Cell-lysate conversion of prion protein into its protease-resistant isoform suggests the participation of a cellular chaperone

A conformational transition between the normal cellular prion protein (PrPC ) and the beta-sheet-rich pathological isoform (PrPSc) is a central event i n the pathogenesis of spongiform encephalopathies. The prion infectious age nt seems to contain mainly, if not exclusively, PrPSc, which has the abilit y to propagate its abnormal conformation by transforming the host PrPC into the pathological isoform. We have developed an in vitro system to induce t he PrPC --> PrPSc conversion by incubating a cell-lysate containing mouse P rPC with partially purified mouse PrPSc. After 48 h of incubation with a 10 -fold molar excess of PrPSc, the cellular protein acquired PH-resistance re sembling a PrPSc-like state. Time course experiments suggest that the conve rsion follows a stepwise mechanism involving kinetic intermediates. The con version was induced by PrPSc extracted from mice infected with two differen t prion strains, each propagating its characteristic Western blot profile. The latter results and the fact that all the cellular components are presen t in the conversion reaction suggest that PrPC-PrPSc interaction is highly specific and required for the conversion. No transformation was observed un der the same conditions using purified proteins without cell-lysate. Howeve r, when PrPC-depleted cell-lysate was added to the purified proteins the co nversion was recovered. These findings provide direct evidence for the part icipation of a chaperone-like activity involved in catalyzing the conversio n of PrPC into PrPSc. (C) 1999 Academic Press.