Letter pubs.acs.org/chemneuro
Inhibition and Degradation of Amyloid Beta (Aβ40) Fibrillation by Designed Small Peptide: A Combined Spectroscopy, Microscopy, and Cell Toxicity Study Anirban Ghosh,† Nibedita Pradhan,‡ Swapna Bera,† Aritreyee Datta,† Janarthanan Krishnamoorthy,§ Nikhil R. Jana,‡ and Anirban Bhunia*,† †
Department of Biophysics, Bose Institute, Kolkata 700054, India Indian Association for the Cultivation of Science, Kolkata 700032, India § Vclinbio Laboratories Pvt Ltd., Sri Ramachandra Medical Center, Chennai 600116, India ‡
S Supporting Information *
ABSTRACT: A designed nontoxic, nonhemolytic 11-residue peptide, NF11 (NAVRWSLMRPF), not only inhibits the aggregation of amyloid beta (Aβ40) protein but also disaggregates the preformed oligomers and mature Aβ fibrils, thereby reducing associated-toxicity. NMR experiments provide evidence of NF11’s ability to inhibit fibril formation, primarily through interaction with the N-terminus region as well as the central hydrophobic cluster of Aβ40. NF11 has micromolar binding affinity toward both monomeric and aggregated species for efficient clearance of toxic aggregates. From these in vitro results, the future development of a next generation peptidomimetic therapeutic agent for amyloid disease may be possible. KEYWORDS: Alzheimer’s disease, amyloid beta (Aβ), peptide, STD NMR, fibril disintegration, toxicity inhibitor designed to disaggregate amyloid fibrils.15 Alternately, the most successful was the “β-sheet breaker”, iAβ5p peptide with the sequence LPFFD, with its c-terminal region being protected from proteolysis. iAβ5p has been shown to inhibit formation of Aβ40 fibrils as well the disaggregation of amyloid plaques.16,17 Recently, synthetically modified peptides have also been used effectively to inhibit as well as disaggregate amyloid fibrils. NH2-D-Trp-Aib-OH is a small dipeptide, with α-aminoisobutyric acid (Aib) replacing proline as a potent β-sheet breaker. This peptide particularly targets Aβ assembly into oligomer formation.18 We report a nonhemolytic and nontoxic 11-residue peptide NF11 (NAVRWSLMRPF) (Scheme S1) capable of inhibiting Aβ40 aggregation and dissolving any preformed Aβ oligomers and fibrils, culminating in a reduced oligomer/fibril-associated toxicity in the neuronal cells. NF11 is a modified analogue of a 9-residue peptide NK9 (NIVNVSLVK), from SARS coronavirus E-protein sequence, previously reported to inhibit insulin fibrillation.19 NF11 was synthesized by solid phase peptide synthesis, purified by HPLC and characterized by mass as well as NMR spectroscopy (see the Supporting Information and Figure S1 for details). First, ThT fluorescence assay was
nraveling the molecular mechanism of amyloid fibril formation entails addressing two important aspects, namely, protein folding and protein aggregation.1,2 Accurate experimental constraints are quintessential to channelize the available information to design molecules for treating amyloid diseases.1,3 Recent studies demonstrate that prefibrillar aggregates (soluble oligomers and protofibrils) are highly cytotoxic compared to mature fibrils.4−6 Over the past decade, a plethora of small molecules, which include homotaurin (3amino-1-propanesulfonic acid), scyllo-inositol, and (−)-epigallocatechin-3-gallate (EGCG), have been shown to be effective in disaggregating amyloid fibrils.7−10 In addition, few potent peptides and peptidomimetics were designed to target the inhibition of amyloid oligomer formation. However, the major research was focused on self-recognition segments (L17VFFA21) of the Aβ protein to improve the activity.11−14 One such peptide is rGffvlkGr-1,5-diaminopentane (the small letter convention used in the sequence refers to the D-form of the amino acid), which was shown to have antiaggregation activity significantly better than that of KLVFF/LPFFD.14 Also the substituted diaminopentane group improved the bioavailability of the peptide across the blood-brain barrier (BBB).4,12,14 H102, a novel peptide with sequence HKQLPFFEED was also shown to exhibit potent inhibitory and disaggregation effect on amyloid fibrillation process.13 Transthyretin based structurally unique cyclic peptides have also been
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© 2017 American Chemical Society
Received: October 14, 2016 Accepted: January 6, 2017 Published: January 6, 2017 718
DOI: 10.1021/acschemneuro.6b00349 ACS Chem. Neurosci. 2017, 8, 718−722
Letter
ACS Chemical Neuroscience
