Hematoxylin Inhibits Amyloid Î²-Protein Fibrillation and Alleviates

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Hematoxylin Inhibits Amyloid #-Protein Fibrillation and Alleviates Amyloid-Induced Cytotoxicity Yilong Tu, Shuai Ma, Fufeng Liu, Yan Sun, and Xiaoyan Dong J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b06878 • Publication Date (Web): 17 Oct 2016 Downloaded from http://pubs.acs.org on October 18, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Hematoxylin Inhibits Amyloid β-Protein Fibrillation and Alleviates Amyloid-Induced Cytotoxicity

Yilong Tu1, Shuai Ma1, Fufeng Liu1,2*, Yan Sun1, and Xiaoyan Dong1*

1

Department of Biochemical Engineering and Key Laboratory of Systems

Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China 2

College of Biotechnology and National and Local United Engineering Lab of

Metabolic Control Fermentation Technology, Tianjin University of Science & Technology, Tianjin 300457, P. R. China

1

ACS Paragon Plus Environment


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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Accumulation and aggregation of amyloid β-protein (Aβ) play an important role in the pathogenesis of Alzheimer’s disease. There has been increased interest in finding new anti-amyloidogenic compounds to inhibit Aβ aggregation. Herein, thioflavin T fluorescent assay and transmission electron microscopy results showed that hematoxylin, a natural organic molecule extracted from caesalpinia sappan, was a powerful inhibitor of Aβ42 fibrillogenesis. Circular dichroism studies revealed hematoxylin reduced the β-sheet content of Aβ42 and made it assemble into antiparallel arrangement, which induced Aβ42 to form off-pathway aggregates. As a result, hematoxylin greatly alleviated Aβ42-induced cytotoxicity. Molecular dynamics simulations revealed the detailed interactions between hematoxylin and Aβ42. Four binding sites of hematoxylin on Aβ42 hexamer were identified, including the N-terminal region, S8GY10 region, turn region and C-terminal region. Notably, abundant hydroxyl groups made hematoxylin prefer to interact with Aβ42 via hydrogen bonds. This also contributed to the formation of π-π stacking and hydrophobic interactions. Taken together, the research proved that hematoxylin was a potential agent against Aβ fibrillogenesis and cytotoxicity.

2


Page 2 of 38

Page 3 of 38

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Alzheimer’s

disease

(AD),

the

most common

form

of

dementia,

is a

neurodegenerative disorder that is manifested by the progressive loss of memory and cognitive functions. The histological picture of AD shows the accumulation of the cerebral intracellular neuroﬁbrillary tangles formed by hyper-phosphorylated tau protein and extracellular senile plaques by amyloid-β protein (Aβ).1, 2, 3 Aβ containing 39-43 residues is normally produced by sequential cleavage of the amyloid precursor protein (APP) by both β- and γ-secretases.4 Upon cleavage, Aβ40 and Aβ42 are the two most common forms. Aβ40 is the most abundant form, while Aβ42 is prone to aggregation.5, 6, 7 It is proven that Aβ self-assembles into a well-ordered aggregates containing cross-β-sheet structure, leading to apoptosis of neuronal cells.8, 9 Therefore, inhibiting the aggregation of Aβ might be an effective therapeutic method for AD. Up till now, many inhibitors including organic molecules,10, 11, 12 peptides13, 14 and nanoparticles,15, 16 have been developed against Aβ aggregation. At present, more extensive attention has been taken to the natural organic molecules because of their high permeability through the blood-brain barrier and low cytotoxicity. Many excellent natural molecules have been discovered to inhibit amyloid fibrillation and reduce the corresponding cytotoxicity, such as anthocyanins (found in various plant foods),17 (-)-epigallocatechin gallate (EGCG) (found in green tea),18,

19

resveratrol

(found in grape skin and seeds),20 carotenoid (found in apricot fruit),21 curcumin (found in rhizome of Zingiberaceae),22, 23 tanshinones (extracted from the roots of Salvia Miltiorrhiza Bunge)24 and tabersonine (extracted from the bean of Voacanga 3



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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

africana).25 However, none of them has been used for the clinical treatment of AD. Therefore, more effective natural molecules with high inhibitory capability on Aβ fibrillation are urgently required. Caesalpinia sappan, a common natural plant, possesses the medicinal abilities as antibacterial and anticoagulant properties and produces some valued reddish dyes, which were used for making herbal drinking water.26, 27 Our previous studies have discovered brazilin (another common component of Caesalpinia sappan) as a dual functional compound in both Aβ fibrillogenesis inhibition and mature fibril remodeling.28 Similar with brazilin (Figure S1), hematoxylin also contains two benzene rings and several hydroxyl groups (Figure 1A), which will form π-π stacking and hydrogen bonds with Aβ42, respectively. Moreover, these interactions have been reported to be crucial to the formation of Aβ42 fibrils.29, 30 Herein, we have studied the inhibitory capacity of hematoxylin on Aβ42 fibrillogenesis and its corresponding cytotoxicity using systematic biochemical, biophysical and cell biological experiments. Then, all-atom molecular dynamics (MD) simulations were used to further explore atomic insights into the detailed interactions between Aβ42 and hematoxylin. Among them, the hydrogen bonds between hematoxylin and Aβ42 were analyzed emphatically. Brazilin was also selected as the control compound for comparative analysis. These works had great implications to the rational design of novel small organic molecules to combat AD.

