Tyrosine generated nanostructures initiate amyloid cross-seeding in

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Tyrosine generated nanostructures initiate amyloid crossseeding in proteins leading to lethal aggregation trap Bibin G Anand, Kailash Prasad Prajapati, Dolat Singh Shekhawat, and Karunakar Kar Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00472 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 6, 2018

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Biochemistry

Tyrosine generated nanostructures initiate amyloid cross-seeding in proteins leading to lethal aggregation trap. Bibin G. Anand ¶, Kailash P. Prajapati, Dolat S. Shekhawat‡, Karunakar Kar* Biophysical and Biomaterials Research Laboratory, Room 310, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India, Phone No. 91-1126704517, email: [email protected], [email protected]

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ABSTRACT

Here, we show that aromatic amino acid tyrosine, under physiologically mimicked condition, readily forms amyloid-like entities which can effectively drive aggregation of different globular proteins and aromatic residues. Tyrosine self-assembly resulted in the formation of cross-β rich regular fibrils as well as spheroidal oligomers. Computational data suggest intermolecular interaction between specifically oriented tyrosine molecules mediated through π-π stacking and H-bonding interactions, mimicking a cross-β like architecture. Both individual protein samples and mixed protein samples underwent an aggregation process in the presence of tyrosine-fibrils, confirming the occurrence of amyloid cross-seeding. The surface of the tyrosine’s amyloid like entities was predicted to trap native protein structures, preferably through hydrophobic and electrostatic interactions initiating an aggregation event. Since tyrosine is a precursor to vital neuromodulators, the inherent cross-seeding potential of the tyrosine-fibrils may have direct relevance to amyloid-linked pathologies.

KEYWORDS. Tyrosine nanostructure, Metabolite self-assembly, Protein aggregation, Amyloid cross-seeding, coaggregation


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Biochemistry

INTRODCTION

Recent revelations on the self-assembly of biologically relevant single metabolites have attracted much interest not only to understand the mechanism of such self-assembly process but also to explore the significance of such events in biological systems. Aromatic residues and metabolites have been observed to undergo spontaneous self-assembly, yielding amyloid like cytotoxic structures (1-4). For example, phenylalanine is known to form amyloid like cytotoxic and hemolytic fibrils (1, 3). Further, it has been reported that phenylalanine-fibrils can induce amyloid cross-seeding in many non-amyloidogenic globular proteins, yielding cytotoxic aggregates of amyloid nature (1). Occurrence of amyloid cross-seeding has been observed among globular proteins and many amyloid linked proteins (5, 6). Recently, our group has shown that Aβ fibrils can effectively trigger aggregation of proteins via amyloid cross-seeding (7).

The same study also showed aggregation of aromatic molecules (including tyrosine)

mediated by Aβ fibrils (7). Hence, it is important to know whether tyrosine generated selfassembled structures are capable of amyloid cross-seeding. Because aromatic residues including tyrosine are important precursors to neuromodulators (8) in the body system, the detailed understanding of tyrosine self-assembly and its possible influence on amyloid cross-seeding in other proteins and metabolites becomes important to clarify the mechanism of many complicated neuronal disorders. Though self-assembly of tyrosine into higher order structures has been recently reported (9, 10), the clear mechanism of tyrosine self-assembly, particularly tyrosine self-assembly under physiological buffer conditions is not clear. Few studies have reported the self-assembly of tyrosine and other metabolites under adverse conditions such as increasing the temperature of a highly concentrated sample followed by slow cooling (10-12). Studies have also revealed the occurrence of neuropathological consequences due to altered homeostasis of


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tyrosine concentration in the body system (13-15) (Supplementary Table S1). It is also not clear whether such tyrosine-related complications are the resultant of amyloid mediated consequences. Understanding the amyloid cross-seeding ability of both protein amyloids and metabolite amyloids would clarify whether these cross-seeding events have any synergetic consequences in biological systems.

