Basified Human Lysozyme: A Potent Inhibitor against Amyloid Î²

Subscriber access provided by Kaohsiung Medical University

Biological and Environmental Phenomena at the Interface

Basified Human Lysozyme: A Potent Inhibitor against Amyloid #-Protein Fibrillogenesis Xi Li, Baolong Xie, and Yan Sun Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03278 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 9, 2018

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 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 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.

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 31 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

Langmuir

Basified Human Lysozyme: A Potent Inhibitor against Amyloid β-Protein Fibrillogenesis

Xi Li,† Baolong Xie, †,‡ Yan Sun*,†

†Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300354, China ‡Institute of Tianjin Seawater Desalination and Multipurpose Utilization, State Oceanic Administration (SOA), Tianjin 300192, China

1

ACS Paragon Plus Environment

Langmuir 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: Aggregation of amyloid β-proteins (Aβ) has been recognized as a key process in the pathogenesis of Alzheimer’s disease (AD), so inhibiting Aβ aggregation is an important strategy for prevention from the onset of and treatment of AD. Our recent work indicated that decreasing the positive charges (or introducing negative charges) on human lysozyme (hLys) was unfavorable to keep the inhibiting capability of hLys on Aβ aggregation. Therefore, we have herein proposed to basify hLys by conversion of the carboxyl groups into amino groups by modification with ethylene diamine. Basified hLys (Lys-B) preparations of three modification degrees (MDs), denoted as hLys-B1 (MD, 1.5), hLys-B2 (MD, 3.3) and hLys-B3 (MD, 4.4), were synthesized for modulating Aβ fibrillogenesis. The hLys-B preparations kept the stability and biocompatibility as native hLys did, while the inhibitory potency of hLys-B on Aβ fibrillogenesis increased with increasing MD. Cytotoxicity analysis showed that the cell viability with 2.5 μM hLys-B3 increased from 62.5% (with 25 μM Aβ only) to 76.1%, similar with the case with 12.5 μM hLys (75.5%); the cell viability with 6.25 μM hLys-B3 increased to 82.0%, similar with the case with 25 μM hLys (80.9%). The results indicate about four to five times increase of the inhibition efficiency of hLys by the amino modification. Mechanistic analysis suggests that such a superior inhibitory capability of hLys-B was attributed to its more widely distributed positive charges, which promoted broad electrostatic interactions between Aβ and hLys-B. Thus, hLys-B suppressed the conformational transition of Aβ to β-sheet structures at low concentrations (e.g., 2.5 μM hLys-B3), leading to the changes in the aggregation pathway and the formation of Aβ species of less cytotoxicity. The findings provided new insights into the development of more potent protein-based inhibitors against Aβ fibrillogenesis.

KEYWORDS: amyloid β-protein; lysozyme; surface modification; aggregation; inhibition; positive charges

2


Page 2 of 31

Page 3 of 31 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

Langmuir

INTRODUCTION Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by loss of memory and other cognitive abilities.1-3 Till now, drug after drug has failed to slow the progression of AD in clinical trials for several reasons.4 The presence of amyloid plaques in brain tissues, which are primarily composed of aggregates of amyloid β-proteins (Aβ), is the main pathological hallmark of AD.5,6 It has been demonstrated that Aβ aggregates can be assembled from different alloforms, such as Aβ40 and Aβ42, which differ in their length and concentrations, and present different aggregation properties and toxicities.7-9 The aggregation of Aβ from soluble monomers into amyloid fibers through toxic oligomers is thought to be directly linked to the disease onset.10 Therefore, a strategy that inhibits Aβ fibrillogenesis can be a potential therapeutic approach for the treatment of AD. At present, a plethora of inhibitors have been reported to interfere with Aβ aggregation and regulate the toxicity, including small organic compounds,11-13 peptides,14,15 proteins,16,17 and nanoparticles.18-20 Among them, peptide- and protein-based inhibitors are advantageous due to their biocompatibility and effective use in vivo. Human lysozyme (hLys) is a natural nonspecific immune protein and is abundant in all body fluids.21 Studies have shown that lysozyme is able to inhibit the aggregation of Aβ40,17 Aβ42,22 and Aβ17-42.23 Besides, co-localization of hLys with Aβ plaques in AD patients has been reported.22 However, at physiological conditions, the inhibition effect of hLys on Aβ aggregation is quite limited.22 Hence, we propose to enhance the inhibition effect of hLys by chemical modifications. Recently, we developed iminodiacetic acid (IDA)-modified hLys (IDA-hLys) to create a bifunctional agent capable of inhibiting Aβ aggregation and modulating Zn2+-mediated Aβ species.24 However, its inhibitory effect on the self-aggregation of Aβ became weaker than native hLys, indicating that introduction of more negative charges on its surface compromised the inhibitory effect of the protein. Moreover, a recent study by Liu et al. revealed that N-trimethyl chitosan chloride of higher positive charge density showed stronger inhibiting activity on Aβ40 aggregation than 3


Langmuir 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

Page 4 of 31

native chitosan.25 These findings inspired us herein to consider modification of hLys by conversion of the carboxyl groups into amino groups to increase the positive charges on the protein surface. The basified hLys (hLys-B) preparations were comprehensively evaluated for their inhibitory potency on Aβ aggregation and cytotoxicity by extensive biophysical and biological analyses, and the mechanism behind the experimental observations was explored to provide new insight into the design of potent inhibitors against Aβ fibrillogenesis.

EXPERIMENTAL RPOCEDURES Materials. Aβ42 and Aβ40 (>95%, lyophilized powder) were purchased from GL Biochem (Shanghai, China). Ethylene diamine, hLys, thioflavin T (ThT), dimethyl sulfoxide

(DMSO),

1,1,1,3,3,3-hexafluoro-2-propanol

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 2-(N-Morpholino)ethanesulfonic

acid

(HFIP), (EDC),

hydrate

(MES)

and

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Human neuroblastoma SH-SY5Y cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS) and Dulbecco's Modified Eagle’s Medium/Ham's F-12 (DMEM/F12) were provided by Invitrogen (Carlsbad, CA, USA). Other chemicals were all of the highest purity available from local sources. Synthesis and Characterization of Basified Human Lysozyme. Three hLys-B preparations with different modification degrees (MDs) (denoted as hLys-B1, hLys-B2 and hLys-B3) were synthesized by reaction between the protein carboxylate and ethylene diamine, as illustrated in Scheme 1. Briefly, 100 mg hLys powder was transferred into a flask containing 0.09 mL ethylene diamine and 10-40 mg EDC in 10 mL 0.1M MES buffer, using HCl to adjust the solution pH to 6.0. The initial EDC concentration in the reaction was tuned to prepare hLys-B of three different MDs. The solution was kept shaking in an incubator at 25 °C and 170 rpm for 12 h. Thereafter, the resultant solution was dialyzed with a dialyzer (molecular weight cut-off, 3500 4


