Amyloid formation characteristics of GNNQQNY from yeast prion protein Sup35 and its seeding with heterogeneous polypeptides

Colloids and Surfaces B: Biointerfaces 149 (2017) 72–79

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Amyloid formation characteristics of GNNQQNY from yeast prion protein Sup35 and its seeding with heterogeneous polypeptides Mamoru Haratake a,∗ , Tohru Takiguchi b , Naho Masuda b , Sakura Yoshida b , Takeshi Fuchigami b , Morio Nakayama b,∗∗ a b

Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan

a r t i c l e

i n f o

Article history: Received 7 July 2016 Received in revised form 3 September 2016 Accepted 6 October 2016 Available online 7 October 2016 Keywords: Prion Yeast Amyloid Aggregation ˇ-Sheet Thioflavin T Seeding

a b s t r a c t Sup35 is a prion-like protein from yeast and shares the ability to transmit its aberrant fold and to aggregate into amyloid fibrils. 7 GNNQQNY13 from the prion-determining domain of Sup35 was reported to form an amyloid. We first investigated the self-aggregation transition behavior of GNNQQNY to the ˇ-sheet amyloid state under various conditions. Mechanical stirring using a magnetic bar resulted in accelerated aggregation of the GNNQQNY. The aggregation rate of GNNQQNY was also dependent on its concentration; the higher the GNNQQNY concentration, the faster the aggregation. Circular dichroism and Fourier transform-infrared spectral data indicated the formation of the ˇ-sheet structure in the GNNQQNY aggregates. The fluorescence experiments using an amyloid-specific thioflavin T also demonstrated that the GNNQQNY aggregates formed the amyloid structures. The amyloid structure of the GNNQQNY aggregates served as seeds for the elongation of the monomeric GNNQQNY in the solution state. We further studied the ability of the GNNQQNY amyloid fibrils to act as seeds for the elongation of the amyloidforming monomeric proteins (albumin, lysozyme and insulin). The cross-seeding experiments suggested that the GNNQQNY aggregate could possibly promote the amyloid fibril formation of heterogeneous insulin. The inverse monomeric GNNQQNY would have a binding capacity for the heterogeneous alreadyformed amyloid-ˇ fibrils on a mice brain section. These basic data could be informative for elucidating the pathogenic and/or propagation mechanisms of prion agents and developing effective therapeutics and/or diagnosis for prion diseases. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Amyloid-related diseases are characterized by the misfolding of proteins into amyloid fibrils and subsequent deposition of amyloid aggregates in the body. More than 30 amyloid-related diseases have been described, among which are Alzheimer’s disease, Parkinson’s disease, prion diseases and type 2 diabetes mellitus [1–3]. Each disease is associated with a particular protein, and aggregates of the proteins are thought to be the direct or indirect origin of the pathological conditions associated with the disease. In some cases, the quantity of material involved is enormous with several

∗ Corresponding author at: Faculty of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan ∗∗ Corresponding author at: Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki, 852-8521 Japan. E-mail addresses: [email protected] (M. Haratake), [email protected] (M. Nakayama). http://dx.doi.org/10.1016/j.colsurfb.2016.10.011 0927-7765/© 2016 Elsevier B.V. All rights reserved.

kilograms of protein being deposited in certain manifestations of systemic amyloidosis [4–7]. The aggregated forms of the proteins have many characteristics in common, although all of which have a unique native folding [8]. A protein-based fibril is identified as an amyloid by its structural and tinctorial properties; amyloid fibrils are unbranched, and bind the dye Congo red. The fibrils also produce a characteristic cross-ˇ X-ray diffraction pattern, consistent with a model in which stacked ˇ-sheets form parallel to the fiber axis having their individual ˇ-strands perpendicular to the fiber axis [9]. However, the process of amyloid formation and its structural details are still unknown [10,11]. Prion diseases are lethal neurodegenerative disorders that occur in humans as well as in animals [4], which involve CreutzfeldtJakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, etc. [12]. A prion is a proteinaceous particle that resists inactivation by procedures that modify nucleic acids [13]. Thus, the protein structure is distinctively passed at the protein, and not at the nucleic acid level. Fundamental to the diseases is the conversion of a normally folded, globular, mainly

