(â)-Epigallocatechin-3-gallate Inhibits Fibrillogenesis of Chicken

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Letter

(-)-Epigallocatechin-3-gallate Inhibits Fibrillogenesis of Chicken Cystatin Na Wang, Jianwei He, Alan K Chang, Yu Wang, Linan Xu, Xiaoying Chong, Xian Lu, Yonghui Sun, Xichun Xia, Hui Li, Bing Zhang, youtao song, Akio Kato, and Gary W Jones J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 26 Jan 2015 Downloaded from http://pubs.acs.org on January 28, 2015

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Journal of Agricultural and Food Chemistry

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(−)-Epigallocatechin-3-gallate Inhibits Fibrillogenesis of Chicken

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Cystatin

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Na Wanga, Jianwei Hea*, Alan K. Changa, Yu Wanga, Linan Xub, Xiaoying Chonga, Xian Lua,

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Yonghui Suna, Xichun Xiaa, Hui Lia, Bing Zhangc, Youtao Songa, Akio Katod and Gary W.

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Jonesb*

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a

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School of Life Science, Liaoning University, Shenyang 110036, China;

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b

Province Key Laboratory of Animal Resource and Epidemic Disease Prevention,

Department of Biology, National University of Ireland Maynooth, Maynooth, Co.

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Kildare, Ireland;

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c

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Road, Heping District, Shenyang 110001, China

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d

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Yamaguchi, Japan

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*Corresponding authors. Email: [email protected], [email protected]

Experimental Center of Functional Subjects, China Medical University, 92 BeiEr

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,

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KEYWORDS: (−)-Epigallocatechin-3-gallate; cystatin; amyloid; molecular dynamics

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simulation; molecular docking

ACS Paragon Plus Environment


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Abstract:

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Previous studies have reported that (-)-epigallocatechin-3-gallate (EGCG), the

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most abundant flavonoid in green tea, can bind to unfolded native polypeptides and

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prevent conversion to amyloid fibrils. To elucidate whether this anti-fibril activity is

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specific to disease-related target proteins or is more generic, we investigated the

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ability of EGCG to inhibit amyloid fibril formation of amyloidogenic mutant chicken

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cystatin I66Q, a generic amyloid-forming model protein that undergoes fibril

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formation through a domain swapping mechanism. We demonstrated that EGCG was

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a potent inhibitor of amyloidogenic cystatin I66Q amyloid fibril formation in vitro.

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Computational analysis suggested that EGCG prevented amyloidogenic cystatin fibril

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formation by stabilizing the molecule in its native-like state as opposed to redirecting

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aggregation toward disordered and amorphous aggregates. Therefore, while EGCG

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appears to be a generic inhibitor of amyloid-fibril formation, the mechanism by which

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it achieves such inhibition may be specific to the target fibril-forming polypeptide.

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Introduction

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Flavonoids have been demonstrated to be active inhibitors of fibrillation by

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amyloidogenic proteins.1, 2Previous studies have reported that EGCG is the most

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abundant flavonoid in green tea, and it can directly bind to unfolded native

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polypeptides and prevent their conversion to amyloid fibrils.3 However, the

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mechanism by which EGCG exerts its effects, as with all anti-amyloidogenic

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flavonoids, remains unclear. Currently, three mechanisms have been proposed to

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explain how EGCG inhibits the formation of amyloid fibril of disease-causing

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proteins: firstly, EGCG redirects the amyloidogenic proteins, such as α-synuclein,

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amyloid-β, PrP and hen egg white lysozyme, into nontoxic, unstructured and

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off-pathway oligomers;3-5 secondly, EGCG remodels mature α-synuclein and

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amyloid-β fibrils into smaller and amorphous protein aggregates that are nontoxic to

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mammalian cells;6 thirdly, EGCG maintains kappa-casein in its pre-fibrillar state

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without redirecting its aggregation pathway.7

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Although amyloid fibril formation is now recognized as a phenomenon

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common to many proteins, and it is rarely possible to form chimeric fibrils composed

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of distinct amyloid proteins or peptides,8 whether the anti-fibril activity of EGCG is

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specific to these disease-related target proteins or is a more generic property of the

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molecule, remains to be established.