coinbutation with NF11 (at 1:10 molar ratio), the Aβ40 fibrillar networks substantially fragmented to short, and sparsely branched fibrils with an average length of 0.14 μm (Figures 1f, S3d−f, and S4b). The inhibitory effect of NF11 was found to be pronounced even after 7 days of incubation displaying a complete disruption of Aβ fibrillar network (average length of 0.03 μm), leading to the formation of amorphous aggregates (Figures 1g, S3g−i, and S4c). Taken together, the qualitative microscopic results are in good agreement with the quantitative ThT fluorescence assay data, signifying substantial inhibition of Aβ40 aggregation by NF11. This motivated us to investigate whether NF11 could disaggregate or dissolve preformed Aβ40 oligomers or fibrils. ThT fluorescence of prefibrillar species (obtained at the end of 3 days during elongation phase) after the addition of NF11 drastically reduced to a nearly basal level within ∼6−8 h, indicating the dissolution of oligomeric species. Moreover, the inhibitory effect of NF11 was observed even during the saturation phase of fibrillation (i.e., at 7 and 12 days post incubation) demonstrating its efficiency to disintegrate the mature fibrillar network (Figure 2a and inset). The drastic changes in the morphology of Aβ40 fibril in the presence of NF11 in contrast to the control Aβ40 fibril indicate that NF11 interacts with the Aβ40 fibril and mediates disaggregation (Figures 2b, i−iv and S5). This observation is further supported by ∼4 fold reduction of β sheet content of Aβ40 fbirl in the presence of NF11 (1:10 molar ratio) from CD seectroscopy (Figure S6). To elucidate the binding affinity of NF11 with the pathophysiologically relevant Aβ40 fibril, steady-state fluorescence experiments were performed. The intrinsic tryptophan fluorescence emission maxima (∼353 nm) of NF11 was blueshifted to ∼331 nm with higher intensity upon addition of increasing concentrations of Aβ40 fibrils (Figure 2c), most likely due to the insertion of NF11 in the hydrophobic macromolecular cavity of Aβ40 cluster. The similar blue shift (∼15 nm) was also observed for tryptophan fluorescence of NF11 in the context of Aβ42 fibril (data not shown). In support of this observation, acrylamide fluorescence quenching showed solvent accessibility the tryptophans of NF11 to be significantly reduced in the presence of Aβ40 fibrils, as manifested by an ∼4-fold reduction in stern volmer constant (Ksv) (Figure S7a). The Aβ40 fibril-NF11 complexation was further supported by fluorescence anisotropy, which showed a consistent concentration-dependent increase in anisotropy values of NF11 from ∼0.02 to 0.06 upon addition of Aβ40 fibril which yield the equilibrium dissociation constants (KD = 11.2 ± 2.8 μM) in the micromolar range (Figure S7b). Since neuronal toxicity is a natural consequence of Aβ aggregation, we further extended our study to investigate the dampening effect of NF11 on the toxicity of different species of Aβ40 fibrillation process in a mouse neuro2A cell line. The MTT assay indicated that low molecular weight (LMW) oligomeric species of Aβ40 (2 days post incubation) are highly toxic compared to the high molecular weight (HMW) oligomers (4 days post incubation) as well as mature fibrils (6 days post incubation) (Figure 2d), as found in previous reports.6,8 Interestingly, treatment with NF11 significantly increase neuronal cell viability and subsequently diminished the toxicity of oligomeric and fbirllar Aβ40 species. This is likely the result of sequestering toxic oligomers by NF11 such that they can no longer effectively engage in the toxic pathways.5,6 On the other hand, NF11 by itself showed neither hemolytic nor cytotoxic effect when tested against human red
performed to compare the kinetics of Aβ40 fibrillation, in the presence as well as in the absence of NF11 (Figures 1a and
Figure 1. (a) Bar diagram showing ThT fluorescence assay of Aβ40 in the absence (blue) and presence of NF11 (red) at a 1:10 molar ratio and at different time period of incubation. The error bars in the bar diagram signifies standard deviations from three consecutive measurements for each set. Fluorescence microscopy images of ThT stained Aβ40 fibril (b, c) and in the presence of 1:10 molar ratios of NF11 (d) after 48 h of postincubation. An enlarged view of the fibrillary network (scale bar = 25 μm) in (b) is marked in the inset (c) with scale bar of 10 μm. HRTEM images of Aβ40 fibrils (scale bar = 1 μm) after 7 days (e) and in the presence of 1:10 molar equiv of NF11 after (f) 2 days (scale bar = 1 μm) and (g) 7 days (scale bar = 0.5 μm) post-treatment, respectively.