MATERIALS AND METHODS 4


Page 4 of 38

Page 5 of 38

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Materials. Aβ42 (＞95%) lyophilized powder was purchased from GL Biochem Ltd. (Shanghai, China). Hematoxylin (HPLC >=98%) and brazilin (HPLC >=98%) were purchased from Yuanye Bio-Technology Co. Ltd. (Shanghai, China). Thioflavin T

(ThT),

dimethyl

sulfoxide

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

(DMSO), (MTT)

and

1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were obtained from Sigma (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco Invitrogen (Grand Island, NY). All other chemicals were the highest purity available from local sources. Aβ42 Preparation. Aβ42 was prepared as described in our previous literature.31 Briefly, lyophilized Aβ42 was dissolved in HFIP to a final concentration 1.0 mg/mL and incubated at 4 oC for 2 h. Thereafter, the solution was sonicated for 2 min and then centrifuged at 16,000 g (4 oC) for 20 min to remove the pre-existing aggregates. Finally, about top 75% of the supernatant was collected and lyophilized by vacuum freeze-drying overnight. Before use, Aβ42 was stored at -20 oC temporarily. Thioflavin T Fluorescence Assay. Aβ42 fibrillization in the presence and absence of the inhibitors was monitored by ThT fluorescence assay. Aβ42 powder was firstly dissolved in NaOH and diluted to a final concentration of 25 µM in phosphate buffer solution (100 mM sodium phosphate, NaCl 10 mM, pH 7.4). Then the samples were incubated with or without different concentrations of inhibitors at 37 °C with continuous shaking at 150 rpm for 24 h. 2 mM ThT store solution was prepared by adding 32.8 mg of ThT powder into 50 mL de-ionized water. Then the store ThT 5



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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

solution was further diluted using Tris buffer (pH 7.4) to reach a final concentration of 25 µM. Thereafter, aliquots of incubation solutions at different time points were then diluted 10 times into ThT solution (25 µM ThT, pH 7.4). The ex situ ThT fluorescent assay was performed by a fluorescence spectrophotometer (LS-55, PerkinElmer) at room temperature. Moreover, the in situ ThT fluorescent assay were performed in triplicate by a fluorescence plate reader (SpectraMax M2e, Molecular Devices, USA) at 15 min reading intervals and 5 s shaking before each read. The wavelengths of excitation and emission were 440 and 480 nm, respectively. The fluorescence intensity of solution without Aβ42 was subtracted as background from each read with Aβ42. Three measurements were performed and all data represent the mean ± standard deviation. Transmission Electron Microscopy (TEM). From each 200 µM incubation solution, 10 µM samples were adsorbed onto formvar carbon-coated copper grids (400-mesh) for 2 min and then stained with 2% phosphotungstic acid for 5 min. The stained samples were examined and photographed on a JEM-100CXII TEM system (JEOL Inc., Tokyo, Japan) operating at 100 kV. Circular Dichroism (CD) Spectroscopy. Far-UV (200-260 nm) CD measurements on 25 µM Aβ42 solution samples with or without 25 µM hematoxylin were performed using a J-810 spectrometer (Jasco, Japan) with a 1 mm path length quartz cuvette at room temperature. The spectra were averaged for three scans with 1 nm bandwidth at a scanning speed of 20 nm/min. The response time is 2 sec and the data pitch is 0.1 nm. The baseline (buffer with and without hematoxylin) was subtracted from the 6


Page 6 of 38

Page 7 of 38

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


result for each sample. The CD spectral data was normalized as molar residue ellipticity. Cell Viability Assay. Human neuroblastoma SH-SY5Y cells were cultured in DMEM/F-12 medium with 20% fetal bovine serum and 2% L-glutamine at a density of ~5000 cell/well (90 µL/well) in 96-well plate. Aβ42 was incubated in PBS buffer with different concentrations of hematoxylin. In the previous preliminary study, it was found that the 24 h-aged Aβ42 aggregates were the most toxicity one (data not shown). Therefore, the 24 h-aged Aβ42 aggregates were selected to asses the cell viability. The 24 h-aged Aβ42 aggregates were added into the plates (10 µL/well) and the cells were incubated for another 48 h. Thereafter, 6.0 mg/L MTT solution was added into a plate (10 µL/well) and incubated for another 4 h. Then the plate was centrifuged to remove the culture medium and DMSO was added to lyse the precipitated cells. Cytotoxicity was measured utilizing a plate reader (TECAN, Austria) by calculating the absorbance signals at 570 nm wavelength. The wells with only medium were subtracted as the background from each reading. The cell viability data were normalized as a percentage of the control group without Aβ42 and inhibitors. The error bars represent the standard deviations of four independent measurements. Simulation System. To probe the molecular interactions between hematoxylin and Aβ, Aβ42 hexamer was selected as the target protein and the 3D model is shown in Figure S2. The initial structure of Aβ42 hexamer was kindly provided by Wei et al.32 The initial structure of hematoxylin and brazilin were generated by the program 7



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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ChemBioDraw (Ultra 13.0 2012) and its GROMOS topology was first made by the GlycoBioChem PRODRG2 Server (http://davapc1.bioch.dundee.ac.uk/cgi-bin/prodrg). Then, the atomic charges and charge groups of these compounds were adjusted based on the GROMOS96 53A6 force field parameter set.33 Molecular Dynamics Simulation. One Aβ42 hexamer was put into a cubic box with periodic boundary conditions. The box size of the hexamer simulation system was 9 nm. Based on the experimental results of ThT fluorescent, TEM, CD and cell viability assay, we can concluded that hematoxylin inhibit Aβ42 fibrillization at the molar ration of 1:1 with Aβ42. Therefore, 6 hematoxylin or brazilin molecules were located randomly around the hexamer. Thereafter, water molecules were added into the box and several solvent molecules were replaced by positive ions (Na+) to neutralize the simulation system. The simple point charge (SPC) model was used to describe water. After 1000 steps energy minimization, the system was equilibrated for 200 ps under an isochoric-isothermal (NVT) ensemble and isothermal–isobaric (NPT) ensemble successively using the Berendsen weak coupling method.34 Then, three MD simulations of 300 ns under different initial conditions were carried out by assigning different initial velocities on each atom of the simulation system. All of the MD simulations were performed at a temperature of 310 K and a pressure of 1 bar. MD Simulation Analysis. The simulation trajectories were analyzed using auxiliary programs provided by GROMACS 5.1.2 package. The snapshots were made by visual molecular dynamics (VMD) software version 1.9.2. The programs include gmx energy for calculating the Lenard Jones and coulomb potential energies between 8


Page 8 of 38

Page 9 of 38

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


small molecules and Aβ42, gmx hbond for the number of hydrogen bonds between Aβ42 and the inhibitors, gmx mindist for the number of contacts between the peptide and the inhibitors with the cut-off of 0.5 nm. The probabilities of contacts and hydrogen bonds in six monomers of hexamer were counted together. The standard error of mean (SEM) was used to describe the statistical error.

