(623at) Inhibition and Deconstruction of Insulin Amyloid Fibrillation

Amyloid fibrils are associated with the pathologies of more than 20 human diseases, including Alzheimer’s, type II diabetes, Parkinson’s and Huntingtons’s [Dobson, 2003; Murphy and Kendrick, 2007]. Moreover, the ability of proteins to form amyloid structures poses problems in its production, storage and delivery[Manning et al., 2010]. Especially for protein drugs like insulin, it is extremely dangerous to undergo amyloid fibrillation. Therefore, it is critical to find new strategies for destroying amyloid fibrils and developing potential inhibitors.

This paper will present our work on the inhibition and deconstruction of insulin amyloid fibrils. we firstly used NH₃-H₂O₂ two-step method to monitor the cleavage and regeneration of disulfide bonds between insulin A-chain and B-chain, and investigated the inhibition effects on insulin aggregation by small polyphenolic molecules and laser irradiation. The main ideas and conclusions are summarized as follows.

(1) The NH₃-H₂O₂ two-step method was investigated to destroy insulin amyloid fibrils and help its conformations back to the native state. ThT fluorescence results showed that insulin fibrils were completely soluble in 25% NH₃·H₂O after 2 h incubation. The secondary structure contents (α-helix, β-sheet, random coil) of insulin monomers and NH₃·H₂O-treated insulin fibrils predicted by K2D method [Whitmore and Wallace, 2008] were 0.80, 0.01, 0.19 and 0.79, 0.00, 0.21, respectively, indicating conformational similarities between them. The HPLC peak of insulin monomer moves to lower retention time with the increase of NH₃·H₂O concentrations. Furthermore, the disulfide bonds between insulin A-chain and B-chain were regenerated by hydrogen peroxide oxidation of sulfydryls.

(2) The polyphenol was studied to inhibit insulin amyloid fibrillation. In the absence of 0.1 M NaCl, the inhibition effects of polyphenols were concentration-dependent. ThT fluorescence results showed that there was no significant decrease in insulin fibrils by adding 50 mM and 100 mM resveratrol and trehalose, while a significant inhibition effect on insulin aggregates was shown with the lag time ~61.9 h by adding low concentration procyanidine (10 mM). The results indicated that procyanidine could provent insulin amyloid fibrillation by inhibiting nucleation. In the presence of 0.1 M NaCl, the kinetics of insulin amyloid fibrillation incubated was similar to that without 0.1 M NaCl, but have shorter lag time. In sum, the inhibition ability of procyanidine, resveratrol and trehalose decreased in turn. They may affect the process of insulin fibrillation by inhibiting insulin nucleation, preventing growth of protofilament and with no significant inhibition effects, respectively.

(3) A fibrillation inhibition experiment using laser beam irradiation is introduced. During heat-induced aggregation, the size distribution of two insulin solutions obtained by online and offline dynamic light scattering were different. The laser-on insulin in the presence of 0.1 M NaCl exhibited fewer fibrils than the laser-off insulin, whereas no insulin fibril under laser irradiation was observed in the absence of 0.1 M NaCl for 45 h incubation. According to the CD results, the laser-irradiated insulin solution maintained mainly an α-helical conformation, but the laser-off insulin solution formed bulk fibrils followed by a significant increase in β-sheet content for 106 h incubation. These findings provide an inhibition method for insulin amyloid fibrillation using the laser irradiation and demonstrate that the online long-time laser measurements should be carefully used in the study of amyloid proteins because they may change the original results.

 References

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4. Whitmore L. and Wallace B. A. Protein secondary structure analyses from circular dichroism spectroscopy: Methods and reference databases, Biopolymers, 89, 392-400, 2008