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The mechanisms of fibril formation and morphology of fibrils are diverse, but

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fibrils do possess a characteristic X-ray diffraction cross-β pattern.9, 10 In contrast to

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Aβ and other amyloid forming proteins, human cystatin C (hCC) is the first



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amyloidogenic protein whose oligomerization was shown to be dependent on domain

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swapping, a mechanism that is ultimately responsible for forming the β-sheet-rich

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architecture seen with protein fibrils.11 Thus, while previous research suggests that

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EGCG may well be a generic inhibitor of amyloid-fibril formation, the ability of

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EGCG to inhibit protein oligomerization reliant upon a domain-swapping mechanism

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has yet to be elucidated.

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Cystatins are a superfamily of cysteine protease inhibitors.12 hCC, the most

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abundant extracellular inhibitor of cysteine protease, exists in almost all human

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tissues and body fluids.13-15 A point mutation of hCC (L68Q) can cause the formation

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of highly stable and domain-swapped dimers at physiological protein concentrations,

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and this is known to be responsible for hereditary cystatin C amyloid angiopathy

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(HCCAA).16 The structural conformation of hCC protein is very similar to that of

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chicken cystatin (cC),17, 18 and the higher thermodynamic stability of cC over hCC

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makes it more suitable for in vitro fibril-forming kinetics studies.19 The I66Q mutation

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of cC corresponds to the amyloidogenic mutation of hCC, L68Q.17

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In this letter we showed that EGCG efficiently inhibited the formation of amyloid

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fibrils by cC I66Q and we also proposed a mechanism of action. Importantly, while

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EGCG might appear to be a generic amyloid fibril-forming inhibitor, our data

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suggested that this molecule may cause inhibition through a variety of different

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mechanisms. As EGCG is a nutraceutical agent, our findings implicate the importance

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of diet and drink habits as playing a major role in guarding against amyloid fibril

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formation and promoting healthy aging.


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Materials and methods

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Proteins and reagents

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EGCG, Thioflavin T (ThT), 1-Anilinonaphthalene-8-sulfonic (ANS) were

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purchased from Sigma-Aldrich (St. Louis, MO, USA). Chicken cystatin mutant I66Q

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(cC I66Q) was expressed in Pichia pastoris, purified and characterized as described

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previously.20

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Preparation of cC mutant I66Q samples

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For fibril formation experiments, samples of cC I66Q solution were prepared by

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dissolving 1 mg cC I66Q powder in 1 mL glycine-hydrochloric acid buffer (50 mM,

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pH 2.0) with and without EGCG. EGCG was added to the cC I66Q samples at the

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desired concentration. The samples were incubated at 65 ℃ with constant agitation

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at 150 rpm during the course of aggregation.

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ThT fluorescence assay

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To determine the formation of amyloid fibrils, phosphate buffer (50 mM Na2HPO4,

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50 mM NaH2PO4, pH 7.0) was used to prepare a ThT stock solution of 1 mM.

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Aliquots of cC I66Q samples taken at different times were diluted with phosphate

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buffer, followed by the addition of 30 µL ThT stock solution. ThT fluorescence

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measurement was conducted by exciting samples at 440 nm and recording the

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emission signals at 485 nm over 120s using a Cary Eclipse fluorescence



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spectrophotometer (Varian, USA).