RESULTS AND DISCUSSION Inhibitory Effect of Hematoxylin on Aβ42 Fibrillation. ThT fluorescence assay is usually applied to quantitatively characterize the aggregation kinetics of amyloid fibrillation. However, many previous studies have proven that ThT assay often was biased by the presence of small molecular inhibitors.14 For example, EGCG and curcumin were reported to compete with ThT for protein binding and affected the quantitative accuracy of Aβ42 fibrils.35 Therefore, the ex situ ThT assay was performed to investigate whether hematoxylin competed for binding sites with ThT. As shown in Figure S3, the fluorescence intensity of pure Aβ42 was defined as 100% and set as a control. In the presence of hematoxylin, the ThT fluorescent intensity of Aβ42 fibrils was about 95%, well within the margin of error of pure Aβ42. That is, the presence of hematoxylin almost did not affect the accuracy of the ThT fluorescence measurement. Therefore, ThT fluorescence assay can be used to study the inhibitory effect of hematoxylin on Aβ42 fibrillogenesis. Based on the above work, the inhibitory effects of hematoxylin and brazilin with different concentrations on Aβ42 fibrillogenesis were studied and shown in Figure 1B. 9



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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

It is clear that hematoxylin had a marked inhibitory effect on Aβ42 fibrillogenesis with an IC50 of 1.6 µM, which is comparatively inhibitory capacity of brazilin (IC50 ~2.3 µM, shown in Figure 1B) and the (-)-epigallocatechin gallate in Phase III clinical trials.28 Then we used transmission electron microscopy (TEM) to further investigate the effect of hematoxylin on the morphology of Aβ42 fibril. In the absence of hematoxylin, large and long fibrils with ~400 nm length and 15 nm width were observed after 24 h incubation (Figure 1C). When hematoxylin was added at a molar ratio of 1:1 to Aβ42, the typical fibrils were not found, but some granulated aggregates were formed and distributed sporadically (Figure 1C). It suggests that hematoxylin could modify the morphologies of Aβ42 aggregates, supporting the results of preceding ThT fluorescent assays (Figure 1B). In order to compare the inhibitory capacity of hematoxylin with brazilin, the aggregation kinetic curves of Aβ42 with different concentrations of the two inhibitors were measured by ThT fluorescent assay (Figure 2). It is clear that both hematoxylin and brazilin prevented greatly on the aggregation of Aβ42 in the selected concentrations. It is noted that hematoxylin had stronger inhibitory capacity than brazilin in the lower concentrations. Especially for hematoxylin at a molar ratio of 0.1:1 to Aβ42, the ThT fluorescent intensity was lower than that of brazilin (Figure 2A). It suggests that hematoxylin had higher inhibitory efficiency than brazilin at low concentrations although the two inhibitors differ in only one hydrogen atom. Moreover, in the studied concentrations from 5 to 25 µM (Figures 2B-2D), 10


Page 10 of 38

Page 11 of 38

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hematoxylin showed a stronger inhibitory capacity than brazilin in the initial 5 h although similar ThT fluorescent intensities were observed for both hematoxylin and brazilin after 10 h incubation. That is, hematoxylin was more efficient than brazilin in the initial phase of Aβ42 aggregation. Generally, small-molecular inhibitors have low bioavailability and strong toxic side effects.36 For example, 10 µM or higher concentrations

of

brazilin

shows

significant

cytotoxicity.28

In

addition,

small-molecular compounds must traverse the blood-brain barrier because the lesions of AD are located in the brain, which makes the concentration of the target compound in a brain very low. Therefore, hematoxylin had more potential for drug development than brazilin. Secondary Structure of Aβ42 Affected by Hematoxylin. To investigate the effect of hematoxylin on the conformational behavior of Aβ42, CD spectroscopy experiments were performed to explore the secondary structure of Aβ42 in the absence and presence of hematoxylin and shown in Figure 3. At the beginning of incubation, the typical unstructured conformation, signified by a negative peak at 200 nm, was observed in the absence and presence of hematoxylin (Figure 3A). After 24 h incubation, the negative peak at 200 nm disappeared and a negative peak at 217 nm appeared which corresponds to the β-sheet structure (Figure 3D). However, the typical CD spectrogram of β-sheet structure was also observed in the presence of hematoxylin at a molar ratio of 1:1 to Aβ42, although the values of the negative valley at 217 nm and the peak at 200 nm had slightly changed. That is, the content of β-sheet of Aβ species mediated by hematoxylin is lower than that of pure Aβ42, which is also 11



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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

consistent with brazilin.28 In order to further analyze the secondary structure affected by hematoxylin, the CD spectra of Aβ42 in the absence and presence of hematoxylin were calculated by the BeStSeL web server (http://bestsel.elte.hu/) and shown in Figure 4.37 From Figure 4A, the secondary structures of pure Aβ42 fibrils were mainly composed of parallel β-sheet (about 37.7%), antiparallel β-sheet (13.2%) and others (about 48.5%). By contrast, 42.5% β-sheet structures were formed in presence of hematoxylin (Figure 4B), which is less than those of pure Aβ42, consistent with the results of CD spectrogram (Figure 3D). It is remarkably notable that these β-sheet structures were stacked in an antiparallel way in the presence of hematoxylin while those of pure Aβ42 fibrils were arranged in parallel. Recent studies have found that the β-sheet of Aβ42 fibrils was placed in a parallel arrangement.38, 39 Therefore, it is concluded that hematoxylin could not only reduce the β-sheet content of Aβ42, but also induce Aβ42 to form the off-pathway aggregates with antiparallel arrangement. Protective Effect of Hematoxylin on the Aβ42-induced Cytotoxicity. In order to investigate the ability of hematoxylin to inhibit Aβ42 aggregates-induced cell death, the MTT cytotoxicity assay was carried out using human neuroblastoma SH-SY5Y cells. Firstly, the toxicity of hematoxylin was detected and shown in Figure S4. The different concentrations of hematoxylin were added to cell culture to determine the maximal tolerance dose for cell. In these experiments, the absorbance of the cell media containing only SH-SY5Y cells rather than Aβ42 or hematoxylin was measured and the value was defined as 100% cell viability. From Figure S4, cell viability 12


