Pressure Denaturation of the Yeast Prion Protein Ure2

Zhou, Jun‐Mei; Zhu, Li; Balny, Claude; Perrett, Sarah

doi:10.1006/bbrc.2001.5556

Cited by 39 publications

(27 citation statements)

References 32 publications

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“…Pressure has also been reported to induce the aggregation of transthyretin, converting the native protein into a tetrameric, amyloidogenic state. However, Zhou et al (2001) found that only the combination of pressure with a denaturing agent (guanidinium hydrochloride) was able to induce an irreversible unfolding in the structure of the yeast prion protein Ure2, a similar phenomenon to that observed in the case of lysozyme. Pressure alone was inefficient, at least up to 600 MPa.…”

mentioning

confidence: 58%

Reduced proteinase K resistance and infectivity of prions after pressure treatment at 60 °C

García¹,

Heindl²,

Voigt

et al. 2004

Journal of General Virology

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High hydrostatic pressure is a mild technology compared with high temperatures and is commonly used for food pasteurization. Crude brain homogenates of terminally diseased hamsters infected with scrapie 263K strain were heated at 60 6C and/or pressurized up to 1000 MPa for 2 h. Prion proteins were analysed for their proteinase K sensitivity using a Western blot technique. PrP Sc pressurized with 500 MPa or above proved to be proteinase K sensitive. To test the remaining infectivity of the pressurized material, hamsters were infected intracerebrally. Results showed a greatly delayed onset of disease (from 80 up to 153 days) when samples had been pressurized at 500 MPa and above. An increase in the survival rate was also observed: 47 % survival over 180 days was seen following infection with homogenates pressurized at 700-1000 MPa.Prion diseases are associated with the accumulation of an isoform of the cellular prion protein, designated PrP Sc , after misfolding in a b-rich aggregated pathogenic multimer, which seems to be the main component of the transmissible form (Prusiner, 1991). The transmissible agent resists conventional autoclaving (Brown et al., 2000). Thus, the European Directive of 1996 recommends the use of at least 133 uC for 20 min to achieve effective inactivation during sterilization processes or rendering. Other suitable treatments, such as the combination of alkali and heat, or the use of hypochlorite solutions, although efficient, are aggressive, with a consequent loss of quality or texture in the treated tissues. Therefore, an interest in assessing the effects of unconventional milder technologies on prion stability and infectivity is growing. Milder processes would provide alternative sterilization procedures for at-risk materials and may reduce economic losses in the rendering industry. Here we report the effects of treatment with high hydrostatic pressure at 60 uC on the proteinase K (PK) sensitivity of hamster PrP Sc and the in vivo infectivity of the highpressure-treated samples.These experiments were performed with a single brain pool of the 263K strain of hamster-adapted scrapie agent. Brains containing PrP Sc were homogenized in a 1 : 10 dilution of PBS, pH 7?4, in a FastPrep cell disrupter (Qbiogene). Sets of duplicate samples were heated at 60 uC and/or pressurized up to 1000 MPa for 2 h independently. Total volumes of 250 ml of homogenate (10 %, w/v) were pressurized in a hydraulic press, U 101 (Polish Academy of Sciences, Warsaw). U 101 is a manually operated twin-piston hydraulic press (100 mm piston length, 80 mm piston movement). The vessel is a cylinder made of steel, with an inside diameter of 16 mm and 150 mm in height. The piston position is monitored with a linear transformer transducer and the pressure-measuring unit is an in-vessel manganin pressure gauge; both are digitally displayed. The pressure-transmitting medium was a 7 : 3 mixture of petroleum ether (boiling point 80-100 uC) and hydraulic oil (SAE 32). Samples were pressurized in polyethylene caps hermetically clos...

