Effects Of Phenolic Substances And Fiber Seeds On Protein Amyloidosis And Its Mechanism

Amyloidosis of human proteins associate with a variety of neurodegenerative diseases, including Alzheimer’s disease, Parkinson disease, mad cow disease, familial vascular amyloidosis, and lysozyme amyloidosis. Protein amyloidosis involves misfolding or partially degrading of the protein, and assembling into amyloid fibrils which are insoluble in intra- and intercellular media. Amyloid fibrils can cause dysfunction of urinary, nervous and cardiovascular systems. Mounting studies demonstrate that some proteins which are unrelated to illness can also transform into amyloid fibrils under certain conditions, suggesting that amyloid fibrillation is a common property of proteins.Amyloid fibrillation of a protein is a nucleation-dependent process. During amyloid formation, native protein monomers undergo unfolding or partially unfolding, which are followed with aggregating of the monomers into oligomers, and growing into protofibrils and eventualy mature fibrils.In the present study, bovine insulin and egg white lysozyme have been used as in vitro models to explore the effects of phenols and fibrillar seeds on the process of amyloid fibrillation of a protein and the molecular mechanism of actions. The works included:screening effective inhibitor on amyloid formation, exploring the inhibitory mechanism through quinone formation and covalent binding, evaluating the seeding effects on protein amyloid fibrillation in the absence and presence of various inhibitors.Experimental methods and results:1. Kinetics of amyloid fibrillation of bovine insulin and lysozymeThe growth kinetics of amyloid fibrils of bovine insulin and lysozyme were detected by using fluorescent probes ThT and ANS, and the fibrillar morphologies were monitored by using transmission electron microscope (TEM). ThT binds specifically to the β-sheet structure in protein or protein assemblies with a singnificant increase in its fluorescent intensity. ANS probes the surficial hydrophobicity of amyloid species by interacting with the hydrophobic domains in protein. The results showed that amyloid fibrillation processes of bovine insulin and lysozyme involved exposure of β-sheet structures and increasing in surface hydrophobicity. Their growth curves of amyloid fibrils appeared as a sigmoidal shape, indicating amyloid formation went though stages of nucleation, elongation and maturation. Bovine insulin incubated for 3 days looked like spherical partical under TEM. At the fourth day, it appeared as sparse, branched short fibrils. Both mature fibrils of insulin and lysozyme showed morphologies with thick, dense, unbranched long fibrils with diameters ranged from 5 to 35 nm.2. Effects of EGCG on protein amyloid fibrillation and mature amyloid fibrilsEpogallocatechin gallate (EGCG) has been proven to be the strongest inhibitor of amyloid formation among the tea polyphenols testing in this study. Moreover, EGCG was able to destabilize mature fibrils. Mass spectrometry demonstrated that bovine insulin was covalently modified by EGCG To further explore the covalent binding, SDS-PAGE and electric blotting-NBT staining were performed. The results showed that, upon interacting with amyloid fibrillar species, EGCG transfored into quinones and reacted with sulfydryl group of the protein.3.Inhibitory effects of monocyclic phenols and benzoquinone on protein amyloid fibrillationThT assay proved that catechol and hydroquinone inhibited protein amyloid fibrillation, similar to the role of benzoquinone. In contrast, phenol and resorcinol showed no effect on amyloid formation. Furthermore, NBT-staining assay indicated that quinoproteins were formed in the samples containing catechol, hydroquinone and benzoquinone, suggesting that transformation into quinones and subsequently binding to peptide chains are key processes in the inhibitory role of a phenol on amyloid formation.4. Seeding effect on protein amyloid fibrillationMature fibrils dissociated under ultrasonic treatment can be used as seeds for accelerating amyloid formation. The seeds act as templates on which protein monomers and oligomers assemble into amyloid fibrils. The efficiency of seeding on amyloid formation depends on the degree of maturation of fibrillar assemblies. Mature fibrils own stronger seeding effect than that of protofibrils.5. The inhibitory role of EGCG on protein amyloid fibrillation in the presence of seedsFibrillar seeds were added into the mixture of EGCG and bovine insulin prior to incubation. The fluorescence intensity of ThT significantly increased with the addition of seeds into the sample. EGCG attenuated the enhancement of amyloid formation induced by seeds. The fluorescence intensity of the sample containing EGCG and seeds decreased gradually from the third day of incubation. On the seventh day, the fluorescence intensity was almost the same as the sample containing EGCG only. TEM determination demonstrated that, in the presence of EGCG and seeds, the resultant aggregates showed sparse and short fibrillar morphology.These facts suggest that both the seeds and EGCG play their roles in the early stage of protein amyloid fibrillation, although EGCG also destabilizes fibrils in the later stage. As the effect of EGCG on protein fibrillation was irreversible, it was difficult for seeds to promote fibril growth when EGCG presented.6. The effect polyethylene glycol on protein amyloid fibrillationPolyethylene glycol (PEG), a hydrophilic polymer, is often utilized to make a crowed environment similar to an intracellular medium. Under a crowding condition, the deffusional rate of protein decreases due to an increase in viscosity of the solvent. As a consequence the fibril assembly of protein is retarded. However, PEG showed no effect on amyloid formation under seeding. In the presence of fibrillar seeds, protein is still able to assemble into fibrils by using the seeds as a template even under a crowding enviorment.ConclusionBovine insulin and lysozyme are able to form amyloid fibrils under the conditions of this study. The growth curves of amyloid fibrils appeared as sigmoidal shapes, corresponding to the phases of nucleation, elongation and maturation. EGCG, catechol and hydroquinone inhibit protein amyloid fibrillation and destabilize mature fibrils. These polyphenols share same mechanism in their anti-amyloidogenic roles. Upon auto-oxidation the phenols transformed into quinones which reacted with sulfydryl groups of protein and altered the pathway of protein aggregation. Fibrillar seeds accelerated protein fibrillation significantly. In the presence of EGCG, seeds failed to promote amyloid formation. In contrast, seeds were able to stimulate protein fibrillation when PEG was added as an amyloid inhibitor. These facts suggest that polyphenols and a crowding reagent inhibit protein amyloid fibrillation through different pathways. PEG plays its role on amyloid assembly in the initial stage. Once seeds are introduced, protein can assemble into mature fibrils by using seeds as templates. The information of the present study not only provides a fresh insight into the anti-amyloidogenic roles of polyphenols, but also shed light on a rational design of novel anti-amyloidogenic drugs with a phenolic structure.