Page 12 of 38

Page 13 of 38

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


decreased with the increasement of hematoxylin’s concentration as expected. When the concentration was below 5 µM, however, hematoxylin did not show a significant cytotoxicity to SH-SY5Y cells. Then we validated the protective capacity of hematoxylin on the Aβ42-induced cytotoxicity. As shown in Figure 5, the cell viability decreased to around 67% after the 24 h incubated Aβ42 species were added into SH-SY5Y cells. When Aβ42 was co-incubated with different concentrations of hematoxylin, it substantially increased the cell viability in a dose-dependent manner. For example, in the presence of 12.5 µM hematoxylin, the cell viability reached the highest value of about 92%. With further increasing of hematoxylin, the cell viability decreased instead. This suggests that the toxicity of hematoxylin was greater than its detoxification effect at higher concentrations because the higher concentrations of hematoxylin showed a significant cytotoxicity (Figure S4). Therefore, hematoxylin can protect SH-SY5Y against Aβ42-induced cytotoxicity at low concentrations. An appropriate concentration was necessary for hematoxylin to inhibit the Aβ42-induced cytotoxicity. Direct Interactions between Hematoxylin and Aβ42. To explore the inhibitory mechanism of hematoxylin on Aβ42 aggregation, direct interactions between hematoxylin and Aβ42 protofibril were analyzed based on the trajectories of all-atom MD simulations. Before the three S-shaped models have been reported38, 40, 41, all of the Aβ fibril models are U-shaped although the salt bridge and turn formed in different residues42. Herein, the U-shaped Aβ42 hexamer with β-sheet structure was selected as the protofibrillar model43, 44 and used in the following MD simulations. In 13



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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

this hexamer model (Figure S2), the U-bend conformation was stabilized by the intermolecular salt bridge between D23 and K28.45, 46 Although the fibrillar models are different in shape (e.g., U- and S-shape) and β-strand length, they share some similarities: the parallel β-strand-turn-β-strand features, the salt bridges via charged residues (especially for D23 and K28), and the hydrophobic grooves. Considering the polymorphic nature of amyloid fibrils, it is very likely that Aβ42 adopts different conformations that facilitate Aβ42 aggregation. Therefore, the slightly different simulation results would be obtained using different Aβ42 fibrillar forms. In order to probe the direct interactions between Aβ42 hexamer and hematoxylin, the intermolecular Lennard-Jones and electrostatic energies between Aβ42 and hematoxylin were firstly calculated and shown in Figure 6. The corresponding energies between brazilin and the Aβ42 hexamer were also calculated and compared with those of hematoxylin. From Figure 6, it is clear that both Lennard-Jones and electrostatic energies contributed greatly to the interactions between hematoxylin and Aβ42, which is consistent with brazilin. However, both Lennard-Jones and electrostatic energies between hematoxylin and Aβ42 hexamer were smaller than those of brazilin, especially for electrostatic energy. For example, the last 150-ns average value of the Lennard-Jones potential energy between hematoxylin and hexamer was about -396.2 kJ/mol (Figure 6A), which is lower than that of brazilin (-329.4 kJ/mol). Thus, the hydrophobic interactions between Aβ42 and hematoxylin were slightly stronger than those between Aβ42 and brazilin. Moreover, the last 150-ns electrostatic energy between hematoxylin and hexamer was about -320.3 14


Page 14 of 38

Page 15 of 38

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


kJ/mol, which is about 1.7 times smaller than that (-209.8 kJ/mol) between brazilin and Aβ42 hexamer. It indicates that both hydrophobic and electrostatic interactions between hematoxylin and Aβ42 hexamer are stronger than those of brazilin. It may be caused by that hematoxylin has one more hydroxyl group than brazilin. Previous studies have found that electrostatic interactions are crucial for the stability of Aβ species.46 Therefore, the stronger electrostatic interactions between hematoxylin and Aβ42 are the major reason why hematoxylin has higher inhibitory activity than brazilin on Aβ42 fibrillogenesis. And the stronger π-π stacking and hydrophobic interactions between hematoxylin and Aβ42 are also induced by the strong electrostatic interactions. Binding Domains of Hematoxylin to Aβ42 Hexamer. To further explore the binding regions of hematoxylin on Aβ42 hexamer, we computed the contact numbers between each residue of Aβ42 hexamer and hematoxylin (Figure 7). Herein, the contact number of 10 was used as the criterion to identify the important residues in interactions between hematoxylin and Aβ42 hexamer. From Figure 7, four binding regions including the N-terminal region (residues 1-4), S8GY10 region, turn region (residues 20-29) and C-terminal region (residues 41-42) were found. And Figure 8 shows the typical binding conformations of hematoxylin and brazilin interacted with Aβ42 hexamer. As for the N-terminal region, hematoxylin preferentially contacted with residues Ala2, Glu3 and Phe4 (Figure 7A). As shown in Figure S5A, the π-π stacking between the side chain of Phe4 and hematoxylin would interfere with the intra-molecular interactions of Aβ fibrils, so Phe4 was the key residue in the 15



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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

N-terminal region. It has been reported that π-π stacking interactions played a crucial role in amyloid fibrils.29,

