Identification of proteins with high affinity for refolded and native PrPC.
PrPC, the cellular prion protein, is widely expressed in most tissues, including brain, muscle and the gastrointestinal tract, but its physiological role remains unclear. During propagation of transmissible spongiform encephalopathies (TSEs), prion protein is converted to the pathological isoform, PrPSc, in a process believed to be mediated by as-yet-unknown host factors. The identification of proteins associated with PrP may provide information about the biology of prions and the pathogenesis of TSEs. In the present work, we report proteins identified from brain tissue based on their ability to bind to recombinant PrP (recPrP) or form multimolecular complexes with native PrPC in the presence of cross-linkers. Immobilized his-tagged recPrP was used as an affinity matrix to isolate PrP-interacting proteins from brain homogenates of normal individuals. In parallel, PrPC-associated proteins were characterized by cross-linking and co-immunoprecipitation assays. The unknown molecules were identified by MS and the results of LC-MS/MS analysis were subsequently verified by Western blot. Both techniques resulted in identification of proteins participating in the formation of cytoskeleton and signal transduction, further supporting the hypothesis that PrP is involved in the organization and function of receptors throughout the nervous system.
Petrakis
Spyros
S
Prion Disease Research Group, Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece.
Sklaviadis
Theodoros
T
D000368:Aged; D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000378:metabolism; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D006801:Humans; D066298:In Vitro Techniques; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D018621:PrPC Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D017096:Prion Diseases / Q000209:etiology / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D025941:Protein Interaction Mapping; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012756:Sheep; D015398:Signal Transduction
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D009419:Nerve Tissue Proteins; D018621:PrPC Proteins; D011994:Recombinant Proteins