Grp78 destabilization of infectious prions is strain-specific and modified by multiple factors including accessory chaperones and pH.

Lethal neurodegenerative prion diseases result from the continuous accumulation of infectious and variably protease-resistant prion protein aggregates (PrP) which are misfolded forms of the normally detergent soluble and protease-sensitive cellular prion protein. Molecular chaperones like Grp78 have been found to reduce the accumulation of PrP, but how different cellular environments and other chaperones influence the ability of Grp78 to modify PrP is poorly understood. In this work, we investigated how pH and protease-mediated structural changes in PrP from two mouse-adapted scrapie prion strains, 22L and 87V, influenced processing by Grp78 in the presence or absence of chaperones Hsp90, DnaJC1, and Stip1. We developed a cell-free in vitro system to monitor chaperone-mediated structural changes to, and disaggregation of, PrP. For both strains, Grp78 was most effective at structurally altering PrP at low pH, especially when additional chaperones were present. While Grp78, DnaJC1, Stip1, and Hsp90 were unable to disaggregate the majority of PrP from either strain, pretreatment of PrP with proteases increased disaggregation of 22L PrP compared to 87V, indicating strain-specific differences in aggregate structure were impacting chaperone activity. Hsp90 also induced structural changes in 87V PrP as indicated by an increase in the susceptibility of its n-terminus to proteases. Our data suggest that, while chaperones like Grp78, DnaJC1, Stip1, and Hsp90 disaggregate only a small fraction of PrP, they may still facilitate its clearance by altering aggregate structure and sensitizing PrP to proteases in a strain and pH-dependent manner.