Effect Of Peptides And Oligosaccharides On The Amyloid Fibrillation Of Insulin

The present work is related to amyloid fibrillation and protein aggregation.We are devoted to the study of the inhibition of peptides and oligosaccharides on protein amyloid fibrillation experimentally and theoretically,which can lead to a deep understanding of the peptides-regulated mechanism during this process and to give a possible general mechanism of the oligosaccharides-regulated mechanism.First of all,we focused on the dose-dependent inhibitory effects of Fc-dipeptides(Fc-FF and Fc-FY)on insulin fibrillation.Both Fc-dipeptides can strongly inhibit insulin fibrillation by extending lag phase time,reducing final fibril formation(beyond 99% by Fc-peptides of 400 μM),decreasing the formation of high-contentβ-sheet structures and reducing the size of insulin fibrils.And Fc-dipeptides showed potent inhibition at all stages of insulin fibrillation and can depolymerize mature fibrils.We investigated the inhibition mechanism by molecular docking and molecular dynamics simulations.The results suggested that Fc-dipeptides can stably combined with the B-chain helical structure of the insulin monomers or oligomers through hydrogen bonding interactions,π-π stacking interaction,etc.,which twisted the three-dimensional structure of insulin and prevented the transformation of the secondary structure of insulin from the α-helical structure to the β-sheet folded structure,thereby inhibiting or completely blocking the formation of insulin fibrils.Compared with Fc-FY,the better inhibitory effect of Fc-FF at concentration below400 μM was mainly resulted from the difference in π-π stacking interaction and hydrogen bonds between Fc-peptides and insulin,according to molecular dynamics analysis.Secondly,we investigated the effects of Fc-tripeptides(Fc-FFY,Fc-FFF,Fc-FFD and Fc-FFK)on the kinetics of insulin aggregation,as well as the morphology of insulin aggregates.Based on a dose-dependent manner,Fc-tripeptides can effectively inhibit insulin fibrillation and the insulin fibrillation pathway is almost completely blocked when incubated with 300 μM Fc-FFY or Fc-FFF.Compared to the dense,long and thick fibrils formed for insulin alone,few,short and thin insulin aggregates were obtained when incubated with Fc-tripeptides.The Zeta potential analysis and molecular simulation mechanism showed all four Fc-tripeptides can stably combined with the B-chain helical structure of the insulin monomers,and theπ-π stacking interaction is the main interaction between Fc-FFY or Fc-FFF and insulin,while Fc-FFD or Fc-FFK combined with insulin mainly through the electrostatic interaction.Compared with Fc-FFD,Fc-FFK,Fc-FF and Fc-FY,Fc-FFY and Fc-FFF have the better ability to inhibit insulin aggregation is stronger.Thirdly,we focused on the effects of different oligosaccharides on the kinetics of insulin aggregation,as well as the structure and morphology of insulin aggregates at all stages.We founded that oligosaccharides can interfere with the process of insulin fibrillation to a certain degree,by reducing the number and length of insulin aggregates formed at various stages.We analyzed the possible oligosaccharides-regulated mechanism concluded that oligosaccharides form a "protective layer" around the insulin oligomer through non-covalent interaction with the insulin oligomer β-sheet structure,preventing short fibers from further assembly into mature long fibers,thereby interfering with the aggregation process of insulin and reducing the formation of mature long fibrils.