Amyloid-like Structures Formed by Single Amino Acid Self-Assemblies

Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Amyloid-like Structures Formed by Single Amino Acid SelfAssemblies of Cysteine and Methionine Nidhi Gour,*,† Chandra Kanth P.,‡,¶ Bharti Koshti,†,¶ Vivekshinh Kshtriya,† Dhruvi Shah,§ Sunita Patel,∥ Reena Agrawal-Rajput,§ and Manoj K. Pandey‡ †

Centre of Engineering and Enterprise, Indian Institute of Advanced Research, Gandhinagar, Gujarat 382426, India Department of Science, School of Technology, Pandit Deendayal Petroleum University, Gandhinagar, Gujarat 382007, India § School of Biological Sciences and Biotechnology, Indian Institute of Advanced Research, Gandhinagar, Gujarat 382426, India ∥ UM-DAE Centre for Excellence in Basic Sciences, Mumbai University Campus, Mumbai, Maharashtra 400098, India

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S Supporting Information *

ABSTRACT: We report for the very first time the discovery of amyloid-like self-assemblies formed by the nonaromatic single amino acids cysteine (Cys) and methionine (Met) under neutral aqueous conditions. The structure formation was assessed and characterized by various microscopic and spectroscopic techniques such as optical microscopy, phase contrast microscopy, scanning electron microscopy, and transmission electron microscopy. The mechanism of self-assembly and the role of hydrogen bonding and thiol interactions of Cys and Met were assessed by Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray diffraction, and solid state NMR along with various control experiments. In addition, molecular dynamics simulations were carried out to gain insight into assembly initiation. Further, Thioflavin T and Congo red binding assays with Cys and Met structures indicated that these single amino acid assemblies may have amyloid-like characteristics. To understand the biological significance of the Cys and Met structures, cytotoxicity assays of the assemblies were performed on human neuroblastoma IMR-32 cells and monkey kidney cells (COS-7). The results revealed that both Cys and Met fibers were cytotoxic. The cell viability assay further supported the hypothesis that aggregation of single amino acid may contribute to the etiology of metabolic disorders like cystinuria and hypermethioninemia. The results presented in this study are striking, and to the best of our knowledge this is the first report which demonstrates that nonaromatic amino acids like Cys and Met can undergo spontaneous self-assembly to form amyloidogenic aggregates. The results presented are also consistent with the established generic amyloid hypothesis and support a new paradigm for the study of the etiology of single amino acid initiated metabolic disorders in amyloid related diseases. KEYWORDS: Single amino acid, amyloid, cysteine, methionine, self-assembly, cytotoxic

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INTRODUCTION Abnormal fibrous insoluble extracellular protein deposits found in organs and tissues are referred as amyloids.1,2 They are structurally dominated by β-sheet structure,3 although recent research also implicates formation of amyloid-like aggregates by nonproteinaceous metabolites4−6 as well as helical structures.7,8 The study of amyloids is important because they play crucial roles in pathologies seen in a range of diseases including Alzheimer’s, the spongiform encephalopathies, Parkinson’s disease, and type II diabetes, all of which are progressive disorders with associated high morbidity and mortality.9,10 Although the frequently observed peptide which aggregates in these diseases is in the Aβ11 and prion protein families,12,13 there are many reports wherein the protein and peptides that are not related to this sequence, and even single amino acids have been found to adopt an amyloid-like morphology.14−23 There is also an increasing interest in © XXXX American Chemical Society

investigating amyloid structures using short peptides as models and using this reductionist approach to identify amyloid inhibitors.14,15 In this regard, Gazit and co-workers were the first to report the utility of dipeptide (FF)16 and, very recently, fibrils composed of the single amino acid phenylalanine as a reductionist model for Aβ42.17 The pioneering work of Gazit and co-workers also includes the very first report of the selfassembly properties of the single amino acid phenylalanine by various biophysical and cell-based assays.18 The assemblies of phenylalanine exhibited amyloid characteristics and were observed to be toxic and the “generic amyloid hypothesis” relating the etiology of phenylketonuria to amyloid associated diseases was proposed.18 As an extension of this hypothesis, Received: June 25, 2018 Accepted: October 31, 2018 Published: November 1, 2018 A