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␣-helical protein (PrPC , ‘C’ for ‘cellular’) into a misfolded, insoluble, largely ˇ-strand rich, pathological form (PrPSc , ‘Sc’ for ‘scrapie’) that can aggregate and accumulate in the brain [14]. Another distinguishing feature of the prion diseases is only infectious in the amyloid-related diseases. In the 1980s, an epidemic of BSE in England that killed over 140,000 cows seems to have been caused by feed containing spinal cords and brains of cattle and/or sheep that may have died of a related disease such as scrapie. In 1996, the variant form of CJD was finally spread to humans through the consumption of meat products from BSE-infected cattle [15,16]. PrPC is a membrane-associated protein occurring in a wide range of eukaryotic cells. The physiological functions of PrPC are still unknown, although the broad distribution among mammalian species and the high conservation of PrPC implies a role of general importance. During the propagation and onset of prion diseases, irreversible structural conversion of endogenous PrPC in the native conformation into PrPSc in an aberrant ‘killer’ conformation is thought to occur and then aggregate into an infectious form with the amyloid structure [4,17]. PrPC and PrPSc are conformationally isomeric forms of each other; the PrPC to PrPSc transition involves conversion of the ␣-helix to ˇ-sheet in large parts of the protein. The killer conformer and its aggregates are thought to be the diseasecausing agents in the prion diseases. The decisive process i.e., the irreversible conversion of PrPC into PrPSc , initiates an ‘autocatalytic’ reaction which leads to the amyloid accumulation in the central nervous system [18]. Actually, inhibitors of pathological amyloid fibril formation could be useful in the development of therapeutics against prions. Currently, the molecular details of the structure transmission from PrPSc to PrPC are still hardly known. Sup35 is a prion-like protein from yeast and shares the ability to transmit its aberrant folding and to aggregate into amyloid fibrils. Its normal cellular function is to terminate translation [19–22]. 7 Gly-Asn-Asn-Gln-Gln-Asn-Tyr13 (GNNQQNY) from the prion-determining domain (residues 1–123) of Sup35 was reported to form an amyloid [1]. This heptapeptide segment can form closely related microcrystals, from which Nelson et al. have determined the atomic structure of the cross-ˇ spine [23,24]. GNNQQNY aggregates could be used as a model of amyloid fibrils in order to obtain basic information for elucidation of the propagation mechanism and the development of therapeutic and preventive agents. In this study, we investigated the self-aggregation transition behavior of GNNQQNY to the ˇ-sheet amyloid state under various conditions, and the ability of GNNQQNY fibrils to serve as seeds for the elongation of several amyloid-forming monomeric proteins.

2. Materials and methods 2.1. Materials Heptapeptide (H-Gly-Asn-Asn-Gln-Gln-Asn-Tyr-OH, GNNQQNY) was obtained from Takara Bio, Inc. (Tokyo, Japan) (Fig. S1). Fluorescamine (FS), human serum albumin (HSA), lysozyme from chicken egg white (LYS) and insulin from bovine pancreas (INS) were purchased from Sigma Co., Ltd. (St. Louis, MO). Thioflavin T (ThT) was from Nacalai Tesque, Inc. (Kyoto, Japan). Water used throughout this study was generated using a Milli-Q Biocel system (Millipore Corp., Billerica, MA). All other chemicals were of commercial reagent grade and used as received. Brain sections from Tg2576 transgenic mice were prepared according to a previously reported procedure [25]. Fluorescently labeled GNNQQNY was prepared as briefly described; FS (0.67 mg/mL) and GNNQQNY (1 mg/mL) were mixed in acetonitrile and then stirred with a magnetic bar at room temperature for 15 min. The resulting mixture was subjected to reverse-phase liquid chromatography to isolate the FS-labeled GNNQQNY (Fig. S2).