Page 5 of 31 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

Langmuir

Da) against deionized water to remove the non-reacted substances and byproducts. The product was freeze-dried under vacuum for 24 h and stored at -20 °C for further use. The molecular weights of hLys and hLys-B in deionized water at 1.0 mg/mL were determined by matrix-assisted laser desorption ionization time-of-flight mass spectroscope (Autoflex Tof/TofIII, Bruker Daltonics Inc., Billerica, MA). The zeta potentials of hLys and hLys-B at different pH values were measured on a Zetasizer Nano (Malvern Instruments, Worcestershire, UK) at 25 μM in deionized water at 37 °C, using NaOH or HCl to adjust pH values. The molecular sizes and intrinsic fluorescence spectra of hLys and hLys-B were respectively analyze by dynamic light scattering with the Zetasizer Nano and fluorescent analysis with a fluorescence spectrometer (Perkin Elmer LS-55) at 25 μM in buffer A (100 mM phosphate buffer, 10 mM NaCl, pH 7.4) at 37 °C. The net charge of hLys at pH 7.4 was calculated from the following equation,26 z = ∑ Ni i

1

1 + 10

pH - pKi

- ∑ Mj j

10

pH - pKj

1 + 10

(1)

pH - pKj

where Ni and Mj are the numbers of basic and acidic groups, respectively, pKi and pKj are the dissociation constants of the basic and acidic groups, respectively. Aβ42 and Aβ40 Monomer Solution Preparation. Aβ42 and Aβ40 monomer solutions were prepared with HFIP to remove pre-existing aggregates as reported previously.24,27 Immediately before a use, the pretreated Aβ42 or Aβ40 monomer was dissolved by 20 mM NaOH at 275 μM, sonicated for 10 min followed by centrifugation (16,000 g) for 20 min. Then, the upper 75% of the supernatant was collected and diluted with buffer A in the absence and presence of different concentrations of hLys/hLys-B to the final Aβ concentration of 25 μM. Thioflavin T Fluorescent Assay. ThT fluorescence has been widely used to diagnose amyloid fibrils that exhibits enhanced fluorescence upon binding to the β-sheet structure.28,29 Aβ42 samples (25 μM) with various concentrations of hLys or hLys-B were incubated at 37 °C and 150 rpm in a shaking incubator. After being incubated for 24 h, aliquots of the incubation solutions were withdrawn and diluted 11 5


Langmuir 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

Page 6 of 31

times into ThT solution (25 μM ThT in 25 mM sodium phosphate buffer, pH 6.0), and the ThT fluorescent intensity was measured by a fluorescence spectrometer (Perking Elmer LS-55, MA) with a slit width of 5 nm at 25 °C. ThT emission was monitored at 480 nm with the excitation wavelength at 440 nm. The fluorescent intensity of the samples without Aβ42 was subtracted as background from each read with Aβ42. Three measurements were performed and the data were averaged. Aggregation Kinetics. Kinetic analysis is a powerful approach to reveal detailed molecular events during the aggregation process.30 The kinetics of Aβ42 and Aβ40 aggregations were monitored by a fluorescent plate reader (TECAN Infinite, Salzburg, Austria). The samples of 200 μL were mixed in a 96-well plate, containing 25 μL Aβ42 or Aβ40 monomers, 25 μM ThT, and different concentrations of hLys or hLys-B in buffer A. The plate was set to automatic recording at 10 min reading intervals and 5 s shaking before read at 37 °C with excitation wavelength at 440 nm and emission wavelength at 480 nm. The fluorescent intensity of the samples without Aβ42/Aβ40 was subtracted as background from each read with Aβ42/Aβ40. As Aβ42 and Aβ40 exhibit different aggregation kinetics and the aggregation kinetics of Aβ40 show a sigmoidal curve, the experimental data were fitted by the following equation,31 y = y0 +

ymax - y0 1+e

(2)

-(t - t1/2)k

where y is the ThT fluorescent intensity at time t, y0 and ymax are the initial and maximum fluorescent intensities, respectively, t1/2 is the time to 50% of the maximum fluorescent intensity, and k is the elongation rate constant. The lag time (Tlag) was then calculated from,31 2

(3)

Tlag = t1/2 - k

Atomic Force Microscope (AFM). Aβ42 (25 μM) samples in the absence and presence of different concentrations of hLys or hLys-B were incubated at 37 °C and 150 rpm in a shaking incubator for 24 h. Aβ42 sample (10 μL) was deposited on a freshly cleaved mica for 2 min, and then washed using ultrapure water to washout salt ions in the sample. Then, the sample was air-dried and observed under a multi-mode 6


Page 7 of 31 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

Langmuir

atomic force microscope (CSPM5500, Benyuan, China) in a tapping mode. Circular Dichroism Spectroscopy. J-810 spectrometer (Jasco, Japan) was used for circular dichroism (CD) measurements. The CD spectra of different concentrations of hLys/hLys-B and 25 μM Aβ42 monomer or fibril solutions with or without inhibitors were recorded in the spectral range 190-260 nm using a quartz cell with 1 mm path length at 37 °C. Preparation of Aβ42 fibril was the same as that in the ThT fluorescence assays in a shaking incubator. The bandwidth was 1 nm and scan speed was 100 nm/min. The CD spectra of solutions without inhibitor/Aβ42 were subtracted as background. All CD spectra were the average of three consecutive scans for each sample. Cell Viability Assay. The MTT assays were performed to examine the cytotoxicity of hLys-B and its effect on Aβ42-induced cytotoxicity. Human neuroblastoma SH-SY5Y cells were cultured in DMEM/F12 supplemented with 20% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C under 5% CO2. Cells were plated in 96-well plates at a density of 5×103 cells per well (80 μL). After incubation for 24 h, the aged Aβ42 aggregates (25 μM) with different concentrations of hLys/hLys-B (20 μL each well) were added. After incubation for additional 24 h, 10 μL MTT (5.5 mg/mL) in buffer A was added into each well and the plates were incubated for another 4 h. The culture medium was centrifuged at 1,500 rpm for 10 min to remove the supernatant. Then, 100 μL DMSO was used to lyse the precipitated cells. After formazan was fully dissolved, the cell viability was calculated from the absorbance at 570 nm measured by a Plate Reader (TECAN GmbH, Salzburg, Austria). Six replicates were performed for each sample, and the data were averaged. The wells containing medium only were subtracted as the background signal from each reading. The cell survival treated with buffer A only was used as control to normalize other data for comparison. Statistical Analysis. In the above ThT and cell viability assays, each sample was measured at least three times, and the mean value and standard errors were calculated. Analysis of variance was carried out for statistical comparisons using one-way ANOVA with Tukey’s post hoc test, and p < 0.05 or less was considered to be 7


Langmuir 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

statistically significant.

