NR AXVC
AU Requena,J.R.; Sajnani,G.; Pastrana,M.A.; Yeves,L.; Onisko,B.; Dynin,I.
TI Limited Proteolysis of PrPsc: Alternating PK Resistant and Sensitive Segments Suggest a Beta Strand-turn-beta Strand Structural Core
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Oral Abstracts FC1.2
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Vortrag
AB
Elucidation of the structure of PrPsc, essential to understand the molecular mechanism of prion transmission, continues to be a major challenge in prion research. Limited proteolysis has been successfully used to obtain information on structural features of insoluble proteins. Proteolytic enzymes cleave proteins at exposed, flexible sites, while sparing rigid ß-sheet stretches. We treated PrPsc isolated from 263K and Drowsy prioninfected hamsters with increasing concentrations of proteinase K (PK). After PK deactivation, PrPsc was denatured, reduced, cleaved at Cys179 with 2-nitro-5thiocyanatonitrobenzoic acid (NTCB), and fragments analyzed by nanoHPLC-MS and MALDI. We detected peptides corresponding to the known amino-terminal cleavages at positions 86, 90, 92, 94, 96, 97, 98, 99 and 101 in both Dy and 263K PrPsc, although with different individual abundances. We also detected novel, albeit minor cleavage points at internal positions corresponding to two discrete regions: 117,119, and 135, 139, 142, in both strains, and detectable at early times. PK concentration-dependence analysis and limited proteolysis after treatment of PrPsc with 2 M guanidine, known to partially but not irreversibly unfold PrPsc, confirmed that only these cleavage sites are susceptible to PK, while interspersed segments remained spared.
These results place experimental constraints on PrPsc structural models, and are suggestive of a structure consisting of short ß-sheet stretches connected by loops and turns, similar to the one recently derived from hydrogen exchange/solid NMR studies for the Het-s prion. The essential commonality of this structural arrangement, a stack of interdigitated pairs of ß-sheet stretches, with essentially unchanged globular domains located in the periphery, extensive to other fibril-forming proteins such as Aß (1-42) or Ure2p (10-39) is further suggested by recognition of PrPsc by generic amyloidconformation specific antibodies. Our data also suggest that the overal structures of Dy and 263K PrPsc are very similar, with differences primarily in the 86-101 region.
AD J.R. Requena, G. Sajnani, M.A. Pastrana, L. Yeves, University of Santiago de Compostela, Medicine, Spain; B. Onisko, I. Dynin, USDA, Western Regional Research Center, USA
SP englisch
PO Schottland