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2.2. Measurements of turbidity and free peptide concentration After gentle pipetting several times, an aliquot of the sample solution was transferred into a 1-cm path quartz cuvette. The turbidity produced by the aggregation was monitored at 400 nm by a V-660 UV–vis spectrophotometer (Jasco Corp., Tokyo, Japan). The peptide solution was treated using an L-80 centrifuge with a SW-40Ti rotor (Beckman Coulter, Inc., Brea, CA) at 10,000 min−1 and 25 ◦ C for 10 min, and then the absorbance of the supernatant was monitored at 276 nm. The free peptide concentration was expressed in% with the initial GNNQQNY concentration (0.33 mg mL−1 ) being defined as 100%. 2.3. Circular dichroism After the sample solutions were appropriately diluted with the buffer solution, circular dichroism spectra were measured by a J725 (Jasco Corp., Tokyo, Japan) using a 1-cm path quartz cuvette from 190 to 400 nm at ambient temperature. 2.4. Fourier transform-infrared spectroscopy The aggregate suspensions were ultrafiltered through an Ultrafree-MC (molecular weight cutoff: 10,000, Millipore Corp.) at 5000 g for desalting. The filtrate (≈10 ␮L) was placed on a Zn-Se prism of PRO410-S attenuated total reflectance (ATR, Jasco Corp.), and allowed to dry at ambient temperature. The Fourier transform infrared spectra were measured by the ATR method using an FT/IR 4200 spectrometer (Jasco Corp.). 2.5. Measurement of ThT fluorescence spectra After the addition of 1 mM ThT dissolved in Milli-Q water, and gentle pipetting several times, an aliquot of the sample solution was transferred into a 1-cm path quartz cuvette, then the fluorescence spectra were measured by a FP-6600 spectrofluorometer (Jasco Corp.) using an excitation wavelength of 450 nm. 2.6. Microscopic observations The peptide aggregates were collected by centrifugation of the samples at 4000 min−1 for 15 min. The obtained precipitates (2.5 ␮L) were mixed with an equal volume of ThT solution (40 ␮M), and washed with water by the centrifugation. Fluorescence images were acquired by a BZ-8100 fluorescence microscope (Keyence Corp., Tokyo, Japan). For the electron microscopy, the specimens were put on a polycarbonate membrane in a SEM Pore (pore size 0.6 ␮m, Jeol Ltd., Tokyo, Japan), then washed with Milli-Q water to remove the inorganic salts. After drying at ambient temperature for 1 day, the specimens were subjected to a gold sputter coating for 1 min. Electron micrograms were acquired using a JSM-7500F scanning microscope equipped with a field emission gun (Jeol Ltd., Tokyo, Japan). 2.7. GNNQQNY aggregation experiments GNNQQNY was dispersed in 0.01 M phosphate buffer (pH 7.4). To completely solubilize the peptide, the resulting solutions were sonicated for ≈10 min and subsequently left in a water-bath at 65 ◦ C (final GNNQQNY concentration: 0.082–1.0 mg mL−1 ). The GNNQQNY solutions were pass through a DISMIC-25CS cellulose acetate membrane filter (pore size: 0.2 ␮m). The obtained clear fresh solution was transferred to a 10-mL Erlenmeyer flask and vigorously stirred by a 1.5-cm length magnetic stirrer bar (≈300 rpm) on a SR-506 (Advantec, Tokyo, Japan) or gently in a water bath PERSONAL-11 (Taitec, Tokyo, Japan) at 37 ◦ C and 60 stroke min−1 .

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The turbidity at 400 nm of the GNNQQNY solutions and the concentrations of the free GNNQQNY were monitored in time-course aggregation experiments.

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A GNNQQNY solution (0.33 mg mL−1 ) was prepared as described in Section 2.7. The obtained clear solution was transferred to a 10mL Erlenmeyer flask and vigorously stirred using a 1.5-cm length magnetic stirrer bar on a SR-506 (Advantec, Tokyo, Japan) for 6 days or longer at ambient temperature. The obtained aggregate solutions were used as seeds for the study of the interaction between the GNNQQNY aggregate and amyloid-forming proteins (HSA, LYS and INS). An HSA solution (15 ␮M) and an LYS solution (30 ␮M) were prepared using 0.01 M phosphate buffer (pH 7.4). An INS solution (250 ␮M) was made using 0.01 M glycine buffer (pH 2.5). These solutions were pass through a DISMIC-25CS cellulose acetate membrane filter (pore size: 0.2 ␮m) before mixing with the GNNQQNY aggregates. An 1: 2 mixture by volume of each fresh protein solution and the GNNQQNY aggregate solution was placed in a 10-mL Erlenmeyer flask and gently stirred in a PERSONAL-11water bath at 37 ◦ C and 60 stroke min−1 . The turbidity at 400 nm of the mixtures and the concentrations of the free proteins were monitored during the time-course aggregation experiments. 3. Results and discussion

