International Journal of Biological Macromolecules 78 (2015) 389–395

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Quinopeptide formation associated with the disruptive effect of epigallocatechin-gallate on lysozyme fibrils Na Cao a,1 , Yu-Jie Zhang a,1 , Shuang Feng b , Cheng-Ming Zeng a,∗ a Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi an 710119, China b School of Chemical Engineering, Guizhou Institute of Technology, Guiyang 550003, China

a r t i c l e

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Article history: Received 2 December 2014 Received in revised form 2 April 2015 Accepted 20 April 2015 Available online 27 April 2015 Keywords: Epigallocatechin-3-gallate Amyloid fibrils Quinopeptides

a b s t r a c t Numerous studies demonstrate that natural polyphenols can inhibit amyloid formation and disrupt preformed amyloid fibrils. In the present study, the fibril-disruptive effects of epigallocatechin-3-gallate (EGCG) were examined using lysozyme as a model protein. The results indicated that EGCG dose dependently inhibited lysozyme fibrillation and modified the peptide chains with quinonoid moieties under acidic conditions, as measured by ThT fluorescence, transmission electron microscopy, and an NBTstaining assay. Moreover, EGCG transformed the preformed lysozyme fibrils to amorphous aggregates through quinopeptide formation. The thiol blocker, N-ethylmaleimide, inhibited the disruptive effect of EGCG on preformed fibrils, suggesting that thiol groups are the binding sites for EGCG. We propose that the formation of quinone intermediates via oxidation and subsequent binding to lysozyme chains are the main processes driving the inhibition of amyloid formation and disruption of preformed fibrils by EGCG. The information presented in this study may provide fresh insight into the link between the antioxidant capacity and anti-amyloid activity of polyphenols. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Amyloid fibrillation of polypeptides is associated with more than 20 human diseases, including Alzheimer’s disease, type 2 diabetes mellitus, Parkinson’s disease, hemodialysis-related amyloid deposition, transmissible spongiform encephalopathies, and a number of systemic amyloidoses [1–4]. Despite their unrelated amino acid sequences, these polypeptides are able to assemble into fibrils with identical amyloid properties including long and unbranched fibrillar morphology, enriched ␤-sheet structure, increased surface hydrophobicity, and the ability to disrupt cellular membranes. Inhibition of amyloid formation and disruption of formed fibrillar assemblies are the therapeutic strategies proposed for the treatment of amyloid-related diseases. Recent investigations have demonstrated that polyphenolic compounds, particularly natural polyphenols, are able to inhibit amyloid formation and disrupt preformed amyloid fibrils. Hydrogen bonding, hydrophobic interactions, and aromatic stacking are suggested to be the driving

∗ Corresponding author. Tel.: +86 29 81530726; fax: +86 29 81530727. E-mail address: [email protected] (C.-M. Zeng). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijbiomac.2015.04.031 0141-8130/© 2015 Elsevier B.V. All rights reserved.

forces of the anti-amyloidogenic role of polyphenols [5–7]. In addition, antioxidant capacity is also thought to be involved in the fibril-disrupting activity of a polyphenol [8,9]. It has been reported that the oxidized form of a polyphenol has a more potent disruptive effect on amyloid fibrils than the reduced form [7,10,11]. In the previous work [12], we found that the inhibition of lysozyme amyloid fibrillation by monocyclic diphenols was associated with the formation of quinoproteins and that quinone intermediates were actually the active form for phenolic compounds to interrupt amyloid structure. Many natural polyphenols have a polycyclic molecular structure, for example, some members of the flavonoid and flavanoid families. It is of significance to determine whether these natural polyphenols share a similar pathway with monocyclic diphenols in their anti-amyloidogenic activity. Epigallocatechin-3-gallate (EGCG, Fig. 1A), a member of the flavanoid family, is the most abundant catechin in green tea and is a potent antioxidant that has been widely investigated due to its benefits on human health. Recent investigations have indicated that EGCG exhibits an inhibitory effect on amyloid formation by the ␤amyloid peptide, ␣-synuclein, and other proteins [13–16]. It has also been reported that the fibril-disrupting efficiency of a tea catechin is positively correlated with its antioxidant capacity [9,17,18]. However, there is no clear understanding of the link between redox property and the corresponding anti-amyloid activity of

