Aggregation and Amyloid Fibril Formation Induced by Chemical Dimerization of Recombinant Prion Protein in Physiological-like Conditions

Prion diseases are caused by the conversion of a cellular protein (PrPC) into a misfolded, aggregated isoform (PrPRes). Misfolding of recombinant PrPC in the absence of PrPRes template, cellular factors, denaturing agents, or at neutral pH has not been achieved. A number of studies indicate that dimerization of PrPC may be a key step in the aggregation process. In an effort to understand the molecular event that may activate misfolding of PrPC in more relevant physiological conditions, we tested if enforced dimerization of PrPC may induce a conformational change reminiscent of the conversion of PrPC to PrPRes. We used a well described inducible dimerization strategy whereby a chimeric PrPC composed of a modified FK506-binding protein (Fv) fused with PrPC and termed Fv-PrP is incubated in the presence of a monomeric FK506 or dimerizing AP20187 ligand. Addition of AP20187 but not FK506 to recombinant Fv-PrP (rFv-PrP) in physiological-like conditions resulted in a rapid conformational change characterized by an increase in beta-sheet structure and simultaneous aggregation of the protein. Aggregates were partially resistant to proteinase K and induced the conversion of soluble rFv-PrP in serial seeding experiments. As judged from thioflavin T binding and electron microscopy, aggregates converted to amyloid fibers. Aggregates were toxic to cultured cells, whereas soluble rFv-PrP and amyloid fibers were harmless. This study strongly supports the proposition that dimerization of PrPC is a key pathological primary event in the conversion of PrPC and may initiate the pathogenesis of prion diseases.