| Principle:Alzheimer’s disease(AD)is a complicated neurodegenerative disease which is characterized by two classic features.One is extracellular amyloid(3(Aβ)plaques caused by the β-amyloid deposits and the other is intracellular neurofibrillary tangles(NFTs)aggregated by hyperphosphorylated Tau.Among lots of hypothesis about the pathogenesis of AD,the Aβ hypothesis advocates that extracellular deposition of Aβ which is caused by overproduction and/or failure to clear of Aβpeptides might be the main pathogenic factor.At present,the research of the factors influencing the aggregation of Aβ42 oligomers is a hot topic.Secretory clusterin,as an extracellar chaperone of Aβ,plays an important role in its formation,but there exist some contradictory viewpoints and research results.Therefore,the purpose of this paper is to investigate the effect of sCLU on aggregation of Aβ42 at the single molecule level in order to elucidate the molecular mechanism of the interaction between the two,and provide an experimental basis for revealing the pathogenesis of AD.Method:(1)Expression,purification and identification of sCLU-His.The pFastBac 1-sCL U-His plasmid was constructed,and the sCLU-His protein was expressed by Bac-To-Bac baculovirus expression system.After sCLU-His was purified by Ni-NTA and gel filtration(GF),it was further characterised by SDS-PAGE,Western blotting and HPLC.(2)The effects of different fluorescent labels on AP42 aggregation.Here,we used six different fluorescent dyes namely BODIPY(?)FL-C5(BP),N-hydroxysuccinimide rhodamine B ester(RB),Rhodamine B isothiocyanate(RITC),6-(fluorescein-5-carboxamido)hexanoic acid succinimidyl ester(5-SFX)、Fluorescein 5(6)-isothiocyanate(5(6)-FITC)and Alexa Fluor 647(Alexa)to label Aβ42 monomer separately(abbreviated as Aβ42/Bp,Aβ42/RB,Aβ42/RITC,Aβ42/5-SFx,Aβ42/5(6)-FITC and AP42/Alex,respectively).After that,the effects of different labels on the aggregation of Aβ42 were investigated by using fluorescence correlation spectroscopy(FCS).(3)The change in kinetics of the influence of sCLU-His on the aggregation of Aβ42.The AP42/RB,Aβ42/5(6)-FITC and AP42/Alexa mentioned above and Aβ42/Atto prepared in previours work were choosn to build an easy(Aβ42/Atto-Aβ42/RB)and difficult(Aβ42/5(6)-FITC～Aβ42/Alexa)aggregation model(Aβ42/Label)respectively.And then fluorescence resonance energy transfer-fluorescence cross correlation spectroscopy(FRET-FCS)was used to detect the change in kinetics of different concentrations of sCLU-His(0.1,1 and 10 μM)on the aggregation of 1 μM AP42/Label.Results:(1)The sCLU-His protein was expressed and purified successfully.The molecular weight is 67.3 kD and the purity is up to 96.8%,which could meet the needs of the further experiments.(2)The experimental results showed that differently labelled Aβ42/Label exhibited different aggregation behaviours.Some fluorescent dyes promoted aggregation of A(342/Label,such as BP,RB and RITC,whereas others inhibited aggregation of Aβ42/Label,such as 5-SFX,5(6)-FITC and Alexa.This difference originated from the different electric charges and hydrophobicity of the fluorescent dyes.(3)The results of FRET-FCS experiments showed that the large complexes could be detected in the groups of sCLU-His with 1 and 10 μM,yet,the fluorescence intensity in the group of Aβ42/Atto～Aβ42/RB was reduced.The results indicated sCLU-His and all Aβ42/Label could form complexes,and sCLU-His inhibited Aβ42/Atto～Aβ42/RB self-aggregating with the molecular mechanism conforming to”Strawberry Model".Conclusion:1.The sCLU-His protein was expressed and purified successfully,which could be used in further experiments;2.Different fluorescent labels had different effects on the aggregation of Aβ42,making it easier or more difficult to aggregate;3.The molecular mechanism of sCLU-His inhibiting the aggregation of Aβ42/Label is accordance with the "Strawberry Model". |
PDF Full Text Request