Research On Small Molecule Inhibitors Against Tau Protein Aggregation And Mechanism Of Suppression

Alzheimer’s disease(AD)is one of the most common neurodegenerative diseases associated with cognitive decline and memory loss in people aged over 65 years,with no mechanism-based therapies developed to date.The foremost histopathological features of AD are extracellular deposits of amyloid(β(Aβ)and the accumulation of intracellular neurofibrillary tangles(NFTs)composed of hyperphosphorylated tau protein.The tau protein is a neuronal microtubuleassociated protein that binds to and stabilizes microtubules in neurons of human central nervous system.In AD brain cells,after being post-translationally modified by the hyperphosphorylation,tau protein detaches from microtubules leading to its self-aggregation,and thus forms oligomeric species,subsequently aggregates into fibrils,which eventually culminates in the formation of NFTs.Most importantly,the accumulation of NFTs by tau protein is declared to play a more pivotal role in the development of AD than Aβ pathology.Full-length tau protein can be divided into four functional regions:the N-terminal domain,the proline-rich domain,the presence of three(3R)or four(4R)of repeat microtubule binding domains(tau-RD),and the C-terminal domain.Like the full-length tau protein,tau-RD protein has also the ability to bind tubulin,promote tubulin assembly,and stabilize microtubules.Being capable of forming filaments by self-aggregating,tau-RD protein is widely used as a model to mimic full-length tau protein function.Therefore,there is great interest in finding inhibitors that may suppress tau-RD protein aggregation or reverse tau-RD mature fibrils.In this thesis,tau-RD protein was selected as the target protein and its aggregation inhibition was studied in vitro and in a yeast model.We used a variety of biochemical techniques to determine the effects of different inhibitors on preventing tau-RD protein aggregation and disintegrating tau-RD filaments;meanwhile,experimental and computational methods were used to explore the interaction mechanism between tau-RD protein and small molecule inhibitors.Insights obtained from the present work may help us in the rational design of novel drugs for the clinical therapeutics of AD.First,the plasmid harboring tau-RD gene was constructed and recombinant tau-RD protein was expressed in Escherichia coli(E.coli),which was purified by Ni2+nitrilotriacetic acid affinity chromatography.In order to assess the fibrillization process of tau-RD protein in vitro,aggregation kinetics induced artificially by heparin and dithiothreitol(DTT)were conducted.The effects of hematoxylin or grape seed proanthocyanidins(GSPs)on the aggregation kinetics,secondary structural changes,fibril morphology and cytotoxicity of tau-RD were comprehensively evaluated in vitro.Soluble tau-RD protein with the molecular weight of 17 kDa was obtained,which indicated the correct protein folding and expression of recombinant tau-RD protein.Actually,heparin induced the aggregation of tau-RD protein in vitro,and 1 mM DTT was also applied as a reducing agent to eliminate the interference of intramolecular disulfide bonds on tau-RD protein aggregation.The results indicated that hematoxylin and GSPs were powerful inhibitors against tau-RD protein fibrillogenesis.Compared with 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid(HEPES)group,about 50%and 40%of tau-RD aggregation were inhibited in the presence of hematoxylin and GSPs at concentrations of 50μM,respectively.Meanwhile,the IC50 of hematoxylin(42 μM)was 55%less than that of GSPs(IC50 of 94 μM).Hematoxylin or GSPs were found to effectively disaggregate tau-RD fibrils.In addition,hematoxylin or GSPs protected human neuroblastoma SH-SY5Y cells from damage caused by tau-RD aggregates.Then,a yeast strain harboring an integrated single copy of the human tau-RD gene fused with an EGFP label was generated,and the inhibition of tau-RD protein aggregation in Saccharomyces cerevisiae(S.cerevisiae)was further explored.Meanwhile,a yeast strain harboring EGFP without tau-RD gene was used as the control strain.The effects of heparin addition on EGFP-tau-RD protein aggregation were determined,and yeast-based experiments were performed to identify the inhibitory effects of hematoxylin or GSPs on tau-RD aggregation.These two recombinant plasmids(pUMRl-11-EGFP-tau-RD and pUMR1-11-EGFP)were transformed into a wild type S.cerevisiae BY4741(MATα his3Δ leu2Δ met15Δ ura3Δ)strain,respectively.The overexpression of tau-RD protein and EGFP fluorescent protein exerted mild growth defects in the strain cells but did not do harm to the subsequent application.The optimal concentration of heparin was fixed at 10 μM and used for following experiments.Herein,high concentration of heparin produced an excessively negative charge that instead inhibited the aggregation of tau-RD protein,while low concentration of heparin did not completely induce tau-RD aggregation.Hematoxylin or GSPs efficiently reduced the accumulation of abnormal tauRD aggregates in yeast with a dose-dependent manner and reflected the amelioration of AD symptoms in vivo.The formation of tau-RD aggregates in S,cerevisiae mainly required the involvement of microtubule structure.To avoid cell damage,the transportation of tau-RD protein and misfolded protein in S.cerevisiae cells necessitated actin skeleton structure.Furthermore,the aggregation mechanism of tau-RD protein and the multiple interactions between tau-RD protein and inhibitors were revealed via fluorescence quenching,surface hydrophobicity measurements,molecular modification and molecular dynamics simulations etc.Hydrogen bonds and hydrophobic forces between tau-RD protein were major reasons why tauRD had high aggregation tendency.Concretely,residues of V306,1308,Y310,V313,S320,G323,L325 and K331 located at R3(The 3rd microtubule-binding repeat composed of 31 amino acids residues from V306 to G336)played important roles in the aggregation process of tau-RD protein.During the interactions of inhibitors with tau-RD protein dominated by hydrogen bonds and hydrophobic forces,they interfered with the stability of tau-RD protein aggregates by changing the contents of secondary structures.The activity of hematoxylin on inhibiting tau-RD protein aggregation was much higher than that of GSPs due to two main reasons.Above all,the LJ potential and coulomb potential between hematoxylin and tau-RD pentamers were lower than that between GSPs and tau-RD pentamers,so that hematoxylin could form a tight binding with tau-RD pentamers.Then,compared with GSPs,hematoxylin has less steric hindrance and was more likely to interact with amino acid residues in tau-RD protein.In summary,the residues of 1308 and Y310 played important roles in the interactions between polyphenol inhibitors and tauRD protein,which would provide some principles for the rational design of AD drugs.Finally,the relationship between liquid-liquid phase separation(LLPS)and tau-RD protein aggregation was attempted by changing the salt concentration of tau-RD protein solution and the mechanism of LLPS was explored.The amino acid sequence analysis of tau-RD protein showed that tau-RD protein had the large probability of LLPS.Combining fluorescence microscopy,microplate reader detection and dynamic light scattering,the results all showed that LLPS occurred with high salt concentration of 4.75 M in tau-RD protein solution.Effects of physiological factors including protein concentration,pH and temperature of solution on LLPS was studied;meanwhile,amphipathic 1,6-hexanediol and small molecule inhibitors were also added.The results showed that the driving force of LLPS occurring in tau-RD protein solution under high salt condition was hydrophobic interaction,and the inhibitory ability of hematoxylin on LLPS in tau-RD under high salt condition was significantly better than that of GSPs.In conclusion,hematoxylin as a natural compound is considered as a promising candidate for further evaluation to be a preventive and therapeutic drug in reducing amyloidogenesis for AD patients.And the inhibitory mechanisms of hematoxylin and GSPs will provide more theoretical references for the further research and the rational design of novel inhibitors,thus the development of drug discovery could be expected.