Here we have used a combination of both computational and biophysical tools to gain more insights into the molecular self-assembly of tyrosine under physiological conditions of buffer and temperature. The study has also focused on the role of tyrosine fibrils on the onset of amyloid cross-seeding. Selected

proteins namely lysozyme, BSA, and myoglobin were used as

convenient model proteins to examine whether tyrosine generated amyloid fibrils can actively induce amyloid cross-seeding process in these proteins under physiologically mimicked conditions of buffer and temperature. We have also used computational tools to explore the molecular interaction between tyrosine generated nanostructures with native structures of proteins.

EXPERIMENTAL METHODS

Reagents- All the chemicals, reagents, proteins, and amino acids used in this study were either purchased from Sigma-Aldrich or from HIMEDIA (India). The protein concentrations were measured considering the respective molar extinction coefficient values viz., 43824 M-1cm1

at 280 nm for BSA, 2.6 mg ml-1 at 280 nm for lysozyme, 12.8 mM-1cm-1 at 550 nm for

myoglobin.


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Biochemistry

Aggregation of tyrosine-Aggregation kinetics of tyrosine was monitored by following the established protocol for Thioflavin T fluorescence assay which shows increased fluorescence intensity during amyloid fibril formation. All experiments were carried out in phosphate buffer saline maintained at pH 7.4 at 37°C and the concentration of Thioflavin T was maintained at 30 µM(13). The Thioflavin T fluorescence intensity at different time points was recorded under ambient conditions, using a fluorescence spectrometer. Thioflavin T added samples were excited at 440 nm and the resultant emission was recorded 495 nm. Turbidity of the aggregating tyrosine samples (2 mM, in PBS at 37°C) were measured by recording the absorbance at 450 nm. The concentration of Tyrosine for these aggregation studies was maintained at 2 mM. The choice of this value for tyrosine concentration was based on previous reports(10) on the self-assembly tyrosine in different conditions. Congo red Assay: Protein amyloid fibril samples were tested for amyloid CR binding using spectroscopic CR assay as described in literature (16, 17). An aliquot of aggregated protein sample was diluted in the reaction buffer PBS (with 10% ethanol) which contained 5x10-6 M CR (3 ml final reaction volume). Ethanol was added to the stock solution to prevent CR micelle (micro aggregate) formation. The CR stock solution was freshly prepared and filtered three times through a 0.2 µm filter before use. The reaction sample was thoroughly mixed and incubated at room temperature for 30 min before recording the spectra. Dynamic Light scattering (DLS)-The self-assembly of tyrosine molecules were detected experimentally by dynamic light scattering approach. The DLS measurement was performed by spectrosize300 from Nano Biochem Technology, Hamburg equipped with an inbuilt Peltier controller unit. Before analysis, the samples were filtered through ~0.1 µm pre-rinsed syringe filters. The concentration of tyrosine sample was also maintained ~1 mM.


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Atomic Force Microscopy (AFM)-To visualize the surface morphology of aggregated samples, AFM measurements were performed by using WITec AFM system, Germany. The aggregated samples were collected and were diluted 10 folds using ultrapure water from which an aliquot of ~20 µL was smeared on freshly cleaved mica. The sample was then air dried and washed thoroughly with ultrapure water. Images were taken using tapping mode (NC-AFM) with a resonance frequency of 300 Hz. All AFM images were captured under ambient condition. Fluorescence microscopy-The fluorescence microscopic images of Thioflavin T stained of tyrosine aggregates, amino acid co-aggregates and protein aggregates were imaged (1) by using fluorescence microscope (Nikon) at 10X magnification. Native Gel Electrophoresis-Native (non- denaturing) polyacrylamide gel electrophoresis for detecting the aggregates was performed in a 10% the acrylamide gel at a constant voltage of 10 Amp with a mini-PROTEIN II Bio-Rad electrophoresis system using a Tris-HCl polyacrylamide gel at 4ºC (1). The main objective of this experiment was to check the formation of the range of higher order entities formed during self-assembly. The gels were then developed by silver staining. The stained gels were visualized by using gel documentation units. Circular Dichroism- CD signals were recorded by Chirascan™ qCD, with attached Peltier temperature controller. The changes in secondary structures in the protein samples in presence of tyrosine fibrils were analyzed by monitoring the CD signals of the samples taken from the aggregation reactions (1). An aliquot of approximately 700 µl of the sample was taken in a CD cell of pathlength 2 mm. Reference solution was the buffer in which the reaction was initiated. All the measurements have been recorded at room temperature. Each CD curve reported was the average representative curve of three acquisitions (between 260 nm–200 nm).