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3.1. GNNQQNY aggregation behavior Aggregation processes of the amyloid-forming proteins are generally characterized by (a) a slow nucleation phase, in which the peptide undergoes a series of unfavorable association steps to form an ordered oligomeric nucleus, (b) a growth phase, in which the nucleus grows to form polymeric aggregates, and (c) a steady state phase, in which the ordered aggregate and the monomer appear to be at equilibrium (nucleation dependent amyloid formation). The aggregation of GNNQQNY was characterized by monitoring the changes in the turbidity at 400 nm and the concentration of the free GNNQQNY of a GNNQQNY solution (0.33 mg mL−1 ). Self-aggregation into amyloid fibrils is generally a slow process, occurring on the time-scales of hours to days. No changes in both the turbidity and the% free GNNQQNY of the GNNQQNY solution were observed when the solution was gently shaken in a water bath at 37 ◦ C for 7 days (Fig. 1a). In contrast, when the GNNQQNY solution at the same concentration was vigorously stirred with a magnetic bar at ambient temperature (≈25 ◦ C), it became opaque at approximately a few hours later. The% free GNNQQNY in this solution decreased in an inverse manner (Fig. 1a). Mechanical stirring apparently resulted in accelerated aggregation of the GNNQQNY. The formation of the GNNQQNY aggregates was reported to critically depend on its concentration [26]. No fibril and/or crystal formation in the GNNQQNY solutions at less than 6 mM (≈5 mg mL−1 ) occurred even when they were left for one year [27]. During the course of our study, the turbidity of the GNNQQNY solution at 0.33 mg mL−1 did not totally increase with a gentle stirring (data not shown). Therefore, the observed aggregation of GNNQQNY at a very low concentration resulted from the intensive agitation using a stirring bar. When the aggregation behavior of GNNQQNY were compared among the GNNQQNY solutions at three different concentrations (0.08, 0.17 and 0.33 mg mL−1 ) with magnetic stirring, the higher GNNQQNY concentration, the faster the turbidity increased (Fig. 1b). Because the GNNQQNY molecule consists of electrically neutral amino acids, van der Wales interactions among the

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Fig. 1. Effect of agitation intensity (a) and concentration (b) on GNNQQNY aggregation. (a) Circle: gentle shaking, triangle: intensive stirring, Closed symbols: turbidity at 400 nm, Open symbols: % free GNNQQNY [Initial GNNQQNY concentration (0.33 mg mL−1 ) was defined as 100%.]. Medium: 10 mM phosphate buffer (pH 7.4). Temperature: ambient (≈25 ◦ C). (b) Initial GNNQQNY concentration: Open circle, 0.08 mg mL−1 ; Grey solid circle, 0.17 mg mL−1 ; Black solid circle, 0.33 mg mL−1 . Agitation: intensive stirring. Medium: 10 mM phosphate buffer (pH 7.4). Temperature: ambient. Data express mean and standard error (n = 5).

molecules can contribute to the aggregation of GNNQQNY. Since the GNNQQNY has electric charges at the both ends resulting in electrostatic interactions between the aggregating peptides, electric dipole, electrostatic charge-charge, charge-dipole and dipole–dipole interactions may also serve as attractive forces in the aggregation of this peptide. Subsequently, the aggregation behavior of GNNQQNY was compared between the GNNQQNY solutions (0.08 mg mL−1 ) at 4 ◦ C and ambient temperature (≈25 ◦ C) (Fig. S3). Since, in general, the higher the temperature, the higher the motility of the molecules, the GNNQQNY solution at 25 ◦ C was expected to become turbid faster than that at 4 ◦ C. However, the turbidity of this solution at 4 ◦ C increased faster than that at 25 ◦ C. It has been reported that GNNQQNY tends to form amyloid fibrils at 4 ◦ C, but nanocrystals at 25 ◦ C [26]. It was also reported that the Sup35 fragment amyloids formed at different temperatures adopt distinct and stably propagating conformations [28,29]. Thus, the structures of the GNNQQNY aggregates are thought to be dependent on the temperature. The GNNQQNY aggregates formed at 4 ◦ C were likely to be somewhat structurally different from those at 25 ◦ C. A low temperature could be preferable to the nucleation and/or subsequent fibril elongation processes, possibly due to the low solubility.