S2).20 A striking dose-dependent inhibition of Aβ40 fibrillation by NF11 was observed with maximum inhibition occurring at a molar ratio of 1:10 (Aβ40:NF11) (Figure S2a). A timedependent plot of the increase in ThT fluorescence for Aβ fibril formation in the presence of NF11 at the aforementioned molar ratio reveals only a marginal (∼0.5 fold) intensity increment compared to 2-fold increase for Aβ fibril formation in the absence of NF11 (Figure 1a). NF11 also shows its drastic inhibitory effect in the aggregation kinetics of another pathologically relevant Aβ42 species (data not shown). Under identical experimental conditions, NF11 does not self-aggregate to form amyloid, thereby ruling out any interference or artifacts in the ThT fluorescence assay (Figure S2b). Moreover, the lag time of Aβ40 fibrillation was prolonged by ∼4 fold in the presence of NF11 as of 1:10 molar ratio and reaching a plateau (Figure S2c). Since the maximum inhibition of Aβ40 fibrillation was observed at 1:10 molar ratio, subsequent experiments were performed at the same molar ratio. Further, to support NF11 mediated inhibition of Aβ40 fibrillation, morphological differences in the supramolecular structures of Aβ40 fibrils in the presence of NF11 were analyzed using fluorescence microscopy and high-resolution transmission electron microscopy (HRTEM) (Figure 1b−g). A dense fibrillar network could be detected for ThT stained control Aβ40 aggregates without NF11 (Figure 1b,c). In contrast, upon incubation with NF11 (Aβ40/NF11 = 1:10) the extent of Aβ fibrillation was largely reduced and restrained in its oligomeric state, as evident from the morphology of spherical conglomerates in fluorescence microscopy images (Figure 1d). Aβ40 alone forms long, dense and branched fibrillar network with a length of 3.0 μm (Figures 1e, S3a−c, and S4a), as previously reported.20 In contrast, upon 719
DOI: 10.1021/acschemneuro.6b00349 ACS Chem. Neurosci. 2017, 8, 718−722
Letter
ACS Chemical Neuroscience
Figure 2. (a) Effect of adding NF11 at different stages of Aβ40 fibrillation pathway, studied by ThT fluorescence. NF11 was added to 3, 7, and 12 days post incubated Aβ40 at 1:10 molar ratio. Inset, NF11 disaggregates the preformed oligomers or fibrils. The blue and red dotted lines represent ThT fluorescence intensity profiles for Aβ40 in the absence of NF11 at 7 and 12 days, respectively. The error bars represent the average ThT fluorescence intensity values from three consecutive measurements and their corresponding standard deviations. (b) Comparison of fluorescence microscopy images of matured Aβ40 fibril in the presence of NF11 (molar ratio = 1:10) (i−iii, 0, 4, and 8 h, respectively) with control fibril (iv). (c) Steady state tryptophan fluorescence emission profile for NF11 alone (black) and in the presence of 1:6 molar equiv of Aβ40 fibril (red) showing 22 nm blue shift. (d) Cytotoxicity of either untreated Aβ40 alone (blue bars) or after treatment with NF11 (Aβ40/NF11 = 1:10) in Neuro2A cell line (red bars) at different time interval. The gray bars represent cell viability of Neuro 2A cells in the absence of Aβ40 and NF11 (control). The concentration of Aβ40 was 3.13 μM in terms of monomer concentration. LMW and HMW stand for low molecular weight and high molecular weight oligomer, respectively. The mean ± SD of three determinations (n = 3) are represented in bars. Bars with different characters are statistically different at *P < 0.05, **P < 0.01, ***P < 0.001 level as analyzed by one-way ANOVA. A criterion of p < 0.05 was employed to determine whether the sample data set and the untreated control data set were statistically different.
