Molecular Mechanism Of Thiol Compounds Inhibiting Insulin Amyloidosis

Proteins are key components in organism and play important roles in biological activities. More than 20 human diseases have been confirmed to be related to amyloid fibrillation of proteins or peptides, including Alzheimer’s disease, mad cow disease, Parkinson’s disease, familial amyloidosis, type 2 diabetes. Recent studies have found that some proteins which are not relate to human diaeases are able to form amyloid fibrils under appropriate conditions, such as low pH value, heating, stirring, co-incubating with cosolvents or denaturants. These self-assembled fibrillar structure showes typical amyloid morphology and exerts toxicity to living cells. Therefore, amyloid fibrillation is a common property of all peptides and proteins. Amyloidosis can cause dysfunctions of cardiovascular, nervous and urinary systems. It is of great significance to explore the molecular mechanism in protein amyloid fibrosis and to develop methods for the treatment of amyloid diseases.Amyloidosis is a complex process involving the formations of different shapes and sizes of intermediates including oligomers, protofibrils and mature fibrils. The formed amyloid fibrils gathered in cell membranes of an organ or tissue, triggering a casecade of events leading to sick symptoms. The molecular mechanisms in amyloid formation, fibrillar cytotoxicity, and pathogenic pathways are still not clear. Further explorations of amyloidosis at molecular level are critical,In the present study, using bovine insulin as a model protein, the roles of three thiol compounds on amyloid fibrosis are investigated. In the first part, the anti-amyloidogenic effects of cysteine (CYS), glutathione (GSH) and dithiothreitol (DTT) were evaluated with a comparison with the effects of their oxidative forms GSSG and cystine. In the second part, the molecular mechanism of thiol compounds in their anti-amyloidogeic activity was investigated by utilizing electrophoresis, high performance liquid chromatography and mass spectrometry.Methods and results:1. Detection of the growth of bovine insulin fibrilsFluorescent probes ThT (Thioflavin T) and ANS (1-anilino-naphthalene 8-sulfonate) have been used to monitor the growth kinetics and surface hydrophobicity of amyloid species of bovine insulin. The conformational change of bovine insulin was analyzed by means of circular dichroism. A decrease in a-helix structure and an increase in β-sheet structure were observed with the fibril growth. The growth curve of amyloid fibrils appeared as a sigmoidal shape; indicating that the bovine insulin fibrillation involved different phases including nucleation, elongation and maturation.2. The influence of thiol compounds on insulin fibrillationThe impacts of thiol compounds on bovine insulin amyloid fibrillation were detected by using fluorescent probes and liquid chromatographic analysis. The addition of CYS, GSH andDTT effectively inhibited fibril growth of insulin. In constract, GSSG and cystine showed no effect on amyloid formation, suggesting that a thiol group is the key factor for a sulfur-containing compound to inhibit insulin fibrillation.Under a transmission electron microscope, the mature fibrils of bovine insulin showed morphology with unbranch, thick and cluster of fine wire mesh structures. In the presence of thiol compounds CYS, GSH and DTT, small worm-like aggregates were observed. On another hand, typical fibrils similar to mature fibrils were formed when GSSG and cystine were incubated with insulin, in accordance with the spectrometric data.SDS-PAGE and DTNB assay were performed to monitor the disulfide status of bovine insulin during amyloid formation. The disulfide bridges remained intact when native insulin converted into amyloid fibrils. In the presence of thiol compounds CYS, GSH and DTT, the disulfide bridges were reduced and small molecular fragments were detected. MALDI-TOF-MS detection comfirms that one of the small molecular fragments is B-chain.3. The molecular mechanism in the inhibiton of insulin fibrillation by thiol compoundsTo further explore the molecular mechanism in the inhibitory role of thiol compounds on insulin fibrillation, an inhibitor was introduced into the sample at later time points after incubation was initiated. The results suggest that a thiol compound plays its role mainly in the early stages of fibril growth. An exchange reaction between SH and S-S occurred in the inhibition of amyloid formation. Upon disulfide breakage insulin decomposed into small fragments, therefore the process of amyloid assembly was interrupted.Conclusion:Bovine insulin is able to form amyloid fibrils under the experimental conditions of this study. The fibril growth is accompanied with a decrease in a-helix, and increases in β-sheets and surficial hydrophobicity. Thiol compounds CYS, GSH and DTT exhibited strong inhibitory effects on insulin fibrillation. In the presence of a thiol compound, insulin decomposed into small peptide fragment through SH/S-S exchange, leading to an interruption of the fibril growth. The information providing in this study unveil a novel pathway by which a thiol compound exerts inhibitory role on amyloid formation.