NR AXHX
AU Almstedt,K.; Hammarström,P.
TI Kinetics of Amyloid Fibril Formation of Recombinant Human Prion Protein under Native Conditions in Vitro
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Protein Misfolding P01.03
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Background: The human prion diseases are characterized by depositions of amyloid plaque from misfolded prion protein (HuPrP) in various regions of the brain depending on disease. Aggregation and amyloidogenesis of HuPrP is hence strongly correlated with prion disease.
Aims: In this work we investigated if it was possible to induce amyloid fibril formation of recHuPrP90-231 starting from native folded protein under physiological salt, pH and temperature.
Results: Intense shaking of the folded protein under these native conditions induced irreversible conversion of native HuPrP90-231 into aggregates composed of amyloidlike fibrils of HuPrP90-231 within hours. Formed aggregates were positive using thioflavin T (ThT) fluorescence and showed Congo red birefringence. Fibrillar morphology was verified by transmission electron microscopy where solid state conversion of amorphous aggregates into amyloid-like structures was observed. Under these conditions the kinetic profile of aggregation of HuPrP90-231 showed that within minutes the protein assembled into large amorphous non-thioflavinophilic aggregates which after >2h converted into amyloid-like fibrils. The amyloid fibrillation kinetics followed a traditional sigmoidal trajectory with a lag phase, a growth phase and an equilibrium phase. Interestingly, the lag phase of the ThT assay showed unusual variability indicating a stochastic onset of fibril conversion. That the lag phase was due to nucleation was evident because seeding with preformed HuPrP90-231 fibrils shortened the lag-phase.
The fibrillation rate of the GSS mutant P105LHuPrP90-231 was faster compared to that of the wild type under identical conditions. The fibrillation rate of the GSS mutant could also be augmented through seeding both using wild type (HuPrP90-231) fibrils and P105LHuPrP90-231 fibrils. The converse reaction was also possible where seeds from P105LHuPrP90-231 efficiently shortened the amyloid fibril formation lag phase of wild type.
Conclusion: Amyloid formation of HuPrP90-231 can be achieved starting from the native protein under gentle conditions without addition of denaturant or altered pH. The process is catalyzed by addition of preformed amyloid seeds. It is plausible that amyloid seeding reflect the mechanism of transmissibility of prion diseases. Elucidating the mechanism of PrP aggregation and amyloidogenesis
AD K. Almstedt, Per Hammarström, Linköping University, IFM-chemistry, Sweden
SP englisch
PO Schottland
EA pdf-Datei und Poster (erweiterter Titel: Amyloid fibril formation of human prion protein (HuPr5P90-231) under native conditions in vitro, zusätzliche Autorin: S. Nyström)