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Updated: Jun 1, 2026
Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay
Published on: March 10, 2015
Strain-specific effects of reducing agents on the cell-free conversion of recombinant prion protein into a
Morikazu Imamura1, Nobuko Kato, Hiroyuki Okada
1Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan. imamuram@affrc.go.jp
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.
Insights
Reducing conditions accelerate prion protein conversion for some strains, not inhibit it. This finding helps classify prion strains based on their interaction with prion protein under varying conditions.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Transmissible spongiform encephalopathies are linked to the pathogenic prion protein (PrPSc).
- Reducing conditions accelerate spontaneous conversion of normal prion protein (PrPC) to a PrPSc-like form.
- The impact of reducing conditions on PrPSc-mediated conversion remains unclear.
Purpose of the Study:
- To investigate the effect of reducing conditions on PrPSc binding and conversion of mouse PrP (MoPrP).
- To determine if reducing conditions affect PrPSc-mediated conversion using a cell-free assay.
- To classify prion strains based on their conversion efficiency under reducing and nonreducing conditions.
Main Methods:
- Utilized a cell-free conversion assay to study prion protein interactions.
- Investigated the binding and conversion of bacterial recombinant MoPrP with PrPSc.
- Assessed the impact of high concentrations of dithiothreitol (DTT) on binding and conversion.
- Examined the behavior of MoPrP and cysteine-less mutants under reducing and nonreducing conditions.
Main Results:
- High dithiothreitol concentrations did not inhibit PrPSc binding or conversion for five prion strains.
- Dithiothreitol significantly accelerated mouse-adapted BSE-seeded conversion.
- Prion strains were classified into three groups based on binding and conversion efficiency.
- This classification correlated with known biological and biochemical strain features.
Conclusions:
- PrPSc-mediated conversion is accelerated by reducing conditions for a subset of prion strains.
- Reducing conditions do not inhibit PrPSc binding or conversion.
- The study provides a new classification system for prion strains based on their behavior under reducing conditions.
