Abstract
During the course of the transmissible spongiform encephalopathy diseases, a protease-resistant ordered aggregate of scrapie prion protein (PrP(Sc)) accumulates in affected animals. From mechanistic and therapeutic points of view, it is relevant to determine the extent to which PrP(Sc) formation and aggregation are reversible. PrP(Sc) 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 alpha-helix as measured by CD spectroscopy, similar to the normal cellular isoform of PrP (PrP(C)). This provided evidence that PrP(Sc) can be induced to revert to a PrP(C)-like conformation with a strong denaturant. To examine the reversibility of PrP(Sc) formation and aggregation under more physiological conditions, PrP(Sc) 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 PrP(Sc) aggregates was detected in aqueous buffers at pH 6 and 7.5. The effective solubility of PrP was <0.7 nm. Treatment of PrP(Sc) with proteinase K (PK) before the analysis did not enhance the dissociation of PrP from the PrP(Sc) aggregates. Treatment with 2.5 m GdnHCl, which partially and reversibly unfolds PrP(Sc), caused only limited dissociation of PrP from the aggregates. The PrP that dissociated from the aggregates over time was entirely PK-sensitive, like PrP(C), 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.
Highlights
Of PrPSc are highly infectious, and infectivity has not been physically separated from PrPSc [8, 9]
To verify that the guanidine-solubilized PrP was unfolded, PrPSc disaggregated in 5 M guanidine hydrochloride (GdnHCl) was analyzed by Fourier Transform Infrared (FTIR) spectroscopy
Protease Sensitivity of Soluble and Aggregate Forms of PrP—In addition to aggregation, another process that occurs during the conversion of PrPC to PrPSc is the acquisition of protease resistance. To determine if this conversion is reversed in the presence of 2.5 M GdnHCl, an experiment similar to that shown in Fig. 5 was performed, except that aliquots of the PrP aggregate mixture were subjected to digestion with proteinase K before the centrifugation that was performed at the beginning and end of the dissociation period
Summary
PrP 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. The transmissible spongiform encephalopathies (TSEs) are progressive neurodegenerative diseases of mammals. The accumulation of PrPSc in TSE-infected animals may be due to an inherent irreversibility of PrP aggregation during some stage of PrPC-to-PrPSc conversion. To gain insight into parameters controlling PrPSc accumulation in TSE-infected animals, experiments were performed to determine if the polymerization/aggregation of PrP that occurs during PrPSc formation is a reversible process. This paper is available on line at http://www.jbc.org suggest that PrP aggregation is not detectably reversible, but partial unfolding can induce a modest level of PrP dissociation
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