Aggregates of scrapie-associated prion protein induce the cell-free conversion of protease-sensitive prion protein to the protease-resistant state

Abstract

Introduction: Scrapie infection instigates the in vivo conversion of normal, protease-sensitive prion protein (PrPc) into a protease-resistant form (PrP Sc) by an unknown mechanism. In vitro studies have indicated that PrP Sc can induce this conversion, consistent with proposals that PrP Sc itself might be the infectious scrapie agent. Using this cell-free model of the PrP C to PrP Sc conversion, we have studied the dependence of conversion on reactant concentration, and the properties of the PrP Sc-derived species that has converting activity. Results: The cell-free conversion of 35S PrP C to the proteinase K-resistant form was dependent on the reaction time and initial concentrations of PrP Sc (above an apparent minimum threshold concentration) and 35S PrP C. Analysis of the physical size of the converting activity indicated that detectable converting activity was associated only with aggregates. Under mildly chaotropic conditions, which partially disaggregated PrP SC and enhanced the converting activity, the active species were heterogeneous in size, but larger than either effectively solubilized PrP or molecular weight standards of ≈2000 kDa. Conclusions: The entity responsible for the converting activity was many times larger than a soluble PrP monomer and required a threshold concentration of PrP Sc. These results are consistent with a nucleated polymerization mechanism of PrP Sc formation and inconsistent with a heterodimer mechanism.