Mechanisms For The Misfolding Of Human Prion Protein Under Physiological Conditions

Transmissible spongiform encephalopathies,also known as prion diseases,is an infectious fatal neurodegenerative disease exiting in most mammal animals.Prion diseases include scrapie,bovine spongiform encephalopathy(B SE),transmissible mink encephalopathy,human Creutzfeldt-Jakob disease(CJD),Variant Creutzfeldt-Jacob disease(vCJD),and Kuru.The key event of such diseases is the conversion from cellular prion protein(PrPC)to scrapie isoform of prion protein(PrPSc),but the mechanism for the conformational change from α-helix to β-sheet is still not clearly clarified.Previous studies reported a large number of reliable results and meaningful conclusions are mainly performed in vitro under denaturing conditions.In the present study,we examined whether prion protein could form amyloid fibrils or fibril-like aggregates in vitro under physiological conditions.Wild-type human prion protein and two pathological mutants were used in our studies as the test samples.ThT fluorescence,Sarcosyl-soluble SDS-PAGE and PK digestion assays,circular dichroism(CD),Fourier transform infrared(FTIR),and other traditional methods were used.We found that compared with that under denaturing conditions,prion protein had the same or similar properties in vitro under physiological conditions(there was no signal detected by CD).Under physiological conditions,the kinetics of fibril formation of prion protein also presented an"S-shaped" curve,which characterized by a lag period(longer),followed by a period of exponential growth and an asymptotic approach to equilibrium.PrP aggregates also had PK-resistance activity.The morphology of PrP aggregates formed under physiological condition was different from that formed under denaturing conditions detected by TEM,containing more β-sheet structure and reticular aggregates formed by human prion protein and detected by Immunoelectron microscopy.Furthermore,in the seed-induced experiments,we found that 1%sonificated-prion aggregates as seeds could induce prion protein aggregation under physiological conditions.Physiological conditions are closer to the biological cell environments,therefore this study will be of great significance for understanding the pathogenesis of human prion protein.