Pressure-assisted dissociation and degradation of "proteinase K-resistant" fibrils prepared by seeding with scrapie-infected hamster prion protein

Abstract

The crucial step for the fatal neurodegenerative prion diseases involves the conversion of a normal cellular protein, PrP(C), into a fibrous pathogenic form, PrP(Sc), which has an unusual stability against heat and resistance against proteinase K digestion. A successful challenge to reverse the reaction from PrP(Sc) into PrP(C) is considered valuable, as it would give a key to dissolving the complex molecular events into thermodynamic and kinetic analyses and may also provide a means to prevent the formation of PrP(Sc) from PrP(C) eventually in vivo. Here we show that, by applying pressures at kbar range, the "proteinase K-resistant" fibrils (rHaPrP(res)) prepared from hamster prion protein (rHaPrP [23-231]) by seeding with brain homogenate of scrapie-infected hamster, becomes easily digestible. The result is consistent with the notion that rHaPrP(res) fibrils are dissociated into rHaPrP monomers under pressure and that the formation of PrP(Sc) from PrP(C) is thermodynamically controlled. Moreover, the efficient degradation of prion fibrils under pressure provides a novel means of eliminating infectious PrP(Sc) from various systems of pathogenic concern.

Keywords: AFM atomic force microscopy; PK proteinase K; PrPC cellular form of prion protein; PrPSc scrapie form of prion protein; QUIC quaking-induced conversion; Recombinant Hamster prion protein; Western blotting; dissociation of prion fibrils; enzymatic degradation of prion fibrils; pressure-assisted dissociation; proteinase K-resistant prion fibrils; rHaPrP recombinant Hamster prion protein; rHaPrPres PK-resistant recombinant Hamster prion protein.

Figure 1.

Illustration of the experimental procedure in the present work. Step1: rHamPrP was treated with brain homogenate of scrapie-infected hamster 263K brain homogenate to produce rHamPrP fibrils (the method of QUIC¹⁴). Step 2: rHamPrP fibrils were treated with proteinase K under different pressures at 25 °C. Step 3. The reaction products were subjected to analysis with western blotting and AFM. See Materials and Methods for more details.

Figure 2.

Atomic force microscopy images of rHaPrP^res fibrils PK-treated under different conditions. (A) A representative image of rHaPrP^res fibrils as prepared by the method of QUIC. (B) Representative images of rHaPrP^res fibrils after PK-treatment for 1 h at respective pressures. See Materials and Methods for more details.

Figure 3.

Western blotting of rHaPrP and rHaPrP^res fibrils PK-treated under different pressures. (A) rHaPrP treated with proteinase K at different pressures at 25 °C for 1 h. (B) rHaPrP^res fibrils treated with proteinase K at different pressures at 25 °C for 1 h See Materials and Methods for more details.

Figure 4.

Illustration of the molecular events in the PK-treatment of rHaPrP^res fibrils. (A) At 0.1 MPa, where no dissociation of fibrous rHaPrP^res takes place, only the flexible N-terminal segments (residues 23 to 140) are cleaved off by proteinase K, leaving the fibril consisting only of the core parts (residues ∼141 to 231). (B) At high pressures (100–400 MPa), fibrous rHaPrP^res dissociates into monomeric rHaPrP one by one, which is then readily degraded by proteinase K.