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Biochemistry

Scanning electron microscopy (SEM)-The morphology of tyrosine aggregates was examined by SEM using Carl Zeiss EVO18 equipment. Samples were drop casted over carbon stubs and then sputtered with gold/palladium for 180 s. Then the prepared samples were imaged at a constant voltage of 20 kV. Molecular Dynamics studies on the self-assembly of Tyrosine molecules-The tyrosine molecules were initially processed and packed in a cubic box model by using PACKMOL (14). The mixture model protocol was selected for the tyrosine and water molecules3. Thus, the obtained solvated molecules were used for the desired simulation studies. The molecular dynamics studies were performed with Discovery Studio 4.0 on a 16 core Dell Precision 5610 Workstation. Charmm36 force field was applied to the solvated cubic box. Simulations were performed at 310 K. The energy minimization for the molecules was repeated twice through a steep descendent algorithm. The obtained molecules were then re-equilibrated for 10,000 picoseconds to a target temperature of 310K. Leap frog verlet algorithm with shake constraints are used to fix the chemical bonds between the atoms of the molecule. To maintain a constant temperature and pressure for various components Langevin dynamics and Berendsen pressure with MOLLY and Impulse algorithm was used.

Molecular docking of tyrosine nanostructure and native protein structures-The trapping affinity of tyrosine nanostructure with the native protein monomer was analyzed by employing ZDOCK 3.0 tool (18). The simulated tyrosine molecules at (10 ns) which yielded nanostructure resembling morphology and such tyrosine assembled entities were selected for this study. The protein structures viz BSA (PDB ID: 4F5S), lysozyme (PDB ID: 193L) and myoglobin (PDB ID:1DWR) were downloaded from Protein database. The molecules were then docked individually with the tyrosine nanostructure using ZDOCK program, an in-depth rigid-


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body search in the six-dimensional rotational and translational space was performed as an automated parameter. Briefly three rotational angles were tested with 6° spacing; the three translational degrees of freedom were sampled with a 1.2 Å spacing. The top 10 docked poses were downloaded from the ZDOCK server and were examined thoroughly and the best pose was selected out of ten using Discovery Studio 4.0 visualizer.

RESULTS.

Self-assembly of tyrosine yields nanostructures of amyloid nature-The self-assembly of tyrosine molecules was studied under mimicked physiological condition of temperature (37°C) and buffer (pH 7.4 in PBS). The concentration of tyrosine was selected based on the previously reported studies (10). A sample of tyrosine (in PBS buffer) was incubated at 37°C and was regularly monitored for the rise in the intensity of fluorescence emission of Thioflavin T, a universally used amyloid sensing dye (1, 19). We also recorded the turbidity signal (20, 21) of the sample at different time points. Both the turbidity reading and Thioflavin T signal confirmed the self-assembly of tyrosine molecules into aggregated structures of amyloid nature (Figure 1a). The aggregation curves displayed an initial lag phase, an elongation (log) phase and a plateau phase (Figure 1a) suggesting a nucleation growth propagation (22) as observed for self-assembly of other aromatic residues (1). The above data indicate the conversion of soluble tyrosine molecules into self-assembled amyloid like entities (Figure 1). Examination of the CD signal of the tyrosine sample before and after aggregation, as shown in Figure 1b, clearly suggest the formation of β-structures during self-assembly of tyrosine molecules.


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Biochemistry

Figure 1. Self-assembly of tyrosine into amyloid like fibrils under mimicked physiological conditions. (a) Aggregation of tyrosine at 2 mM in PBS (at 37°C) as monitored by Thioflavin T reading () and by the rise in the turbidity (absorbance at 450 nm) signal (); (b) Circular dichroism spectra of tyrosine sample showing the conformational switch towards βsheet structures during tyrosine self-assembly; (c) Scanning electron microscopic image of tyrosine fibrils. Scale bar 1µm; (d) Dynamic light scattering data showing radius distribution (~10-100 nm) for tyrosine generated nanostructures of 2 mM tyrosine 15 m time point; (e) and (f) AFM images confirm the presence of regular fibrils as well as spheroidal oligomers, as labeled; (g) Native PAGE data confirming the presence of both low and high molecular weight structures generated due to self-assembly of tyrosine. (h) Thioflavin T stained fluorescence images obtained for tyrosine higher order structures.


