Dual Nature of the Infectious Prion Protein Revealed by High Pressure

Abstract

Crude brain homogenates of terminally diseased hamsters infected with the 263K strain of scrapie (PrPSc) and purified prion fibrils were heated or pressurized at 800 megapascals and 60 °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 PrPSc 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 log10 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 β-structures (PrPScΔPsen) and extremely pressure-resistant β-structures (PrPScΔPres). Remarkably, after the first centrifugation step at 540,000 × 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 PrPSc under pressure, suggesting a kind of semicrystalline polymer structure. Crude brain homogenates of terminally diseased hamsters infected with the 263K strain of scrapie (PrPSc) and purified prion fibrils were heated or pressurized at 800 megapascals and 60 °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 PrPSc 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 log10 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 β-structures (PrPScΔPsen) and extremely pressure-resistant β-structures (PrPScΔPres). Remarkably, after the first centrifugation step at 540,000 × 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 PrPSc under pressure, suggesting a kind of semicrystalline polymer structure. Transmissible spongiform encephalopathies (TSEs) 1The abbreviations used are: TSE, transmissible spongiform encephalopathy; PrP, prion protein; iPrP, isolated PrP; MPa, megapascal; PBS, phosphate-buffered saline.1The abbreviations used are: TSE, transmissible spongiform encephalopathy; PrP, prion protein; iPrP, isolated PrP; MPa, megapascal; PBS, phosphate-buffered saline. are associated with the accumulation of an isoform of the cellular prion protein, designated PrPSc in brain. Evidence indicates that misfolding of the cellular isoform into a β-sheet-rich aggregated pathogenic self-propagating multimer is the main step of infection (1Prusiner S.B. Science. 1991; 252: 81-91Crossref Scopus (1732) Google Scholar). Even though detailed studies on the structure of the cellular prion protein (PrPC) have been performed (e.g. with NMR (2Riek R. Hornemann S. Wider G. Glockshuber R. Wuthrich K. FEBS Letts. 1997; 413: 282-288Crossref PubMed Scopus (657) Google Scholar)) and the amino acid sequence is known to be preserved in both isoforms, many features of prions are intriguing because PrPSc-causing TSE is known to be insoluble. Up to now, studies of the PrPC structure after expression of recombinant cellular prions (2Riek R. Hornemann S. Wider G. Glockshuber R. Wuthrich K. FEBS Letts. 1997; 413: 282-288Crossref PubMed Scopus (657) Google Scholar), the purification of native PrPSc after immunoaffinity chromatography and elution at basic pH (3McKinley M.P. Braunfeld M.B. Bellinger C.G. Prusiner S.B. J. Infect. Dis. 1986; 154: 110-120Crossref PubMed Scopus (46) Google Scholar), or the isolation of proteinase K-resistant materials (iPrPres) (4Turk E. Teplow D.B. Hood L.E. Prusiner S.B. Eur. J. Biochem. 1988; 176: 21-30Crossref PubMed Scopus (266) Google Scholar) have been prerequisites for performing studies on the structure of infectious prions. Thus, existent models are mainly derived from spectroscopic data from studies of such structures. Moreover, treatments with heat, salts, denaturants, and extreme pH revealed new conformational states of prions that were different from native. And so far, when a conformational change leads to a reduction in infectivity, inactivation is irreversible (5Riesner D. J. Neurovirol. 2002; 8: 8-20Crossref PubMed Scopus (9) Google Scholar). The β-rich secondary structure most likely provides infectious prions with a tremendous resistance to conventional autoclaving, which leads to the need for severe inactivation strategies, such as autoclaving at 133 °C for at least 20 min, the combination of alkali and heat, or the use of hypochlorite solutions. Those strategies, although efficient, are excessively aggressive, cause a consequent loss in quality and texture in the treated tissues, and are not applicable to foodstuffs. Consequently, our interest in assessing the effects of unconventional milder technologies on prion stability and prion infectivity arises from the necessity of finding milder techniques that provide alternative sterilization procedures for risk materials. High hydrostatic pressure is a mild processing technology with a promising potential in food pasteurization and sterilization. By avoiding heat, undesirable alterations in foods such as vitamin loss and changes in taste and color can be minimized (6San Martin M.F. Barbosa-Canovas G.V. Swanson B.G. Crit. Rev. Food Sci. Nutr. 2002; 42: 627-645Crossref PubMed Scopus (400) Google Scholar, 7Fernández García A. Butz P. Tauscher B. J. Food Sci. 2001; 66: 1033-1038Crossref Scopus (107) Google Scholar). Moreover, the use of high pressure to assist thermal food processing, exploiting the unique effect of adiabatic heating and cooling, has already been