DOI: 10.1021/acschemneuro.8b00310 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience they have also reported the formation of apoptosis-inducing amyloid fibrils by the self-assembly of tryptophan19 and formation of antibodies against toxic tyrosine assemblies.20 Recently, the group further revealed the self-assembly of nonproteinaceous metabolites to amyloid-like toxic aggregates and further extended their generic amyloid hypothesis to the etiology of metabolic disorders.4 Following the work of Gazit and co-workers, several other research groups have observed similar results for single amino acid assemblies and these findings implicate the involvement of self-assembly of single amino acids in the etiology of metabolic disorders such as phenylketonuria and other amyloid associated diseases.21−23 Due to the significance of these studies and our previous research interest in biomolecular/amyloid self-assembly,24−26 we were motivated to examine the self-assembling propensities of individual amino acids in solution to gain further insights into their structure formation. In the present study, we report for the very first time the self-assembled structures formed by the nonaromatic single amino acid Cys and Met. The role of aromatic interactions in the amyloid formation is wellestablished.27−30 However, there are very few reports, wherein aggregation of nonaromatic peptides into amyloid-like assemblies has been reported.31,32 To the best of our knowledge, the present study reports for the very first time that even non aromatic single amino acid may assemble to amyloid-like structures. Herein, we report self-assembling behavior of nonaromatic amino acids Cys and Met to amyloid-like aggregates. The Cys and Met assemblies were characterized in detail by various microscopy and spectral analyses. The nature of the interactions which lead to the selfassembly of Cys and Met were characterized by various physicochemical methods including Fourier transform infrared (FTIR) spectroscopy, NMR, X-ray diffraction (XRD), and thermogravimetric analysis (TGA) along with assessment of the influence of co-incubation of the assemblies in aprotic solvents, exposure to urea and β-mercaptoethanol, oxygen purging and pH variation. The prevalent solution structure and its amyloid nature were assessed by co-incubating Cys and Met assemblies with amyloid binding dyes Thioflavin T (ThT) and Congo Red (CR). Cytotoxicity assays on human neuroblastoma, IMR-32 and COS-7 kidney cells suggested that both Cys and Met structures are cytotoxic. The cytotoxicity of Cys assemblies occurred at a concentration similar to that observed for the toxic phenylalanine and tryptophan fibrils on IMR32 cells.18,19 The studies presented herein, therefore, might be of crucial interest in assessing the etiology/pathology of metabolic disorders associated with amyloids. Moreover, this study provides a very simple and facile methodology for the design of novel nanostructures through a bottom-up approach.

Figure 1. Self-assembled structures formed by Cys and Met. (a) Chemical structure of L-cysteine. (b) SEM of a 3 mM solution of Cys. (c) Optical microscopy image of a 3 mM solution of Cys. (d) Chemical structure of L-methionine. (e) SEM micrograph of a 3 mM solution of Met. (f) Optical microscopy image of a 3 mM solution of Met.

(SEM) image of the structures formed in the 3 mM solution of Cys and Met. The self-assemblies formed by both Cys and Met showed very long fiber-like aggregates. The self-assembling behavior of Cys was particularly interesting since it showed neuronal fibril like morphologies (fractal assemblies) with long fibers originating from single focus (Figure 1b). It was found that these Cys assemblies could be observed in the concentration range of 3−10 mM. However, as the concentration was increased the fibers appeared more thick. Below 3 mM, self-assemblies were not observed while increasing the concentration above 10 mM resulted in very thick assemblies which appeared crystalline (Figure S3, SI). SEM studies of Cys aggregates at the 3 mM concentration showed that the diameter of the fiber varied from 200 nm to a few micrometers and the length was on the order of several micrometers. Figure 1e displays an analogous SEM image for the solution prepared with Met. The results show that Met also assembles into fiber-like structures, However, compared to Cys, the fibers appear more distinct and less aggregated (i.e., no fractal-like structures). The fibers also show branching and ribbonlike structures, implying that Met might assemble via multiple pathways (which will be the subject of future studies). The length ranged from several micrometers with diameters varying from 200 nm to a few micrometers. Figure 1c and f shows representative optical microscopy images of Cys and Met and reveals structure formations complementary to that observed by SEM which were large enough to be readily observed by optical microscopy. Met assemblies were observed in the concentration range of 1−10 mM. But below 1 mM, selfassemblies were not observed. Above 10 mM, they appeared thicker (Figure S3, SI). Studies of the Cys and Met assemblies as a function of pH showed neutral conditions were optimal for their formation and that the structures are disrupted in slightly acidic or alkaline environments (Figures S4 and S5, SI). Cys and Met assemblies were also analyzed by phase contrast microscopy. Phase contract microscopy can be very useful in providing preliminary information about surface characteristics and may indicate whether the material has crystalline properties. The images of Cys and Met at 40× magnification indicated that the assemblies are amorphous in

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RESULTS AND DISCUSSION The self-assembling properties of all 21 amino acids were assessed first by optical microscopy. Of the 21 L-amino acids, only five, namely, phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), cysteine (Cys), and methionine (Met) showed fiber-like assemblies in optical microscopy (Figures S1 and S2 in the Supporting Information (SI)). The selfassembling properties of Phe, Tyr, and Trp into fibrillar structures was previously reported.18−20 However, the aggregation properties of the nonaromatic amino acids Cys and Met were unknown. Hence, we were motivated to characterize the structures formed by Cys and Met in more detail. Figure 1b displays a scanning electron microscopy B


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Figure 2. (a) TEM image of Cys fibers at low magnification (500 nm). Inset: SAED image of Cys. (b) TEM image of individual Met fiber at higher magnification (200 nm). Inset: SAED image. (c) XRD diffractogram of Cys and Met reveals broadening and disappearance of the diffraction peak after self-assembly indicating a change in molecular packing and a transition from crystalline to an amorphous state.

Figure 3. (a) Solid state 1H NMR of Cys non-assembled. (b) Solid state 1H NMR of Cys self-assembled. (c) ATR-FTIR of non-assembled and selfassembled Cys. (d) TGA of non-assembled and self-assembled Cys.

