| Protein is one of the substances necessary for life activities; the biological function of a protein depends on its normal conformation. It has been reported that changes unnormaly in the protein conformation will result in amyloid fibrillation or aggregation, depositing in tissues and triggering damages of organs. Investigations have demonstrated that amyloid fibrillation is related to more than20human disease, including Alzheimer’s desease, Down’s syndrome and several familial systemic amyloidosis. Numerous observations have indicated that the primary interacting site of amyloid fibrils is the target-cell membrane. As a consequence of fibril interaction, the structure and properties of the membrane are altered, and a cascade of events leading to cell death is triggered. However, the pathogenic mechanism of amyloid fibrils remains unclear. There is no effective drug developed and used in the clinical treatment of amyloidogenesis. It is of great significance to investigate the molecular mechanism of amyloid formation and to screen effective medicinal candidates against amyloidogenesis.In the present study, utilizing hen egg white lysozyme as an in vitro model, the processes of protein amyloid fibrillation in the presence of inhibitors and the fibrillar seeds have been explored. First of all, the growth pattern of lysozyme fibrils under seeding was investigated. Then the inhibitory role of an inhibitor on amyloid formation was explored under seeding or non-seeding conditions, with an enphasis on the inhibitory role of dithiothretol (DTT) on the amyloid-accelerating compact of lysozyme fibrillar seeds.Methods and results1. The kinetic characterestics of lysozyme amyloid formation probed by fluorescent probesLysozyme was incubated at pH2.0under65℃. Samples were taken at a fixed time interval. The transformation of the secondary structure of lysozyme along with incubation was analyzed by ThT fluorescence. ThT is a fluorescent reagent binding specifically to the β-sheet structures in amyloid fibrils. The fluorescence intensity is proportional to the amount of β-sheet in the fibrils, quantitating the generation of lysozyme amyloid fibrils. The results showed that amyloid formation of lysozyme was associated with a decrease in a-helix and an increase in β-sheet structures. The growth kinetics of lysozyme fibrils was shown as an "S-shaped" curve, indicating that lysozyme fibrillation was consists of a cascade of events including association/dissociation of the protein oligomer, nucleation, elongation and maturation of the fibrillar species.2. Seeding effects on lysozyme amyloid fibrillationLysozyme fibrillar seeds were prepared by ultrasonicating the mature fibrils. The seeds were analyzed quantitatively by using a spectrometric assay based on Coomassie Brilliant Blue stainning. The seeds were added to fresh lysozyme prior to incubation with a ratio of3%(w/w). The fluorescent data showed that the seeds promoted lysozyme amyloid formation significantly.3. Seeding of lysozyme fibrillation in the presence of simple phenolsThe inhibitory roles of several simple phenols, including catechol, hydroquinone, benzoquinone and phenol were investigated under seeding and non-seeding conditions. The results demonstrated that the seeds promoted amyloid formation either in the presence or in the absence of phenols, although the inhibitors were able to attenuate the seeding effect. Upon incubating with an inhibitor, the seeding efficiency decreased in an order of phenol, catechol, benzoquinone and hydroquinone.4. The inhibitory effects of other compounds on lysozyme fibrillationSeveral other compounds, including DTT, PEG2000(polyethylene glycol2000), sodium sulfate, tea catechins EGC (epigallocatechin) and ECG (epicatechin gallate) have also been used as amyloid inhibitors in this study. ThT fluorescent data showed that DTT, PEG and the catechins inhibited amyloid formation of lysozyme in a dose-dependent manner. The inhibitory role of sodium sulfate on amyloid formation did not demonstrate a regular dose-effect relationship.5. Seeding of lysozyme fibrillation in the presence of DTT, PEG, catechins and sodium sulfateThe inhibitory roles of DTT, PEG, catechins and sodium sulfate on lysozyme fibrillation were investigated under seeding and non-seeding conditions. The results showed that lysozyme fibrillation under seeding was inhibited when DTT and catechins were served as inhibitors, although fibril growth was observed when the inhibitors were applied at a low concentration. In contrast, PEG did not ablished the seeding effect on amyloid formation when it was applied at low concentrations. Sodium sulfate showed no effect on lysozyme fibrillation under seeding.6. The timing effect of introducing DTT and fibrillar seeds on lysozyme fibrillationIn order to determine the seeding effect on different stages of fibril growth, the seeds were introduced and mixed with lysozyme after incubation was initiated. The results showed that fibril growth was accelerated by the seeds introduced after1-9days of incubation. The earlier the seeds were introduced, the more seeding effect achieved. The timing effect of introducing DTT on lysozyme fibrillation under seeding was also investigated. The resultant data demonstrated that DTT inhibited strongly amyloid formation under seeding when the compound was introduced in the early stages of incubation. After5days of incubation, the addition of DTT showed only weak inhibitory role on lysozyem fibrillation.7. The hemolysis and aggregation of red blood cellsTo evaluate the fibrillar cytotoxicity, human red blood cells were separated and used as an in vitro model to analyze the hemolytic role of lysozyme fibrils. Incubation of the cells with mature fibrils resulted in hemolysis. The fibrils or aggregates prepared in the presence of DTT and seeds showed lower hemolytic rate than the mature fibrils, corresponding to lower fibrillar cytotoxicity.8. Electrophoretic analysis of lysozyme fibrillation in the presence of seeds and inhibitorsNative PAGE and SDS-PAGE were utilized to analyze the fibrillar species produced during the incubation of lysozyme with seeds and inhibitors. Oligomers, protofibrils and hydrolytic fragments can be separated by electropheresis. The results demonstrated that the inhibitors were able to delay the generation of the fibrillar species, whereas the seeds showed a promoting effect, similar as the ThT data. Quinoproteins were detected by the NBT assay when the catechins were served as inhibitors.9. Morphology of amyloid fibrils and aggregates of lysozymeTEM was performed to visualize the morphologies of lysozyme fibrils and aggregates prepared in the presence of inhibitors and fibrillar seeds. After5days of incubation, short and flexible fibrils can be observed in the control sample. In contrast, dense fibrils were found in the seeding sample and only amorphous aggregates were visualized in the sample contaning DTT.ConclusionThe present study investigates the seeding effects on lysozyme fibrillation in the presence of inhibitors. ThT fluorescent data showed that the seeds promoted significantly lysozyme amyloid formation and that fibril growth could be accelerated by introducing the seeds on1-9days after incubation was initiated. DTT, PEG and the catechins inhibited amyloid formation of lysozyme in a dose-dependent manner. In the presence of fibrillar seeds, the inhibitory effects of inhibitors were abolished with an exception that high dose of DTT was able to attenuate amyloid formation under seeding. |
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