Enzymatic Degradation of PrPSc by a Protease Secreted from Aeropyrum pernix K1

BACKGROUND: An R30 fraction from the growth medium of Aeropyrum pernix was analyzed for the protease that can digest the pathological prion protein isoform (PrP(Sc)) from different species (human, bovine, deer and mouse). METHODOLOGY/PRINCIPAL FINDINGS: Degradation of the PrP(Sc) isoform by the R30 fraction and the purified protease was evaluated using the 6H4 anti-PrP monoclonal antibody. Fragments from the N-terminal and C-terminal of PrP(Sc) were also monitored by Western blotting using the EB8 anti-PrP monoclonal antibody, and by dot blotting using the C7/5 anti-PrP monoclonal antibody, respectively. For detection of smaller peptides from incomplete digestion of PrP(Sc), the EB8 monoclonal antibody was used after precipitation with sodium phosphotungstate. Characterization of the purified active protease from the R30 fraction was achieved, through purification by fast protein liquid chromatography, and identification by tandem mass spectrometry the serine metalloprotease pernisine. SDS-PAGE and zymography show the purified pernisine plus its proregion with a molecular weight of ca. 45 kDa, and the mature purified pernisine as ca. 23 kDa. The purified pernisine was active between 58 °C and 99 °C, and between pH 3.5 and 8.0. The temperature and pH optima of the enzymatic activity of the purified pernisine in the presence of 1 mM CaCl(2) were 105 °C ± 0.5 °C and pH 6.5 ± 0.2, respectively. CONCLUSIONS/SIGNIFICANCE: Our study has identified and characterized pernisine as a thermostable serine metalloprotease that is secreted from A. pernix and that can digest the pathological prion protein PrP(Sc)

Distribution and amount of cathepsin B in gentamicin-induced acute kidney injury in rats

The aim of our study was to investigate how the distribution and amount of cathepsin B change during acute kidney injury. The research was done on a rat model of acute kidney injury that was induced by nephrotoxic antibiotic gentamicin. Gentamicin was injected at a dose of 40 mg/kg body weight (the first treated group) and 80 mg/kg body weight (the second treated group) for 14 days. Control groups received injections of physiological saline only. One day after the last injection, animals were euthanized, dissected and kidney samples were taken and fixed in 10% buffered formalin. Tissue sections were stained with haematoxylin and eosin, periodic Acid Schiff (PAS) and Oil-red-O. Immunohistochemistry was used for the demonstration of cathepsin B. Vacuolar degeneration of the proximal convoluted tubules was the most prominent pathologic lesion found in the first treated group, while necrosis prevailed in the second treated group in the same localisation. In both treated groups significantly weaker immunohistochemical reaction for cathepsin B was noticed in the proximal convoluted tubules in comparison to the control groups (P 0.05), as well. However, more numerous cathepsin B-positive large granules appeared in the proximal straight tubules of the second treated group then in the second control group (P < 0.05). We can conclude that the amount of cathepsin B in the affected proximal convoluted tubules significantly decreases along the increased severity of the histopathological lesions of the proximal convoluted tubuls, the amount of enzyme in the well preserved proximal straight tubules increases and more cathepsin B-positive large granules appear in the cytoplasm