Selected area electron diffraction (SAED) analysis revealed a lack of any crystalline properties and confirmed the amorphous nature of the assemblies (Figure 2a inset). Figure 2b shows a representative TEM image of Met fibers. The diameter of individual Met fibers was ∼10 nm, and since there was no contrast between the periphery and the center, it can be surmised that structures lack tubular properties and that the assemblies are in fact febrile and compact. The SAED analysis

nature and lack any crystalline properties (Figure S6, SI). Furthermore, characterization by dynamic light scattering (DLS) showed that the aggregates are also present in the solution state (Figure S7, SI). To analyze the morphology of the structures formed by the Cys and Met assemblies in more detail, transmission electron microscopy (TEM) studies were performed. Figure 2a shows representative TEM images of a 3 mM solution of Cys containing the fiber like assemblies. C


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Figure 4. (a) Solid state 1H NMR of Met non-assembled. (b) Solid state 1H NMR of Met self-assembled. (c) ATR-FTIR of non-assembled and self-assembled Met. (d) TGA of non-assembled and self-assembled Met.

and merging of adjacent peaks, which suggests involvement of the thiol group in self-assembly.37,38 However, extensive solid state NMR spectroscopic studies are required to precisely determine the actual mechanism of self-assembly. Additionally, to validate the preliminary data obtained from solid state NMR, ATR FTIR studies were performed on self-assembled and non-assembled Cys. In the non-assembled Cys peaks at 3173.6 and 2968.0 cm−1, were assigned to NH stretching and CH stretching vibrations, respectively. After self-assembly, the NH stretching peak became broader with a 20 cm−1 shift. The peak at 2109 cm−1 was assigned to the NH asymmetric stretching vibration arising from its ionic form. As the concentration was increased from 3 mM to 10 mM the NH stretching peaks became broader and merged with the adjacent peaks (Figure 3c) further implicating its engagement in intermolecular hydrogen bonding. Peaks at 2552.8 and 635.6 cm−1 were assigned to −SH and C−S stretching, respectively.38,39 After self-assembly, both peaks were shifted to lower wavenumbers due to intermolecular interactions induced by self-assembly. Similarly, C−O stretching vibrations also slightly shifted to lower wave numbers. Carbonyl stretching and NH deformation peaks at 1579.3 and 1538.3 cm−1, respectively, exhibited distinct change after self-assembly. A peak broadening effect observed in the carbonyl stretching peak at 1579.3 cm−1 and escalation in intensity of NH deformation peak at 1538.3 cm−1 as the concentration of Cys increased, indicate the presence of intermolecular hydrogen bonding (Figure S8, SI). Peaks near 3400 cm−1 assigned to the hydroxyl group also exhibited broadening. These findings show that the thiol, carboxylic acid, and amine groups are involved in the selfassembly process. Interestingly, no characteristic peaks of S−S vibrations were recognized in the self-assembled Cys spectrum which suggests that Cys alone and not cystine (oxidized

also revealed the amorphous nature of the assemblies and confirmed that the Met aggregates were not crystalline artifacts. Additionally, XRD data clearly revealed that the nature of Cys and Met changed from crystalline to amorphous after the assembly process.33,34 Cys and Met each showed characteristic diffraction peaks before assembly which were reduced to a broad “halo” after assembly. The study further confirmed that Cys and Met structures assessed by microscopy analysis are indeed formed by the process of self-assembly and not by crystallization in agreement with the results of the SAED studies (Figure 2c). Another interesting observation was that in the SEM and TEM studies, both Cys and Met assemblies tended to melt down as voltage or exposure time of the e-beam was increased. This observation further supports the conclusion that both Cys and Met structures are indeed soft in nature and are formed exclusively via a self-assembly process. The melting might occur due to the release of trace water present in assemblies.43 To understand the role of intermolecular interactions in the self-assembly, we resorted to a series of comparative analyses of non-assembled and self-assembled Cys and Met using solid state ATR-FTIR, NMR, and TGA. Solid state 1H NMR studies were conducted to reveal intermolecular interactions after selfassembly.35 Solid state 1H NMR spectra of non-assembled Cys exhibited signals at 9.39, 5.77, 3.71, 2.62, and 1.91 ppm assigned to carboxylic acid, amine, CH, CH2, and SH protons, respectively (Figure 3a).36 Self-assembled Cys showed broad signals at 14.62, 6.05, and 2.51 ppm which were assigned to carboxylic acid, amine and the merging of the peaks from CH, CH2, and SH protons (Figure 3b). The downfield shifts and peak broadening effects indicates self-assembly and intermolecular interactions such as hydrogen bonding between carboxylic acid and amine protons.37 The thiol signal at 1.91 ppm is barely recognizable due to the increased broadening D


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loss in the self-assembled state. This may be attributed to increased intermolecular interactions which delay the weight loss process and the soft nature of fibers which may result in lowering of sublimation points. Met exhibited little difference in thermal behavior after self-assembly in comparison to Cys, which may be due to the absence of strong S−H hydrogen bonding interactions in case of Met. TGA characterization thus gave further insights about differences in thermal behavior, which are a consequence of the increased number of molecular interactions and change in molecular packing induced by selfassembly.40,41 In our previous studies, we have reported structural transitions in self-assemblies owing to disruption of hydrogen bonding on co-incubation with urea.25 To probe the role of hydrogen bonding further, Cys and Met assemblies were coincubated with urea. A noticeable change in the structure of the Cys assemblies can be visualized (Figure 5a). Met fibers

