Reversibility of Scrapie Inactivation Is Enhanced by Copper

Abstract

The only known difference between the cellular (PrP C ) and scrapie-specific (PrP Sc ) isoforms of the prion protein is conformational. Because disruption of PrP Sc structure decreases scrapie infectivity, restoration of the disease-specific conformation should restore infectivity. In this study, disruption of PrP Sc (as monitored by the loss of proteinase K resistance) by guanidine hydrochloride (GdnHCl) resulted in decreased infectivity. Upon dilution of the GdnHCl, protease resistance of PrP was restored and infectivity was regained. The addition of copper facilitated restoration of both infectivity and protease resistance of PrP in a subset of samples that did not renature by the simple dilution of the GdnHCl. These data demonstrate that loss of scrapie infectivity can be a reversible process and that copper can enhance this restoration of proteinase K resistance and infectivity. PrP1 is a highly conserved, host-encoded sialoglycoprotein that may play a role in normal synaptic function and circadian rhythms (1, 2). This host protein (PrP C ) is present in the brain and other tissues and is sensitive to proteinase K (PK) digestion (3). During the course of a scrapie infection, PrP C undergoes a post-translational modification to a disease-specific isoform (PrP Sc ) that has increased resistance to limited PK digestion. This modification appears to be solely conformational with PrP Sc having a higher ␤-sheet content than PrP C (4 -7). In vitro cell culture studies have demonstrated that PrP C is the precursor to PrP Sc (8, 9). Inhibition of the migration of PrP C to the cell surface blocks the formation of PrP Sc , confirming that PrP Sc arises via alternate or misprocessing of PrP Sc (8). PrP Sc is resistant to limited PK digestion (10, 11), but upon treatment with GdnHCl, the PrP denatures (12) and becomes sensitive to protease digestion (13). The loss of PK resistance correlates well with loss of infectivity of the PrP Sc -enriched preparation. In this study, we examined the ability of GdnHCl-denatured, PrP Sc -enriched preparations to renature. Renaturation was monitored by Western blot analysis for the presence of PK-resistant PrP and animal bioassay for infectivity. The capacity of PrP to bind copper led us to determine the effect of this transition metal on the renaturation process. EXPERIMENTAL PROCEDURESDenaturation/Renaturation Reactions-Brains were removed from clinically affected hamsters infected with the 263K strain of hamsteradapted scrapie agent, flash frozen, and stored at Ϫ80°C. PrP Sc -enriched fractions were prepared to the P4 pellet step following a modification of Bolton et al. (14) that excludes the PK digestion. The P4 pellet was resuspended in buffered saline containing glycerol (10 mM Tris, pH 7.4, 130 mM NaCl, 5% glycerol), and the protein concentration was determined by the BCA protein assay (Pierce). The reaction conditions for the denaturation and subsequent renaturation were based upon the cell-free conversion reactions described by Kocisko et al. (13). PrP Sc -enriched preparat...

A role for intermolecular disulfide bonds in prion diseases?

“…Finally, redox reactions with disulfide bonds are catalyzed by metal ions, which may be related to the experimental observation that the PrP C 3 PrP Sc conversion is catalyzed by increasing the number of metal-ion-binding octapeptides in the unstructured N-terminal segment of the prion protein (27), although this N-terminal segment is not necessary for the conversion (28). Additional experiments have demonstrated directly that bound metal ions can influence the conversion (29,30).…”

Disruption of copper homeostasis due to a mutation of Atp7a delays the onset of prion disease

“…Binding of copper to PrP destabilizes the native α-helical PrP and leads to an increase in β-sheet structure and conversion to the proteinaseresistant form (9)(10)(11)(12)(13)), yet it has been reported that copper inhibits PrP res amplification (14). Copper is known to promote prion infectivity (15), yet it is also important in limiting neuropathology caused by reactive oxygen species (16). Although copper chelation delays disease onset in mice (17), similar delays are observed in mice and hamsters maintained on a high-copper diet (18,19).…”

Sequence Variation of Bovine Prion Protein Gene in Japanese Cattle (Holstein and Japanese Black)

“…PrP Sc is generated from the endogenous cellular prion protein PrP C , encoded by the prion protein gene (PRNP), by post-translational modification leading to conformational changes [26,27]. It has been hypothesized that PrP C plays a role in copper metabolism [20,32], but the normal functions of PrP C in cells are unclear.…”