reported effective in the inactivation of prion proteins when the temperature is shifted up to 135–142 °C (8Brown P. Meyer R. Cardone F. Pocchiari M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6093-6097Crossref PubMed Scopus (71) Google Scholar). Furthermore, high hydrostatic pressure is a thermodynamic parameter providing useful information when studying protein denaturation and reaction mechanisms (9Royer C.A. Biochim. Biophys. Acta. 2002; 1595: 201-209Crossref PubMed Scopus (349) Google Scholar). Protein response to pressure is driven by the volume change of the protein solvent system, which is associated with conformational changes, protein folding-unfolding, and aggregation. Pressure shifts the conformational transitions of a protein toward the state with the lower volume; therefore, unexpected effects that are different from temperature or chemical denaturation can be seen under pressure (10Heremans K. Smeller L. Biochim. Biophys. Acta. 1998; 1386: 353-370Crossref PubMed Scopus (417) Google Scholar). The largest contribution of pressure is at the level of weak (non-covalent) interactions; it is accepted that hydrogen bonds are slightly stabilized under pressure and that pressure disfavors hydrophobic and electrostatic interactions, which are mainly responsible for protein aggregation (11Balny C. J. Phys. Condens. Matter. 2004; 16: 1245-1253Crossref Scopus (47) Google Scholar). For example, mild high pressure treatments induce the conversion of the non-infectious cellular prion protein into a proteinase K-resistant form (12Kuwata K. Hua L. Yamada H. Legname G. Prusiner S. Akasaka A. James T.L. Biochemistry. 2002; 41: 12277-12283Crossref PubMed Scopus (147) Google Scholar, 13Torrent J. Alvarez Martinez M.T. Heitz F. Liautard J.P. Balny C. Lange R. Biochemistry. 2003; 42: 1318-1325Crossref PubMed Scopus (85) Google Scholar). Additionally, an impressive reduction in the infectivity of native hamster prion proteins has been reported in PBS buffer (14Fernández García A. Heindl P. Voigt H. Büttner M. Wienhold D. Butz P. Stärke J. Tauscher B. Pfaff E. J. Gen. Virol. 2004; 85: 261-264Crossref PubMed Scopus (39) Google Scholar) at relatively low pressures and with temperatures below customary sterilization parameters. An equivalent reduction in the proteinase K resistance was observed at pressure-holding times that would still be realistic (e.g. 30 min) (15Fernández García A. Heindl P. Tauber N. Butz P. Voigt H. Büttner M. Tauscher B. Pfaff E. Proceedings of the International Conference on Engineering and Food. Montpellier, France2004: 303-306Google Scholar) when trying to employ this technology at the industrial level. However, this irreversible effect is astonishing because usually only extremely high pressures are able to rearrange secondary structures of proteins, and the pressure stability of hydrogen bridges and β-sheets should remain resistant to such high pressure treatments. This work was a phenomenological study using the proteinase K resistance of native prion proteins as an analytical measure for the effects of pressure, contrasting data with the remaining infectivity and aiming at a first estimation of the effects of pH on the structure of prion proteins under pressure. Additionally, pressure effects on brain homogenates containing N-truncated prions were analyzed to verify the recently postulated pressure-induced proteinase K sensitivity of prion proteins after treatments at 60 °C (14Fernández García A. Heindl P. Voigt H. Büttner M. Wienhold D. Butz P. Stärke J. Tauscher B. Pfaff E. J. Gen. Virol. 2004; 85: 261-264Crossref PubMed Scopus (39) Google Scholar). Furthermore, this work also focused on the assay of the proteinase K resistance of N-truncated purified prions after pressure treatments in different matrices, aiming at establishing whether those are acceptable models for explaining pressure effects on native prions. Results were expected to be useful as a basis for the application of high pressure as an alternative technology for the mild inactivation of infectious prions at temperatures below the known denaturing conditions. Native PrPSc—Experiments on native PrPSc were performed with several brain pools of the 263K strain of the scrapie from terminally diseased hamsters containing PrPSc were in phosphate-buffered saline buffer, pH 7.4, buffer, pH PBS buffer, pH or water in a of were heated at 60 °C or pressurized at 800 and 60 °C of was performed on the N-truncated prion protein after with proteinase K for 60 at of hamster brain homogenates in infected with the 263K strain of were heated at 60 °C or pressurized at 800 and 60 °C after inactivation of the at °C for of of was performed a slightly of the in H. H. PubMed Scopus Google of brain in PBS buffer, pH 7.4, pressurized at 800 at 60 °C for or brain was in a containing pH 7.4, and for at × The was for 20 at 540,000 × and the was with a containing and pH and for at 540,000 × The was during 60 with a proteinase K to proteins. the was for at × g and in of PBS, pH 7.4, pH or water. The was