Molecular Mechanism Of Phenolic Compounds On The Protein Amyloid Fibrosis Effect

Protein amyloid deposition is one of the important characteristics of some human diseases. Inhibition of amyloid formation and disruption of the formed fibrillar assemblies are the therapeutic strategies proposed for the treatment of amyloid-related diseases. Natural polyphenol compounds can act as potential therapeutic reagent for these diseases due to their effective role on amyloid aggregates. Increasing evidence has demonstrated that the prooxidant effect directly or indirectly involves in the process of catabolism of polyphenol compounds. The prooxidant potential of a polyphenol in biological system includs modifying proteins, inducing the generation of reactive oxygen species and breaking DNA strands. Numerous phenolic compounds have been reported to have an inhibitory role on amyloid formation of proteins due to their polyhydroxyl structures and redox properties. A large number of experiments and epidemiological studies have shown that tea polyphenols, particularly tea catechin EGCG, having a relatively wide range of pharmacological activity, can prevent chronic diseases such as heart disease, diabetes, neurodegenerative diseases and cancer. A growing number of research results show that the prooxidant property of EGCG is attributed to its inhibition of growth of leukemia cells and induction of apoptosis of cancer cells. Despite extensive investigations on the inhibitory effects of polyphenols on amyloidogenesis, the detailed molecular mechanism is still unclear and needs further investigation.Utilizing lysozyme and bovine serum albumin (BSA) as model proteins, the role of a phenolic compound on protein amyloid fibrillation has been investigated in vitro in terms of (1) screening of effective inhibitor for protein amyloid fibrillation;(2) the molecular mechanism by which a polyphenol disrupts amyloid structure through covalent and/or non-covalent interaction;(3) exploring the role of oxidative metabolic intermediate of polyphenol on amyloid fibrillation, particularlly the role of quinoprotein and (4) the the structure-activity relationship of polyphenol against amyloid formation. The present investigation will provide a theoretical insight in pharmacological effects of natural polyphenol on amyloidosis on the basis of interactions between polyphenol and amyloid fibrils.Methods and results: 1. Kinetics of amyloid fibrillation of lysozyme and bovine serum albuminThe growth kinetics and surface hydrophobicitiy of lysozyme and BSA during their amyloid fibrillation were monitored by using fluorescent probes ThT and ANS. ThT fluorescence detected the growth curves of protein fibrillation, and ANS fluorescence probed the surficial hyfrophobicity of amyloid species. The results showed that the conformation of lysozyme was changed along with the incubation of the protein, including a decrease of the content of a-helices, increases of P-sheet structures and surfacial hydrophobicity. The growth curve of lysozyme amyloid fibrils appeared like a sigmoidal shape; indicating that the lysozyme fibrillation involved different phases including nucleation, elongation and maturation. In contrast, the growth curve of BSA fibrillation didn’t have a sigmoidal shape, suggesting a different pathway in the process of amyloid formation of the protein. The content of P-sheets increased whereas ANS intensity decreased along with incubation of BSA.2. Morphology of lysozyme fibrilsMorphology of lysozyme fibrils was detected by polarized light microscope. Under polarized light, the Congo Red-stained amyloid fibrils of lysozyme showed a no-branch stripe structure with a pale bright yellow color. Lysozyme fibrils were diminished when EGCG was added as an inhibitor.3. Effect of EGCG on matureed lysozyme amyloid fibrilsMatured lysozyme fibrils were incubated with100μg/ml EGCG for6h and followed by acid treating and centrifugation. UV spectroscopic analysis demonstrated that most of the protein was deposited. Meanwhile the deposited part showed different absorbance spectra. These facts suggest that EGCG could disrupt lysozyme amyloid fibrils and transform the fibrils into amorphous aggregates.4. Effect of EGCG on fibril growth of lysozymeEGCG was added into lysozyme at different incubation times from0-9days. ThT fluorescence indicated that EGCG played an inhibitory role at all stages of amyloid formation of lysozyme in a dose-dependent manner.5. Effects of other phenolic compounds on lysozyme fibrillationOther chemicals, including tea catechins EC, EGC, ECG, diphenols catechol, resorcinol, hydroquinone, phenol and benzoquinone were also investigated for a comparison with the effects of EGCG. ThT data indicated that except EC, phenol and resorcinol all the compounds showed a dose-dependent inhibitory role on protein amyloid fibrillation owing to their ability to form quinone intermediates.6. Molecular mechanism of inhibition of amyloid fibrillation by phenolsTo explore the role of quinone intermediates in protein amyloid fibrillation, SDS-PAGE was performed and the gelbands were electrically transferred onto a polyvinylidene fluoride membrane prior to detection of quinoproteins by NBT staining. Samples which could inhibit proteins fibrillation were found to form a blue-purple insoluble formazan upon NBT-staining with a staining level in accord with the order of inhibitory effenciency. Quinoprotein was not found in the sanples in which sulfhydryl-blocking reagent N-Ethylmaleimide (NEM) and sulfhydryl reagent dithiothreitol (DTT) were added. This fact suggests that quinoprotein is formed in a reaction between sulfhydryl group of a peptide and quinone intermediates. We suggest that the formation of reactive quinone intermediates is a prerequisite for a phenolic compound to inhibit amyloid fibrillation of a protein.ConclusionBased on the experimental data, lysozyme and BSA are able to form amyloid fibrils under the conditions of the present work. EGCG could disrupt the amyloid structures and transform the fibrils into amorphous aggregates. Some polyphenolic compounds showed also inbibitory effect on protein amyloid fibrillation with varying effeciencies in a dose-dependent manner. The molecular mechanismma might be: quinone intermediates, produced by phenolic compound via a prooxidant process, modified peptide chain at sulfhydryl group. Therefore the fibrillar structure was disrupted and amyloid fibrillation was inhibited. The formation of a quinone intermediate is a prerequisite for a phenolic compound to inhibit protein amyloid fibrillation. The information of the present study not only provides a fresh insight into the anti-amyloidogenic role of polyphenols, but also shed light on a rational design of novel anti-amyloidogenic drugs with a phenolic structure.