The Inhibitory Mechanism Of A-type EGCG Dimer On Amyloid Aggregates Formation

The purpose of this paper was to study the underlying mechanism of the stronger anti-amyloidogenic effect of A-type EGCG dimer than that of EGCG.The stronger inhibitory and disaggregative effects of A-type EGCG dimer on bovine insulin amyloid fibrils than that of EGCG were confirmed by multi-dimensional methods.And then the crucial inhibiting period of A-type EGCG dimer on the formation of bovine insulin amyloid fibrils was studied.In addition,the PC12 cell model was established to study the inhibitory effects of EGCG and A-type EGCG dimer on the bovine insulin induced cytotoxicity,and the underlying mechanisms were also further investigated.We chose A?40 polypeptide as the research model and employed multiple techniques such as ESI-MS,NMR and molecular docking to explore the binding mode and binding sites between A-type EGCG dimer and A?40 polypeptide.The main results were shown as follows:1.The inhibitory effects of EGCG and A-type EGCG dimer on the formation of bovine insulin amyloid fibrilsThe mature bovine insulin amyloid fibrils were formed after incubating the bovine insulin alone in 20 % acetic acid at 60 ? for 8 h.Simultaneous incubation of EGCG and A-type EGCG dimer with bovine insulin resulted in an obvious attenuation on the amyloid fibrils formation and the inhibition effects of EGCG and A-type EGCG dimer were demonstrated to be concentration-dependent.Compared to EGCG,A-type EGCG dimer exhibited more potent inhibitory effects on the formation of amyloid fibrils,and the formation of bovine insulin amyloid fibrils was completely inhibited upon the addition of 0.35 m M A-type EGCG dimer.The results of TEM agreed with that of ThT fluorescence assay well.In the presence of 0.70 m M A-type EGCG dimer,we could only observe the formation of amorphous aggregates.DLS analysis indicated that A-type EGCG dimer effectively stabilized insulin monomers with the d H at about 2.33 nm and induced the formation of amorphous aggregates with the d H at about 295 nm.Results from ANS fluorescence assay,FTIR and CD demonstrated that A-type EGCG dimer protected the native conformation of bovine insulin by inhibiting the secondary structure changes and the exposure of hydrophobic amino acid residues.Thus,we proposed that the interaction between A-type EGCG dimer and bovine insulin monomers resulted in the inhibition of structural changes of bovine insulin,which was probably one of mechanisms of the anti-amyloidogenic effect by A-type EGCG dimer.To explore the crucial period of inhibiting the formation of bovine insulin amyloid fibrils by A-type EGCG dimer,0.70 m M A-type EGCG dimer was added at different incubation time during the course of bovine insulin aggregation,and the results showed that the exposure of hydrophobic amino acid residues to the surface of protein and the process of protofibrils and amyloid fibrils formation were almost completely inhibited by 0.70 m M A-type EGCG dimer added at 0 h and 2 h.This suggested that A-type EGCG could not only interact with bovine insulin monomer,but also bind with bovine insulin oligomers and disrupt the structure of oligomers,thus resulting in the disability of protofibrils formation by bovine insulin oligomers.However,the formation of bovine insulin amyloid fibrils was only partially inhibited by 0.70 m M A-type EGCG dimer added at 4 h.Thus,these results indicated that the lag phase was the crucial inhibiting period of A-type EGCG dimer on the formation of bovine insulin amyloid fibrils.2.The disaggregative effects of EGCG and A-type EGCG dimer on the preformed bovine insulin amyloid fibrilsThe results of ThT and ANS fluorescence assay indicated that the A-type EGCG dimer significantly decreased the exposure of hydrophobic amino acid residues on the surface of bovine insulin amyloid fibrils and exhibited stronger disaggregative effect on the preformed bovine insulin amyloid fibrils.And the stronger disaggregative effect of A-type EGCG dimer was further confirmed by TEM.We used the SDS-PAGE and Bradford assay to investigate the disaggregative products of preformed bovine insulin amyloid fibrils by EGCG and A-type EGCG,the results indicated that after incubating the amyloid fibrils with EGCG and A-type EGCG dimer,a great number of aggregates were observed with the molecular mass spanning from 10 k Da to 250 k Da,and co-incubating the preformed amyloid fibrils with 0.7 m M A-type EGCG dimer led to a 6.53 fold of soluble protein increase in the supernatant than EGCG.These results not only demonstrated that A-type EGCG dimer exhibited stronger disaggregative effects on the preformed bovine insulin amyloid fibrils,but also facilitated the formation of soluble insulin oligomers.3.The inhibitory effects and mechanisms of EGCG and A-type EGCG dimer on the bovine insulin amyloid fibrils induced cytotoxicity in PC12 cellPC12 cell model was used to investigate the effects of polyphenols on the amyloid fibrils induced cytotoxicity.The results of MTT indicated that the cytotoxicity of bovine insulin amyloid fibrils was dramatically inhibited by EGCG and A-type EGCG dimer.Notably,A-type EGCG dimer exhibited stronger inhibitory effects on the cytotoxicity induced by bovine insulin amyloid fibrils than EGCG.The experimental results suggested that A-type EGCG dimer showed more potent ability to protect the integrity of PC12 cell membrane,reduce the excessive Ca2+,ROS and NO level,increase the mitochondrial membrane potential(MMP)and the activities of CAT,SOD,GSH-Px and inhibit the over-expression of AIF and Endo G.Thus,based on the above results,we proposed that eliminating the excessive ROS in PC12 cells and increasing the activities of antioxidant enzymes were the molecular mechanisms behind the inhibitory effects of A-type EGCG dimer on the cytotoxicity of bovine insulin amyloid fibrils.4.The mechanisms behind the stronger inhibitory effects of A-type EGCG dimer on the formation of A?40 amyloid fibrilsBy ThT fluorescence assay,we observed that A-type EGCG dimer showed stronger inhibitory effects on the A?40 amyloid fibrils formation than EGCG monomer,and the process of A?40 amyloid fibrils formation was completely inhibited upon the addition of 30 ?M A-type EGCG dimer.These results were clearly confirmed by TEM.Amorphous aggregates with no fibrillary species were observed in the presence of 30 ?M A-type EGCG dimer.The results of PICUP and MTT demonstrated that A-type EGCG dimer prevented the formation of A?40 oligomers and the cytotoxicity induced by A?40 oligomers.Moreover,the results from the fluorescence quenching,ESI-MS,NMR and molecular docking suggested that EGCG and A-type EGCG dimer bound to A?40 polypeptide and formed the non-covalent complex mainly by hydrophobic force.In contrast to EGCG,A-type EGCG dimer could bind with more amino acid residues which possessed high aggregation-propensity and interact with A?40 peptide through more non-covalent interactions such as hydrophobic interactions and hydrogen bonds.It was demonstrated that the galloyl group was a more important functional group in the chemical structure of polyphenol than the 3'-hydroxyl group on the inhibition of A?40 fibrillization by the studies of structure-activity relationship.