title: Strain-specific effects of reducing agents on the cell-free conversion of recombinant prion protein into a protease-resistant form.
abstract: The pathogenic isoform (PrP(Sc) ) of the host-encoded normal cellular prion protein (PrP(C) ) is believed to be the infectious agent of transmissible spongiform encephalopathies. Spontaneous conversion of α-helix-rich recombinant PrP into the PrP(Sc) -like β-sheet-rich form or aggregation of cytosolic PrP has been found to be accelerated under reducing conditions. However, the effect of reducing conditions on PrP(Sc) -mediated conversion of PrP(C) into PrP(Sc) has remained unknown. In this study, the effect of reducing conditions on the binding of bacterial recombinant mouse PrP (MoPrP) with PrP(Sc) and the conversion of MoPrP into proteinase K-resistant PrP (PrP(res) ) using a cell-free conversion assay was investigated. High concentrations of dithiothreitol did not inhibit either the binding or conversion reactions of PrP(Sc) from five prion strains. Indeed, dithiothreitol significantly accelerated mouse-adapted BSE-seeded conversion. These data suggest that conversion of PrP(Sc) derived from a subset of prion strains is accelerated under reducing conditions, as has previously been shown for spontaneous conversion. Furthermore, the five prion strains used could be classified into three groups according to their efficiency at binding and conversion of MoPrP and cysteine-less mutants under both reducing and nonreducing conditions. The resulting classification is similar to that derived from biological and biochemical strain-specific features.
authors.affiliation: Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan. imamuram@affrc.go.jp
authors.forename: Morikazu
authors.initials: M
authors.lastname: Imamura
authors.forename: Nobuko
authors.initials: N
authors.lastname: Kato
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Okada
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Iwamaru
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Shimizu
authors.forename: Shirou
authors.initials: S
authors.lastname: Mohri
authors.forename: Takashi
authors.initials: T
authors.lastname: Yokoyama
mesh_terms: D000818:Animals; D004229:Dithiothreitol / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D011328:Prions / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D059748:Proteolysis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019163:Reducing Agents / Q000378:metabolism*
chemical_list: D011328:Prions; D011994:Recombinant Proteins; D019163:Reducing Agents; D019286:Endopeptidase K; D004229:Dithiothreitol