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Ellipticity signal obtained from our CD measurements for soluble tyrosine sample showed a positive peak at ~222 nm due to absorbance originated from n–π* transition in the carboxylic group and π-π* transition in the benzene group of tyrosine (12, 23, 24). However, we observed a decrease in the positive peak (~222 nm) and slight appearance of negative peak near ~218 nm, suggesting the formation of β-structures. Further, structural properties as examined by scanning electron microscopy (SEM) (Figure 1c), fluorescence microscopy (Figure 1h) and atomic force microscopy (AFM) tools (Figure 1e and 1f, Figure S2,S3) clearly indicated the presence of highly ordered fibrillar structures as well as spheroidal oligomers. The analysis of width and diameter of the tyrosine fibrils is shown in Figure S2 and S3. Though we detected the occurrence of spheroidal Tyr-oligomers, most of the area under AFM visualization showed the presence of mature fibrils as shown in Figure 1e. Our native PAGE data revealed the formation of both low and high molecular weight aggregated species (Figure 1g). Our DLS data also revealed rapid formation of tyrosine generated higher order structures (hydrodynamic radius ranging from 10-100 nm) within five minutes of making the tyrosine solution in PBS buffer (Figure 1d). The DLS signal, at 15 min time point, showed the presence of species with Rh value mostly centered around 100 nm and presence of ~1µm peak indicating slight appearance of higher order structures (Supplementary Figure S7). Such DLS data confirm possible structural tie up between individual tyrosine molecules initiating a self-assembly process. We could not obtain the DLS signal for the matured aggregated samples at later time points due to saturation. Simulation of Tyrosine self-assembly-To gain more insights into the type of preferred interactions between tyrosine molecules during their self-assembly into regular ordered fibrils of amyloid nature, we performed molecular simulation studies on tyrosine assembly. The tyrosine molecules were initially packed in a cubic box as a homogeneous mixture along with water


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Biochemistry

a

b

Figure 2. Simulated structure of tyrosine-nanostructures (at 10 ns). (a) View of the simulated nanostructure displaying a dimension of ~50Å. (b) Snap shots of intermolecular interactions between each tyrosine molecules.

molecules. The discovery studio (DS 4.0) was used for in silico self-assembly experiment at 310 K (37 °C). We detected the formation of well-organized self-assembled spheroidal tyrosine generated nanostructures generated at 10,000 picoseconds (Figure 2). Such structures were found to be stabilized through viable π−π interactions as well as strong hydrogen bonds yielding crossβ like frameworks during tyrosine-self-assembly. Recently, it was proposed that aromatic residue


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Phe can promote quantum dot like spheroidal structures (25) via self-assembly process, displaying spheroidal oligomer type morphology. These self-assembled spheroidal tyrosine nanostructures were then visualized by removing the water molecules (Figure 2) which further revealed the formation of a hollow sphere with hydrophobic exterior and hydrophilic core which can readily accommodate water molecules inside (Figure 2). Analysis of the unique arrangement of the tyrosine residues in the simulated structures (Figure 2) suggests the expulsion of water molecules due to intermolecular interaction between aromatic moieties (26). This assumption is consistent with earlier reports on π−π stacking between aromatic residues during their amyloid formation and such interaction has been predicted to increase the overall entropy of the system (26). From our in silico experiment we predict that the onset of self-assembly may possibly begin with the interaction between two tyrosine molecules via π−π stacking and such initial arrangement would preferably allow a step wise addition of tyrosine monomers in the confined cuboidal geometry. Moreover, our prediction about the formation of tyrosine’s spheroidal oligomers is strongly supported by our DLS measurements that showed formation oligomeric species with a hydrodynamic radius about ~10-100 nm (Figure 1d). Interestingly, our AFM data also revealed the presence of spheroidal oligomers within ~40 -60 nm of size (Figure 1e, 1f). Amyloid cross-seeding ability of Tyrosine nanostructures-The human system is a huge reservoir for various proteins and amino acids and these biomolecules perform various biological processes and maintenance of the homeostasis of such metabolites is vital to the normal physiology of the body system. We questioned what effects of tyrosine amyloid formation may possibly have on the integrity of this symbiotic coexistence of various amino acids and proteins. We checked the effect of preformed tyrosine fibrils on the soluble mixture of globular proteins


