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Title: Isolation and characterization of a proteinase K sensitive PrPSc fraction

Author
item PASTRANA, MIGUEL - UNIV OF SANTIAGO, SPAIN
item SAJNANI, GUSTAVO - UNIV SANTIAGO, SPAIN
item Onisko, Bruce
item CASTILLA, JOAQUIN - UNIVERSITY OF TEXAS
item MORALES, RODRIGO - UNIVERSITY OF TEXAS
item SOTO, CLAUDIO - UNIVERSITY OF TEXAS
item REQUENA, JESUS - UNIV OF SANTIAGO, SPAIN

Submitted to: Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/1/2006
Publication Date: 12/6/2006
Citation: Pastrana, M.A., Sajnani, G., Onisko, B.C., Castilla, J., Morales, R., Soto, C., Requena, J.R. 2006. Isolation and characterization of a proteinase K sensitive PrPSc fraction. Biochemistry. Volume 45(51); 15710-15717

Interpretive Summary: Prions are the cause of fatal brain diseases such as scrapie in sheep and bovine spongiform encephalopathy (BSE) is cows. Prions have been shown to be present in blood by experimentally transfusing blood from infected animals to uninfected animals. At present there are no reliable methods to diagnose TSEs in live animals. We report in this paper research that has for the first time separated two different forms of prions, those that are digested by proteases and those that are not. All tests for prions need to take in to account the exisentence of both types of prions.

Technical Abstract: Recent studies have shown that a sizeable fraction of PrPSc present in prion-infected tissues is,contrary to previous conceptions, sensitive to digestion by proteinase K (PK). This finding has important implications in the context of diagnosis of prion disease, as PK has been extensively used in attempts to distinguish between PrPSc and PrPC. Even more importantly, PK-sensitive PrPSc (sPrPSc) might be essential to understand the process of conversion and aggregation of PrPC leading to infectivity. We have isolated a fraction of sPrPSc. This material was obtained by differential centrifugation, at an intermediate speed, of Syrian hamster PrPSc obtained through a conventional procedure based on ultracentrifugation in the presence of detergents. PK-sensitive PrPSc is completely degraded under standard conditions (50 g/ml of proteinase K at 37º C for 1 h) and can also be digested with trypsin. Centrifugation in a sucrose gradient showed sPrPSc to correspond to the lower molecular weight fractions of the continuous range of oligomers that constitute PrPSc. PK-sensitive PrPSc has the ability to convert PrPC into protease-resistant PrPSc, as assessed by the protein misfolding cyclic amplification assay (PMCA). Limited proteolysis of sPrPSc using trypsin allows identification of regions that are particularly susceptible to digestion, i.e., are partially exposed and flexible; we have identified as such the regions around residues K110, R136, R151, K220 and R229. PK-sensitive PrPSc isolates should prove useful for structural studies, to help understand fundamental issues of the molecular biology of PrPSc, and in the quest to design tests to detect pre-clinical prion disease.