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2. Materials and methods 2.1. Chemicals (−)-Epigallocatechin-3-gallate (EGCG, MW 458.4), hen egg white lysozyme (MW 14.3 kDa), thioflavin T (ThT), Nethylmaleimide (NEM), 1-anilino-naphthalene 8-sulfonate (ANS), and nitroblue tetrazolium (NBT) were purchased from Sigma–Aldrich (St Louis, MO, USA). Electrophoresis reagents were from Bio-Rad (Hercules, CA, USA). All other reagents were of analytical grade. 2.2. Preparation and characterization of lysozyme fibrils Lysozyme fibrils were prepared according to our previous reports with minor modifications [9,19]. Briefly, hen egg white lysozyme was dissolved in HCl solution (10 mM, pH 2.0) with or without EGCG to a final concentration of 10 mg/mL (0.7 mM). EGCG (25 mM in water) was added according to the following molar ratios of EGCG to lysozyme (c EGCG/c lysozyme): 1:1, 1:2, and 1:7, respectively. The mixture was incubated for 12–14 days at 65 ◦ C in a water bath without agitation. Lysozyme fibril growth was monitored by ThT fluorescence, ANS fluorescence, and transmission electron microscopy (TEM). ThT fluorescence was measured in a mixture of 33 ␮g/mL lysozyme and 10 ␮M ThT with excitation at 440 nm and emission at 484 nm in a Perkin Elmer LS55 spectrofluorimeter. The emission spectra of ANS fluorescence in the presence of lysozyme fibrils were recorded between 400 and 600 nm using an excitation wavelength of 350 nm [19]. We confirmed that EGCG had no obvious effects on ThT and ANS fluorescence under the experimental conditions of this study. For TEM measurements, an aliquot of lysozyme fibrils was diluted 20-fold with water and dropped onto copper-mesh grids. Samples were negatively stained with 2% (w/v) uranyl acetate and air-dried at room temperature. Observations were carried out using a JEOL JEM-2100 electron microscope with an accelerating voltage of 80 kV. For seeding experiments, mature lysozyme fibrils were sonicated for 15 min in a water bath (37 ◦ C) and added to fresh lysozyme solutions at a ratio of 3% (seeds/fresh lysozyme; w/w). 2.3. Gel electrophoresis and NBT staining assay

Fig. 1. (A) Molecular structure of EGCG. (B) ThT curves of lysozyme fibril growth in the absence () and presence of EGCG at the following molar ratios of EGCG to lysozyme (0.7 mM): 1:7 (), 1:2 (), and 1:1 (). (C) ANS fluorescence of lysozyme fibril growth in the absence () and presence of EGCG at molar ratios of EGCG to lysozyme (0.7 mM): 1:7 () and 1:1 ().

polyphenols. In the present study, the anti-amyloidogenic effects of EGCG were evaluated in vitro using lysozyme as a model protein. The results suggest that EGCG inhibits lysozyme amyloid fibrillation and disrupts preformed fibrils in a dose-dependent manner. Quinopeptide formation was observed in both the inhibition of amyloid formation and disruption of preformed fibrils by EGCG. We further identified thiol groups originating from the breakage of disulfide bonds as the target of EGCG binding to lysozyme chains.

SDS–PAGE was performed in tricine buffer (pH 8.2) using a 5% stacking gel and a 15% separating gel. Bands were visualized by Coomassie brilliant blue R-250 staining. For the blotting assay, the gel bands were transferred onto a polyvinylidene fluoride membrane (0.45 ␮m, Millipore) with a mini transfer cell (GE Healthcare). Quinopeptides were detected by staining the membrane with NBT (0.24 mM in 2 M potassium glycinate, pH 10). The blotting membrane was immersed in the glycinate/NBT solution for 45 min in the dark, resulting in a blue-purple stain of quinopeptide bands and no staining of other peptides. 2.4. Chromatographic analysis of EGCG stability in the inhibition of amyloid formation Chromatographic separation was achieved on an Inerstil ODS column (4.6 × 250 mm, 5 ␮m; GL Sciences, Japan) using a Shimadzu LC-20A system (Kyoto, Japan) at ambient temperature. Samples were filtered over 0.22 ␮m filters (Millipore) prior to injection. The mobile phase consisted of 0.1% formic acid in 40% methanol in water. The injection volume was 20 ␮L, and the flow rate was maintained at 0.5 mL/min in an isocratic elution mode. EGCG and its degrade species were detected at 280 nm.

