Dual Nature of the Infectious Prion Protein Revealed by High Pressure

Avelina Fernandez Garcia,Philipp Heindl,Heiner Voigt,Mathias Büttner,Peter Butz,Nora Tauber,Bernhard Tauscher,Eberhard Pfaff

doi:10.1074/jbc.m410679200

Avelina Fernandez Garcia, Philipp Heindl + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m410679200

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Abstract

Crude brain homogenates of terminally diseased hamsters infected with the 263K strain of scrapie (PrP(Sc)) and purified prion fibrils were heated or pressurized at 800 megapascals and 60 degrees C for 2 h in different buffers and in water. Prion proteins (PrP) were analyzed for their proteinase K resistance in immunoblots and for their infectivity in hamster bioassays. A notable decrease in the proteinase K resistance of unpurified prion proteins, probably because of pressure-induced changes in the protein conformation of native PrP(Sc) or the N-truncated PrP-(27-30), could be demonstrated when pressurized at initially neutral conditions in several buffers and in water but not in a slightly acidic pH. A subsequent 6-7 log(10) reduction of infectious units/g in phosphate-buffered saline buffer, pH 7.4, was found. The proteinase K-resistant core was also not detectable after purification of prions extracted from pressurized samples, confirming pressure effects at the level of the secondary structure of prion proteins. However, opposite results were found after pressurizing purified prions, arguing for the existence of pressure-sensitive beta-structures (PrP(Sc)(DeltaPsen)) and extremely pressure-resistant beta-structures (PrP(Sc)(DeltaPres)). Remarkably, after the first centrifugation step at 540,000 x g during isolation, prions remained proteinase K-resistant when pressurized in all tested buffers and in water. It is known that purified fibrils retain infectivity, but the isolated protein (full and N-truncated) behaved differently from native PrP(Sc) under pressure, suggesting a kind of semicrystalline polymer structure.

Highlights

Transmissible spongiform encephalopathies (TSEs)1 are associated with the accumulation of an isoform of the cellular prion protein, designated PrPSc in brain
Cellular prion protein (PrPC) have been performed (e.g. with NMR [2]) and the amino acid sequence is known to be preserved in both isoforms, many features of prions are intriguing because PrPSccausing TSE is known to be insoluble
In opposition to this observation, an extensive fraction of native PrPSc remained proteinase K-resistant after pressurization in PBS, pH 5.6 (Fig. 1d, PK), because the resistant prion protein was detected, illustrating a protective effect because of the acidic pH, which is not reverted during pressurization

Summary

EXPERIMENTAL PROCEDURES

Native PrPSc—Experiments on native PrPSc were performed with several brain pools of the 263K strain of the hamster-adapted scrapie agent. PrP-(27–30)—Another set of experiments was performed on the Ntruncated prion protein (PrP-(27–30)) after digestion with proteinase K (73 ␮g/ml for 60 min at 37 °C) of hamster brain homogenates (10Ϫ1 w/v in PBS) infected with the 263K strain of scrapie. Sets of duplicate samples were each heated at 60 °C or pressurized at 800 MPa and 60 °C independently after proteinase K digestion of the isolates or, in a second approach, after every centrifugation step. Detection of Hamster Prion Proteins on Immunoblots—After pressure treatments, samples (15 ␮l of iPrPres or brain homogenates containing PrPSc or PrP-(27–30)) were digested with proteinase K (73 ␮g/ml final concentration in brain homogenates and 46 ␮g/ml in iPrPres, Sigma) for 60 min at 37 °C. Results are presented as log ID50 units/g, where ID50 units/g is the infectious titer/g hamster brain in brain homogenates or the infectious titer/500 ␮l of isolated resuspended material in purified samples

RESULTS

82 Ϯ 7 87 Ϯ 6 169 Ϯ 37

DISCUSSION