Reversibility of scrapie-associated prion protein aggregation

During the course of the transmissible spongiform encephalopathy diseases, a protease-resistant ordered aggregate of scrapie prion protein (PrPSc) accumulates in affected animals. From mechanistic and therapeutic points of view, it is relevant to determine the extent to which PrPSc formation and aggregation are reversible. PrPSc solubilized with 5 M guanidine hydrochloride (GdnHCl) was unfolded to a predominantly random coil conformation. Upon dilution of GdnHCl, PrP refolded into a conformation that was high in a-helix as measured by CD spectroscopy, similar to the normal cellular isoform. of PrP (PrPC). This provided evidence that PrPSc can be induced to revert to a PrPC-like conformation with a strong denaturant. To examine the reversibility of PrPSc formation and aggregation under more physiological conditions, PrPSc aggregates were washed and resuspended in buffers lacking GdnHCl and monitored over time for the appearance of soluble PrP. No dissociation of PrP from the PrPSc aggregates was detected in aqueous buffers at pH 6 and 7.5. The effective solubility of PrP was <0.7 nM. Treatment of PrPSc with proteinase K (PK) before the analysis did not enhance the dissociation of PrP from the PrPSc aggregates. Treatment with 2.5 M GdnHCl, which partially and reversibly unfolds PrPSc, caused only limited dissociation of PrP from the aggregates. The PrP that dissociated from the aggregates over time was entirely PK-sensitive, like PrPC, whereas all of the aggregated PrP was partially PK-resistant. PrP also dissociated from aggregates of protease-resistant PrP generated in a cell-free conversion reaction, but only if treated with GdnHCl. Overall, the results suggest that PrP aggregation is not appreciably reversible under physiological conditions' but dissociation and refolding can be enhanced by treatments with GdnHCl.