dimer) is undergoing self-assembly via hydrogen bonding and dipolar interactions. Additionally, to assess the thermodynamics and thermal stability of assemblies, TGA of Cys before and after selfassembly was also pursued. Cys before assembly shows one step weight loss of 76.84% from 210.5 to 249.2 °C which could be attributed to the degradation or sublimation of the compound (Figure 3d).41A sharp weight loss pattern without any steps in TGA of non-assembled Cys is mostly indicative of sublimation. At the same time, self-assembled Cys shows a two step degradation pattern which is clearly noticeable in the DTG graph (Figure S9, SI). After self-assembly the intermolecular interactions were so strong that the sublimation or degradation process was delayed. Weight loss of 70.43% was observed from 187 to 264 °C. Weight loss of 2.1% in selfassembled Cys could be loss of crystalline water trapped inside porous structures formed by self-assembly and to a change in molecular packing as indicated by the XRD results (Figure 2c). The solid state 1H NMR spectrum of non-assembled Met shows signals at 14.38, 6.01, 3.86, 3.34, and 2.26 ppm assigned to carboxylic acid, amine, CH, S−CH2, and C−CH2 protons respectively (Figure 4a).36 After self-assembly, only three distinguishable signals at 18.48, 10.46, and 3.34 ppm were observed and these signals may be assigned to protons associated with carboxylic acid, amine and CH groups (Figure 4b). Downfield shifts of the amine and carboxylic acid protons and extensive peak broadening were also observed. This indicates that Met self-assembly is the result of intermolecular interactions between amine and acid groups. Furthermore, ATR-FTIR analysis of Met also revealed results consistent with those observed by NMR. In the ATR-FTIR spectrum, peak broadening and chemical shift changes were observed after self-assembly, although the magnitudes of the shifts were less when compared to those for Cys. Peaks for the NH stretching vibrations at 3173.6 and 2109.1 cm−1 become broader and shifted with increasing concentrations of Met (Figure S10, SI) indicating intermolecular hydrogen bonding. The peaks at 2552.8 cm−1 arising from the −SH stretch shifted to 2544 cm−1 while peaks at 1139.3 and 635.6 cm−1 assigned to C−O stretching and C−S stretching, respectively, also showed peaks shifts to lower wavenumbers. Most importantly, these peaks also become broader as the concentration of Cys and Met increased which is a fundamental characteristic of intermolecular hydrogen bonding (Figures 4 and S10, SI). The minimal change for the peak positions of OH, NH, SH, and CS related vibrations indicated that no new covalent bonds were formed and that Cys and Met assemblies are formed exclusively via noncovalent intermolecular interactions like hydrogen bonding between amine thiol and carbonyl functional groups.40 Changes in the profile of the peaks were reduced for Met as compared to Cys which may be attributed to the absence of a free SH group which can facilitate induction of self-assembly. This hypothesis is consistent with the results of microscopy, which show that the fibers of Cys are much denser than those for Met (Figure 1). TGA analysis of Met was also performed before and after assembly, and the respective thermograms are shown in Figures 4d and S11. Both Cys and Met are prone to sublimation at higher temperatures. Non-assembled Met exhibited steep weight loss of 90.3% from 270 to 305 °C, whereas self-assembled Met shows weight loss of only 88.38% from 25 to 289 °C. Moreover, both Cys and Met exhibited slow degradation rate but early onset temperature for weight

Figure 5. (a) SEM of Cys with urea. (b) SEM of Cys with βmercaptoethanol showing globular structure (lower magnification). Inset: globular structures are formed by broken fibers of Cys (higher magnification). (c,d) Optical microscopy images of 3 mM Cys where: (c) before oxygen purging long, fibers could be seen along with fractal structures and (d) after oxygen purging for 24 h, only fractal-like assemblies were observed.

were also disrupted completely on co-incubation with urea suggesting a crucial role for hydrogen bonding in both Cys and Met aggregation (Figures S12 and S13, SI). Further, to assess the role of thiol interactions in Cys assembly, co-incubation studies with β-mercaptoethanol were performed. Incubation with β-mercaptoethanol lead to disruption of Cys assemblies and globular structures were observed (Figures 5b, S14, and S15, SI). Careful analysis of the globular structures revealed that they are made up of small broken fibrils (Figure 5b). This study thus suggests a crucial role for disulfide bridges along with thiol interactions in the structure formation of Cys. Coincubation of β-mercaptoethanol with Met on the other hand did not reveal any structural changes likely because of the lack of free thiol (Figure S16, SI). Gazit and co-workers have already reported the self-assembling propensity of cystine (an oxidized dimer of Cys where −SH exist as S−S) to amyloidlike fibrillar aggregates.4 They have further implicated selfE


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Figure 6. Snapshots from Cys, Met, and Phe MD simulations showing small molecular associations in the dotted circle.