with at °C for of were heated at 60 °C or pressurized at 800 and 60 °C after proteinase K of the in a after centrifugation High Pressure of of hamster brain or were pressurized in a of is a The is a of with a and a The is with a and the pressure is an pressure both are The was a of and were isolated in to The effects of adiabatic heating were minimized because of the pressure to the pressures least and by of the temperature in the using a to the was 2 of Prion on pressure of or brain homogenates containing PrPSc or were with proteinase K in brain homogenates and in for 60 at of were were denaturation at the were by on proteins were to were by the in and in PBS with were with the PrPSc to in with were excessively with PBS with and with in PBS with was on a using the were performed with the temperature materials an S.B. Martinez PubMed Scopus Google Scholar). were into of or The was of hamster brain or of isolated or heated h at 60 of the brain homogenates were as were observed for of scrapie during a of and after were and at were to the proteinase K-resistant form of Results are as log10 units/g is the infectious hamster brain in brain homogenates or the infectious of isolated in purified K and of Native PrPSc in brain homogenates for the 263K strain of scrapie were to 800 °C for 2 h in different buffers or in water. the were with or proteinase K to the effect of pressure on the resistance of PrPSc to with the are reported in of heated only were all for PrPSc after 60 °C for 2 h and from the not The accumulation of β-rich proteinase K-resistant is associated with prions and used for TSE Results that the native structure of PrPSc was not when pressurized in the neutral pH for in pH a pressure buffer, but also not in pressure such as PBS buffer, pH or water The sensitivity of the prion structure to pressure is because the core of native PrPSc was not on immunoblots after pressure in of the to this an of native PrPSc remained proteinase K-resistant after in PBS, pH because the resistant prion protein was a effect because of the acidic which is not during The found in homogenates at initially neutral pH the of effects on the core of prion proteins, although are not to because of the likely hydrogen bridges in volume the protein secondary structure (10Heremans K. Smeller L. Biochim. Biophys. Acta. 1998; 1386: 353-370Crossref PubMed Scopus (417) Google Scholar). that structure are by of Consequently, only containing proteinase K-resistant prions are the homogenates pressurized in or PBS buffer or in water should have potential this were performed in hamsters by of the treated PBS of scrapie in this were observed for up to homogenates and those treated at 60 °C pressure to TSE after were after of homogenates treated at 800 at 60 °C for 2 confirming results of our (14Fernández García A. Heindl P. Voigt H. Büttner M. Wienhold D. Butz P. Stärke J. Tauscher B. Pfaff E. J. Gen. Virol. 2004; 85: 261-264Crossref PubMed Scopus (39) Google Scholar). This an reduction in the brain in PBS homogenates and a the reduction and the decrease in the on the immunoblots reported analyzed of terminally diseased at the of the reported were for of the not reduction of pressurized °C for at 60 °C for in a new K of at 2 the results after of the N-truncated with native infectious prions were found to be pressure-sensitive at neutral and could be by after with proteinase K not However, pressure is able to the structure of proteins protein D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M. Biochim. Biophys. Acta. 2002; Scopus Google Scholar), which form not by to during However, that immunoblots of the N-truncated form of PrPSc were the arguing that prion proteins were not aggregated after adiabatic treatments because were able to the Furthermore, a change in the protein structure to a of the and to in could also be after the results in to proteinase K-resistant was on the immunoblots after with proteinase K confirming the postulated proteinase K and of from Native prion proteins were purified from high brain homogenates during the first and in PBS buffer, pH 7.4, after the in H. H. PubMed Scopus Google The of immunoblots from brain homogenates and is reported in purified from were resistant to and for PrPSc because the were results of purified from pressurized with the pressurized brain homogenates and was on the immunoblots after isolation and proteinase K results the already pressure-induced sensitivity to proteinase K that could only be by changes in the protein conformation because to protein aggregation could already be K of at were performed in hamsters by of the purified materials. in this the first of after log10 with The by was log10 from pressurized an acceptable with the reduction in the detectable core resistant to reported prion proteins (iPrPres) were pressurized by in neutral and PBS or water. Results with or proteinase K with are in to the pressure effects on the proteinase K resistance and the infectivity of native PrPSc the of infectious prions were after of the core in