RESULT AND DISCUSSION Characteristics of hLys-B. Analysis by mass spectroscopy can provide the molecular weights for calculation of the MDs of hLys-B preparations. As shown in Figure S1 and Table 1, the molecular weight of the three hLys-B preparations increased over hLys by 63.7, 138.7 and 183.8 Da, respectively. Since coupling one ethylene diamine molecule onto hLys increases the molecular weight by 42.12 Da (Scheme 1), the average MDs were then determined and listed in Table 1. The ζ potentials of hLys and hLys-B as a function of pH were measured. As shown in Figure S2, hLys-B displayed higher ζ potential values than native hLys at the whole pH range (7.0-11.0), and the ζ potential value increased with the average MD of hLys-B. The isoelectric point of hLys-B3 increased from 10.2 of native hLys to 10.6, indicating that the modification increased the positive charges on hLys-B surface. Although the molecular weights and ζ potential values of the hLys-B preparations changed significantly from hLys, their molecular sizes and intrinsic fluorescence spectra had an inappreciable change (Table 1 and Figures S3 and S4). This confirms that the modification had little influence on the structure of the protein. Inhibitory Effect on Aβ Aggregation. First, we examined the inhibitory effect of hLys-B on Aβ aggregation using ThT fluorescence assays. Figure S5 shows the ThT fluorescence kinetics for the inhibition of Aβ42 aggregation. Aβ42 aggregation exhibited a fast growth phase and a stationary state, which was in agreement with previous studies.26,32 It can be seen that the growth rates of the ThT fluorescence intensities (FIs) decreased remarkably and the steady ThT levels also decreased obviously in the presence of hLys or hLys-B. Moreover, the inhibitory effect of hLys/hLys-B on Aβ42 fibrillization showed a dose-dependent manner (Figure S5). Among the four inhibitors, hLys-B3, which has the most positive charges on surface, exhibited the best inhibition effect, implying that the increased positive charges on hLys-B surface is in great favor of the inhibition on Aβ42 aggregation. 8


Page 8 of 31

Page 9 of 31 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

Langmuir

For the fact that the steady state was reached in about 24 h, we carried out ThT fluorescence assays after incubation for 24 h (Figure 1). Similar inhibitory effects of hLys or hLys-B as observed in the kinetic assays (Figure S5) on Aβ42 aggregation were observed. It is worth noting that the hLys-B preparations displayed much stronger inhibitory potency than hLys. The inhibitory effect of hLys-B3 at 2.5 μM (ThT FI decreased by 67%) is almost equivalent to that of hLys at 12.5 μM (ThT FI decreased by 63%). Namely, hLys-B3 at 1/5 of hLys concentration worked similarly with hLys. The results further confirmed that more positive charges on the surface of hLys-B contributed to the occurrence of the enhanced inhibition effect. To gain an insight into the inhibition effect of hLys-B on the nucleation of Aβ, kinetic assays for the inhibition of Aβ40 aggregation were performed (Figure 2 and Table 2). Different from Aβ42, Aβ40 displays longer lag phase time as frequently reported in literature,24,33,34 which benefits in analyzing the inhibition effect of hLys-B on the nucleation of Aβ. As demonstrated in Figure 2 and Table 2, the lag phase time increased and the final ThT FI reduced with increasing hLys/hLys-B concentration. Noticeably, the inhibitory effect of hLys-B3 was remarkably higher than native hLys and the other two hLys-B preparations at the same concentration. When hLys-B3 concentration reached 12.5 μM, amyloid formation was fully inhibited in the experimental range (167 h) (Figure 2D). It is considered that the depletion of Aβ40 monomers in a high affinity complex with hLys-B would reduce the nucleation in the same way as having a lower concentration of Aβ40 monomers available for the aggregation process.35 This would lead to a reduction in the total amount of fibrils at equilibrium.35 The results revealed that the stronger potency of hLys-B with more positive charges than native hLys on prolonging Aβ40 nucleation. Moreover, AFM was used to observe the morphology of Aβ42 aggregates after incubation for 24 h (Figures 3). Aβ42 alone formed long, serried and entangled fibrils (Figure 3A), similar as observed in previous reports.36-38 In the presence of hLys at concentrations of 2.5 and 6.25 μM, fibrils were still observed (Figure 3B1 and 3B2), indicating that hLys at low concentrations have limited effect on fibril formation for its weak inhibitory effect. Under the function of hLys-B1, some small amorphous 9


Langmuir 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

structures still appeared at the two concentrations (Figure 3C1 and 3C2). However, fibrils fully disappeared in the presence of 2.5 μM hLys-B2 and hLys-B3 (Figure 3D1 and 3E1). The results demonstrated that the hLys-B preparations prominently inhibited Aβ42 fibrillation and altered the aggregate morphology. From the above results, it can be concluded that the hLys-B preparations efficiently inhibited Aβ fibrillation and obviously changed the aggregation pathway, demonstrating that introduction of more positive charges benefited in the inhibitory potency. Effects of hLys-B on the Secondary Structure of Aβ42. In order to investigate the effects of hLys-B on the conformational transition of Aβ42 upon aggregation, CD spectroscopy was applied to analyze the secondary structure of Aβ42. The structures of hLys and hLys-B were analyzed first. As shown in Figures S6 and S7, hLys and hLys-B have largely α-helical structure and the structure retained stable within 24 h, consistent with previous study.17 At an hLys/hLys-B concentration of 6.25 μM (Aβ42:hLys/hLys-B=1:0.25) or higher, the CD signal of hLys/hLys-B is rather high (Figures S6 and S7), so it influences the detection of the CD signal of Aβ42 when it is co-incubated with Aβ42 (Figure S8). This made it impossible to detect the effect of hLys/hLys-B on the secondary structure of Aβ42 at an hLys/hLys-B concentration of 6.25 μM or higher. Therefore, we analyzed the inhibitory effect of hLys-B on the secondary structure of Aβ42 at 2.5 μM hLys/hLys-B. As shown in Figure 4A, the initial secondary structure of Aβ42 was random coil with a representative negative peak below 200 nm. After 24-h incubation, the spectrum for Aβ42 displayed a positive peak at 195 nm and a negative peak at 216 nm, which corresponds to the β-sheet structure (Figure 4B). This demonstrated that Aβ42 converted it structure from random coils to β-sheet, the same as that reported in the literatures.39-41 In the present of hLys, the spectrum showed two negative peaks at 195 and 216 nm and the intensity of the peak at 216 nm was approximately the same as that of Aβ42 alone system (Figure 4B), corresponding to a mixed structure of a majority of β-sheet and some random coils. It indicates that hLys at 2.5 μM was unable to prevent the formation of β-sheet structure. However, when incubating Aβ42 with hLys-B of the same concentration (2.5 μM), the 10