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2.5. EGCG transforms lysozyme fibrils to amorphous aggregates The transformation of lysozyme fibrils to amorphous aggregates by EGCG was quantitatively measured according to the method previously reported [9]. Aliquots of preformed fibrils at 12 days and an EGCG stock solution were added to Tris–HCl buffer (10 mM, pH 7.5) with or without 5 mM N-ethylmaleimide (NEM). The final concentration of lysozyme in the mixture was 70 ␮M. After incubating at 37 ◦ C for 6 h, the samples were centrifuged at 7000 × g for 10 min to separate the precipitates. Aliquots of the supernatant were obtained and the protein content was quantitatively measured using the Bradford assay [20]. Both the supernatant and precipitates were subjected to SDS–PAGE analysis and the NBT staining assay as described earlier. The precipitates were dissolved in 0.1 M HCl prior to electrophoresis.

3. Results 3.1. Inhibitory effects of EGCG on lysozyme fibrillation Incubation of lysozyme at low pH and elevated temperature resulted in the formation of amyloid fibrils. Increasing the temperature, ionic strength, or lysozyme concentration significantly shortened the lag time of fibril growth [19,21]. In the present study, we incubated 10 mg/mL (0.7 mM) lysozyme in a 10 mM HCl solution (pH 2.0) at 65 ◦ C in the presence or absence of EGCG. The growth of amyloid fibrils was monitored and characterized by ThT fluorescence, ANS fluorescence, and transmission electron microscopy. ThT is an amyloid fibril-specific dye that allows the monitoring of protein fibril growth when it binds specifically to the highly ordered ␤-sheet structure of amyloid fibrils [22]. The fluorescent profile of ThT bound to fibrillar lysozyme is characterized by a lag phase, followed by a sigmoid-like elongation phase, and a saturation phase, as shown in Fig. 1B. EGCG inhibited lysozyme fibrillation in a dose-dependent manner, resulting in a significant decrease in the final intensity of ThT fluorescence. No increase in ThT fluorescence was observed when lysozyme was incubated with EGCG for 14 days at molar ratios 1:1 and 1:2 of EGCG/lysozyme, indicating that fibril formation was completely inhibited. ANS is a specific fluorescent dye for probing changes in surface hydrophobicity of protein molecules. Upon binding to a hydrophobic region of protein, the intensity of ANS fluorescence is significantly enhanced with a blue-shift of the maximum emission wavelength [19]. Incubation of lysozyme resulted in a significant increase in the ANS fluorescence (Fig. 1C) and a shift in the maximum emission wavelength from 520 to 473 nm, reflecting an increase in the solvent-exposed hydrophobic interior of the protein during fibril growth. In the presence of EGCG, significant depreciation in ANS fluorescence (Fig. 1C) was recorded, suggesting that EGCG inhibited exposure of the hydrophobic interior of the protein, subsequently inhibiting fibril assembly. Fig. 2 shows the TEM images of the lysozyme fibrils prepared in the absence and presence of EGCG. In the absence of EGCG, the mature lysozyme fibrils had a typical amyloid morphology characterized by long, net-like, dense, and partly bundled fibrils (Fig. 2A). In the presence of EGCG, the lysozyme assemblies had different morphologies depending on the concentration of EGCG added. Dispersed, short fibrils and amorphous particles were observed when EGCG was added to lysozyme at a molar ratio of 1:7 (Fig. 2B), whereas only amorphous aggregates were observed in the sample containing equimolar concentrations of EGCG and lysozyme (Fig. 2C); these data indicate that, consistent with the ThT data, EGCG inhibited amyloid formation of lysozyme in a dosedependent manner.

Fig. 2. TEM images of lysozyme assemblies. Samples were prepared by incubating lysozyme (0.7 mM) at 65 ◦ C for 12 days in the absence (A) and presence of EGCG at molar ratios of EGCG to lysozyme: 1:7 (B) and 1:1 (C).

To explore the role of EGCG in different fibril growth stages, the compound was introduced and mixed with lysozyme at 0–7 days of incubation. As shown in Fig. 3A, after fibril growth was triggered, the addition of EGCG resulted in a decrease in ThT fluorescence, suggesting that the formed fibrils were disrupted upon interacting with EGCG. In other words, EGCG was able to transform the fibrils to a non-amyloid structure.