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Biochemistry

Figure 3. Study of amyloid cross-seeding mediated through tyrosine nanostructures. (a) Thioflavin T reading of mixed protein samples (BSA + myoglobin + lysozyme, at 2 µM each) confirming tyrosine mediated amyloid cross-seeding of mixed globular proteins (▲); control reaction of mixed proteins without Tyr-fibrils (); Thioflavin T assay indicating Tyr-fibril induced coaggregation of a mixed amino acids sample (Phe +Trp, at 0.1 mM each ) (▼); control reaction of mixed amino acids without Tyr-fibrils (). Inset shows the aggregation of individual proteins samples in the presence of Tyr-seeds (10%w/w), as labeled. Concentration of each protein was maintained at ~5 µM. (b) Native PAGE data confirming the presence of both low and high molecular weight structures of proteins and amino acid samples generated via amyloid cross-seeding. (c) Absorbance spectra of CR solution in the presence (▬) and absence (▬) of protein fibrils and protein fibrils alone (▬). Inset shows the difference spectra obtained by subtracting the individual spectra of CR and protein fibrils from the spectrum of [CR + protein fibril] sample. The maximal point of difference spectrum is ~541 nm. (d) Aggregation of mixed


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protein sample (BSA+myo+lys, each at ~2 µM) in the presence of Tyr-fibrils at different doses: 5 % w/w (), 15 % w/w () and 30 % w/w (▼) (e) Thioflavin T stained fluorescence images obtained for protein fibrils and amino acid fibrils; scale bar 10 µm. (f) AFM images confirming the formation of mature fibrils and spheroidal oligomers generated from aggregation of both proteins and amino acids, as labeled. (g) Circular dichroism spectra of proteins (▬) and amino acids (▬) showing the conformational switch towards β-sheet structures after aggregation.

like BSA, myoglobin and lysozyme. Concentration values of the protein samples were chosen on the basis of our previous work on phenylalanine induced cross-seeding of proteins (1). Here, we observed that the preformed tyrosine fibrils were able to initiate amyloid cross-seeding in a mixture of globular proteins as well as in aromatic amino acids (Figure 3a). Individual protein samples were also found to undergo aggregation in the presence of tyrosine fibrils (Figure 3a inset and Figure S4). The kinetics of individual protein aggregation reactions did not differ much from each other. The resultant protein fibrils were found to be of regular structures and the Native PAGE data also confirmed the formation of higher order structures (Figure 3b, 3e, 3f) of proteins. Our data on Thioflavin T-stained aggregates (Figure 3e) and CD signal of protein aggregates (Figure 3g) point to the formation of amyloid like β-sheet rich protein fibrils (27). Amyloid nature of resultant protein fibrils was further confirmed by our Congo red assay (16, 17) which also showed typical amyloid specific peak at 541 nm (Inset, Figure 3c). Analysis of AFM data, as shown in supplementary Figure S2,S5 and S6 suggests typical dimensions as seen for protein fibrils formed due to amyloid cross-seeding (7). This observation validates our


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Biochemistry

Figure 4. Docked complexes of the tyrosine-nanostructures with native protein structures using Z-DOCK tool (a) BSA (PDB ID 4F5S); (b) myoglobin (PDB ID 1DWR) and (c) Lysozyme (PDB ID 193L). (d) Analysis of Asp4 residue of myoglobin when it interacts with


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Tyr-nanostructures reveals loss of intramolecular H-bonds and gain of intermolecular noncovalent interactions. Detailed list of all the interactions are given in supplementary information (FigureS8 -S10).