assembly of cystine to amyloid-like fibrils to their generic amyloid hypothesis and explained its crucial significance in the etiology of cystinuria.4 Our studies are thus consistent with the theory of Gazit and co-workers and suggest that Cys assembly might also be of crucial significance in cystinuria since Cys will have a tendency to oxidize to cystine in nonreductive organelles such as ER, vesicles, and Golgi.42 However, cysteine (the unoxidized monomer) is the predominant form present in the cytoplasm and nucleus.42 Thus, Cysteine may also assemble into toxic fibers inside cells and these toxic fibers may cause the pathology associated with cystinuria. Oxygen purging experiments were done on the structures by Cys assembly. Figure 5c and d reveals optical microscopy images of Cys before and after oxygen purging. It can be seen that, before oxygen purging, Cys also showed long fiber-like morphologies along with fractal structures. However, after oxygen purging, the aggregates appear with the fractal-like morphology exclusively. This was a very interesting result, as it clearly revealed the essential role of disulfide bridges in causing fractal like assemblies. Since Met does not possess any free thiol the assemblies formed by Met are only fibril like and not fractal. As a control experiment oxygen was also purged in Met, but it did not induce any morphological transition (Figure S17, SI). Additionally, to understand the role of water in the formation of these self-assemblies, Cys and Met were incubated in an aprotic solvent mixture composed of 5% DMSO: DCM and THF (Figure S18, SI).43 Since these solvents are aprotic, they will not act as hydrogen bond donors, unlike water. Interestingly, under these conditions, no assembly was observed. However, as trace water was added to THF, the Met self-assembly ensued, while the formation of globular structures made from small fibrils could be seen in the case of Cys (Figure S18, SI). Hence these studies provided crucial evidence regarding role of water molecules in the structure formations of Cys and Met. In addition, to get atomistic insights into Cys and Met assembly initiation, molecular dynamics (MD) simulations were performed.44 The results for Phe were used as a comparison because Phe is already known to form amyloidlike fibrils both in experiments and in MD simulations.18 MD simulations are performed only for 100 ns due to time constraints. The simulations are still in progress, and the results will be the subject of a future communication. In the present study, we monitored the radius of gyration which initially was

increasing and then stabilized after about 10 ns (Figure S19, SI). Therefore, we considered the 10−100 ns equilibration data for analysis purpose. In amyloid fibril formation, intermolecular interactions play a very crucial role. The preliminary data suggested that there is dynamic association of amino acids that is driven by intermolecular hydrogen bonds and van der Waals interactions as assessed by the number of residue contacts determined from carbon−carbon contacts (Figure S20, SI). The snapshot at 69.5 ns showed association of five Cys molecules in the Cys MD simulation while two molecular associations having three and two molecules of Met at about 51.8 ns were observed in the Met MD simulations, In the Phe MD simulation association of three molecules was observed around 55.8 ns. The number of intermolecular hydrogen bonding interactions in Cys varied from 1 to 11 whereas in Met and Phe it varied from 1 to 8 (Figures 6 and S20, SI). The number of intermolecular hydrogen bonds in the Cys MD simulation was greater than that observed in the Met and Phe simulations, indicating its greater propensity to aggregate. The hydrogen bonds were observed between the carbonyl oxygen and amide proton. The number of intermolecular residue contacts was less in Cys as compared to that observed in Met and Phe simulations. This suggests that van der Waals interactions are favored more in Met and Phe due to the long hydrophobic side chain in Met and the aromatic nature of Phe (Figure S20E). The events observed in the simulations therefore capture the preliminary phenomenon observed during initiation of amyloid fibril formation. Self-assembly is a spontaneous process of organization of molecules into well-ordered structures mainly driven by electrostatic interactions, hydrophobic attraction, hydrogen bonding, and π−π stacking interactions.45−52 The results obtained via FTIR, solid state NMR, TGA, and preliminary MD simulations of Cys and Met along with control experiments done using varying solvents and urea suggest the crucial role of intermolecular hydrogen bonding in the selfassembly of both Cys and Met. A nonpolar amino acid, like Met which lacks an aromatic side chain, may assemble into fiber-like aggregates by hydrophobic interactions and possibly via backbone hydrogen bonding between the amine nitrogen and the carboxylic acid hydrogen present in an adjoining amino acid molecule.49 Polar amino acids like Cys may assemble mainly due to hydrogen bonding and thiol interactions. In addition, these molecules will have a tendency F


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Figure 7. (a) Cys fibers bind with ThT and show green fluorescence. (b) Met fibers reveal green fluorescence on binding to ThT. (c) Fluorescence spectroscopy of Cys and Met in the presence of ThT reveal enhanced fluorescence characteristic of amyloid fibrils. (d) CR binding assay with different single amino acids based assemblies (3 mM) and FF (3 mM) showed an increase in intensity, further suggesting amyloid characteristics for both Cys and Met.

Figure 8. MTT assay of Cys and Met on human neuroblastoma IMR-32 and COS7 cells. (a) Cys fiber toxicity in IMR 32 cells. (b) Met fiber toxicity in IMR 32 cells. (c) Cys fiber toxicity in COS7 cells. (a) Met fiber toxicity in COS7 cells.