Figure 3. (a) 2D 1H/15N-SOFAST HMQC spectra (Bruker Avance III 500 MHz NMR spectrometer, equipped with SMART probe, at 277 K) of 15N labeled Aβ40 (80 μM), before (red) and after (blue) addition of NF11 at 1:10 molar ratio. Overlapping residues with similar chemical shift are marked with an asterisk (*). The experiment was performed in 20 mM sodium phosphate buffer (pH 7.2), 50 mM of NaCl. (b) Bar plot showing the chemical shift perturbation (CSP) of Aβ40 due to NF11 binding. The gray shaded region represents the average CSP of all residues. The most perturbed residues are marked with red color. (c) Reference (i) and STD NMR spectra (onresonance = −1 ppm, off-resonance = 40 ppm, for 2 s saturation time) of NF11 in the presence (ii) and in the absence (iii) of Aβ40 fibril. The binding and nonbinding region is marked with sky blue and violet color, respectively. d) HADDOCK derived model of Aβ40-NF11 complex (central hydrophobic cluster is highlighted).
blood cells or Neuro2A or CHO cell lines, respectively (Figure S8). To get structural insights into the atomic level interaction between NF11 and Aβ40, we performed 2D 1H/15N bandselective optimized-flip-angle short-transient (SOFAST) heteronuclear multiple quantum coherence (HMQC) NMR experiment for Aβ40 in the presence of NF11 (Figure 3a) up to a molar ratio of 1:30 (Figure S9). The addition of NF11 to Aβ40 triggered substantial residue specific chemical shift perturbation (CSP) of amide N−H, due to fast to intermediate chemical exchange regime present between the free and the NF11-Aβ40 complex. The degree of perturbation was largest for the Nterminal residues, i.e., R5, S8, G9, Y10 E11, H13, H14, and Q15 and the central hydrophobic cluster residues comprising K16, L17, and F19. Moderate perturbation was seen for the C-ter residues such as M35 and V36 (Figure 3b). The side chain of Q15, N27, and imidazole ring of H13 and H14 also exhibited significant CSP. NF11 was also able to interact with Aβ42,
perturbing almost similar residues as Aβ40; e.g., N-terminal residues (R5, S8, G9, Y10 E11, V12, H13, H14, and Q15) as well as central hydrophobic residues (K16, L17, V18, and F19, F20) of Aβ42 showed stronger interaction, confirmed by line broadening of amide groups in the 2D 1H/15N SOFAST HMQC spectra (data not shown). For higher molar ratios of Aβ40/NF11, the magnitude of CSP increased concomitantly; however, the CSP pattern almost remained the same (Figures S9 and S10). An average equilibrium dissociation constant (KD) of 0.41 ± 0.05 mM was evaluated for NF11-Aβ40 monomer complex based on a single site binding model from the CSP values of G9 (Figure S11). On the contrary, no major conformational change was observed for NF11 in the presence of Aβ40 fibril, which is confirmed by both the 1H NMR and trNOESY spectra of NF11 in the context of Aβ40 fibril (Figure S12). This is due to the 720
DOI: 10.1021/acschemneuro.6b00349 ACS Chem. Neurosci. 2017, 8, 718−722
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ACS Chemical Neuroscience
with a field emission gun operating at 200 kV. To elucidate the quantitative morphological characterization, we measured the fibril length using ImageJ.22 Dissolution of Aβ40 Fibril by NF11 Peptide. Aggregates or fibrils at different time points of aggregation (3, 7, and 12 days) were incubated with NF11 (1:10 molar ratio) for up to 48 h. Dissolution kinetics was studied by ThT based assay as already described. Finally, the morpholoy of the solutions was viewed via TEM. Fluorescence Experiments. Change in intrinsic tryptophan fluorescence of NF11 in the presence of Aβ fibrils were executed with a Hitachi F-7000 FL spectrometer at 25 °C. The excitation wavelength was fixed at 295 nm, and emission spectra were recorded with a range of 300−400 nm with 5 nm slit width for