Page 10 of 31

Page 11 of 31 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

Langmuir

negative peak at 216 nm changed greatly (Figure 4B), implying that the β-sheet reduction by hLys-B was more significant than that by hLys. The negative peak below 200 nm suggested that there were still random coils in the present of hLys-B (Figure 4B). The results indicate that the hLys-B preparations prevented the conformational transition of Aβ42 from forming β-sheet structure. Effects on Aβ42-Induced Cytotoxicity. The effects of the hLys-B on the cytotoxicity of Aβ42 aggregates were evaluated using MTT assays with SH-SY5Y cells. As a control, MTT assays were performed with 2.5-25 μM hLys/hLys-B. Figure S9 shows that hLys and hLys-B had negligible effect on the cell viability, indicating their biocompatibility. The addition of 25 μM Aβ42 aggregates to the cell culture system resulted in the reduction of cell viability to 62.5% (Figure 5). When different hLys or hLys-B preparations at various concentrations were added to the Aβ42 solution, the cell viability increased to different extent in a dose-dependent manner. Remarkably, hLys-B3 exhibited the best cell protection potency. As compared to the Aβ42-treated group, equimolar hLys-B3 (25 μM) significantly attenuated the cytotoxicity, increasing the cell viability by about 51% (Figure 5). The cell viability with Aβ42 and 2.5 μM hLys-B3 (76.1%) was similar with that of Aβ42 and 12.5 μM hLys (75.5%); the cell viability with Aβ42 and 6.25 μM hLys-B3 (82.0%) was similar with that of Aβ42 and 25 μM hLys (80.9%). It means that hLys-B3 at 1/4 to 1/5 of hLys concentration worked similarly with hLys in the toxicity assays, similar to that observed in the ThT assays (Figure 1). The results indicate that the hLys-B preparations effectively mitigated Aβ42-induced cytotoxicity and the favorable effect was attributed to the extra positive charges on hLys-B surface. Mechanistic Discussion. To explore the working mechanism of hLys-B with enhanced inhibition effects on Aβ fibrillogenesis, the effect of NaCl concentration on Aβ42 aggregation was detected by ThT kinetic assays (Figures S10), with hLys-B3 as a representative. Figure S10A shows the kinetics of Aβ42 aggregation at different NaCl concentrations in the absence of an inhibitor. At low NaCl concentrations (10 to 200 mM), the growth rate of the ThT FI increased and the steady-state ThT levels increased with increasing NaCl concentration (Figure S10A). The results indicate that 11


Langmuir 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

increasing NaCl concentration promoted Aβ aggregation, which is in accordance with those reported previously.42-44 The promotion effect on aggregation by increasing salt concentration would be caused by the shielding of electrostatic interactions and enhancement of hydrophobic interactions. With further increase in NaCl concentration to 400 and 600 mM, the ThT FI increased at first, then decreased (Figure S10A), which is quite different from the aggregation kinetics at the physiological condition. AFM images of Aβ42 aggregates at 400 and 600 mM NaCl were thus acquired to check what happened in the aggregation (Figure S11). As can be seen, some nonfibrillar aggregates were observed, which are distinctly different from the serried and entangled fibrils formed at the physiological salt concentration (Figure 3A). This indicates that at the high salt concentrations, the Aβ42 aggregation pathway completely changed. Similar phenomenon that high salt concentrations made fibril formation unfavorable and favor, instead, nonfibrillar aggregation pathways, was also observed in the amyloid fibril growth of β2-microglobulin.45 It was reported that there was an optimal salt concentration required for efficient fibril growth, suggesting that counterion interaction was crucial in amyloid formation, and a critical balance in hydrophobic and hydrophilic interactions might be necessary in selecting out specific populations of denatured states that are amyloidogenic in nature.45 In the presence of an inhibitor (hLys/hLys-B3), the changing tendency of Aβ42 aggregation kinetics as a function of NaCl concentration (Figure S10B, S10C) was similar to that without an inhibitor (Figure S10A). To gain more details about the effect of NaCl at different concentrations, Figure S10 was redrawn to compare the effect of the two inhibitors at the same NaCl concentration, as shown in Figure S12. It is evident from Figure S12 that, the higher the NaCl concentration is, the less the effect of hLys and hLys-B show on inhibiting Aβ42 aggregation. To see more clearly, as shown in Figure 6, each sample was normalized by setting the maximum value of Aβ42 only at the same NaCl concentration as 100%. Because the ThT fluorescence of Aβ42 aggregation kinetics at high salt concentration increased and then decreased with time (Figure S12D and S12E), which made it unable to be normalized by the method, only the samples under low salt concentrations (i.e., 10 to 200 mM) were plotted in 12