47

So residue Phe4 played an important role in the

interactions between hematoxylin and Aβ42 hexamer. On the contrary, the contact number between brazilin and N-terminal region of Aβ hexamer was much less than hematoxylin (Table 1 and Figure 7B). As for S8GY10 region, the contact number between brazilin and S8GY10 region of Aβ hexamer was slightly less than that of hematoxylin (Table 1). Of them, residue Y10 played an important role in the interactions with both hematoxylin and brazilin (Figure 7). It is shown in Figure S5B that hematoxylin bound to the aromatic ring in the side chain of Tyr10. Therefore, Tyr10 was a key residue in S8GY10 region. The turn region is composed of 10 residues F20AEDVGSNKG29. Of them, two charged residues D23 and K28 can form intermolecular salt bridge in Aβ aggregates. From Table 1, it is clear that the contact number between the turn region and hematoxylin are 173, which is higher than that of brazilin. From Figure 7A, hematoxylin preferentially bound to residues Phe20, Glu22, Ser26, Asn27, Lys28 and Gly29, while brazilin preferred to interact with residues Phe20, Glu22, Val24, Gly25, Ser26, Asn27 and Lys28. It is notable that the contacts between charged residues and hematoxylin were more than those of brazilin although residues Glu22 and Lys28 interacted with both hematoxylin and brazilin. It is caused by that hematoxylin has one more hydroxyl group than brazilin, which makes hematoxylin tend to interact with charged residues such as Glu22 and Lys28 via hydrogen bonds. Compared with other three regions, the C-terminal region had less contact number 16


Page 16 of 38

Page 17 of 38

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with hematoxylin and brazilin. For example, only hydrophobic residue Ala42 in the C-terminal region had strong interactions with hematoxylin and brazilin (Figures 7A and 7B). Moreover, the contact number between hematoxylin and Aβ42 was higher than that of brazilin. It has been reported that the hydrophobic C-terminals was important for the stability of β-sheet structure in oligomer,48 while the salt bridge between Lys28 and Ala42 was significant to the self-assemblies of Aβ42.38 Thus the external hydrophobic interactions between Ala42 and the inhibitors might disrupt the intermolecular salt bridge and disassembly the preformed fibrils. Based on the above analysis, the contact number between hematoxylin and Aβ42 hexamer is higher than that of brazilin. However, hematoxylin would interact with the charged residues more easily than brazilin. For instance, hematoxylin preferentially interacted with the negatively charged residue Glu22 and positively charged residue Lys28 in turn region. As for the N-terminal region, hematoxylin had more contacts with the negative charged residues (Asp1 and Glu3) than those of brazilin. Thus, it is concluded that the electrostatic interactions between hematoxylin and Aβ42 hexamer were greater than those of brazilin. Hydrogen Bonds Analysis. From above analysis, it is clear that the electrostatic interactions were crucial to the hematoxylin binding with Aβ42. It is known that hematoxylin as well as brazilin didn’t contain charged group, and they only donated or accepted hydrogen atoms via hydroxyl groups to form hydrogen bonds with Aβ. Hydrogen bonds are known as the weak electrostatic interactions. In addition, comparing hematoxylin in Figure 1A and brazilin in Figure S1, hematoxylin has one 17



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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

more phenolic hydroxyl group, so hematoxylin would interact with Aβ42 via more hydrogen bonds. So, we calculated the number of hydrogen bonds between Aβ42 and hematoxylin as well as brazilin, respectively (Table 1). It is clear that the average number of hydrogen bonds between Aβ42 and hematoxylin was about 11, which was higher than that of brazilin (~7). A detailed hydrogen bonding distribution of hematoxylin and brazilin on Aβ42 hexamer was calculated and shown in Figure 9. It is clear that hematoxylin preferentially interacted with the N-terminal, turn and C-terminal regions. For example, residues Ala2, Glu22, Ser26 and Asn27 formed more than 1 hydrogen bond, especially for Glu22 which has the maximum amount of hydrogen bonds. So the hydrogen bonds in these positions of Glu22 and Asn27 would hinder the formation of the salt bridge between Asp23 and Lys28, which led to the destruction of U-bend conformation. However, brazilin only interacted with the turn region of Aβ42 hexamer and only residues Asp23 and Ser26 formed more than 1 hydrogen bond. Moreover, it is found that both hematoxylin and brazilin interacted with both the turn and C-terminal regions via hydrogen bonds, and the number of hydrogen bonds between hematoxylin and Aβ42 was similar with that of brazilin. As for the turn region, hydrogen bonds were stably formed between these compounds and the residues Ser26 and Asn27. However, hematoxylin formed more than one hydrogen bonds with residue Ala2 in the N-terminal region, but brazilin did not. So these hydrogen bonds made hematoxylin bind to the N-terminal region more efficiently (Figure 7), which disturbed the intermolecular interactions of the N-terminal and 18


Page 18 of 38

Page 19 of 38

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


modulated the pathway of Aβ42 aggregation. Based on the above analysis, it is concluded that hematoxylin formed more hydrogen bonds than brazilin with Aβ42 hexamer because hematoxylin has more one hydroxyl groups than brazilin. Moreover, strong hydrogen bonding between hematoxylin and Aβ42 is the main reason why that hematoxylin had a better inhibitory capacity than brazilin at low concentrations, which is consistent with the above ThT fluorescent analysis (Figure 2A). This result is also supported by the thermodynamics analysis between EGCG and amyloid proteins in our previous studies.49 We found that hydrogen bonds between EGCG and Aβ played an important role in the lower concentrations, and the predominant interactions gradually shifts from hydrogen bonds to hydrophobic interactions with the increase of the concentrations of small molecular inhibitor.50

CONCLUSIONS In this work, ThT fluorescence analysis and TEM results confirmed that hematoxylin prevented Aβ42 fibrillogenesis in a dose-dependent manner. Hematoxylin had a stronger inhibitory capacity than brazilin, especially at lower concentrations. CD spectrum results showed that hematoxylin reduced slightly the content of β-sheet structures. Importantly, hematoxylin could modulate the arrangement of β-sheet of mature Aβ42 fibrils, which is the main reason for the formation of amorphous aggregates. The cell viability data showed that hematoxylin significantly alleviated the cytotoxicity at a low concentration of 12.5 µM. 19



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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

MD simulations showed that, like brazilin, hematoxylin interacted with Aβ42 via both electrostatic and hydrophobic interactions. However, the electrostatic interactions between Aβ42 and hematoxylin were stronger than those of brazilin although the hydrophobic interactions between Aβ42 and hematoxylin were similar with those of brazilin. Moreover, four primary binding regions including N-terminal region, S8GY10 region, turn region and C-terminal region on Aβ42 hexamer were identified based on the contact number of hematoxylin and Aβ42 hexamer. The contact number between hematoxylin and Aβ42 hexamer is higher than that of brazilin. Unlike brazilin, hematoxylin preferentially interacted via hydrogen bonds with the charged residues (i.e., Glu22 and Lys28) and had stronger electrostatic interactions than brazilin, which also contributed to the formation of π-π stacking and hydrophobic interactions. This is why hematoxylin had a stronger inhibitory effect than brazilin. Moreover, hematoxylin possesses the advantages of high nature content, low in separation cost and low toxicity, which play a crucial role in pharmaceutical production. All of these results indicated that hematoxylin was a potential compound for therapeutic treatment of AD.

