Protein conformation determines the sensibility to high pressure treatment of infectious scrapie prions.

Application of high pressure can be used for gentle pasteurizing of food, minimizing undesirable alterations such as vitamin losses and changes in taste and color. In addition, pressure has become a useful tool for investigating structural changes in proteins. Treatments of proteins with high pressure can reveal conformations that are not obtainable by other physical variables like temperature, since pressure favors structural transitions accompanied with smaller volumes. Here, we discuss both the potential use of high pressure to inactivate infectious TSE material and the application of this thermodynamic parameter for the investigation of prion folding. This review summarizes our findings on the effects of pressure on the structure of native infectious scrapie prions in hamster brain homogenates and on the structure of infectious prion rods isolated from diseased hamsters brains. Native prions were found to be pressure sensitive, whereas isolated prions revealed an extreme pressure-resistant structure. The discussion will be focused on the different pressure behavior of these prion isoforms, which points out differences in the protein structure that have not been taken into consideration before.

Dual nature of the infectious prion protein revealed by high pressure.

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.

Interaction of selenium compounds with zinc finger proteins involved in DNA repair.

As an essential element, selenium is present in enzymes from several families, including glutathione peroxidases, and is thought to exert anticarcinogenic properties. A remarkable feature of selenium consists of its ability to oxidize thiols under reducing conditions. Thus, one mode of action recently suggested is the oxidation of thiol groups of metallothionein, thereby providing zinc for essential reactions. However, tetrahedral zinc ion complexation to four thiolates, similar to that found in metallothionein, is present in one of the major classes of transcription factors and other so-called zinc finger proteins. Within this study we investigated the effect of selenium compounds on the activity of the formamidopyrimidine-DNA glycosylase (Fpg), a zinc finger protein involved in base excision repair, and on the DNA-binding capacity and integrity of xeroderma pigmentosum group A protein (XPA), a zinc finger protein essential for nucleotide excision repair. The reducible selenium compounds phenylseleninic acid, phenylselenyl chloride, selenocystine, ebselen, and 2-nitrophenylselenocyanate caused a concentration-dependent decrease of Fpg activity, while no inhibition was detected with fully reduced selenomethionine, methylselenocysteine or some sulfur-containing analogs. Furthermore, reducible selenium compounds interfered with XPA-DNA binding and released zinc from the zinc finger motif, XPAzf. Zinc release was even evident at high glutathione/oxidised glutathine ratios prevailing under cellular conditions. Finally, comparative studies with metallothionein and XPAzf revealed similar or even accelerated zinc release from XPAzf. Altogether, the results indicate that zinc finger motifs are highly reactive towards oxidizing selenium compounds. This could affect gene expression, DNA repair and, thus, genomic stability.

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

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 degrees C 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.