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Incubation time (days) Fig. 3. Fibril-disrupting effects of EGCG in the process of lysozyme fibril growth. (A) ThT curves of lysozyme fibril growth in the absence () and presence of EGCG at a 1:1 molar ratio of EGCG to lysozyme (0.7 mM). EGCG was introduced into lysozyme samples at days 0 (), 3 (), 5 (), and 7 () of the incubation. (B) Seeding assay. Incubation of lysozyme (0.7 mM) without fibril seeds (); with fibril seeds (); and with fibril seeds + EGCG at a 1:1 molar ratio of EGCG to lysozyme ().

It is well established that the addition of preformed fibril seeds at the beginning of the incubation accelerates fibril formation and abolishes the nucleation phase. To characterize the possible interaction of EGCG with the oligomers and nuclei formed at the early stage of fibril formation, we performed a seeding assay. As shown in Fig. 3B, addition of mature fibril seeds (3% of native lysozyme, w/w) abolished the lag phase and accelerated fibril formation. The seeding effect was inhibited by EGCG, suggesting that fibril assembly on the seeding templates was interrupted due to the interaction between EGCG and lysozyme. TEM images of the seeded samples at day 12 displayed a morphology similar to regular amyloid fibrils when no EGCG was present. The seeded lysozyme sample with EGCG, however, exhibited predominantly amorphous particles similar to those shown in Fig. 2C (data not shown). It is worth noting that EGCG has been reported [23] to inhibit lysozyme fibrillation in an alkali-salt medium (pH 12.75) instead of the acidic medium (pH 2.0) used in the present work. Typically, lysozyme fibrils are prepared at pH 2.0 to study fibrillar properties and cytotoxicity [19,21,24–28]. Fibrils prepared in an acidic medium can be disassembled by SDS into lysozyme monomers and hydrolysis-originated fragments (discussed subsequently). In contrast, at a high pH, lysozyme transforms into aggregates by intermolecular disulfide cross-linking [29,30]. Although the molecular mechanisms of lysozyme fibrillation were different under acidic and alkaline conditions, EGCG exhibited an inhibitory effect on amyloid formation under both conditions.

3.2. EGCG modifies lysozyme with quinonoid substances during amyloid inhibition There is considerable evidence showing that reactive quinonoid species can covalently bind to proteins [31–33]. When incubated with proteins, EGCG can be oxidized into reactive quinone or semiquinone species and then react with peptide side chains to form quinoproteins [33]. Our previous study demonstrated that the inhibition of lysozyme fibrillation by catechol and hydroquinone was associated with the formation of quinoproteins and that quinone intermediates were indeed the active form mediating the interruption of lysozyme amyloid fibrillation by phenolic compounds [12]. To determine whether quinone intermediates were formed during the incubation of lysozyme with EGCG, NBT-staining assays were performed to detect quinone-modified peptides. At an alkaline pH, quinones and related quinonoid substances can catalyze redox cycling, resulting in the reduction of NBT to the bluepurple insoluble formazan, allowing the detection of quinoproteins on a blotting membrane [34]. Fig. 4A shows the SDS–PAGE patterns of lysozyme incubated for 12 days in the absence and presence of EGCG. In the presence of SDS, the fibrillar species were disaggregated and separated into lysozyme monomers and small peptide fragments which originated from acidic hydrolysis of the protein [12,35]. In the presence of EGCG, the SDS–PAGE patterns (lanes 3–5, Fig. 4A) were similar to those of the control sample (lane 2, Fig. 4A), indicating that fragmentation of lysozyme via acidic hydrolysis is not related to amyloid formation. A parallel SDS–PAGE experiment was performed, and the gel bands were electrically transferred onto a polyvinylidene fluoride membrane prior to the detection of quinopeptides by NBT staining. As shown in Fig. 4B, quinone-modified peptides were observed in lanes 3–5, corresponding to samples containing EGCG at molar ratios of EGCG/lysozyme at 1:1, 1:2, and 1:7, respectively. The extent of NBT staining depended on the amount of EGCG applied in the sample. This indicates that EGCG was oxidized to form reactive quinonoid intermediates, which bound to the peptide chains of lysozyme in a dose-dependent manner. The results of Figs. 1B and 4B suggest that the more lysozyme was modified with quinones, the less amyloid fibrils formed. The modification of peptide chains may alter the interacting forces of intra- and