ASSOCIATED CONTENT Supporting Information Additional Figures S1-S5 are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

20


Page 20 of 38

Page 21 of 38

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


AUTHOR INFORMATION Corresponding Authors *Tel:

+86

22

27404981;

Fax:

+86

22

27403389;

E-mail

addresses:

[email protected]; [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank for Prof. Guanghong Wei providing the all-atom model of Aβ42 hexamer. This work was funded by the National Natural Science Foundation of China (Nos. 21376172 and 21576199).

REFERENCES 1.

Yankner, B. A.; Duffy, L. K.; Kirschner, D. A. Neurotrophic and Neurotoxic Effects of Amyloid

Beta Protein: Reversal by Tachykinin Neuropeptides. Science 1990, 250, 279-282. 2.

Hardy, J. A.; Higgins, G. A. Alzheimer's Disease: the Amyloid Cascade Hypothesis. Science 1992,

256, 184-185. 3.

Carter, J.; Anderton, B. Molecular Pathology of Alzheimer's Disease. Br. J. Hosp. Med. 1994, 51,

522-528. 4.

Hardy, J. The Alzheimer Family of Diseases: Many Etiologies, One Pathogenesis? Proc. Natl.

Acad. Sci. U. S.A. 1997, 94, 2095-2097. 5.

Hardy, J.; Selkoe, D. J. The Amyloid Hypothesis of Alzheimer's Disease: Progress and Problems

on the Road to Therapeutics. Science 2002, 297, 353-356. 6.

Roher, A. E.; Lowenson, J. D.; Clarke, S.; Woods, A. S.; Cotter, R. J.; Gowing, E.; Ball, M. J.

Beta-Amyloid-(1-42) is a Major Component of Cerebrovascular Amyloid Deposits: Implications for the Pathology of Alzheimer Disease. Proc. Natl. Acad. Sci. U. S.A. 1993, 90, 10836-10840. 7.

Iwatsubo, T.; Odaka, A.; Suzuki, N.; Mizusawa, H.; Nukina, N.; Ihara, Y. Visualization of Abeta

42(43) and Abeta 40 in Senile Plaques with End-specific Abeta Monoclonals: Evidence that an Initially Deposited Species is Abeta 42(43). Neuron 1994, 13, 45-53. 21



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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

8.

Chiti, F.; Dobson, C. M. Protein Misfolding, Functional Amyloid, and Human Disease. Annu. Eev.

Biochem. 2006, 75, 333-366. 9.

Terzi, E.; Holzemann, G.; Seelig, J. Interaction of Alzheimer Beta-amyloid Peptide(1-40) with

Lipid Membranes. Biochemistry 1997, 36, 14845-14852. 10. Hawkes, C. A.; Deng, L. H.; Shaw, J. E.; Nitz, M.; McLaurin, J. Small Molecule Beta-amyloid Inhibitors that Stabilize Protofibrillar Structures in Vitro Improve Cognition and Pathology in a Mouse Model of Alzheimer's Disease. Eur. J. Neurosci. 2010, 31, 203-213. 11. Mangialasche, F.; Solomon, A.; Winblad, B.; Mecocci, P.; Kivipelto, M. Alzheimer's Disease: Clinical Trials and Drug Development. Lancet Neurol. 2010, 9, 702-716. 12. Ngo, S. T.; Li, M. S. Curcumin Binds to Abeta1-40 Peptides and Fibrils Stronger than Ibuprofen and Naproxen. J. Phys. Chem. B 2012, 116, 10165-10175. 13. Funke, S. A.; Willbold, D. Peptides for Therapy and Diagnosis of Alzheimer's Disease. Curr. Pharm. Design 2012, 18, 755-767. 14. Turner, J. P.; Lutz-Rechtin, T.; Moore, K. A.; Rogers, L.; Bhave, O.; Moss, M. A.; Servoss, S. L. Rationally Designed Peptoids Modulate Aggregation of Amyloid-beta 40. ACS Chem. Neurosc. 2014, 5, 552-558. 15. Cabaleiro-Lago, C.; Quinlan-Pluck, F.; Lynch, I.; Lindman, S.; Minogue, A. M.; Thulin, E.; Walsh, D. M.; Dawson, K. A.; Linse, S. Inhibition of Amyloid Beta Protein Fibrillation by Polymeric Nanoparticles. J. Am. Chem. Soc. 2008, 130, 15437-15443. 16. Zhang, M.; Mao, X.; Yu, Y.; Wang, C. X.; Yang, Y. L.; Wang, C. Nanomaterials for Reducing Amyloid Cytotoxicity. Adv. Mater. 2013, 25, 3780-3801. 17. Shih, P. H.; Wu, C. H.; Yeh, C. T.; Yen, G. C. Protective Effects of Anthocyanins against Amyloid Beta-peptide-induced Damage in Neuro-2A cells. J. Agric. Food Chem. 2011, 59, 1683-1689. 18. Ehrnhoefer, D. E.; Bieschke, J.; Boeddrich, A.; Herbst, M.; Masino, L.; Lurz, R.; Engemann, S.; Pastore, A.; Wanker, E. E. EGCG Redirects Amyloidogenic Polypeptides into Unstructured, Off-pathway Oligomers. Nat. Struct. Mol. Bio. 2008, 15, 558-566. 19. Wang, N.; He, J.; Chang, A. K.; Wang, Y.; Xu, L.; Chong, X.; Lu, X.; Sun, Y.; Xia, X.; Li, H.; et al. (-)-epigallocatechin-3-gallate Inhibits Fibrillogenesis of Chicken Cystatin. J. Agric. Food. Chem. 2015, 63, 1347-1351. 20. Feng, Y.; Wang, X. P.; Yang, S. G.; Wang, Y. J.; Zhang, X.; Du, X. T.; Sun, X. X.; Zhao, M.; Huang, L.; Liu, R. T. Resveratrol Inhibits Beta-amyloid Oligomeric Cytotoxicity but does not Prevent Oligomer Formation. Neurotoxicology 2009, 30, 986-995. 21. Katayama, S.; Ogawa, H.; Nakamura, S. Apricot Carotenoids Possess Potent Anti-amyloidogenic Activity in Vitro. J. Agric. Food. Chem. 2011, 59, 12691-12696. 22. Yang, F.; Lim, G. P.; Begum, A. N.; Ubeda, O. J.; Simmons, M. R.; Ambegaokar, S. S.; Chen, P. P.; Kayed, R.; Glabe, C. G.; Frautschy, S. A.; et al. Curcumin Inhibits Formation of Amyloid Beta Oligomers and Fibrils, Binds Plaques, and Reduces Amyloid in Vivo. J. Biol. Chem. 2005, 280, 5892-5901. 23. Kumaraswamy, P.; Sethuraman, S.; Krishnan, U. M. Mechanistic Insights of Curcumin Interactions with the Core-recognition Motif of Beta-amyloid Peptide. J. Agric. Food Chem. 2013, 61, 3278-3285. 24. Wang, Q.; Yu, X.; Patal, K.; Hu, R.; Chuang, S.; Zhang, G.; Zheng, J. Tanshinones Inhibit Amyloid Aggregation by Amyloid-beta Peptide, Disaggregate Amyloid Fibrils, and Protect Cultured Cells. ACS Chem. Neurosci. 2013, 4, 1004-1015. 22