accepted that regardless of the manner of binding, CR displays an increase in absorbance intensity and a red-shift on binding with amyloid.54,55 Spectra obtained for Cys and Met with CR (Figure 7d) revealed a slight red shift and an increase in absorbance. As a control, we co-incubated other single amino acids namely, Phe, Tyr and Trp and also the dipeptide FF which has characteristic amyloid properties. All the reported single amino acids and FF exhibited an increase in absorbance and a similar red shift with CR. These results suggest that CR binds to structures formed by Cys and Met and hence the assemblies may have amyloid-like characteristics. It is very well accepted that amyloid assemblies are neurotoxic in nature and accumulation of Aβ peptide induces cell death.56,57 The MTT assay is a reliable in vitro assay for assessment of the effects of neurotoxic compounds and has been used in cultures of different neuroblastoma cells, for the quantitative measurement of Aβ toxicity.56,57 The MTT assay analysis revealed that the fibrillar structures of Cys and Met

to form hydrogen bonds with polar solvents like water. The role of water molecules in the formation of such self-assembled nanotubes/nanofibers has already been reported.43,50 With the fibers of Cys and Met thoroughly characterized, we next investigated the predominant solution structures of Cys and Met assemblies. Hence, ThT and CR binding assays were performed. ThT exhibits remarkable enhancement in its fluorescence on binding with amyloid.53 The fluorescence spectra of ThT with Cys and Met indeed revealed fluorescence enhancement and confirmed amyloid-like structure formation by these single amino acids (Figure 7c). Since ThT binds to hydrophobic pockets of β sheets present in amyloid fibers, it further confirmed Cys and Met amyloid fibers in solution. Further the structures were also stained with ThT as assessed by fluorescence microscopy (Figure 7a, b).The amyloid nature of the assemblies was further confirmed using CR binding assay in solution. Although the manner of binding of CR with amyloidogenic peptides is still under debate, it is universally G


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was used for preparing all solutions (Millipore). No additional organic solvent was added. Congo Red, Thioflavin T, and β-mercaptoethanol were purchased from Sigma. Sodium hydroxide pellets and urea were purchased from SRL, while hydrochloric acid was from Merck. All glassware was cleaned thoroughly, rinsed with solvents and Aqua regia (1:3 concentrated nitric acid and hydrochloric acid), and oven-dried prior to use Sample Preparation and Analysis. Optical Microscopy. All 21 L-amino acids were dissolved at concentrations of 3 mM in DI water and were visualized using a Leica DM2500 upright fluorescence microscope in bright-field mode. Samples were made by drop-casting 20 μL on a clean glass slide. The sample was allowed to dry at room temperature and visualized under 40× and 100× lens. Scanning Electron Microscopy (SEM). SEM images were taken using a Nova Nano FEG-SEM 450 microscope (with accelerating voltage ranging from 5−15 kV). SEM samples were prepared on silicon wafers. Cys and Met (10 μL of a 3 mM solution) were dispensed and dried at room temperature. The samples were analyzed without gold coating under low vacuum. Transmission Electron Microscopy (TEM). Samples were viewed using a transmission electron microscope (model: Tecnai 20 and make: Philips, Holland) operating at an accelerating voltage of 200 Kv. A 10 μL aliquot of the amino acid solution was placed on a 400 mesh copper grid coated with a thin layer of carbon. After 1 min, excess fluid was removed and the grid was stained with 2% uranyl acetate in water. Excess staining solution was removed from the grid after 2 min. X-ray Diffraction (XRD) Analysis. X-ray diffraction experiments were performed on Pan-analytical X-pert Pro instrument. Samples were evenly dispersed over the substrate holder and scanned in the range of 2θ 10−80°. Samples for XRD were prepared by lyophilizing a 3 mM solution of Cys and Met prepared 12 h before lyophilization. Attenuated Total Reflection Fourier Transform Infrared (ATRFTIR) Spectroscopy. ATR-FTIR spectra were recorded on a Bruker Spectrum-II spectrophotometer mounted with a diamond ATR crystal. Spectra were recorded between 4000 and 400 cm−1 wave numbers. Solid State 1H NMR Spectroscopy. Solid state 1H NMR was performed on a Bruker 500 MHz solid state NMR spectrophotometer. About 100 mg of solid sample was placed in the sample holder, and spectra were recorded at 5 kHz energy. Thermogravimetric Analysis (TGA). TGA was recorded on a PerkinElmer Pyris-1 TGA. Solid samples were placed in an alumina crucible, and thermograms were recorded from 30 to 900 °C at a 10 °C heating rate under mild nitrogen flow. Congo Red (CR) and Thioflavin T (ThT) Binding Assay. For the CR absorbance measurements, 4 mL of 3 mM amino acid solution was incubated with 100 μL of a 10 μM solution of CR at room temperature. UV−vis spectra of the resulting solutions were recorded after 15 h using a PerkinElmer LAMBDA 35 spectrophotometer. ThT binding assays were done by previously reported methodology. 4 ThT fluorescence assay and ThT staining were recorded in Fluoromax−2, spectrofluorimeter and Leica DM2500 microscope, respectively. Co-Incubation Assay with Urea and β-Mercaptoethanol. Coincubation studies of Cys and Met with urea and β-mercaptoethanol were done using an equal ratio of Cys and Met with respect to urea and β-mercaptoethanol. The final concentration of amino acid was 3 mM, and both urea and β-mercaptoethanol were also 3 mM. The coincubation was done by mixing for 30 min followed by incubation of 1 h. Oxygen Purging Experiment. Cys (3 mM solution) was prepared by dissolving the desired amount of Cys solid in deionized water followed by sonication for 5 min. Molecular oxygen was purged using a balloon for 1 h and the sample was drop-casted on a glass slide for visualization by microscopy. MD Simulations. Three separate simulations were performed on Met, Cys, and Phe. The starting structure of each simulation was built from 20 molecules of each type of amino acid. The amino acids in the starting structure were placed in two layers separated by 10 Å, and the adjacent molecules were placed 5 Å apart following the approach of

