Skip to main content
ARS Home » Research » Publications at this Location » Publication #197224
Title: INSIGHTS OF PRPSC STRUCTURE OBTAINED BY LIMITED PROTEOLYSIS AND MASS SPECTROMETRY

Author
item SAJNANI, GUSTAVO - UNIV OF SANTIAGO
item PASTRANA, M - UNIV OF SANTIAGO
item Dynin, Irina
item REQUENA, JESUS - UNIV OF SANTIAGO
item Onisko, Bruce

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 9/30/2006
Publication Date: 10/4/2006
Citation: Sajnani, G., Pastrana, M., Dynin, I.A., Requena, J.R., Onisko, B.C. 2006. Insights of prpsc structure obtained by limited proteolysis and mass spectrometry. [Abstract]. Prion 2006. Poster # S-18.

Interpretive Summary:

Technical Abstract: Elucidation of the structure of PrPsc, essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research, and is hampered by the insolubility and polymeric character of PrPsc. Limited proteolysis is a useful tool to obtain insight on structural features of proteins: proteolytic enzymes cleave proteins more readily at exposed sites, preferentially contained in loops, and do not cleave beta sheet stretches. We treated PrPsc from brains of hamsters infected with 263K and Drowsy prions with different concentrations of proteinase K (PK). After PK deactivation, PrPsc was then denatured and cleaved at Cys179 with 2-nitro-5-thiocyanotonitrobenzoic acid (NTCB), and fragments analyzed by nanoHPLC-MS and MALDI. For 263K the known cleavages at positions preceding Gly90, Gly86 and Gly92 were observed. For Drowsy, cleavages at positions preceding Gly92, Gln98 and Lys101 were seen. Additionally, discrete cleavage points were detected at more internal positions, including those preceding Ala117, Gly119 and Ser 135. In parallel, a sub fraction of Prpsc corresponding to smaller oligomers that exhibit increased sensitivity to proteases were treated with trypsin, and fragments analyzed by mass spectrometry. These experiments showed preferential cleavage after Arg136 (confirming the relative protease sensitivity of the region around Ser 135, and at Arg 151. Our results indicate that besides the "classic" amino terminal PK cleavage points, PrPsc contains in its middle region, regions that show some degree of susceptibility to proteases and must therefore correspond to sub-domains with some degree of structural flexibility. Interspersed with domains of high resistance to proteases. These results are compatible with a structure consisting of short beta sheet stretches connected by short loops and turns.