both.23 A neutral quencher acrylamide was used for quenching of free NF11 and Aβ40 bound NF11. The fluorescence anisotropy (r) values were plotted against the ligand concentration (in μM) and fitted in a standard single site ligand binding equation for binding affinity elucidation. Fluorescence Microscopy. Aliquots of 10 μL of the stock solutions were placed on a glass slide. After air-drying, the images were taken using a 63× objective in oil immersion in a confocal microscope (Leica TCSSP8 and the LAS AF Version 2.1.0 built-in 4316 software, Leica Microsystems GmbH, Germany). Cytotoxicity Assay of Aβ40 Fibril Prepared in the Presence of NF11 in Neuro2A Cell Line. Aliquots were taken out from the fibrillation mixture after 2, 4, and 6 days, and the cytotoxicity of these aliquots was checked by MTT assay using the mouse Neuro2A cell line. All the experiments were carried out three times, and the data are expressed as mean ± standard deviation (SD) (n = 3). For statistical analysis purpose, differences between the control and the experimental groups were analyzed using one-way analysis of variance (ANOVA) method. NMR Experiments. All NMR experiments were executed on a Bruker Avance III 500 MHz spectrometer, equipped with a 5 mm SMART probe and in a Shigemi tube (SHIGEMI Inc.) at 277 or 298 K. For each titration of NF11 to 15N labeled Aβ40 (∼80 μM), 2D SOFAST HMQC spectra was acquired. The saturation transfer difference (STD) NMR experiment was performed for NF11 alone and in the presence of Aβ40 fibril at a fibril to peptide ratio of 1:400.24 The off- and on-resonance saturation was attained with a train of selective Gaussian soft pulse consecutively for a duration of 2 s at 40 and −1 ppm, respectively, at 30 dB power in accordance with the previous literature.25 Docking Study with HADDOCK. NF11 was docked with a possible experimental structure of one of the Aβ40 monomer conformations (PDB id: 2LFM)26 using High Ambiguity Driven protein−protein Docking (HADDOCK).27 Here active residues in the Aβ40 and NF11 were provided as an input based on SOFAST-HMQC derived chemical shift perturbation values (CSP) and the STD NMR studies, respectively (see NMR Experiments subsection and the Supporting Informaiton for details). Representative docked complexes were obtained by considering the HADDOCK score and i-I-RMSD according to the HADDOCK server.28
fact that the binding interaction falls within the fast exchange regime on NMR time scale. Thus, we resorted to STD NMR for NF11 in the presence of Aβ40 fibril species to identify the binding site residues (Figure 3c). Interestingly, indole ring protons (of W5) and phenyl ring protons (of F11) of NF11 showed strong STD signals implying their closer proximity to Aβ40. Additionally, aliphatic protons CβHs, CγHs, and CδHs of R4, R8 and CβHs of A2, CγHs of V3, CδHs of L7, and CβHs of W5 and F11 also exhibited moderate STD effect. However, absence of peaks in control STD of NF11 indicates that sustainable saturation transfer takes place only through Aβ40 to NF11 (Figure 3c, iii). Using binding site residues of Aβ40 and NF11 identified from CSP as constraints, HADDOCK simulation was performed. The docking pose suggests that NF11 prefers to bind near the central hydrophobic cluster (L17-A21) and C-ter hydrophobic region (I31−V40) both of which are implicated in Aβ fibrillation and toxicity.5 The complex is stabilized by several salt bridges and cation−π interactions such as R4 (NF11)-Y10 (Aβ40); R9 (NF11)-D23 (Aβ40); W5 (NF11)-R5/Q15 (Aβ40), and so forth. The structural details of the complex obtained through constrained docking (Figure 3d) can be used in future studies to understand the elusive “off-pathway” oligomers, that leads to the formation of nontoxic intermediates, instead of the “on-pathway” toxic oligomer