Formation, Inhibition And Destruction Of Insulin Amyloid Fibrillation

At least 20 amyloid proteins are implicted in the pathologies of various human diseases such as Alzheimer's, type II diabetes, Parkinson's and Huntingtons's. The denatured proteins forms aggregates which cause problems to the production, storage and transport of protein drugs, are quite dangerous to drug intaken. Therefore, it is critical to develop related inhibitors as well as to improve detection techniques. Our studies have verified the mechanism of insulin aggregation, and we first established THz TDS method used for detecting insulin amyloid fibrillation. In addition, we destructed the fibrils and regenerated the disulfide bonds of insulin by NH₃-H₂O₂ two-step method, and investigated the inhibition effects on insulin aggregation by small polyphenolic molecules and laser irradiation. The main ideas and conclusions are summarized as follows.Firstly, to study the changes in secondary, tertiary, and quaternary structures and the alteration in the collective vibrational mode density of states during the amyloid fibrillation, bovine insulin in 20% acetic acid was incubated at 60 oC, and its multi-level structures were followed by various biophysical techniques including CD, ThT, DLS, EM and THz absorption spectroscopy. There is an apparent increase in both the absorbance and refractive index in THz spectra, thus THz technique is expected to provide a quick and non-invasive way of looking into amyloid fibrillation. Secondly, the NH₃-H₂O₂ two-step method was investigated to regenerate insulin monomers and recover the structure. The key parameters were identified via comparision with NH₃-H₂O₂ coupling methond, and the model of insulin fibrils disaggregation by adding aqueous ammonia was supposed to disrupting the hydrophobic environment and intrinsic hydrogen bonds.Thirdly, the polyphenol was studied to inhibit insulin amyloid fibrillation. The inhibition ability of procyanidine, resveratrol and trehalose decreased in turn. They may effect the process of insulin fibrillation by inhibiting insulin nucleation, preventing growth of protofilament and with no significant inhibition effects, respectively.Fourthly, a fibrillation inhibition experiment using laser beam irradiation is introduced. Laser can prevent insulin from forming mature fibrils via nucleation inhibition. Importantly, it should be noted that online laser measurements such as dynamic light scattering and ThT fluorescence should be carefully monitored when samples are prone to aggregate or disaggregate. As shown in our results, continuous irradiation of the laser beam induced a comparable steady state of the unaggregated insulin, thereby preventing further fibrillation and, consequently, potentially curing related amyloid diseases.