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Time (day) Fig. 3. Effect of seeds on GNNQQNY aggregation. (a) Without seeds, (b) With seeds. Closed circle: Turbidity, Open circle: % Free GNNQQNY. Initial GNNQQNY concentration: 1.00 mg mL−1 . Medium: 10 mM phosphate buffer (pH 7.4). Mean particle size of GNNQQNY aggregates used as seeds: 1.900 ± 0.125 ␮m.

Fig. 2. Fluorescence image of GNNQQNY aggregate after treatment of ThT (a) and scanning electron micrographs of GNNQQNY aggregate (b). (a) Initial GNNQQNY concentration: 0.17 mg mL−1 , ThT concentration: 20 ␮M. Filter: Excitation, 450–490 nm; Emission, 510–560 nm. Objective magnification: ×40. Scale bar: 10 ␮m. (b) Scale bar: 1 ␮m. Magnification: ×20,000. Initial GNNQQNY concentration: 0.33 mg mL−1 . The GNNQQNY aggregates were prepared by the intensive stirring for 8 days at ambient temperature. Accelerating voltage: 5 kV.

3.2. Characterization of GNNQQNY aggregates GNNQQNY can form a cross-ˇ structure that is commonly found in the structures of amyloid fibrils [21,23]. To confirm the secondary structure of the formed GNNQQNY aggregates, circular dichroism (CD) and Fourier transform-infrared (FT-IR) spectra of the GNNQQNY solution were measured. Polypeptides that are formed ˇ-sheet structures, such as poly(Leu-Glu-Leu-Glu) and poly(Lys-Leu-Lys-Leu), provide two typical absorption bands in their far-ultraviolet CD spectra due to the amide structure; a positive band at around 198 nm and a negative band at around 215 nm [30–32]. A similar trend in the absorption bands was observed in the CD spectrum of the GNNQQNY solution (Fig. S4a). FT-IR spectroscopy is also frequently used in the analysis of the secondary structure of polypeptides. The FT-IR spectral characteristics in the wavenumber range between 1600 and 1700 cm−1 (the amide I band due to C O stretching) can reflect the difference in the mode of the hydrogen bonding of the polypeptide main chain. Absorp-

tion bands assigned to the hydrogen-bonded amide C O in ␣-helix and ˇ-sheet structures appear at 1650–1658 and 1620–1640 cm−1 , respectively [32,33]. In the FT-IR spectrum of the GNNQQNY aggregates, a peak of the characteristic absorption band was detected at 1625 cm−1 (Fig. S4b). These CD and FT-IR spectra indicated the formation of the ˇ-sheet structure in the GNNQQNY aggregates. The GNNQQNY aggregates were investigated using the fluorescence of thioflavin T (ThT). Upon binding of the amyloid fibrils, ThT displays a large shift in the excitation maximum (from 385 to 450 nm) and the emission maximum (from 445 to 482 nm). ThT is widely used as a sensitive and efficient reporter molecule due to such fluorescence spectral changes upon fibril binding [34–36]. Neither the GNNQQNY aggregates nor ThT solution itself emitted such a distinctive fluorescence when separately excited at 450 nm. On the other hand, the mixture of the two solutions gave rise to a fluorescence spectrum with the emission peak at 483 nm (Fig. S5). Subsequently, the GNNQQNY aggregates in the mixture were separated from the ThT solution by centrifugation and then subjected to fluorescence microscopy. When excited with a 450–490 nm filter block, the fluorescence through a 510–560 nm filter block was observed throughout the specimen. The ThT fluorescence image of the GNNQQNY aggregates was almost completely consistent with their transmission image, that is, ThT molecules were bound to the surface of the GNNQQNY aggregates (Fig. 2a). These fluorescence data also demonstrated that the GNNQQNY aggregates formed the amyloid structures.