formation.21 In summary, our study revealed that NF11 has remarkable inhibitory effects on aggregation kinetics of Aβ40/Aβ42 during oligomerization and fibril elongation stages. Based on our data, we propose two distinct mechanisms for inhibition of Aβ aggregation. First, NF11 binds to the monomer and perturbs the monomer−oligomer equilibrium and subsequently affects the critical nuclei development in the lag phase of Aβ fibrillation. Second, NF11 may also bind to the oligomers, further leading to a diminution of the sub- and near-critical oligomer concentrations leading to the formation of nontoxic “off-pathway” species.6,21 Thus, it can restrict the elongation pathway by blocking the sites critical for the monomer addition to the growing edges of the Aβ40 aggregates.6 Therefore, NF11 can be used as a double-edged sword that on one hand diminishes the amount of preformed mature fibrillar entity, while on the other hand mediates the inhibition of the cytotoxic amyloidogenic oligomeric population. Conversely, our designed peptide NF11 can also be used as a potential preclinical AD detection agent given its ability to sequester Aβ40 oligomers in vitro. Further studies on the inhibitory effects of NF11 using amyloid plaques ex vivo, and its potential to cross the bloodbrain barrier in vivo may serve as a platform for in-depth understanding of therapeutic strategies against AD and design of the potent lead peptides.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.6b00349. Detailed experimental methods and materials and design of peptide (PDF)
METHODS
Amyloid Fibrillation Study by ThT Fluorescence Experiment. Stock solutions of Aβ40 and NF11 were prepared in anhydrous DMSO and water, respectively, according to the required concentration. Four separate sets of experimental solutions were prepared with varying molar ratios of Aβ40/NF11 in acetate buffer solution, containing 137 mM NaCl and 2.68 mM KCl, pH 5.20 All the four sets were then incubated at 37 °C for 12 days and the extent of fibrillation was studied by ThT based fluorescence assay at different time intervals. The fluorescence intensity was measured at an excitation/ emission of 440/485 nm, respectively. Transmission Electron Microscopy (TEM). Aliquots of 10 μL of the experimental solutions were taken on TEM grids. After drying the grids, samples were viewed using an FEI Tecnai G2 F20 microscope
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AUTHOR INFORMATION
Corresponding Author
*Tel: +91-33-2569 3336. E-mail:
[email protected];
[email protected]. ORCID
Nikhil R. Jana: 0000-0002-4595-6917 721
DOI: 10.1021/acschemneuro.6b00349 ACS Chem. Neurosci. 2017, 8, 718−722
Letter
ACS Chemical Neuroscience
(11) Bett, C. K., Serem, W. K., Fontenot, K. R., Hammer, R. P., and Garno, J. C. (2010) Effects of peptides derived from terminal modifications of the aβ central hydrophobic core on aβ fibrillization. ACS Chem. Neurosci. 1, 661−678. (12) Taylor, M., Moore, S., Mayes, J., Parkin, E., Beeg, M., Canovi, M., Gobbi, M., Mann, D. M., and Allsop, D. (2010) Development of a proteolytically stable retro-inverso peptide inhibitor of beta-amyloid oligomerization as a potential novel treatment for Alzheimer’s disease. Biochemistry 49, 3261−3272. (13) Lin, L. X., Bo, X. Y., Tan, Y. Z., Sun, F. X., Song, M., Zhao, J., Ma, Z. H., Li, M., Zheng, K. J., and Xu, S. M. (2014) Feasibility of βsheet breaker peptide-H102 treatment for Alzheimer’s disease based on β-amyloid hypothesis. PLoS One 9, e112052. (14) Matharu, B., El-Agnaf, O., Razvi, A., and Austen, B. M. (2010) Development of retro-inverso peptides as anti-aggregation drugs for βamyloid in Alzheimer’s disease. Peptides 31, 1866−1872. (15) Lu, X., Brickson, C. R., and Murphy, R. M. (2016) TANGOInspired Design of Anti-Amyloid Cyclic Peptides. ACS Chem. Neurosci. 