Page 22 of 38

Page 23 of 38

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25. Kai, T.; Zhang, L.; Wang, X.; Jing, A.; Zhao, B.; Yu, X.; Zheng, J.; Zhou, F. Tabersonine Inhibits Amyloid Fibril Formation and Cytotoxicity of Abeta(1-42). ACS Chem. Neurosci. 2015, 6, 879-888. 26. Kim, B.; Kim, S. H.; Jeong, S. J.; Sohn, E. J.; Jung, J. H.; Lee, M. H.; Kim, S. H. Brazilin Induces Apoptosis and G2/M Arrest via Inactivation of Histone Deacetylase in Multiple Myeloma U266 Cells. J. Agric. Food Chem. 2012, 60, 9882-9889. 27. Lee, C. C.; Wang, C. N.; Kang, J. J.; Liao, J. W.; Chiang, B. L.; Chen, H. C.; Hu, C. M.; Lin, C. D.; Huang, S. H.; Lai, Y. T. Antiallergic Asthma Properties of Brazilin Through Inhibition of TH2 Responses in T Cells and in a Murine Model of Asthma. J. Agric. Food Chem. 2012, 60, 9405-9414. 28. Du, W. J.; Guo, J. J.; Gao, M. T.; Hu, S. Q.; Dong, X. Y.; Han, Y. F.; Liu, F. F.; Jiang, S.; Sun, Y. Brazilin Inhibits Amyloid Beta-protein Fibrillogenesis, Remodels Amyloid Fibrils and Reduces Amyloid Cytotoxicity. Sci. Rep. 2015, 5, 7992. 29. Tracz, S. M.; Abedini, A.; Driscoll, M.; Raleigh, D. P. Role of Aromatic Interactions in Amyloid Formation by Peptides Derived from Human Amylin. Biochemistry 2004, 43, 15901-15908. 30. Lemkul, J. A.; Bevan, D. R. Destabilizing Alzheimer's Abeta(42) Protofibrils with Morin: Mechanistic Insights from Molecular Dynamics Simulations. Biochemistry 2010, 49, 3935-3946. 31. Xiong, N.; Dong, X. Y.; Zheng, J.; Liu, F. F.; Sun, Y. Design of LVFFARK and LVFFARK-functionalized Nanoparticles for Inhibiting Amyloid beta-protein Fibrillation and Cytotoxicity. ACS Appl. Mater. Inter. 2015, 7, 5650-5662. 32. Zhou, X.; Xi, W.; Luo, Y.; Cao, S.; Wei, G. Interactions of a Water-soluble Fullerene Derivative with Amyloid-beta Protofibrils: Dynamics, Binding Mechanism, and the Resulting Salt-bridge Desruption. J. Phys. Chem. B 2014, 118, 6733-6741. 33. Oostenbrink, C.; Villa, A.; Mark, A. E.; Van Gunsteren, W. F. A Biomolecular Force Field Based on the Free Enthalpy of Hydration and Solvation: The GROMOS Force-field Parameter Sets 53A5 and 53A6. J. Comput. Chem. 2004, 25, 1656-1676. 34. Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS 4: Algorithms for Highly Efficient, Load-balanced, and Scalable Molecular Simulation. J Chem. Theory Comput. 2008, 4, 435-447. 35. Hudson, S. A.; Ecroyd, H.; Kee, T. W.; Carver, J. A. The Thioflavin T Fluorescence Assay for Amyloid Fibril Detection can be Biased by the Presence of Exogenous Compounds. FEBS J. 2009, 276, 5960-5972. 36. Anand, P.; Kunnumakkara, A. B.; Newman, R. A.; Aggarwal, B. B. Bioavailability of Curcumin: Problems and Promises. Mol. Pharm. 2007, 4, 807-818. 37. Micsonai, A.; Wien, F.; Kernya, L.; Lee, Y. H.; Goto, Y.; Refregiers, M.; Kardos, J. Accurate Secondary Structure Prediction and Fold Recognition for Circular Dichroism Spectroscopy. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, E3095-3103. 38. Xiao, Y.; Ma, B.; McElheny, D.; Parthasarathy, S.; Long, F.; Hoshi, M.; Nussinov, R.; Ishii, Y. Abeta(1-42) Fibril Structure Illuminates Self-recognition and Replication of Amyloid in Alzheimer's Disease. Nat. Struct. Mol. Biol. 2015, 22, 499-505. 39. Serpell, L. C.; Sunde, M.; Benson, M. D.; Tennent, G. A.; Pepys, M. B.; Fraser, P. E. The Protofilament Substructure of Amyloid Fibrils. J. Mol. Biol. 2000, 300, 1033-1039. 40. Colvin, M. T.; Silvers, R.; Ni, Q. Z.; Can, T. V.; Sergeyev, I.; Rosay, M.; Donovan, K. J.; Michael, B.; Wall, J.; Linse, S.; Griffin, R. G. Atomic Resolution Structure of Monomorphic Abeta42 Amyloid Fibrils. J. Am. Chem. Soc. 2016, 138, 9663-9674. 41. Walti, M. A.; Ravotti, F.; Arai, H.; Glabe, C. G.; Wall, J. S.; Bockmann, A.; Guntert, P.; Meier, B. 23