Page 12 of 31

Page 13 of 31 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

Langmuir

Figure 6. It is obvious from Figure 6 that lower NaCl concentration favors hLys and hLys-B to work as stronger inhibitors. The results indicate that electrostatic interactions were crucial for the inhibitory effect of hLys/hLys-B on Aβ42 aggregation for electrostatic interaction increases with decreasing ionic strength.46 Since hLys-B3 has more positive charges, it showed stronger inhibitory potency than hLys at the different salt concentrations (Figures 6 and S12). This suggested that extra positive charges contributed to the electrostatic interactions between hLys-B and Aβ42, leading to the enhancement of the inhibition effect on Aβ42 aggregation. Based on the above results, we herein discuss more details about the inhibition mechanisms of hLys and hLys-B on Aβ aggregation as well as their differences. For better understanding of the inhibition mechanism, the structures of Aβ and hLys were analyzed and represented in Figure S13. It should be noted that Figure S13A and S13B show the model of Aβ42 in an apolar microenvironment and partially folded structure of Aβ40 in aqueous solution. The monomer and dimer forms of Aβ40/Aβ42 have been described as random coil in aqueous solution,47,48 and a number of molecular structures of Aβ40/Aβ42 monomers, oligomers, protofibrils and amyloid fibrils in aqueous solutions have been reported in a review paper.49 It can be seen that Aβ has a negatively charged N-terminus at pH 7.4 and two hydrophobic parts including the central hydrophobic core and the C-terminus (Figure S13A, S13B).50 Under the same condition, the surface of hLys has heterogeneous distributions of hydrophobic patches and positive-potential areas (Figure S13C).24 According to eq. 1 and Table S1, the net charge of hLys at pH 7.4 was calculated to be 7.9 and those of hLys-B1, hLys-B2 and hLys-B3 were estimated at about 11, 14.5 and 17, respectively, because the amounts of carboxyl groups converted into amino groups were 1.5, 3.3 and 4.4, respectively (Table 1). Therefore, the hLys-B preparations, particularly B-Lys3, has much larger positively charged areas than hLys (Figure S13C). Aβ is an acidic protein, which has an isoelectric point of 5.5 and carries negative charges at pH 7.4 (Figure S13A, S13B).51 The net charge of Aβ was calculated to be -2.8 from eq. 1. Since Aβ and hLys/hLys-B carry different types of charges, they are electrostatically attracted from each other. This is the basis for the 13


Langmuir 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

inhibition mechanism for the effect of hLys/hLys-B on Aβ aggregation. Aβ aggregation is a complex self-assembly process, involving β-sheet formation, oligomerization and fibrillation.52,53 In the presence of hLys, the binding of Aβ onto the surface of hLys could change the pathway of Aβ aggregation, thereby inhibiting the fibrillation and cytotoxicity of Aβ. Molecular dynamics simulations have identified the most likely binding sites of hLys to Aβ, using the monomeric structure of Aβ40 in sodium dodecyl sulfate, which was determined by nuclear magnetic resonance (pdb ID:1BA4), and the crystal structure of human lysozyme (pdb ID:1REX).17 Three different binding sites on hLys (i.e., A, B and C, see below) were found to interact with the N terminus of Aβ, its residues Q15-V24 and its C terminus. Those binding sites were R62, Y63 and W64 of hLys at site A, the helical residues (D102-R107) and the loop residues (Q117-R122) of hLys at site B and a few hydrophobic residues (such as R119 and S24) of hLys at the C site. Taken together, hLys stabilizes the N-terminus of Aβ by electrostatic interactions and interacts with the C-terminus of Aβ via a hydrophobic surface.17 Electrostatic and hydrophobic interactions may be the key for hLys to prevent Aβ aggregation. Besides, the above assays confirmed that electrostatic interaction played a dominant role in the inhibitory effect of hLys on Aβ aggregation (Figure 6). However, the positive charges on the surface of native hLys is insufficient, so the inhibitory effect of hLys on Aβ aggregation was limited. Moreover, we previously reported that IDA-hLys, which has less positive charges than native hLys, showed a weaker inhibitory effect on Aβ40 aggregation than hLys. It indicates that introduction of more negative charges on surface was against the inhibitory effect of hLys. In the presence of hLys-B, more widely distributed positive charges would give rise to broad electrostatic interactions between Aβ and hLys-B. The enhanced electrostatic interactions remarkably changed Aβ conformations that are different from β-sheet structure at a concentration as low as 2.5 μM (Figure 4B). So the on-pathway fibrillogenesis that needs a β-sheet structured Aβ conformation was suppressed by hLys-B (particularly by hLys-B3), as revealed by the ThT fluorescent assays (Figures 1 and 2) and AFM observations (Figure 3). Thus, hLys-B greatly alleviated the amyloid cytotoxicity (Figure 5). Besides, hLys-B3 of 14


Page 14 of 31

Page 15 of 31 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

Langmuir

the most positive charges showed the most prominent inhibitory effect of the three hLys-B preparations; hLys-B3 at 1/4 to 1/5 of hLys concentration worked similarly with hLys (Figures 1 and 5). Taken together, basified modification of hLys resulted in the development of a potent agent against Aβ fibrillogenesis.

CONCLUSIONS In this work, we have demonstrated basified hLys as a novel and potent protein-based inhibitor against Aβ fibrillogenesis. By conversion of 1.5 to 4.4 carboxyl groups into amino groups, the hLys-B preparations retained the stability and biocompatibility of native hLys. It was found that the inhibition effect of hLys-B increased with increasing MD, and the hLys-B preparations significantly slowed down Aβ aggregation and altered the aggregation pathway by suppressing the conformational transition to β-sheet structure, leading to the formation of aggregation species of less cytotoxicity. Particularly, hLys-B3 carrying the most positive charges of the basified preparations was demonstrated in cell viability assays with cultured cells to work similarly with hLys at 1/4 to 1/5 of hLys concentrations. The significant improvement of inhibitory capability of hLys-B was attributed to its more widely distributed positive charges, which promote broad electrostatic interactions with Aβ. The findings would benefit in the design of more potent inhibitors of Aβ aggregation and cytotoxicity.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx Mass spectra, zeta potentials, size distributions, and fluorescence intensities of hLys and hLys-B, ThT fluorescence kinetic assays for the inhibition of Aβ42 aggregation, AFM measurements of the Aβ42 aggregates, far-UV circular dichroism spectra of hLys/IDA-hLys and Aβ42, cytotoxicity assays of hLys and IDA-hLys, 15


Langmuir 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

effect of NaCl concentration on the inhibition effect of hLys-B, surface models of Aβ42, Aβ40 and hLys, and pKa of each charge group for lysozyme.

AUTHOR IMFORMATION Corresponding Author *E-mail: [email protected]. Tel: +86 22 27403389. Fax: +86 22 27403389. ORCID Yan Sun: 0000-0001-5256-9571 Author Contributions Y.S. designed the research; X.L. performed the experiments; X.L. and B.X. analyzed the data; X.L., B.X., and Y.S. wrote the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was funded by the National Natural Science Foundation of China (Nos. 91634119 and 21621004).