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Wave lengt h (nm ) Fig. 4. Changes in turbidity and circular dichroism spectra of INS. Medium: 50 mM acetate buffer (pH 4.0). (a) Open square: without seeds, Closed circle: with seeds. Initial INS concentration: 1.45 mg mL−1 . (b) Dashed line: before seeding, Broken line: without seeds, Solid line: with seeds. Initial INS concentration: 0.15 mg mL−1 .

The formed GNNQQNY aggregates were further subjected to electron scanning microscopic observations (Fig. 2b). An unbranched needle-like morphology of the GNNQQNY aggregates was found, which is a distinctive structural feature of the amyloid structure [26]. The diameters of the GNNQQNY aggregates were estimated in the range of 30–200 nm. Thus, the scanning electron micrographs suggested that the GNNQQNY aggregates are composed of several aligned nanometer-sized fibrils (≈10 nm in diameter). 3.3. Effect of homogeneous-seeding on GNNQQNY aggregation Because most peptide conformational changes are much faster than the amyloid formation, perturbations of the monomer conformational dynamics are quite unlikely to affect the overall aggregation rate. The slow step in the amyloid formation seems to be the formation of an ordered oligomeric nucleus. As a nucleation event from soluble monomers and/or oligomers is the slowest phase in the process of amyloid formation, there is a lag time before the fibril growth process occurs. Thus, during the lag time, the addition of an exogenous seed results in accelerating the elongation of the aggregates [37–39]. Seeding is a key phenomenon in all forms of the amyloid and is most likely the mechanism by which amyloid deposits spread in a tissue and, in the case of systemic amyloidosis,

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Fig. 5. Changes in ThT fluorescence intensity at 3-day after mixing of GNNQQNY, HSA, LYS and INS with GNNQQNY aggregates. Medium: (a), (b) and (c), 10 mM phosphate buffer (pH 7.4); (d), 50 mM acetate buffer (pH 4.0). Initial concentration: GNNQQNY, 0.33 mg mL−1 ; INS, 0.07 mg mL−1 . ThT concentration: 20 ␮M. Excitation: 450 nm, Fluorescence intensity (FI) at 484 nm was calculated by (FI48h /FI0h ) × 100.

from one organ to another. The propagation of the GNNQQNY amyloid structure was examined by monitoring the turbidity at 400 nm and the% free GNNQQNY of a GNNQQNY solution (1.00 mg mL−1 ). Both parameters of the GNNQQNY solution were not changed at all, when gently shaken at 37 ◦ C for 3 days (Fig. 3a). On the other hand, the addition of the GNNQQNY aggregates to the GNNQQNY solution at the same concentration resulted in an increase in the turbidity and an inverse decrease in the free GNNQQNY in only a few hours (Fig. 3b). ThT was added to the obtained turbid GNNQQNY solution, then the generated GNNQQNY aggregates were observed under a fluorescence microscope. The ThT fluorescence was observed in the entire surface of the GNNQQNY aggregates (Fig. S6), as well as that for the seeds shown in Fig. 2a. These results clearly demonstrated that the amyloid structure of the added GNNQQNY aggregates served as seeds for the elongation of the monomeric GNNQQNY in the solution state. Homogeneous-seeding of the amyloid formation, like the seeding for crystallization, seems extremely discriminating because it relies on a complementarity between the growth face of the GNNQQNY aggregates and the monomeric peptide. 3.4. Interaction between GNNQQNY aggregate and amyloid-forming heterogeneous proteins A seed of a different constitution can function as a heterogeneous seed, provided that its growth face is complementary to the native material. During the process of the prion propagation, a killer conformation of the proteins in the transmitting agents is thought to transmit to the native conformers. Almost every human protein has segments that can form the amyloid structures, the sticky aggregates known for their role in prion diseases. Therefore, such transmitting agents could possibly be formed with endogenous proteins and peptides in infected individuals, and the resulting mixed amyloids may be implicated in the prion propagation and/or onset of the diseases. Cross-seeding (heterogeneous-seeding) among the amyloid fibrils would suggest the possibility that one amyloid fibril could influence the heterogeneous protein species. From this viewpoint, it can be informative to study the interactions of the GNNQQNY aggregates with endogenous proteins under physiological conditions [3,7]. To test whether the GNNQQNY aggregates non-specifically interact with heterogeneous proteins, the aggregation behavior of several amyloid-forming proteins in the presence of the GNNQQNY aggregate was examined. Human serum albumin (HSA), lysozyme from chicken egg white (LYS) and insulin from bovine pancreas (INS) are known to form amyloid fibrils under certain conditions [40–43]. The turbidity at 400 nm of the protein solutions with and without the GNNQQNY aggregates was monitored with gentle shaking at