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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

H.; Riek, R. Atomic-resolution Structure of a Disease-relevant Abeta(1-42) Amyloid fibril. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E4976-4984. 42. Tycko, R. Amyloid polymorphism: structural basis and neurobiological relevance. Neuron 2015, 86, 632-645. 43. Zhou, X. Y.; Xi, W. H.; Luo, Y.; Cao, S. Q.; Wei, G. H. Interactions of a Water-Soluble Fullerene Derivative with Amyloid-beta Protofibrils: Dynamics, Binding Mechanism, and the Resulting Salt Bridge Disruption. J. Phys. Chem. B 2014, 118, 6733-6741. 44. Wu, C.; Scott, J.; Shea, J. E. Binding of Congo Red to Amyloid Proto Fibrils of the Alzheimer Abeta(9-40) Peptide Probed by Molecular Dynamics Simulations. Biophys. J. 2012, 103, 550-557. 45. Bertini, I.; Gonnelli, L.; Luchinat, C.; Mao, J. F.; Nesi, A. A New Structural Model of Abeta(40) Fibrils. J. Am. Chem. Soc. 2011, 133, 16013-16022. 46. Marshall, K. E.; Morris, K. L.; Charlton, D.; O'Reilly, N.; Lewis, L.; Walden, H.; Serpell, L. C. Hydrophobic, Aromatic, and Electrostatic Interactions Play a Central Role in Amyloid Fibril Formation and Stability. Biochemistry 2011, 50, 2061-2071. 47. Gazit, E. A Possible Role for Pi-stacking in the Self-assembly of Amyloid Fibrils. FASEB J. 2002, 16, 77-83. 48. Zheng, J.; Jang, H.; Ma, B.; Tsai, C. J.; Nussinov, R. Modeling the Alzheimer Abeta17-42 Fibril Architecture: Tight Intermolecular Sheet-sheet Association and Intramolecular Hydrated Cavities. Biophys. J. 2007, 93, 3046-3057. 49. Wang, S. H.; Liu, F. F.; Dong, X. Y.; Sun, Y. Calorimetric and Spectroscopic Studies of the Interactions between Insulin and (-)-epigallocatechin-3-gallate. Biochem. Eng. J. 2012, 62, 70-78. 50. Wang, S. H.; Liu, F. F.; Dong, X. Y.; Sun, Y. Thermodynamic Analysis of the Molecular Interactions between Amyloid Beta-peptide 42 and (-)-epigallocatechin-3-gallate. J. Phys. Chem. B 2010, 114, 11576-11583.

24


Page 24 of 38

Page 25 of 38

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table Table 1. Statistics of the number of residue-residue contacts and hydrogen bonds in each region Inhibitor

N-terminal

S8GY10

Turn

C-terminal

Total

Contact

Hematoxylin

73

40

173

27

335

number

Brazilin

16

29

135

16

224

Hydrogen

Hematoxylin

3

0

6

1

11

bonds

Brazilin

0

0

5

1

7

25



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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure captions Figure 1. The inhibitory effect of hematoxylin on Aβ42 fibrillogenesis. (A) Chemical structure of hematoxylin. (B) ThT fluorescence of Aβ42 (25 µM) incubated with various concentrations of hematoxylin and brazilin for 24 h. (C) TEM images of Aβ42 in the absence and presence of hematoxylin at a molar ratio of 1:1 to Aβ42 after 24 h incubation.

Figure 2. Time-dependent ThT fluorescence changes for Aβ42 incubated with various concentrations of hematoxylin and brazilin. Hematoxylin and brazilin were added at a molar ratio of 0.1 (A), 0.2 (B), 0.5 (C) and 1 (D) to Aβ42 (25 µM). The ThT fluorescence measurements were performed in triplicate by a fluorescence plate reader at 15 min reading intervals and 5 s shaking before each read.

Figure 3. Effect of hematoxylin on Aβ42 fibrillogenesis as measured by CD spectrum after 0 h (A), 2 h (B), 4 h (C), 24 h (D). The concentrations of Aβ42 and hematoxylin were both 25 µM.

Figure 4. The proportion of different secondary structures of Aβ42 fibrils incubated in the absence (A) and presence (B) of hematoxylin at a molar ratio 1:1 to Aβ42 (25 µM) for 24 h analyzed by BeStSeL web server.

26


Page 26 of 38

Page 27 of 38

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Viability of SH-SY5Y cells after addition of aged Aβ42 incubated with varying concentrations of hematoxylin for 24 h. The cell viability treated with PBS buffer only was used as a control. The error bars were standard deviations of four different replicates. ***p

Hematoxylin Inhibits Amyloid Î²-Protein Fibrillation and Alleviates

Recommend Documents