REFERENCES (1) Albert, M. S.; DeKosky, S. T.; Dickson, D.; Dubois, B.; Feldman, H. H.; Fox, N. C.; Gamst, A.; Holtzman, D. M.; Jagust, W. J.; Petersen, R. C.; Snyder, P. J.; Carrillo, M. C.; Thies, B.; Phelps, C. H. The diagnosis of mild cognitive impairment due to Alzheimer’s disease: Recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimer's Dement. 2011, 7, 270-279. (2) Ferreira, S. T.; Klein, W. L. The Aβ oligomer hypothesis for synapse failure and memory loss in Alzheimer’s disease. Neurobiol. Learn. Mem. 2011, 96, 529-543. (3) Jakob-Roetne, R.; Jacobsen, H. Alzheimer's disease: from pathology to therapeutic approaches. Angew. Chem., Int. Ed. 2009, 48, 3030-3059. 16


Page 16 of 31

Page 17 of 31 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

Langmuir

(4) Doig, A. J.; del Castillo-Frias, M. P.; Berthoumieu, O.; Tarus, B.; Nasica-Labouze, J.; Sterpone, F.; Nguyen, P. H.; Hooper, N. M.; Faller, P.; Derreumaux, P. Why is research on amyloid-β failing to give new drugs for Alzheimer’s disease? ACS Chem. Neurosci. 2017, 8, 1435-1437. (5) Karran, E.; Mercken, M.; De Strooper, B. The amyloid cascade hypothesis for Alzheimer's disease: an appraisal for the development of therapeutics. Nat. Rev. Drug Discovery 2011, 10, 698-712. (6) Jiang, D.; Rauda, I.; Han, S.; Chen, S.; Zhou, F. Aggregation pathways of the amyloid β (1-42) peptide depend on its colloidal stability and ordered β-sheet stacking. Langmuir, 2012, 28, 12711-12721. (7) Bitan, G.; Kirkitadze, M. D.; Lomakin, A.; Vollers, S. S.; Benedek, G. B.; Teplow, D. B. Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ42 oligomerize through distinct pathways. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 330-335. (8) Chen, Y. R.; Glabe, C. G. Distinct Early Folding and Aggregation Properties of Alzheimer Amyloid-β Peptides Aβ40 and Aβ42 STABLE TRIMER OR TETRAMER FORMATION BY Aβ42. J. Biol. Chem. 2006, 281, 24414-24422. (9) Yan, Y.; Wang, C. Aβ42 is more rigid than Aβ40 at the C terminus: implications for Aβ aggregation and toxicity. J. Biol. Chem. 2006, 364, 853-862. (10) Rauk, A. The chemistry of Alzheimer’s disease. Chem. Soc. Rev. 2009, 38, 2698-2715. (11) Bieschke, J.; Herbst, M.; Wiglenda, T.; Friedrich, R. P.; Boeddrich, A.; Schiele, F.; Kleckers, D.; Amo, J. M. L.; Grüning, B. A.; Wang, Q.; Schmidt, M. R; Lurz, R.; Anwyl, R.; Schnoegl, S.; Fändrich, M.; Frank, R. F.; Reif, B.; Günther, S.; Walsh, D. M.; Wanker, E. E. Small-molecule conversion of toxic oligomers to nontoxic β-sheet-rich amyloid fibrils. Nat. Chem. Biol. 2012, 8, 93-101. (12) 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. Biol. 2008, 15, 558-566. (13) Liu, F. F.; Ji, L.; Dong, X. Y.; Sun, Y. Molecular insight into the inhibition 17


Langmuir 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

Page 18 of 31

effect of trehalose on the nucleation and elongation of amyloid β-peptide oligomers. J. Phys. Chem. B 2009, 113, 11320-11329. (14) Soto, C.; Sigurdsson, E. M.; Morelli, L.; Kumar, R. A.; Castaño, E. M.; Frangione, B. β-sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: implications for Alzheimer's therapy. Nat. Med. 1998, 4, 822-826. (15) Fülöp, L.; Zarándi, M.; Datki, Z.; Soós, K.; Penke, B. β-Amyloid-derived pentapeptide RIIGLa inhibits Aβ1–42 aggregation and toxicity. Biochem. Biophys. Res. Commun. 2004, 324, 64-69. (16) Milojevic, J.; Raditsis, A.; Melacini, G. Human serum albumin inhibits Aβ fibrillization through a “monomer-competitor” mechanism. Biophys. J. 2009, 97, 2585-2594. (17) Luo, J.; Wärmländer, S. K.; Gräslund, A.; Abrahams, J. P. Human lysozyme inhibits the in vitro aggregation of Aβ peptides, which in vivo are associated with Alzheimer's disease. Chem. Commun. 2013, 49, 6507-6509. (18) Cabaleiro-Lago, C.; Quinlan-Pluck, F.; Lynch, I.; Dawson, K. A.; Linse, S. Dual effect of amino modified polystyrene nanoparticles on amyloid β protein fibrillation. ACS Chem. Neurosci. 2010, 1, 279-287. (19) Cabaleiro-Lago, C.; Quinlan-Pluck, F.; Lynch, I.; Lindman, S.; Minogue, A. M.; Thulin, E.; Thulin, E.; Dawson, K. A.; Linse, S. Inhibition of amyloid β protein fibrillation by polymeric nanoparticles. J. Am. Chem. Soc. 2008, 130, 15437-15443. (20) Skaat, H.; Chen, R.; Grinberg, I.; Margel, S. Engineered polymer nanoparticles containing

hydrophobic

dipeptide

for

inhibition

of

amyloid-β

fibrillation.

Biomacromolecules 2012, 13, 2662-2670. (21) Yang, B.; Wang, J.; Tang, B.; Liu, Y.; Guo, C.; Yang, P.; Yu, T.; Li, R.; Zhao, J.; Dai, Y.; Li, N. Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle. PloS one 2011, 6, No. e17593. (22) Helmfors, L.; Boman, A.; Civitelli, L.; Nath, S.; Sandin, L.; Janefjord, C.; McCann, H.; Zetterberg, H.; Blennow, K.; Halliday, G.; Brorsson, A. C.; Kagedal, K. Protective properties of lysozyme on β-amyloid pathology: implications for Alzheimer disease. Neurobiol. Dis. 2015, 83, 122-133. 18


Page 19 of 31 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

Langmuir

(23) Das, P.; Kang, S. G.; Temple, S.; Belfort, G. Interaction of amyloid inhibitor proteins with amyloid beta peptides: insight from molecular dynamics simulations. PloS one 2014, 9, No. e113041. (24) Li, X.; Xie, B.; Dong, X.; Sun, Y. Bifunctionality of Iminodiacetic Acid-Modified

