Proteins/metabolism; Prions/*metabolism; Protein Binding; Research Support, Non-U.S. Gov't; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/*methods
AD Molecular Medicine, Ottawa Health Research Institute, Lab N1, Box 221, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada. astrom@ohri.ca
SP englisch
PO Deutschland