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Fig. 5. HPLC-UV chromatograms of EGCG and its degrading products. Lysozyme (0.7 mM) was dissolved in 10 mM HCl solution (pH 2.0) with or without addition of EGCG (0.7 mM). Samples were diluted 5-folds in 10 mM HCl prior to filtration and LC injection. (A) EGCG + lysozyme before incubation; (B) EGCG + lysozyme after incubation at 65 ◦ C for 4 days; (C) EGCG + lysozyme after incubation for 12 days; (D) lysozyme before incubation; (E) lysozyme after incubation for 12 days; and (F) 10 mM HCl. The abscissa represents retention time in minutes; the vertical ordinate represents absorbance at 280 nm in counts.

inter-peptide chains, thereby interrupting the process of amyloid fibrillation. The chemical instability of EGCG has been extensively investigated in a neutral or alkaline medium. Dube et al. [36] reported that there was ca. 80% degradation of EGCG in a phosphate buffer (pH 7.4) after incubating for 2 h at 37 ◦ C. Besides the degradation under neutral and alkaline conditions, EGCG is also found to degrade slowly in an acidic medium [37]. The data of this study suggested that EGCG degraded and transformed into quinonoid substances at pH 2 upon incubating with lysozyme at 65 ◦ C. The degradation of EGCG during the incubation was detected by liquid

chromatographic analysis. Under the chromatographic condition described in the experimental section, EGCG was eluted at a retention time of 13.8 min (Fig. 5A). The absorbance of EGCG decreased with increases of incubation time. After incubating for 4 days, more than 50% reduction in EGCG with a generation of new peak at 10.4 min was recorded (Fig. 5B). After incubating for 12 days, less than 10% of intact EGCG remained in the solution (Fig. 5C). No interference was observed from lysozyme in the retention time window of EGCG and its degrading products. Fig. 5D and E shows the chromatographic profiles of lysozyme in 10 mM HCl before and after incubation, respectively.

Fig. 6. (A) SDS–PAGE of mature lysozyme fibrils treated with EGCG. Lane 1, native lysozyme (L, 14.3 kDa) and bovine insulin (I, 5.7 kDa) were added as molecular weight markers; lane 2, lysozyme fibrils without EGCG; lane 3, lysozyme fibrils (70 ␮M) treated with 70 ␮M EGCG (the supernatant after centrifugation); lane 4, lysozyme fibrils treated with EGCG (the precipitates after centrifugation). (B) SDS–PAGE of lysozyme fibrils treated with EGCG in the absence and presence of NEM. Lane 5, lysozyme fibrils without EGCG; lane 6, lysozyme fibrils (70 ␮M) treated with 5 mM NEM in the absence of EGCG; lane 7, lysozyme fibrils treated with EGCG (70 ␮M) in the presence of 5 mM NEM (the supernatant after centrifugation); lane 8, lysozyme fibrils treated with70 ␮M EGCG in the presence of 5 mM NEM (the precipitate after centrifugation). (C) and (D) show the NBT-staining results of (A) and (B), respectively.