water and and buffer, pH and a preserved proteinase K resistance after pressure The proteinase K resistance was for pressurized in PBS buffer, pH although denaturation and in of the pressurized proteinase could be on the immunoblots in the of proteinase or in the of proteinase K and the of prion proteins was a pressure effect on the protein structure in PBS buffer, which was probably by protein aggregation. PBS and pH in the acidic during pressure the aggregation of purified prion which is already known to be the for treatments of native PrPSc with at pressure. results on the resistance to with proteinase K were when was with a scrapie brain in PBS buffer, pH and that brain as and changes to aggregation were by pressure because the of prion proteins were on the immunoblots the denaturing native and purified PrPSc to a study of the pressure effects on the protein structure during the isolation For were and pressurized after centrifugation step during Results with and proteinase K are reported in the high of not induce changes in the prion protein which could have to an pressure and could be with native PrPSc after the first centrifugation at × g However, subsequent centrifugation at 540,000 × g to a effect in the protein which was because of the resistance to after pressure at 800 for 2 h at 60 °C and with proteinase K and the centrifugation step at × the pressure-induced aggregation of was and prions were not on immunoblots of samples, with the already in of high pressure for the inactivation of pathogenic have been J. Barbosa-Canovas G.V. Crit. Rev. Food Sci. Nutr. 2003; PubMed Scopus Google Scholar). have been found to be the most and only of and extreme high pressures are able to at a are resistant to conventional the of prions our for a inactivation when to high pressures at mild a (14Fernández García A. Heindl P. Voigt H. Büttner M. Wienhold D. Butz P. Stärke J. Tauscher B. Pfaff E. J. Gen. Virol. 2004; 85: 261-264Crossref PubMed Scopus (39) Google Scholar), reported an in the for the of and postulated a decrease in proteinase K resistance in of the immunoblots after pressure treatments of brain the pressure of native prion proteins has been in several buffers and in water. The of brain homogenates was to induce changes in the protein structure during the pressure up and the This a loss of the detectable PrPSc core resistant to to The pH of such as PBS containing buffer and shifts during into the acidic for S. E. Scopus Google Scholar). 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Protein Sci. PubMed Scopus Google Scholar). This would the results of immunoblots and the reported proteinase K but the pressure-induced of the secondary structure of prion proteins, and of the would have to be results are when the effects of pressure on purified brain materials. The conformation of purified prion proteins not to be when to high pressures because the main to native infectious prions, the proteinase K resistance of the is preserved after pressure. the of purified pressurized prions, results not with during isolation should be centrifugation at 540,000 × g several at the of centrifugation extremely high pressures during centrifugation have the inactivation of infectious prions. However, a during centrifugation is that the of proteins is and the of hydrophobic proteins to the of a hydrophobic core at high proteins are aggregated and not a semicrystalline structure. a structure has been reported after isolation of H. S. A. Prusiner S. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar) and was the basis for the performed on which the most accepted on the structure of prion proteins. not to be is the that an by to be in the form of a R. Rev. Biophys. PubMed Scopus Google for could during results that PrPSc a structure different from native during The of a or the high aggregation and the of are probably the that a protein conformation that is not pressure-sensitive and not to Those proteins would still be infectious but would differently from the native PrPSc and would probably have a structure. This would that models on prion been on a protein could when trying to the native prion the structure of as a basis for and for prion to the native and prions J. Protein Sci. PubMed Scopus Google Scholar) has already been Those an during the loss of which is with an resistance to in isolated prions in the state that could to the extreme pressure resistance reported work J. R. M.P. D. R. J. 2004; PubMed Scopus Google Scholar) the of and aggregation on the pressure sensitivity of the secondary structure of recombinant prions. our results probably for the existence of pressure-sensitive β-structures (PrPScΔPsen) and extremely pressure-resistant β-structures because of the high aggregation and low in purified prions. that native prion proteins not have the structure as purified prions in the from in buffers different from PBS and in food at neutral pH are under and the realistic of this technology in risk materials below the customary denaturation of for during the of this