Prion Protein Inhibits Amyloid-? Protein Fibrillization

Alzheimer's disease(AD)is a complex neurodegenerative disease and one of the most common dementias.Its pathological features include senile plaques(SP)formed by the deposition of amyloid-?protein(A?)and neurofibrillary tangle(NFT)formed by hyperphosphorylated tau protein.The key step in AD is that A?oligomers act on the surface of neurons in the central nervous system and induce changes in signal transduction,leading to synaptic dysfunction and synaptic damage.Cellular prion protein(PrP~C)is a glycosylphosphatidylinositol-anchored protein located in cell membrane lipid rafts.A large body of evidence suggests that protein misfolding and aggregation are mechanisms of many neurodegenerative diseases,such as prion diseases and AD.Studies have shown that PrP~C plays an important role in the pathogenesis of AD and may be a potential intervention site for AD treatment.This study was designed to investigate the effects of wild-type and mutant prion protein(PrP)on A?fibrillization.It were expressed that wild-type human full-length PrP,AD-associated mutant PrP and non-AD-associated mutant PrP.ThT fluorescence reaction was used to detect the inhibition of A?fibrillization by different types of PrP.First,we constructed mutant PrP expression vectors including S97N,F198V,P102L,and Y145stop expression vectors.Prokaryotic expression of wild-type full-length human PrP WT(PrP23-231)and the above four mutant prions.Lysing the cells to obtain PrP.The protein was purified by denaturing and denaturing on a nickel column,and the purification effect was examined by SDS-PAGE.After dialysis and concentration,the protein concentration was measured using G250.The PCR results indicated that the construction of the mutant PrP expression vector was successful.The plasmid was transformed into the cells to express a large amount of wild-type PrP and mutant PrP.When the protein was extracted by ultrasonic disruption,PrP was mainly detected as inclusion bodies.More than 90%purity of PrP solution was obtained after purification.After concentration,we can obtain high concentration of PrP aqueous solution.The WT,S97N,F198V,P102L,and Y145stop PrP were freeze-dried to obtain protein powder.The protein was dispersed by hexafluoroisopropanol to obtain monomeric A?and PrP.Aggregation of wild-type and mutant PrP was detected by ThT fluorescence.ThT fluorescence was used to detect the effect of wild-type and mutant PrP on inhibition of A?fibrillization at concentrations where PrP was not aggregated.The experimental results showed that the A?fluorescence intensity was similar in the S97N,F198V and WT samples,indicating that the effect of these two mutation sites was not obvious.The lowest A?fluorescence intensity was in the Y145stop mutant group,indicating that the Y145stop mutation has the best fibrillization inhibition effect,and this site is most sensitive to the influence of A?fibrillization.The highest fluorescence intensity of A?was in the P102L mutation group,and the P102L mutation PrP had the lowest inhibitory effect on A?fibrillization.