previous reports on the occurrence of efficient cross-seeding between several proteins and peptides during their amyloid aggregation (1, 6, 7). We also tried to check whether amyloid cross-seeding efficacy of tyrosine-fibrils is dose dependent. Hence, we examined the effect of different seed amounts (varied from 5% to 30% w/w) in a sample containing protein monomers (maintained at same concentration of 2 µM). Aggregation profiles as obtained from Thioflavin T reading assay (Figure 3d) reflect that the reaction kinetics increases with the increasing seed concentration. To address the question of whether the amyloid cross-seeding efficacy is different for different proteins, we also compared aggregation reactions of individual protein samples (lysozme, BSA and myoglobin, at 5 µM each) in the presence of Tyr-fibrils. The obtained data (Inset Figure 3a, Figure S4) did not show much difference in the aggregation kinetics, suggesting that the cross-seeding efficacy of Tyrnanostructures is not protein specific. Similar aggressive aggregation of several globular proteins of different sequences driven by Phe-fibrils and Aβ fibrils mediated amyloid cross-seeding has been reported (1, 7).

Interaction between tyrosine nanostructures with native structures of proteins-To explore the possible interaction between tyrosine generated amyloid like entities with native protein structures, we performed Z-DOCK 3.0, an online tool for studying protein-protein interaction (18). The aim was to examine whether tyrosine generated nanostructures, as shown in Figure 2


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Biochemistry

have any affinity for the native structures of proteins. The results (Figure 4, Figure S8-S11) revealed that these tyrosine nanostructures can effectively trap the dynamic native conformers of proteins through viable non-covalent interactions. The analysis of the docked images (as shown in Figure 4) are given in supplementary Figure S1 which clearly suggests the involvement of both hydrophobic and charged amino acids (28) for Tyrosine-assembled nanostructure and protein complex formation. Next, we looked at the contacts between the protein species and the Tyr-nanostructure and examined the conformational changes within the tyrosine entities after complex formation. We selectively examined three tyrosine residues (labeled as tyrosine44, tyrosine13 and tyrosine 32) of the Tyr-nanostructure (Figure 2). The analysis of these residues as displayed in Figure S11 suggests alteration of non-covalent interactions between the tyrosine residues of the Tyr nanostructure after its interaction with the native structures of proteins. Another important question was whether there is any alteration in the protein molecule’s intramolecular interactions when it is bound to the Tyr-nanostructures. To address this issue, we also looked at selected residues of the docked proteins. The detailed list of interactions within the protein molecule and with the Tyr-nanostructure is given in the supplementary information (Figure S8 to S10). The analysis of data (Figures S8-S10) points to the formation of additional intermolecular interactions through additional non-covalent contacts and sometimes through replacement of intramolecular interactions. For example, Asp4 of native myoglobin displays three contacts with Lys79, Trp7, Gln8 via electrostatic electrostatic and H-Bonding interactions (left panel, Figure 4d). After docking of myoglobin with Tyr-nanostructure, Asp4 of myoglobin was observed to be making electrostatic and H-bonding interactions with the Tyr residue by losing its intramolecular electrostatic and H-bonding interactions with Lys79, Trp7 and Gln8. The electrostatic interaction between delta oxygen (OD1) of Asp4 and Lys79 is lost as the same


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OD1 makes new H-bond with the tyrosine (Figure 4d, Figure S9). Also, many residues which do not participate in intramolecular interaction of myoglobin were found to make viable noncovalent contacts in the docked complex. Similarly, Leu149 of docked myoglobin loses intramolecular hydrophobic interaction with Ala94 while making contacts with tyrosine of the Tyr-nanostructure (Figure S9). More importantly, these residues (Asp4, Ala94 and Leu149) exist in the unstructured stretches of myoglobin which suggests that flexibility of the interacting tract may possibly facilitate the trapping of proteins.

