NR AWNA
AU Sajnani,G.; Pastrana,M.; Dynin,I.; Requena,J.R.; Onisko,B.
TI Insights of PrPsc structure obtained by limited proteolysis and mass spectrometry
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions S-18
PT Konferenz-Poster
AB 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 if contained in loops, and do not cleave ß-sheet stretches. We treated PrPsc isolated from brains of hamsters infected with 263K and Drowsy prions with different concentrations of proteinase K (PK). After PK deactivation, PrPsc was denatured, reduced, and cleaved at Cys179 with 2-nitro-5-thiocyanatonitrobenzoic 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. But also, additional discrete cleavage points were detected at more internal positions, including those preceding Ala117, Gly119 and Ser135. In parallel, a subfraction 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 Ser135), and at Arg151. Our results indicate that besides the "classic" amino terminal PK cleavage points, PrPsc contains, in its middle core, 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 ß-sheet stretches connected by short loops and turns.
AD G. Sajnani, M. Pastrana, J.R. Requena: Prion research unit, School of Medicine, University of Santiago, Santiago de Compostela, Galiza, Spain; I. Dynin, B. Onisko: Western Regional Research Center, USDA, Albany, California, USA
SP englisch
PO Italien