Lysozyme

on

Inhibiting

Zn2+-Mediated

Amyloid

β-Protein

Aggregation. Langmuir 2018, 34, 5106-5115. (25) Liu, H.; Ojha, B.; Morris, C.; Jiang, M.; Wojcikiewicz, E. P.; Rao, P. P.; Du, D. Positively charged chitosan and N-trimethyl chitosan inhibit Aβ40 fibrillogenesis. Biomacromolecules, 2015, 16, 2363-2373. (26) Xie, B.; Li, X.; Dong, X. Y.; Sun, Y. Insight into the inhibition effect of acidulated serum albumin on amyloid β-protein fibrillogenesis and cytotoxicity. Langmuir 2014, 30, 9789-9796. (27) Wang, Q.; Shah, N.; Zhao, J.; Wang, C.; Zhao, C.; Liu, L.; Li, L.; Zhou, F.; Zheng, J. Structural, morphological, and kinetic studies of β-amyloid peptide aggregation on self-assembled monolayers. Phys. Chem. Chem. Phys. 2011, 13, 15200-15210. (28) Khurana, R.; Coleman, C.; Ionescu-Zanetti, C.; Carter, S. A.; Krishna, V.; Grover, R. K.; Roy, R.; Singh, S. Mechanism of thioflavin T binding to amyloid fibrils. J. Struct. Biol. 2005, 151, 229-238. (29) LeVine, H. Quantification of β-sheet amyloid fibril structures with thioflavin T. Methods Enzymol. 1999, 309, 274-284. (30) Cohen, S. I. A.; Cukalevski, R.; Michaels, T. C. T.; Šarić, A.; Törnquist, M.; Vendruscolo, M.; Dobson, C. M.; Buell, A. K.; Knowles, T. P. J.; Linse, S. Distinct thermodynamic signatures of oligomer generation in the aggregation of the amyloid-β peptide. Nat. Chem. 2018, 10, 523-531. (31) Cabaleiro-Lago, C.; Szczepankiewicz, O.; Linse, S. The effect of nanoparticles on amyloid aggregation depends on the protein stability and intrinsic aggregation rate. Langmuir 2012, 28, 1852-1857. (32) Xiong, N.; Dong, X. Y.; Zheng, J.; Liu, F. F.; Sun, Y. Design of LVFFARK and LVFFARK-functionalized nanoparticles for inhibiting amyloid β-protein 19


Langmuir 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

fibrillation and cytotoxicity. ACS Appl. Mater. Interfaces 2015, 7, 5650-5662. (33) Jiang, Z.; Dong, X.; Liu, H.; Wang, Y.; Zhang, L.; Sun, Y. Multifunctionality of self-assembled nanogels of curcumin-hyaluronic acid conjugates on inhibiting amyloid β-protein fibrillation and cytotoxicity. React. Funct. Polym. 2016, 104, 22-29. (34) 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. Neurosci. 2014, 5, 552-558. (35) Assarsson, A.; Hellstrand, E.; Cabaleiro-Lago, C.; Linse, S. Charge dependent retardation of amyloid β aggregation by hydrophilic proteins. ACS Chem. Neurosci. 2014, 5, 266-274. (36) Liu, R.; Barkhordarian, H.; Emadi, S.; Park, C. B.; Sierks, M. R. Trehalose differentially inhibits aggregation and neurotoxicity of beta-amyloid 40 and 42. Neurobiol. Dis. 2005, 20, 74-81. (37) Wang, P.; Liao, W.; Fang, J.; Liu, Q.; Yao, J.; Hu, M.; Ding, K. A glucan isolated from flowers of Lonicera japonica Thunb. Inhibits aggregation and neurotoxicity of Aβ42. Carbohydr. Polym. 2014, 110, 142-147. (38) Zhao, Z.; Zhu, L.; Li, H.; Cheng, P.; Peng, J.; Yin, Y.; Yang, Y.; Wang C.; Hu Z.; Yang Y. Antiamyloidogenic Activity of Aβ42-Binding Peptoid in Modulating Amyloid Oligomerization. small 2017, 13, 1602857. (39) Gu, L.; Liu, C.; Guo, Z. Structural insights into Aβ42 oligomers using site-directed spin labeling. J. Biol. Chem. 2013, 288, 18673-18683. (40) Li, H.; Monien, B. H.; Fradinger, E. A.; Urbanc, B.; Bitan, G. Biophysical characterization of Aβ42 C-terminal fragments: inhibitors of Aβ42 neurotoxicity. Biochemistry 2010, 49, 1259-1267. (41) Tew, D. J.; Bottomley, S. P.; Smith, D. P.; Ciccotosto, G. D.; Babon, J.; Hinds, M. G.; Masters, C. L.; Cappai, R.; Barnham, K. J. Stabilization of neurotoxic soluble β-sheet-rich conformations of the Alzheimer's disease amyloid-β peptide. Biophys. J. 2008, 94, 2752-2766. (42) Shen, C. L.; Scott, G. L.; Merchant, F.; Murphy, R. M. Light scattering 20


Page 20 of 31

Page 21 of 31 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

Langmuir

analysis of fibril growth from the amino-terminal fragment beta (1-28) of beta-amyloid peptide. Biophys. J. 1993, 65, 2383-2395. (43) Nichols, M. R.; Moss, M. A.; Reed, D. K.; Lin, W. L.; Mukhopadhyay, R.; Hoh, J. H.; Rosenberry, T. L. Growth of β-amyloid (1-40) protofibrils by monomer elongation and lateral association. Characterization of distinct products by light scattering and atomic force microscopy. Biochemistry, 2002, 41, 6115-6127. (44) Klement, K.; Wieligmann, K.; Meinhardt, J.; Hortschansky, P.; Richter, W.; Fändrich, M. Effect of different salt ions on the propensity of aggregation and on the structure of Alzheimer’s Aβ (1-40) amyloid fibrils. J. Mol. Biol. 2007, 373, 1321-1333. (45) Raman, B.; Chatani, E.; Kihara, M.; Ban, T.; Sakai, M.; Hasegawa, K.; Naiki, H.; Rao, C. M.; Goto, Y. Critical balance of electrostatic and hydrophobic interactions is required for β2-microglobulin amyloid fibril growth and stability. Biochemistry, 2005, 44, 1288-1299. (46) Hu, Y.; Guo, T.; Ye, X.; Li, Q.; Guo, M.; Liu, H.; Wu, Z. Dye adsorption by resins: effect of ionic strength on hydrophobic and electrostatic interactions. Chem. Eng. J. 2013, 228, 392-397. (47) Roche, J.; Shen, Y.; Lee, J. H.; Ying, J.; Bax, A. Monomeric Aβ1–40 and Aβ1–42 peptides in solution adopt very similar Ramachandran map distributions that closely resemble random coil. Biochemistry, 2016, 55, 762-775. (48) Man, V. H.; Nguyen, P. H.; Derreumaux, P. High-resolution structures of the amyloid-β 1–42 dimers from the comparison of four atomistic force fields. J. Phys. Chem. B 2017, 121, 5977-5987. (49) Nasica-Labouze, J.; Nguyen, P. H.; Sterpone, F.; Berthoumieu, O.; Buchete, N. V.; Cote, S.; Simone, A. D.; Doig, A. J.; Faller, P.; Garcia, A.; Laio, A.; Li,