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confidence: 58%

Reduced proteinase K resistance and infectivity of prions after pressure treatment at 60 °C

García¹,

Heindl²,

Voigt

et al. 2004

Journal of General Virology

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“…The majority of folding studies that have been carried out have used the chemical denaturant and salt, guanidinium chloride (GdmCl). This is because milder (and arguably more physiological) denaturants such as acid, alkali, urea (up to 8 M) [27] or pressure (up to 600 Mpa) [105,111] are each insufficient to unfold the protein; and temperature denaturation is irreversible [27]. In order to guarantee that the thermodynamic parameters obtained from denaturation experiments are meaningful and can be interpreted, it is important to demonstrate reversibility of unfolding; typically this is done by gradually unfolding the protein and comparing the results obtained when the experiment is done in reverse i.e.…”

Section: Comparison Of Different Denaturation Methodsmentioning

confidence: 99%

“…The lower propensity of mutants such as Δ15-42Ure2 (see Fig. 2) to undergo non-specific aggregation and to form amyloid makes them particularly useful for use in rigorous folding studies [27,35,36,105,112].…”

Section: Comparison Of Different Denaturation Methodsmentioning

confidence: 99%

“…Subsequent biophysical analysis of the purified Ure2 protein has shown that the structural properties of the N-terminal and C-terminal regions are also distinct. The N-terminal domain is extremely sensitive to protease digestion [26][27][28]104] and progressive deletion of the N-terminal domain has no effect on the dimerisation, stability or folding kinetics of the protein in vitro [27,33,35,36,105] (see Fig. 6).…”

Section: Role Of the N-terminal Prion Domain In Ure2 Structure And Fomentioning

confidence: 99%

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The yeast prion protein Ure2: Structure, function and folding

Lian

Jiang

Zhang

et al. 2006

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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The Saccharomyces cerevisiae protein Ure2 functions as a regulator of nitrogen metabolism and as a glutathione-dependent peroxidase. Ure2 also has the characteristics of a prion, in that it can undergo a heritable conformational change to an aggregated state; the prion form of Ure2 loses the regulatory function, but the enzymatic function appears to be maintained. A number of factors are found to affect the prion properties of Ure2, including mutation and expression levels of molecular chaperones, and the effect of these factors on structure and stability are being investigated. The relationship between structure, function and folding for the yeast prion Ure2 are discussed.

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“…The potential of high pressure to investigate protein misfolding diseases, including Alzheimer's disease and prion diseases, has been noted (14,15), and there are reports of reversible disaggregation of transthyretin and amyloid A subjected to pressures of 350 and 1,200 MPa, respectively (16,17). In another study, the normal form of the yeast prion (URE 2) suffered only limited structural change under pressures as high as 600 MPa (18).…”

Section: Discussionmentioning

confidence: 98%

Ultra-high-pressure inactivation of prion infectivity in processed meat: A practical method to prevent human infection

Brown

Meyer

Cardone

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Bovine spongiform encephalopathy contamination of the human food chain most likely resulted from nervous system tissue in mechanically recovered meat used in the manufacture of processed meats. We spiked hot dogs with 263K hamster-adapted scrapie brain (10% wt͞wt) to produce an infectivity level of Ϸ9 log10 mean lethal doses (LD50) per g of paste homogenate. Aliquots were subjected to short pressure pulses of 690, 1,000, and 1,200 MPa at running temperatures of 121-137°C. Western blots of PrPres were found to be useful indicators of infectivity levels, which at all tested pressures were significantly reduced as compared with untreated controls: from Ϸ10 3 LD50 per g at 690 MPa to Ϸ10 6 LD50 per g at 1,200 MPa. The application of commercially practical conditions of temperature and pressure could ensure the safety of processed meats from bovine spongiform encephalopathy contamination, and could also be used to study phase transitions of the prion protein from its normal to misfolded state.food processing ͉ scrapie ͉ bovine spongiform encephalopathy ͉ new variant Creutzfeldt-Jakob disease ͉ prion disease

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Pressure Denaturation of the Yeast Prion Protein Ure2

Cited by 39 publications

References 32 publications

Reduced proteinase K resistance and infectivity of prions after pressure treatment at 60 °C

Reduced proteinase K resistance and infectivity of prions after pressure treatment at 60 °C

The yeast prion protein Ure2: Structure, function and folding

Ultra-high-pressure inactivation of prion infectivity in processed meat: A practical method to prevent human infection

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