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The degradation of EGCG is a complex process probably involving oxidation, hydrolysis, polymerization, and other chemical changes. Due to difficulties of qualification and complexity of the products, it is a complicated task to get detailed information about the degradation of EGCG. The auto-oxidation or oxidation of EGCG results in the formation of several products such as quinones and dimer quinones. Possible mechanisms for EGCG in vitro oxidation have been illustrated by Sang et al. [38]. The NBT assay of the present study indicates that EGCG transformed into reactive quinonoid intermediates and subsequently bound to the peptide chains of lysozyme. Under an acidic condition, the reaction rate of oxidation and quinone formation of EGCG is limited. However, the unfolding and disulfide breakage of lysozyme during incubation can provide effective binding sites for EGCG quinones, resulting in a shift of EGCG reaction to quinone formation. Nevertheless, the inhibitory role of the remaining intact EGCG and other degrading products on lysozyme fibrillation cannot be excluded and remains further investigation. 3.3. EGCG disrupts preformed lysozyme fibrils through quinopeptide formation One fibril-disruptive role of a natural polyphenol is to transform amyloid fibrils into amorphous aggregates. Transformation of amyloid fibrils into amorphous aggregates is accompanied by a decrease in the amyloid structure and fibril cytotoxicity [9,39,40]. In the previous work, we found that the fibril-disruptive activity of tea catechins depended on both their hydrophobicity and antioxidant capacity [9]. However, the detailed underlying molecular mechanism is still unclear. In this study, SDS–PAGE and NBT-staining assays were performed to analyze the EGCG-induced amorphous aggregates of lysozyme fibrils. The preformed fibrils were incubated with EGCG, and the resultant amorphous aggregates were separated by centrifugation and solubilized in 0.1 M HCl. Both the precipitates and supernatant were subjected to SDS–PAGE and NBT-staining assay. As depicted in Fig. 6A, the acidic hydrolysisinduced peptide fragments with molecular weights around 6 kDa were predominant in both the supernatant (lane 3) and the deposited aggregates (lane 4). The bands of lysozyme monomer and other peptide fragments were weak in lanes 3 and 4, probably due to the low staining efficiency of Coomassie brilliant blue on the EGCG-bound amorphous components. A parallel SDS–PAGE gel was subsequently electrically blotted on a membrane for NBT staining. The results showed that the components in the supernatant (lane 3, Fig. 6C) and the deposited aggregates (lane 4, Fig. 6C) were stained by NBT, suggesting that quinopeptides were formed when EGCG transformed lysozyme fibrils to amorphous aggregates. NEM is chemically reactive with thiol groups and is often used as a thiol blocker in protein chemistry. Our previous work confirmed that EGCG induced aggregation of cytoskeletal proteins by covalently binding with the thiol groups of peptide chains and that NEM inhibited EGCG-induced aggregation of membrane proteins [33]. In the present study, we evaluated the role of thiol groups in the EGCG-induced fibril deposition. As shown in Fig. 7, EGCG dose dependently transforms lysozyme fibrils into amorphous aggregates (the blank columns). After incubation with EGCG (70 ␮M) at 37 ◦ C for 6 h, 15.3% of the fibrils (70 ␮M) were transformed into amorphous deposits. Interestingly, NEM attenuated the disruptive effect of EGCG on lysozyme fibrils. In the presence of excess NEM (5 mM), the formation of amorphous deposits was inhibited significantly (Fig. 7, the shaded columns). SDS–PAGE results demonstrate that, in the presence of NEM, the bands of EGCG-treated fibrils (lanes 7 and 8, Fig. 6B) exhibit a pattern similar to the bands of untreated fibrils (lane 5, Fig. 6B) and to the bands of fibrils treated with NEM only (lane 6, Fig. 6B). A parallel NBT-staining assay was

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also performed. As depicted in Fig. 6D, only weak stains could be observed in lanes 7 and 8, to which the supernatant (lane 7) and deposited aggregates (lane 8) of EGCG-treated fibrils were applied, respectively, indicating that quinopeptide formation was inhibited by NEM. The weak stains in lanes 7 and 8 probably originated from the binding of quinone substances to other side chains, for instance, the amine group of lysine [41–43]. These findings suggest that EGCG disrupts preformed fibrils primarily by binding to the thiol groups of lysozyme chains. According to Fig. 7, NEM could not completely inhibit the disruptive effect of EGCG on lysozyme fibrils. In addition to quinone binding, other driving forces, probably weak interactions, may be also involved in the fibril-disruptive role of EGCG. The weak interactions, including hydrophobic interactions, aromatic stacking, and hydrogen bonding, have been suggested to play important roles in the anti-amyloidogenic effect of a polyphenol containing a polyhydroxylated and polycyclic structure. 4. Discussion Amyloid fibrillation of lysozyme consists of a cascade of events including unfolding and association of the protein monomers, nucleation, elongation, and maturation of the fibrillar species, corresponding to different stages of the growth curve shown in Fig. 1B. Under the experimental conditions in the present study, lysozyme transformed into mature amyloid fibrils in 12 days with a lag time of approximately 4 days. During the early stage of incubation, lysozyme molecules underwent unfolding and their hydrophobic interior was exposed to the solvent. This process is thermodynamically unfavorable. The positive shift in free energy was compensated by the formation of lysozyme aggregates, which can serve as nuclei for amyloid assembly. In the presence of EGCG, exposure of the hydrophobic interior in the lysozyme molecule was inhibited, leading to a decrease in ANS fluorescence and the inhibition of fibril growth. The inhibition of lysozyme fibrillation by EGCG can be attributed to the binding of the compound to peptide chains. Early reports emphasized the role of weak forces (non-covalent interactions) between natural polyphenols and peptide chains in amyloid inhibition and fibril disruption [5–7]. Recent investigations demonstrated that covalent binding also plays an important role in the inhibition of protein fibrillation by polyphenols. It has been suggested that quinone or quinonoid substances mediate the inhibitory effect of polyphenols on amyloid formation [11,41]. Our previous work demonstrated that the formation of reactive

