NR AXPO
AU Kalnov,S.; Grigoriev,V.; Pokidishev,A.; Tsibezov,V.; Balandina,M.; Gibadulin,R.; Verkhovsky,O.
TI Spontaneous Fibrillisation of Rec.Bovine PrP(102-230) in Vitro
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Protein Misfolding P01.46
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
The conformational conversion of the normal cellular isoform of the prion protein, PrPc ,into an abnormal pathological isoform, PrPSC, underlies a group of fatal neurodegenerative disorder known as transmissible spongiform encephalopathies (TSE) or prion diseases. In the present work we developed conversion protocols for generating amyloid fibrils from bovine rPrP encompassing residues 102-240. The morphological features and partial resistance to PK-digestion of fibrils have been demonstrated. Rec.Prp was expressed in Eschercha coli system and purified using nickel-nitrilotriacetic acid metal-affinity chromatography (Qiagen). Aliquots withdrawn during time-course of incubation after each 24 hour. The kinetic of fibrils formation was monitoring by electron microscopy (EM).
Self-assembly of fibrillar structures had hierarchical character-from thin flexible helical protofibrils (PF) to muture ribbon-like fibrils. The presented results allow to propose stepwise mode of rec.bovine PrP fibrils assembly:(i) formation of long helical PF(diameter~7nm, length up to 5µm) by mean interaction undetectable by EM protein monomers;(ii) generation of the higher-order, compact fibrils (diameter ~12nm and length up to 4µm) as a result of lateral interactions between several PF by twisting each around other;(iii) fragmentation of the last followed by association of fragments into short ribbon- like structures revealing high polymorphism( diameter~ 16-25nm and length~80-160nm). The end-product of assembly of rec.bovine PrP had intrinsic fragility, i.e. the ability of fibrils to fragment into shorter pieces. This mechanical propertiy of fibrils likely to be linked to their low conformation stability. All kinds of fibrils revealed partial resistance to limited PK-digestion. After treatment with PK fibrils were underwent incomplete destruction and fragments MW 14 kD were produced. Our findings suggest a structural similarity of rec.bovine PrP amyloid fibrils and nvCJD SAFs and their low conformation stability. These data could be part of reason for transmission BSE to humans. Amyloid fibrils of rec.bovine PrP could have the potential technological utility for generating new antibodies and as model for treatment of prion diseases.
AD S. Kalnov, M. Balandina, O. Verkhovsky, Ivanovsky Virology Institute, NARVAC R&D, Russia; V. Grigoriev, A. Pokidishev, V. Tsibezov, R. Gibadulin, Ivanovsky Virology Institute, Russia
SP englisch
PO Schottland