M. Haratake et al. / Colloids and Surfaces B: Biointerfaces 149 (2017) 72–79

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(c) Fig. 7. Fluorescence images of Tg2576 mice brain sections after treatment of fluorescamine-labeled GNNQQNY (a) and ThT (b). Fluorescamine-labeled GNNQQNY concentration: 50 ␮M, ThT concentration: 2.5 ␮M. Filter: Excitation, 450–490 nm; Emission, 510–560 nm. Objective magnification: ×20. Scale bar: 10 ␮m.

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Fig. 6. Effect of GNNQQNY aggregates on aggregation of INS at pH 2.5. (a) Turbidity and ThT fluorescence intensity, ThT concentration: 2.5 ␮M, Excitation: 450 nm, Fluorescence intensity at 484 nm was calculated by (FI48h /FI0h ) × 100, (b) CD spectra, (c) FT-IR spectra. Dashed line: before incubation, Broken line: day 2, Solid line: day 3. Initial INS concentration: 1.45 mg mL−1 . Medium: 10 mM glycine-HCl solution (pH 2.5).

37 ◦ C for 3 days. No remarkable difference in the turbidity change between the INS solutions with and without the GNNQQNY aggregates was observed (Fig. 4a). The CD spectra of the two INS solutions at day 3 were nearly identical to that of a fresh INS solution (Fig. 4b). Similar trends in the turbidity and CD spectral changes were observed for the HSA and LYS solutions (Fig. S7a-1, a-2, b1 and b-2). The ThT fluorescence assay was further carried out (Fig. 5). The addition of the GNNQQNY aggregates resulted in a six-fold increase in the relative fluorescence intensity of the initial GNNQQNY solution, while no increases in those of the three protein solutions were observed. These spectral data implied that the GNNQQNY aggregates do not induce heterogeneous aggrega-

tion and changes in the secondary structures of the three proteins. Therefore, the GNNQQNY aggregates seem to be poor seeds for the amyloid formation of HSA, LYS and INS. Next, a similar cross-seeding experiment was carried out for INS at pH 2.5 and 37 ◦ C. The amyloid fibril formation of INS is fairly accelerated at a lower pH compared to that at pH 4 [44]. Actually, both the turbidity and ThT fluorescence intensity at 484 nm of the INS solution increased at day 3 (Fig. S8a). The addition of the GNNQQNY aggregates to the INS solution produced increases in the turbidity and the ThT fluorescence at day 2 (Fig. 6a). In the CD spectrum of INS at day 0, a negative band at around 222 nm was observed, which shifted to 215 nm at day 3 (Fig. S8b). Such a spectral change was detected at only day 2 in the presence of the GNNQQNY aggregates (Fig. 6b). A similar trend in this time-course experiment was found in the FT-IR spectrum; the amide I band was shifted from 1650 cm−1 to 1625 cm−1 at day 2 in the absence of the GNNQQNY aggregates (Fig. S8c), while it was shifted at day 3 in the presence of the GNNQQNY aggregates (Fig. 6c). Thus, cross-seeding of the GNNQQNY aggregate appeared to potentially promote the amyloid fibril formation of the heterogeneous INS. 3.5. Interaction between GNNQQNY and amyloid-ˇ plaques on Tg2576 mice brain section Double immunestaining for Aˇ and PrP in the frontal cortex was reported for familial early-onset AD with a concomitant Aˇ and prion brain pathology [45]. To examine the interactions