7, 1264−1274. (16) Soto, C., Kindy, M. S., Baumann, M., and Frangione, B. (1996) Inhibition of Alzheimer’s amyloidosis by peptides that prevent betasheet conformation. Biochem. Biophys. Res. Commun. 226, 672−680. (17) De Bona, P., Giuffrida, M. L., Caraci, F., Copani, A., Pignataro, B., Attanasio, F., Cataldo, S., Pappalardo, G., and Rizzarelli, E. (2009) Design and synthesis of new trehalose-conjugated pentapeptides as inhibitors of Abeta(1−42) fibrillogenesis and toxicity. J. Pept. Sci. 15, 220−228. (18) Frydman-Marom, A., Rechter, M., Shefler, I., Bram, Y., Shalev, D. E., and Gazit, E. (2009) Cognitive-performance recovery of Alzheimer’s disease model mice by modulation of early soluble amyloidal assemblies. Angew. Chem., Int. Ed. 48, 1981−1986. (19) Banerjee, V., Kar, R. K., Datta, A., Parthasarathi, K., Chatterjee, S., Das, K. P., and Bhunia, A. (2013) Use of a small peptide fragment as an inhibitor of insulin fibrillation process: a study by high and low resolution spectroscopy. PLoS One 8, e72318. (20) Palmal, S., Maity, A. R., Singh, B. K., Basu, S., Jana, N. R., and Jana, N. R. (2014) Inhibition of Amyloid Fibril Growth and Dissolution of Amyloid Fibrils by Curcumin−Gold Nanoparticles. Chem. - Eur. J. 20, 6184−6191. (21) Ehrnhoefer, D. E., Bieschke, J., Boeddrich, A., Herbst, M., Masino, L., Lurz, R., Engemann, S., Pastore, A., and Wanker, E. E. (2008) EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nat. Struct. Mol. Biol. 15, 558−566. (22) Abràmoff, M. D., Magalhães, P. J., and Ram, S. J. (2004) Image processing with ImageJ. Biophotonics Int. 11, 36−42. (23) Ghosh, A., Datta, A., Jana, J., Kar, R. K., Chatterjee, C., Chatterjee, S., and Bhunia, A. (2014) Sequence context induced antimicrobial activity: insight into lipopolysaccharide permeabilization. Mol. BioSyst. 10, 1596−1612. (24) Meyer, B., and Peters, T. (2003) NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors. Angew. Chem., Int. Ed. 42, 864−890. (25) Yesuvadian, R., Krishnamoorthy, J., Ramamoorthy, A., and Bhunia, A. (2014) Potent γ-secretase inhibitors/modulators interact with amyloid-β fibrils but do not inhibit fibrillation: A high-resolution NMR study. Biochem. Biophys. Res. Commun. 447, 590−595. (26) Vivekanandan, S., Brender, J. R., Lee, S. Y., and Ramamoorthy, A. (2011) A partially folded structure of amyloid-beta(1−40) in an aqueous environment. Biochem. Biophys. Res. Commun. 411, 312−316. (27) De Vries, S. J., van Dijk, M., and Bonvin, A. M. (2010) The HADDOCK web server for data-driven biomolecular docking. Nat. Protoc. 5, 883−897. (28) Dominguez, C., Boelens, R., and Bonvin, A. M. (2003) HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J. Am. Chem. Soc. 125, 1731− 1737.
Anirban Bhunia: 0000-0002-8752-2842 Author Contributions
A.B. conceived, designed, as well as funded the research work; A.G. conducted most of the biophysical experiments including NMR and wrote the manuscript; N.P. conducted cell biology and HRTEM experiments under the supervision of N.R.J.; S.B. performed fluorescence microscopy experiments; J.K. prepared the samples and helped in NMR experiments; A.D. performed hemolytic assay; A.B. edited the manuscript. All authors reviewed the manuscript. Funding
A.B. would like to thank Plan Project-II, Bose Institute, India for financial support. A.G., A.D., S.B., and N.P. thank CSIR, UGC, and IACS for fellowships, respectively. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS CIF of Bose Institute is greatly acknowledged. The authors also thank Dr. Jeffrey R. Brender for advice. DEDICATION Dedicated to Prof. Dr. Thomas Peters, University of Lübeck, Germany on the occasion of his 60th birthday. REFERENCES
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DOI: 10.1021/acschemneuro.6b00349 ACS Chem. Neurosci. 2017, 8, 718−722
