Research On Suppression Of Amyloid β Aggregation Using The Minichaperone(GroEL 191-345 Apical Domain)

The accumulation and aggregation of amyloid beta protein(Aβ)plays an important role in the pathogenesis of Alzheimer’s disease(AD).Therefore,it is meaningful and significant to develop novel suppressors that can effectively suppress Aβ aggegation.The minichaperone(residues 191-345 in the apical domain of GroEL)derived from the E.coli GroEL/GroES system is a potential suppressor for Aβaggregation,which can bind to the target protein through hydrophobic interactions with good biocompatibility while requiring no cofactors.In this paper,interactions between the minichaperone and Aβ42 were studied in detail via diverse characterizations including thioflavin T fluorescence spectra,TEM,gel filtration chromatography analysis(ana-SEC),and cytotoxicity experiments.Meanwhile,as a comparison,the suppression effects of polyphenol suppressors such as curcumin,quercetin and epigallocatechin gallate(EGCG)were also studied.Firstly,in order to obtain Aβ42 peptides with no impurities such as racemates and metal ions,the plasmids of pET-GroES-Ub-Aβ42 and pET-Usp-cc were constructed in this experiment.Recombinant Aβ42 fusion protein and Usp catalytic core(Usp-cc)were then successfully expressed in E.coli,and inclusion bodies of Aβ42 fusion protein were prepared.Then,inclusion bodies were processed by cell precipitation dissolution using urea,Ni-NTA column affinity chromatography,Usp-cc cleavage of ubiquitin fragments and prep-size exclusion chromatography,and recombinant Aβ42 peptide with high purity was solubly obtained.Subsequently,the effects of curcumin,EGCG and quercetin on the aggregation kinetics,fibril morphology,soluble component compositions and cytotoxicity of Aβ42 were characterized.Compared with PBS group,the results showed that curcumin,EGCG and quercetin reduced the ex-situ ThT fluorescence intensity at 24 h by 65%,63%,and 66%,respectively.Meanwhile,the time required to achieve the aggregation stabilization state was shortened to be less than 4 h,because these suppressors can destroy the highly ordered structure of Aβ42 oligomer by depolymerizing it into monomers.And the fibril length was also decreased from hundreds of nanometers to 10-50 nm.Unfortunately,these three suppressors could not significantly relieve the cytotoxicity of Aβ42 and showed some limitationsFinally,the suppression effects and pathway of the minichaperones on Aβ42 were studied to reveal the mechanism of interactions.The results showed that the minichaperone not only reduced the aggregation level of Aβ42,but also shifted the on-pathway aggregates to off-pathway aggregates.Thus the molecular weights of aggregates and fibrils morphology were changed significantly.Compared with PBS group,the addition of the minichaperone reduced in situ ThT fluorescence response value of Aβ42 solution up to 72%after 16 h of aging,decreased the length of Aβ42 fibril from above 200 nm to<50 nm after 24 h of aging,and resulted in more than 20%increase of SH-SY5Y cell viability.In summary,the minichaperone presents good application prospects as a result of preventing the formation of Aβ fibrils and reducing Aβ in vitro cytotoxicity.Compared with previously reported Aβ42 aggregation suppressors,the minichaperone presents good biocompatibility.Above all,the minichaperone not only shows distinct suppression effects,but also can shift Aβ42 pathway to form low cytotoxic aggregates.The excellent performance of the minichaperone provides an inspiring idea for the design and development of novel suppressors,and also has laid the foundation for the clinical application of peptide and protein suppressors.