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ANS binding assay

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A 0.4 mM stock solution of ANS was prepared by dissolving ANS in PBS (pH

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7.0). The ANS stock solution was stored at 4 ℃. Aliquots of cC I66Q solution with or

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without EGCG taken at different times were mixed with an aliquot of ANS solution,

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followed by the addition of PBS (pH7.0) to a final volume of 3 mL. After incubation

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at room temperature in the dark for 30 min, the samples were subjected to

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fluorescence assay using an excitation wavelength of 380 nm and an emission

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wavelength between 400 nm and 600 nm. Both the ANS fluorescence intensity and

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the average emission wavelength were recorded to account for the changes in

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intensity and spectrum.

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Transmission electron microscopy

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Cystatin samples with or without EGCG were diluted five-fold and 10 µL of

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each sample were dispensed onto coated copper-mesh grid. The grids were negatively

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stained with 1% (w/v) phosphotungstic acid and then observed under a Hitachi

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H-7650 transmission electron microscope (Techcomp Ltd., Tokyo, Japan) with an

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accelerating voltage of 80 kV.

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MTT assay PC12 cells were purchased from American Type Culture Collection. PC12 cells


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were maintained in DMEM medium with 10% horse serum, 5% fetal bovine serum

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and 1% penicillin/streptomycin antibiotics. Cells were cultured in a 5% CO2

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atmosphere at 37℃, and then harvested and plated in 96-well plates (Beijing Dingguo

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Changsheng Biotechnology Co., Ltd.) at a density of 104 cells/well. The plates were

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incubated at 37℃ for 24h. Subsequently, cC I66Q with and without EGCG was

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incubated at 65 ℃ for 35 days, aliquots of the samples were collected after

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centrifugation at 6000 ×g for 1h. The concentrated cC I66Q samples were dissolved in

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PBS buffer (pH 7.0, 50 mM), and the protein concentration in each sample was

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determined by BCA (a kit bought from Beijing Dingguo Changsheng Biotechnology

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Co., Ltd.) assay. The cC 166Q samples, with and without ECGC, were separately

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added to the cells to give a final concentration of cC 166Q in the cells ranging from 0

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to 500 ng/mL. The final concentration of cC I66Q samples (both with or without

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EGCG) in each well was 0, 1, 5, 50, 500 ng/mL. The plates were then incubated with

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the protein samples for 48h at 37℃, and cell viability was determined using MTT

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toxicity assay by adding 10 µL of 5mg/mL MTT (Beijing Dingguo Changsheng

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Biotechnology Co., Ltd.) reagent to each well, followed by further incubation for 3h.

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After that, the medium was removed and replaced with 100 µL DMSO. After shaking

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for 10min at room temperature, the absorbance of the plate was measured at 490 nm

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using aniMarkMicroplate Reader (Bio-RAD).

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Molecular Dynamic Simulations (MD) and Docking Studies MD simulations were carried out using the GROMACS 4.0.7 software



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package.21 The crystal structure of chicken cystatin (PDB entry 1CEW) was

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downloaded from the Protein Data Bank.22 The model of I66Q monomer was

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constructed using the Swiss-Pdb Viewer software package. Before the docking, MD

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simulations of cC I66Q monomer at 338K (65℃) and pH 2.0 (amyloid fibril forming

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conditions) were carried out for 20ns to equilibrate (data not shown).

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Docking studies on the interaction between EGCG and cC I66Q were carried

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out using AutoDock 4.2.5.1 (Molecular Graphics Laboratory, The Scripps Research

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Institute). The 3D structure of EGCG was downloaded from Chem Spider database

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(http://www.chemspider.com/). Both cC I66Q and EGCG molecules were prepared

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using AutoDockTools 1.5.6 ( (c) 1999-2011 Molecular Graphics Laboratory, The

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Scripps Research Institute)before docking, The docking were carried out with number

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of 60×60×60 0.375 Å spacing grids covering the entire surface of cC I66Q.

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Lamarckian Genetic Algorithm, which is considered one of the most appropriate

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docking methods available in AutoDock, was used in the docking analysis.