were cytotoxic (Figures 8 and S21). The cytotoxicity analysis was done at six different concentrations of amino acids: 1, 2, 4, 6, 8, and 10 mM to assess the dose dependent cytotoxicity following previously reported methodology.19 Alanine was used as a negative control since it does not aggregate and is not cytotoxic effect even at very high concentrations (4 mg/mL, 20 mM),18,19 Phenylalanine was used as a positive control since its toxicity has been established.18 Figure 8a reveals the results of cytotoxicty analysis of Cys and shows that cell viability was decreased by a remarkable 71.3% at 10 mM. Similarly, Figure 8b shows that cell viability was reduced at higher concentrations of Met in a dose dependent manner. Higher concentrations of amino acids may be required to induce cytotoxicity since the cells are incubated with the fibers for a short time as compared to physiological conditions.18 A comparative analysis of toxicity caused by single amino acid based assemblies was also done and it revealed that the cytotoxicity induced by the Cys fibers at the 10 mM concentration is comparable to what has been observed for Phe and Trp fibers (Figure S21, SI). Met also reduced cell viability by 26.5% at 8 mM and 36.8% at 10 mM concentrations, respectively. Symptoms of both cystinuria and hypermethioninemia are accompanied by renal dysfunction and formation of kidney stones.58,59 Hence, the toxicity of both Cys and Met were also analyzed on COS-7 monkey kidney cells. The toxicity assays revealed that both Cys and Met structures were cytotoxic and the cell viability in the presence of Met and Cys fibers were only 60% and 46% respectively (Figure 8c, d). A control experiment was also performed using the normal mouse macrophage cell line, Raw 264.7, Surprisingly it did not show any toxicity (data not shown), indicating that the toxicity of these amino acid fibrils may be tissue/cell specific. The finding suggests that the etiology of single amino acid metabolic disorders like cystinuria and hypermethioninemia may be related to amyloid associated diseases and needs to be studied further. Our future endeavors will focus on further studies using other cell based and histochemical analyses to confirm the role of Cys and Met aggregation in the etiology of these diseases.

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CONCLUSION In summary, we report for the very first time the spontaneous structure formation by self-assembly of the single amino acids Cys and Met. The structures were characterized in detail by various microscopy and spectroscopic analyses. Amyloid binding assays and cell viability analyses revealed that Cys and Met aggregates may have an amyloid nature and are cytotoxic to human neuroblastoma cells. These results are thus highly significant. To the best of our knowledge, only amino acids with aromatic side chains (i.e., Phe, Tyr, and Trp) are reported to be capable of forming spontaneous self-assemblies. Future endeavors include further characterization and more critical analysis of the mechanism of self-assembly via computational and NMR studies, and assessing its implications in amino acid metabolic disorders like cystinuria and hypermethioniemia.

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METHODS

General. All 21 L-amino acids were purchased as part of a reference grade kit of L-amino acids offered by Sisco Research Laboratories (SRL, India) and used without further purification. The purity of all the amino acids was at a minimum 99%. Deionized water H



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Gazit et al.18 All MD simulations were performed using the software GROMACS (version 5.0.4) with explicit solvent. Full details can be accessed in the Supporting Information.