M. S.; Melchionna, S.; Mousseau, N.; Mu, Y.; Paravastu, A.; Pasquali, S.; Rosenman, D.J.; Strodel, B.; Tarus, B.; Viles, J. H.; Zhang, T.; Wang, C.; Derreumaux, P. Amyloid β protein and Alzheimer’s disease: When computer simulations complement experimental studies. Chem. Rev. 2015, 115, 3518-3563. (50) Nagel-Steger, L.; Owen, M. C.; Strodel, B. An account of amyloid oligomers: 21


Langmuir 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

facts and figures obtained from experiments and simulations. ChemBioChem, 2016, 17, 657-676. (51) Wood, S. J.; Maleeff, B.; Hart, T.; Wetzel, R. Physical, morphological and functional differences between pH 5.8 and 7.4 aggregates of the Alzheimer's amyloid peptide Aβ. J. Mol. Biol. 1996, 256, 870-877. (52) Hortschansky, P.; Schroeckh, V.; Christopeit, T.; Zandomeneghi, G.; Fändrich, M. The aggregation kinetics of Alzheimer's β-amyloid peptide is controlled by stochastic nucleation. Protein Sci. 2005, 14, 1753-1759. (53) Scheidt, H. A.; Morgado, I.; Rothemund, S.; Huster, D.; Fändrich, M. Solid-State NMR Spectroscopic Investigation of Aβ Protofibrils: Implication of a β-Sheet Remodeling upon Maturation into Terminal Amyloid Fibrils. Angew. Chem., Int. Ed. 2011, 50, 2837-2840.

22


Page 22 of 31

Page 23 of 31 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

Langmuir

Table 1. Physicochemical Properties of hLys and hLys-B Preparations at pH 7.4. Molecular weight

MD

Size

(Da)

(-)

(nm)

hLys

14697.1

-

4.6 ± 0.3

hLys-B1

14760.8

1.5

4.6 ± 0.2

hLys-B2

14835.8

3.3

4.7 ± 0.5

hLys-B3

14880.9

4.4

4.8 ± 0.3

Agent

Table 2. Lag Times and Reduction of Final ThT FI for Aggregation Kinetics of Aβ40 (25 μM) at Different Conditions. Inhibitor

None

hLys

hLys-B1

hLys-B2

hLys-B3

Tlag

Aβ

(h)

Aβ:Ia=1:0.1

30.1±0.7

42.8±0.5

59.2±0.6

91.6±0.8

Aβ:I=1:0.25

38.1±0.4

51.7±0.8

81.6±0.6

140.9±0.9

Aβ:I=1:0.5

83.2±0.9

139.8±1.0 149.5±1.2

＞166.7

Reduction

Aβ:I=1:0.1

22.4±2.5

54.4±3.2

62.5±2.1

74.3±1.4

of FI

Aβ:I=1:0.25

34.8±3.1

77.4±2.8

85.2±2.5

89.6±2.2

(%)

Aβ:I=1:0.5

73.1±1.2

92.6±2.6

95.0±2.7

100

a

17.9±0.5

I stands for inhibitor.

23


Langmuir 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

Scheme 1. Reaction Scheme for the Modification of hLys with Ethylene Diamine.

24


Page 24 of 31

Page 25 of 31 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

Langmuir

Figure 1. Normalized ThT fluorescence of Aβ42 incubated in the absence and presence of various concentrations of hLys/hLys-B for 24 h. Aβ42 concentration was 25 μM. ***, p < 0.001 as compared to Aβ only. The values of p < 0.001, p < 0.01 and p < 0.05 for the specific pairs of data sets are marked with ###, ## and #, respectively.

25


Langmuir 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 2. ThT fluorescence kinetic assays for the inhibition of Aβ40 aggregation. Aβ40 concentration was 25 μM. All experiments were in buffer A at 37 °C. The solid lines were calculated from eq. 2.

26


Page 26 of 31

Page 27 of 31 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

Langmuir

Figure 3. AFM measurements of the (A) Aβ42, (B) Aβ42 + hLys, (C) Aβ42 + hLys-B1, (D) Aβ42 + hLys-B2, (E) Aβ42 + hLys-B3 after 24 h of incubation at 37 °C. Aβ42 concentration was 25 μM, and the inhibitor was 2.5 μM in image (B1) to (E1), 6.25 μM in (B2) to (E2) or 12.5 μM in (B3) to (E3).

27


Langmuir 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 4. Circular dichroism spectra of Aβ42 (25 μM) incubated for (A) 0 h and (B) 24 h with hLys/hLys-B (2.5 μM) in buffer A at 37 °C.

28


Page 28 of 31

Page 29 of 31 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

Langmuir

Figure 5. Viability of SH-SY5Y cells incubated with Aβ42 aggregates prepared in the absence and presence of hLys and hLys-B preparations. The samples obtained by pre-incubation of Aβ42 at different conditions. The concentration of aged-Aβ42 was 25 μM in the absence and presence of an inhibitor at the mole ratio to Aβ42 as indicated in the abscissa. The cell viability treated with buffer A was set to 100%. ***, p < 0.001, **, p < 0.01, and *, p < 0.05 as compared to the Aβ42 only group (column 2). The values of p < 0.001, p < 0.01, p < 0.05 for the specific pairs of data sets are marked with ###, ## and #, respectively.

29


Langmuir 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 6. Effect of NaCl concentration on the inhibition effects of hLys and hLys-B3 by ThT fluorescence kinetic assays. Each line was normalized by setting the maximum value of Aβ42 only at the same NaCl concentration as 100%. The concentrations of Aβ42 and inhibitors were 25 μM and 12.5 μM, respectively.

30


Page 30 of 31

Page 31 of 31 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

Langmuir

Table of Contents Graphic

31


Basified Human Lysozyme: A Potent Inhibitor against Amyloid Î²

Recommend Documents