Aggregates of scrapie-associated prion protein induce the cell-free conversion of protease-sensitive prion protein to the protease-resistant state.
BACKGROUND
Scrapie infection instigates the in vivo conversion of normal, protease-sensitive prion protein (PrPC) into a protease-resistant form (PrPSc) by an unknown mechanism. In vitro studies have indicated that PrPSc can induce this conversion, consistent with proposals that PrPSc itself might be the infectious scrapie agent. Using this cell-free model of the PrPC to PrPSc conversion, we have studied the dependence of conversion on reactant concentration, and the properties of the PrPSc-derived species that has converting activity.


RESULTS
The cell-free conversion of 35S PrPC to the proteinase K-resistant form was dependent on the reaction time and initial concentrations of PrPSc (above an apparent minimum threshold concentration) and 35S PrPC. 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 PrPSc and enhanced the converting activity, the active species were heterogeneous in size, but larger than either effectively solubilized PrP or molecular weight standards of approximately 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 PrPSc. These results are consistent with a nucleated polymerization mechanism of PrPSc formation and inconsistent with a heterodimer mechanism.
Caughey
B
B
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratory, NIAID, NIH, Hamilton, MT 59840, USA.
Kocisko
D A
DA
Raymond
G J
GJ
Lansbury
P T
PT
D000818:Animals; D015153:Blotting, Western; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011328:Prions / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D012608:Scrapie / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014461:Ultracentrifugation; D014462:Ultrafiltration
D011328:Prions; D013462:Sulfur Radioisotopes; D010450:Endopeptidases