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 (PrP(C)) into a misfolded, aggregated isoform (PrP(Res)). Misfolding of recombinant PrP(C) in the absence of PrP(Res) template, cellular factors, denaturing agents, or at neutral pH has not been achieved. A number of studies indicate that dimerization of PrP(C) may be a key step in the aggregation process. In an effort to understand the molecular event that may activate misfolding of PrP(C) in more relevant physiological conditions, we tested if enforced dimerization of PrP(C) may induce a conformational change reminiscent of the conversion of PrP(C) to PrP(Res). We used a well described inducible dimerization strategy whereby a chimeric PrP(C) composed of a modified FK506-binding protein (Fv) fused with PrP(C) 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 PrP(C) is a key pathological primary event in the conversion of PrP(C) and may initiate the pathogenesis of prion diseases.
Roostaee
Alireza
A
Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Côté
Sébastien
S
Roucou
Xavier
X
D000682:Amyloid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D019281:Dimerization; D006801:Humans; D018621:PrPC Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017096:Prion Diseases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000737:chemistry; D022021:Tacrolimus Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C463061:AP20187; D000682:Amyloid; D018621:PrPC Proteins; D011994:Recombinant Proteins; D022021:Tacrolimus Binding Proteins; D016559:Tacrolimus