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REFERENCES

(1) Gazit, E. (2002) The “correctly folded” state of proteins: is it a metastable state? Angew. Chem., Int. Ed. 41 (2), 257−259. (2) Knowles, T. P., Vendruscolo, M., and Dobson, C. M. (2014) The amyloid state and its association with protein misfolding diseases. Nat. Rev. Mol. Cell Biol. 15 (6), 384−396. (3) Rambaran, R. N., and Serpell, L. C. (2008) Amyloid fibrils: abnormal protein assembly. Prion 2 (3), 112−117. (4) Shaham-Niv, S., Adler-Abramovich, L., Schnaider, L., and Gazit, E. (2015) Extension of the generic amyloid hypothesis to nonproteinaceous metabolite assemblies. Sci. Adv. 1 (7), e1500137. (5) Sade, D., Shaham-Niv, S., Arnon, Z. A., Tavassoly, O., and Gazit, E. (2018) Seeding of proteins into amyloid structures by metabolite assemblies may clarify certain unexplained epidemiological associations. Open Biol. 8 (1), 170229. (6) Gazit, E. (2016) Metabolite amyloids: a new paradigm for inborn error of metabolism disorders. J. Inherited Metab. Dis. 39 (4), 483−488. (7) Tayeb-Fligelman, E., Tabachnikov, O., Moshe, A., GoldshmidtTran, O., Sawaya, M. R., Coquelle, N., Colletier, J.-P., and Landau, M. (2017) The cytotoxic Staphylococcus aureus PSMα3 reveals a cross-α amyloid-like fibril. Science 355 (6327), 831−833. (8) Stroobants, K., Kumita, J. R., Harris, N. J., Chirgadze, D. Y., Dobson, C. M., Booth, P. J., and Vendruscolo, M. (2017) Amyloidlike fibrils from an α-helical transmembrane protein. Biochemistry 56 (25), 3225−3233. (9) Ross, C. A., and Poirier, M. A. (2004) Protein aggregation and neurodegenerative disease. Nat. Med. 10 (7), S10. (10) Maltsev, A. V., Bystryak, S., and Galzitskaya, O. V. (2011) The role of β-amyloid peptide in neurodegenerative diseases. Ageing Res. Rev. 10 (4), 440−452. (11) Huang, H. C., and Jiang, Z. F. (2011) Amyloid-β protein precursor family members: A review from homology to biological function. J. Alzheimer's Dis. 26 (4), 607−626. (12) Watts, J. C., and Westaway, D. (2007) The prion protein family: diversity, rivalry, and dysfunction. Biochim. Biophys. Acta, Mol. Basis Dis. 1772 (6), 654−672. (13) Younan, N. D., Chen, K. F., Rose, R. S., Crowther, D. C., and Viles, J. H. (2018) Prion protein stabilizes amyloid-β (Aβ) oligomers and enhances Aβ neurotoxicity in a Drosophila model of Alzheimer disease. J. Biol. Chem., 13090−13099. (14) Gazit, E. (2018) Reductionist Approach in Peptide-Based Nanotechnology. Annu. Rev. Biochem. 87, 533−553. (15) Makam, P., and Gazit, E. (2018) Minimalistic peptide supramolecular co-assembly: expanding the conformational space for nanotechnology. Chem. Soc. Rev. 47 (10), 3406−3420. (16) Brahmachari, S., Arnon, Z. A., Frydman-Marom, A., Gazit, E., and Adler-Abramovich, L. (2017) Diphenylalanine as a Reductionist Model for the Mechanistic Characterization of β-Amyloid Modulators. ACS Nano 11 (6), 5960−5969. (17) Shaham-Niv, S., Rehak, P., Zaguri, D., Levin, A., AdlerAbramovich, L., Vuković, L., Kral, P., and Gazit, E. (2018) Differential inhibition of metabolite amyloid formation by generic fibrillationmodifying polyphenols. Communications Chemistry 1 (1), 25. (18) Adler-Abramovich, L., Vaks, L., Carny, O., Trudler, D., Magno, A., Caflisch, A., Frenkel, D., and Gazit, E. (2012) Phenylalanine assembly into toxic fibrils suggests amyloid etiology in phenylketonuria. Nat. Chem. Biol. 8 (8), 701. (19) Shaham-Niv, S., Rehak, P., Vuković, L., Adler-Abramovich, L., Král, P., and Gazit, E. (2017) Formation of apoptosis-inducing amyloid fibrils by tryptophan. Isr. J. Chem. 57 (7−8), 729−737. (20) Zaguri, D., Kreiser, T., Shaham-Niv, S., and Gazit, E. (2018) Antibodies towards Tyrosine Amyloid-Like Fibrils Allow Toxicity Modulation and Cellular Imaging of the Assemblies. Molecules 23 (6), 1273. (21) Singh, V., Rai, R. K., Arora, A., Sinha, N., and Thakur, A. K. (2015) Therapeutic implication of L-phenylalanine aggregation mechanism and its modulation by D-phenylalanine in phenylketonuria. Sci. Rep. 4, 3875.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.8b00310.

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Research Article

Full details of the MD simulations as well as the materials and methods used; optical microscopy images and the results of FTIR, DTG, and solvent dependence studies, cytotoxicity assay. The additional figures (Figures S1-S21) are presented for supporting the results discussed (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. Fax: +91 79 30514110. Tel: +91 79 30514142. ORCID

Nidhi Gour: 0000-0003-2479-2113 Author Contributions ¶

B.K. and C.K.P. contributed equally. N.G. proposed the concept and drafted the article along with C.K.P. N.G., B.K., and V.S.K. worked on data characterization and analysis by OM, SEM, TEM, and biophysical assays like CR and ThT binding. N.G., C.K.P., and M.P. worked on data characterization and analysis by FTIR, XRD, DLS, NMR, TGA, and CR assays. D.S. and R.A.-R. exclusively performed, interpreted, and drafted the cytotoxicity data. S.P. performed MD simulations.

Funding

The work was supported by the DST SERB extramural research fund (Project No. EMR/2016/003186) received by N.G. and M.K.P. B.K. and C.K.P. also greatly acknowledge the support from DST and the SERB research grant (EMR/2016/ 003186) for funding and fellowships. D.S. thanks GSBTM for the fellowship. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We greatly acknowledge Prof. Ehud Gazit and Dr. Shira Shaham-Niv of Tel Aviv University, Israel for the useful discussion and feedback of our work. We thank the SICART SEM, TEM and TGA facility and Dr. Sanjay Patel and Dr. Vikas Patel for their assistance with Imaging. We also thank Dr. Virupakshi Soppina (IIT Gandhinagar) for sharing COS-7 cells.

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ABBREVIATIONS Cys, cysteine; Met, methionine; Phe, phenylalanine; Tyr, tyrosine; Trp, tryptophan; FF, di-L-phenylalanine; ThT, Thioflavin T; CR, Congo red; SEM, scanning electron microscopy; TEM, transmission electron microscopy; XRD, X-ray diffraction; TGA, thermogravimetric analysis; DTG, derivative of thermogravimetric; NMR, nuclear magnetic resonance; ATR, attenuated total reflectance; FTIR, Fourier transform infrared spectroscopy I


Research Article


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Amyloid-like Structures Formed by Single Amino Acid Self-Assemblies

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