DICUSSION

This study has examined both experimentally and computationally the intrinsic property of tyrosine molecules to self-assemble via unique arrangement, resembling β-sheet-like structures. The tyrosine molecule is considered as a precursor for the synthesis of vital neurotransmitters including dopamine (8, 29) and GABA (gamma-aminobutyric acid) (30). Tyrosine accumulation could therefore be a determining factor by directly or indirectly influencing the normal neurophysiology of our body system. Further, tyrosine amyloid formation process may adversely affect the homeostasis of such neuromodulators that originate from tyrosine molecule leading to neuronal defects. The formation of amyloid like entities such as tyrosine fibrils which can readily drive aggregation of other globular proteins and aromatic metabolites (as summarized in Figure 5) suggests lethal consequences including both toxic gain of amyloid aggregates and the loss of functional biomacromolecules. The ability of an amyloid like entity to trigger amyloid crossseeding has recently been reported in both proteins and metabolite systems. Though earlier reports have proposed the requirement of sequence identity between cross-seeding species (31),


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Biochemistry

Figure 5. Schematic representation of self-assembly of tyrosine residues yielding fibrillar entities capable of amyloid cross-seeding in globular proteins and aromatic metabolites.

recent experimental evidences have however revealed the occurrence of co-assembly and crossseeding among proteins without much sequence identity (1, 6).

Amyloid cross-seeding

experiments on polyQ peptides by Wetzel’s group have even revealed efficient cross-seeding of D-polyQ

peptides by L-polyQ seeds and vice versa, in both cell models and in vitro models (32).

The same study has suggested that formation of H-bonds between D- and L-versions of the polyQ peptide (33, 34) was important for such chirality independent amyloid cross-seeding (32). Hence, in the current observation where tyrosine was found to induce amyloid cross-seeding, it appears that sequence similarity may not be a critical factor for initiating such aggregation process. It is possible that, rather than the sequence identity, the fundamental intermolecular interactions driven through both hydrophobic and charged residues play a critical role in the mechanism of


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metabolite induced amyloid cross-seeding. Our computational (Figure 4, Figure S8-S11) and experimental data further validate this hypothesis of common driving force mediated through hydrophobic and electrostatic interactions to initiate amyloid-cross-seeding. The surface of tyrosine generated higher order structures may have sufficient number of exposed sticky groups that can effectively trap native protein species, preferably through viable non-covalent interactions, as predicted by our computational data (Figure 4, Figure S8-S11). Such trapping of protein species would certainly increase the local concentration of the proteins sufficient enough to possibly begin the aggregation process (35). The data obtained from circular dichroism and Thioflavin T binding assay also propose the formation of β-structures during aggregation, suggesting the possible conformational switching of native protein structures (bound to tyrosine fibrils) to β-sheet frameworks. Our computational data also clearly revealed possible alterations in some intramolecular interactions within the protein’s native structure when it interacts with the Tyr assembled entity (Figure 4d, Figure S8-S11). In fact, we did not see any distinct lag phases in our tyrosine-induced aggregation profiles of proteins and such observation suggests that tyrosine fibrils may act as efficient nuclei (36) to begin aggregation. Such hypothesis is supported by aggregation data obtained from our amyloid cross-seeding experiments at different doses of Tyr-seeds (Figure 3d). Hence, the observation that tyrosine fibrils have intrinsic property to trap proteins and drive their amyloid aggregation may possibly broaden our knowledge on the significance of metabolite amyloid formation and its relevance to many complicated neurodegenerative diseases.

ASSOCIATED CONTENT Supporting Information Supplementary information: Table S1 and Figure S1-S11.


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AUTHOR INFORMATION *Corresponding Author Correspondence

and

requests

for

materials

should

be

addressed

to

K.K.

email:

[email protected] and [email protected] Present address: ¶B. G. Anand: Department of Medicine, University of Alberta, Edmonton, Canada; ‡D. S. Shekhawat: Department of Pediatrics, All India Institute of Medical Sciences, Jodhpu, Rajasthan India. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interests. Funding Sources DST-PURSE II, UGC RN, UGC DRS SAP-I, UPOE II-JNU and UGC start-up GRANT

ACKNOWLEDGMENT We thank Jawaharlal Nehru University for providing us the required facilities (ARIF- JNU and CIF-School of Life Sciences). Authors thank DST-PURSE II, UGC RN, UGC DRS SAP-I, UPOE II-JNU and UGC start-up GRANT for funding support. We thank Prof. Gourinath for help with DLS facility. KPP thanks CISR for fellowship support. We thank Dr. G. Bagler for help with the computational studies.


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Tyrosine generated nanostructures initiate amyloid cross-seeding in

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