| Objective:Tau protein is a microtubule-associated protein whose gene is located at the17th position of the autosome.Its physiological function is mainly to assemble and stabilize the microtubule cytoskeleton.Hyperphophorylated tau protein would self-aggregate into fibrillary tangles that are one of the most important characteristics of AD.Therefore,targeting tau aggregation is an effective strategy for the treatment of neurodegenerative diseases such as Alzheimer’s disease(AD).This thesis aims to screen one or several natural compounds as potential drugs for AD in terms of tau aggregation inhibitor.Methods:The effects of several natural compounds on the aggregation of tau K18 protein induced by heparin sodium were detected by thioflavin-T(ThT)fluorescence.Then the morphology of tau K18 fibers was observed by transmission electron microscope(TEM).The fluorescence spectroscopy and biofilm interference technology(BLI)were used to measure the binding between small molecule compounds and tau K18 protein.The circular dichroism(CD)was used to detec the effect of small molecule compounds on the protein secondary structure.UV spectrum was utilized to observe the effects of the compounds on the tubulin assembly induced by tau K18.Furthermore,the mechanism of interaction between small molecule compounds and tau K18 was predicted by computational molecular docking technology or dynamic simulation.The tau441 plasmid with flag tag was transfected into HEK293 cells using lentiviral packaging technology,and the expression of tau protein in cells was analyzed by Western-blot.The b End.3 cell as a template was used to construct a blood-brain barrier model to detect whether small molecules can cross the blood-brain barrier.Result:1)Preliminary screening of drugs by ThT experiment reveale that only three flavonol compounds have inhibitory effects on tau K18 protein,namely fisetin,quercetin,and myricetin.The IC50 of these three compounds are 38μM,32μM and 44μM.2)Furthermore,fisetin was added at different stages of tau aggregation.The results show that the compound can inhibit the aggregation of tau K18 protein and depolymerize tau K18 fibrils.Two different experimental methods,fluorescence spectroscopy and biolayer interference technique,revealed that fisetin directly binds to tau K18 protein with K_D value 22.6μM and 61.5μM respectively.CD spectra indicated that the fisetin-bound tau K18 retains same secondary structure.UV spectra showed that the addition of fisetin doesn’t destroy tubulin assembly induced by tau K18.Computational molecular docking simulation confirmed the interaction of fisetin and tau K18.The fisetin phenolic hydroxyl group interactes with residues His268,Val309,Lys311,and Ser320 of monomeric tau K18,and Lys331E and Lys331G in the tau fiber mainly by hydrogen bonds.In addition,fisetin increases the expression of tau protein in HEK293-flagtau441 and can also penetrate the blood-brain barrier model.3)ThT and TEM results showed that quercetin and myricetin can inhibit the aggregation of tau K18 protein and depolymerize tau fibers,but dihydroquercetin and dihydromyricetin cannot.The CD spectra showed that these four compounds have no effect on the secondary structure of tau K18.In addition,BLI results showed that quercetin,myricetin and dihydromyricetin could bind to tau K18 protein.Molecular dynamic simulation results confirmed and deepened experimental data.They displayed that fisetin,quercetin and myricetin can inhibit the formation of tau peptide dimers in varying degrees.In addition,myricetin destroyed the trimer of tau fiber core most obviously.Conclusion:1)Only three compounds(all flavonols)inhibit the abnormal aggregation of tau K18 protein in vitro among the 19 natural compounds.The inhibitory effects of these three compounds are ranked as follows:quercetin>fisetin>myricetin.2)Fisetin inhibits the aggregation of tau protein by hydrogen bonds,and can depolymerize tau fibrils.Fisetin doesn’t affect tau K18’s secondary structure and ability to assemble microtubules,and improves tau protein expression at the cellular level.3)Quercetin and myricetin inhibit protein aggregation and depolymerize fibrils,but dihydroquercetin and dihydromyricetin doesnot,although dihydromyricetin could bind to tau K18.Further analysis reveal that the inhibitory effect of flavonoids on tau K18 aggregation is proportional to the binding affinity. |