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Results and discussion

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Analysis of cystatin fibril formation using ThT ﬂuorescence

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The recombinant variant cC I66Q has been reported to form amyloid fibrils

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following incubation with continuous agitation at high temperature and low pH.23

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After incubation of cC I66Q with and without EGCG at pH 2.0 and 65℃, inhibition

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of cC I66Q fibril formation by EGCG was determined using the ThT-binding


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fluorescence assay. A consistent increase in the ThT fluorescence intensity occurred

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when cC I66Q was incubated alone, indicating that the formation of amyloid fibrils

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proceeded rapidly and without a lag phase (Figure 1A). However, cC I66Q

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co-incubated with 1 mM EGCG showed a significant decrease in ThT fluorescence

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across the whole time frame of the experiment. The ThT fluorescence intensity of cC

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I66Q with EGCG reached a maximum (25.7 a.u.) on the 6th day, indicating that the

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inhibitory effect exerted by EGCG on the formation of cystatin fibril started from the

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very beginning of the log phase and continued to the end of the log phase.

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To determine the effective concentrations of EGCG for inhibiting cC I66Q fibril

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formation, the reaction was allowed to occur in the presence of different

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concentrations (1, 10, 100 and 1000 µM) of EGCG (Figure 1B). Reduction in ThT

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fluorescence was dependent on EGCG concentration, with significant reduction of

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fluorescence at 10 µM (P< 0.05).

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Structural changes of cC I66Q during fibril formation

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ANS is a fluorescent dye that probes exposed hydrophobic surfaces of proteins.

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Upon binding to the aromatic hydrophobic amino acids of cC, the chemical change of

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ANS would cause an increase in the intensity of the light to be emitted, with the

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emission maximum showing a blue shift.19, 24 As shown in Figure 2, the intensity of

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ANS fluorescence exhibited by cC I66Q fibrils showed a prominent increase in blue

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shift from 500 to 480 nm. In contrast, the addition of EGCG to cC I66Q resulted in a

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large reduction in ANS fluorescence intensity and almost no blue shift. This result



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indicates that the addition of EGCG prevented the accessible exposure hydrophobic

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regions of cC I66Q. In addition, this result indicated that binding of EGCG to cC

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probably occured at the monomeric level.

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Physical analysis of cC I66Q fibril formation and anti-fibrillogenic activity of

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EGCG

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TEM was used to observe the inhibitory effect of EGCG on cC I66Q fibril

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formation. After incubation at 65℃ and in glycine-hydrochloric acid buffer (pH 2.0)

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for 35 days, cC I66Q formed long and regular mature amyloid fibrils in the absence

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of EGCG (Figure 3A and 3B). The width of the detected fibroid material was

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calculated to be in the range of 50 Å to 100 Å, which closely matched the widths of

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the fibers observed for many amyloidogenic proteins, including those of human

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cystatins.17 In the presence of EGCG, cC I66Q formed oligomeric aggregates instead

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of amyloid fibrils, indicating that EGCG either redirected the amyloidogenic cC

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mutant I66Q toward unstructured off-pathway oligomers, or maintained the proteins

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in the pre-fibrillar state without redirecting them toward the aggregation pathway,

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thus, inhibiting the process of fibrillation (Figure 3C, D).23

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MTT analysis of the cellular toxicity of cC I66Q amyloid fibril and oligomers

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The mutation of hCC L68Q is associated with massive amyloid deposition within

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small arteries and arterioles of the leptomeninges, cerebral cortex, basal ganglia,

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brainstem, and cerebellum, resulting in severe cellular toxicity within the brain.25


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Therefore, after observing the morphology of the cC I66Q amyloid fibril and

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oligomer formations in the presence of EGCG, we assessed the cellular toxicity of cC

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I66Q on PC12 cells (neuronal cell model) using the MTT assay. After 48h of

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incubation, cell viability decreased significantly (P

(â)-Epigallocatechin-3-gallate Inhibits Fibrillogenesis of Chicken

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