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quinone intermediates was a prerequisite for a monocyclic phenolic compound to inhibit amyloid fibrillation of lysozyme [12]. In the present study, quinone-modified peptide chains of lysozyme were detected in the samples treated with EGCG. The NBT-staining assay suggested that the inhibitory effect of EGCG on amyloid formation was related to the formation of quinopeptides. Another important result of this study is that quinopeptide formation is the main process in EGCG-induced transformation of lysozyme fibrils to amorphous aggregates. When incubated with the fibrils, EGCG was transformed to quinone intermediates that interacted with the thiol groups originating from the breakage of disulfide bonds in lysozyme, resulting in the disruption of the amyloid fibrillar structure. This was verified by the finding that the thiol blocker, NEM, attenuated EGCG-induced fibril deposition and inhibited quinopeptide formation. Previous investigation showed that the oxidative form of polyphenols showed more potent inhibitory effect on amyloid formation than the reductive form [7,10,11]. The authors attributed this phenomenon to that oxidized polyphenol possessed higher binding affinity for the growing ends of fibrils than fresh compound. On the basis of results of the present study, we suggest that the formation of quinone species and their subsequent specific binding to the peptide chains are crucial for EGCG-disrupting lysozyme fibrils. Native lysozyme is a small globular protein with a single chain comprising 129 amino acid residues and four disulfide linkages. During amyloid formation, the disulfide bonds in lysozyme become more accessible to the solvent [19] and free thiols may be produced due to disulfide breakage, particularly in the presence of thiol-reacting reagents or reducing compounds [44–46]. In another experiment, the production of free thiols in the process of lysozyme fibrillation was confirmed by the Ellman method [47]. Interestingly, co-incubation of lysozyme with EGCG significantly decreased the level of free thiols (data not shown). This suggests that free thiols may be a binding site for EGCG in the process of lysozyme fibrillation. Binding of thiols with active EGCG quinones results in the modification of lysozyme peptide chains, leading to the disruption of amyloid structures in both fibril growth and deposition. However, whether quinopeptide formation is a main factor in EGCG-mediated inhibition of lysozyme fibrillation requires further investigation. Indeed, NEM (5 mM) had no effect on lysozyme fibrillation either in the presence or in the absence of EGCG (data not shown). The reaction between NEM and thiols occurs optimally at a neutral pH range of 6.5–7.5, higher than the pH value of the solvent used in the preparation of lysozyme fibrils in this study. Therefore, under these experimental conditions, NEM was not able to compete with EGCG to bind thiols during lysozyme fibrillation. Of the anti-amyloidogenic natural polyphenols, only EGCG is currently being evaluated in the clinic due to its benefits in the treatment of amyloid disorders. However, the exact molecular mechanism of action of EGCG on amyloidosis has not been elucidated. The results of the present study suggest that quinopeptide formation is a key process in the inhibition of lysozyme fibrillation and disruption of preformed amyloid fibrils by EGCG. The data in this study may provide insight into the link between the antioxidant potency and anti-amyloid activity of EGCG. In conclusion, the present study shows that EGCG inhibits lysozyme fibrillation and disrupts preformed fibrils by modifying the peptide chains with quinone substances. When incubated with lysozyme, EGCG is oxidized to form quinonoid intermediates that interact with lysozyme chains, resulting in the formation of quinopeptides. The thiol blocker, NEM, inhibits EGCG-induced fibril disruption and quinopeptide formation, suggesting that thiol groups are the binding sites for EGCG quinones. We suggest that quinopeptide formation plays a crucial role in the inhibition of amyloid formation and disruption of mature amyloid fibrils by EGCG.

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Acknowledgments This work was supported by the Fundamental Research Funds for the Central Universities (GK20133001). We thank Miss Gai-Tao Li for her invaluable assistance in performing NBT assays. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]

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