Molecular Mechanism Of Lysozyme Amyloid Fibrosis

More than 20 human diseases have been recognized to be associated with amyloid fibrils of proteins and peptides, including prion disease, Alzheimer's disease, Parkinson disease, familial vascular amyloidosis and lysozyme amyloidosis. Recent studies have demonstrated that some proteins which are not related to human diseases are also able to form amyloid fibrils under appropriate conditions; leading to a postulation that amyloid fibrillation is a common feature of all proteins and peptides. Proteins are key components in organism and play important roles in biological systems. Therefore, further elucidation of the molecular mechanism of amyloid fibrillation of proteins and development of effective methods for the treatment of amyloid diseases become challenging tasks of the scientific research.Amyloid fibrillation of a protein is a complex process. In the duration of amyloidogenesis peptide monomers tranform and self-assembly into a cascade of intermediates with different sizes and shapes, including oligomers, profibers and mature amyloid fibers. These fibrillar aggregates deposit on cell membranes and cause organ dysfunction or disorder; leading to amyloidosis disease. The mechanism by which the fibrillar species of a protein induces cell damage has been studied extensively, although the molecular mechanism of amyloid fibrillation and the fibrillar pathogenesis is still obscure and remains further explored.In the present work by using hen egg white lysozyme as a model protein, the roles of disulfides and sulfhydryls in the amyloid fibrillation of a protein are investigated. In PartⅠ, the conformation change of lysozyme during amyloid fibrillation was analyzed by tracking disulfide exposure and analyzingα-helix andβ-sheet elements of the protein. In PartⅡ, the inhibitory effects of sulfhydryl reagents, glutathione (GSH), cysteine and dithiothreitol (DTT), on the amyloid fibrillation of lysozyme were determined to elucidate the roles of disulfide and free sulfhydryl in amyloidogenesis of a protein.Methods and resultsPartⅠ1. Detection of the growth of lysozyme fibrilsFluorescent probes ThT (Thioflavin T) and ANS (1-anilino- naphthalene 8-sulfonate) have been used to monitor the growth kinetics and the surface hydrophobicity of lysozyme fibrils. The conformational change of lysozyme was analyzed by means of circular dichroism. The results showed that the hydrophobic regions of lysozyme exposed gradually during the fibril growth, accompanied by a decrease ofα-helix structure and an increase ofβ-sheet structure. The growth curve of amyloid fibrils appeared as a sigmoidal shape; indicating that the lysozyme fibrillation involved different phases including nucleation, elongation and maturation. 2. Disulfide labeling and determinationTo detect the exposing order of the disulfide bonds of lysozyme in the fibrillation process, the incubated samples were treated by DTT to reduce the disulfide bonds into free sulfhydryls and labeled by blocking reagents NEM (N-ethylmaleimide) and IAM (Iodoacetamide). The resultant peptides were subjected to enzymatic digestion and MALDI-TOF-MS analysis. The results indicated that the cystein residues 64,115 and 127 were located hydrophilic surface of the protein. The next exposed were cystein residues 6 and 30, whereas cystein residues 76,80 and 94 were exposed only in the late phase of lysozyme fibrillation; suggesting these cystein residues were buried in the hydrophobic regions of the protein. These results indicated that the surfacial hydrophobicity of lysozyme is increased upon amyloid fibrillation, consistent with the data of ANS assay.PartⅡ1. Fibril growth in the presence of sulfhydryl reagentsThT fluorescent was utilized to monitor the fibrillation of lysozyme in the presence of sulfhydryl reagents. The results suggested that reduced glutathione, cysteine and DTT were able to inhibit amyloid fibrillation of lysozyme in a dose-dependent manner. In contrast, the oxidized form of GSH had no effect on the lysozyme fibrillation, indicating that free sulfhydryl is a prerequisite for these compounds to inhibit amyloid fibrillation.ANS assay demonstrated that GSH also inhibited the exposure of the buried hydrophobic region of lysozyme.Circular dichroism is an effective tool to detect the secondary structure of proteins. The results showed that GSH increased the a-helix content of native lysozyme. Upon incubation with GSH, lysozyme transformed into amyloid morphology with a decrease of a-helix and an increase ofβ-sheet structure.2. The fibrillar morphologyThe fibrillar morphology was monitored by polarizing microscopy and transmission electron microscopy. Mature fibrils showed bright yellow under polarizing microscopy. Fibrillar filaments were observed by Transmission electron microscopy. In the presence of GSH, fibrillation of lysozyme was inhibited and amorphous aggregates were observed in the samples that high concentration of GSH was applied.3. The effect of GSH on the cytotoxicity of lysozyme amyloid fibrilsHuman erythrocytes were used as an in vitro model to determine the disruptive effect of lysozyme fibrils on cell membranes. Lysozyme fibrils were able to induce hemolysis of erythrocytes. GSH inhibited the fibrillation of lysozyme and consequently attenuated the fibrillar cytotoxicity of the protein. The inhibitory effect of GSH was dose-dependent, consistent with the results of ThT assay. 4. The molecular mechanism of the roles of GSHSDS-PAGE results demonstrated that GSH increased the formation of hydrolytic products of lysozyme upon incubation. Hydrolysis resulted in degrading lysozyme into small peptides and consequently the fibrillation was inhibited. Moreover, lysozyme fibrillation can also be inhibited by the interactions between disulfide and free sulfhydryl groups.ConclusionLysozyme can form amyloid fibrils under the conditions of the present work. Amyloid fibrillation of lysozyme was associated with exposure of the buried hydrophobic regions and changes of the secondary structures, as demonstrated by disulfide-labeling-MS analysis, circular dichroism, ThT and ANS assays. The increase in surface hydrophobicity of lysozyme assemblies resulted in an increase in cytotoxicity. Sulfhydryl reagents, reduced glutathione, cysteine and DTT inhibited amyloid fibrillation of lysozyme in a dose-dependent manner. High concentration of GSH inhibited the amyloid fibrillation, altered the fibrillar morphology and attenuated the fibrillar cytotoxicity of lysozyme. The molecular mechanism involved possibly that GSH enhanced hydrolytic rate of lysozyme and therefore the fibrillation was altered.