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of the GNNQQNY with heterogeneous Aˇ amyloid plaques using the brain section of the Tg2576 transgenic mice, the monomeric soluble GNNQQNY was fluorescently labeled with fluorescamine (FS) through its terminal amino group. The Tg2576 transgenic mice overexpress a mutant variant of the human amyloid precursor protein (Lys670Asn, Met671Leu) and develop extracellular Aˇ plaque deposits [46]. Many fluorescently positive dots from FS were detected on the brain section (Fig. 7a). Staining of an adjacent section with ThT identified the locations of the Aˇ amyloid plaques (Fig. 7b). The locations of the fluorescently positive dots from FS corresponded to those of ThT, although there were some tiny dots that deviated away from the ThT fluorescence. Thus, GNNQQNY would have a binding capacity for the heterogeneous Aˇ plaques in the mice brain in addition to having it for INS. 4. Conclusions The self-aggregation transition behavior of GNNQQNY from the yeast prion protein Sup35 to the ˇ-sheet amyloid fibril was investigated under various conditions. Mechanical stirring using a magnetic bar resulted in accelerated aggregation of the GNNQQNY. The aggregation rate of GNNQQNY was also dependent on its concentration; the higher the GNNQQNY concentration, the faster the aggregation. The CD, FT-IR and ThT fluorescence spectral data indicated the formation of the ˇ-sheet structure in the GNNQQNY aggregates. Scanning electron microscopic observations clarified that the GNNQQNY aggregates are composed of several aligned, nanometer-sized fibrils. The amyloid structure of the GNNQQNY aggregates served as seeds for the elongation of the monomeric GNNQQNY in the solution state (homogeneousseeding of GNNQQNY amyloid formation). We further studied the ability of the GNNQQNY amyloid fibrils to act as seeds for the elongation of several amyloid-forming monomeric proteins. Crossseeding experiments implied that the GNNQQNY aggregates could possibly promote the amyloid fibril formation of the heterogeneous INS. The inverse monomeric GNNQQNY would have a binding capacity for the heterogeneous already-formed Aˇ fibrils. These basic data could be informative for elucidating the pathogenic and/or propagation mechanisms of prion agents and developing effective therapeutics and/or diagnosis for prion diseases. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.10. 011. References [1] M. Balbirnie, R. Grothe, D.S. Eisenberg, An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated beta-sheet structure for amyloid, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 2375–2380. [2] B. O’Nuallain, A.D. Williams, P. Westermark, R. Wetzel, Seeding specificity in amyloid growth induced by heterologous fibrils, J. Biol. Chem. 279 (2004) 17490–17499. [3] R. Morales, L.D. Estrada, R. Diaz-Espinoza, D. Morales-Scheihing, M.C. Jara, J. Castilla, C. Soto, Molecular cross talk between misfolded proteins in animal models of Alzheimer’s and prion diseases, J. Neurosci. 30 (2010) 4528–4535. [4] S.B. Prusiner, Prions, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 13363–13383. [5] C.M. Dobson, Protein misfolding, evolution and disease, Trends Biochem. Sci. 24 (1999) 329–332. [6] J. Collinge, Prion diseases of humans and animals: their causes and molecular basis, Annu. Rev. Neurosci. 24 (2001) 519–550. [7] T. Konno, Amyloid-induced aggregation and precipitation of soluble proteins: an electrostatic contribution of the Alzheimer’s ␤(25–35) amyloid fibril, Biochemistry 40 (2001) 2148–2154. [8] M. Sunde, C.C.F. Blake, The structure of amyloid fibrils by electron microscopy and X-ray diffraction, Adv. Protein Chem. 50 (1997) 123–159. [9] W.T. Astbury, S. Dickson, K. Bailey, The X-ray interpretation of denaturation and the structure of the seed globulins, Biochem. J. 29 (1935) 2351–2361. [10] C.M. Dobson, Protein folding and misfolding, Nature 426 (2003) 884–890.

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