Study of a Bifunctional AÎ² Aggregation Inhibitor ... - ACS Publications

Subscriber access provided by MAHIDOL UNIVERSITY (UniNet)

Article

The Study of a Bifunctional A# Aggregation Inhibitor with the Abilities of Anti-amyloid-# and Copper Chelation Qian Zhang, Xiaoyu Hu, Wei Wang, and Zhi Yuan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01603 • Publication Date (Web): 12 Jan 2016 Downloaded from http://pubs.acs.org on January 18, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

The Study of a Bifunctional Aβ Aggregation Inhibitor with the Abilities of Anti-amyloid-β and Copper Chelation Qian Zhang,a Xiaoyu Hu,a Wei Wang,a Zhi Yuan* a,b a

Key Laboratory of Functional Polymer Materials of Ministry of Education, Institute of Polymer Chemistry, Nankai University, Tianjin 300071, China b

Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, China

KEYWORDS: Alzheimer’s disease, amyloid-β peptide, copper ion, chelate, peptide inhibitor

ABSTRACT:

In

this

study,

a

bifunctional

Aβ

aggregation

inhibitor

peptide,

GGHRYYAAFFARR (GR), with the abilities to bind copper and anti-amyloid was designed to inhibit the neurotoxicity of the Aβ-Cu(II) complex. The thioflavin T (ThT) assay, turbidimetric analysis, transmission electron microscopy (TEM), and (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) (MTT) assay were used to study its potential inhibitory effect on Aβ aggregation. Our findings indicate that GGH was the specific chelating sequence and that the RYYAAFFARR (RR) component acted as an aggregation inhibitor. More importantly, GR significantly decreased the cytotoxicity of the Aβ-Cu(II) complex. The cell viability improved to

ACS Paragon Plus Environment

1

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 26

88%, which was higher than with the single functional peptide GGH and RR by 39% and 20%, respectively. Moreover, the qualitative effect of Cu(II) on the Aβ-Cu(II) complex was also studied. Our results indicate that Cu(II) induces the formation of the β-sheet structure with a subequimolar Cu(II):Aβ molar ratio (0.25 : 1) but led to increased ROS production at a supraequimolar ratio.

Introduction Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases. According to the amyloid cascade hypothesis, the amyloid-β peptides (Aβ) aggregate and the formation of amyloid plaques are the main etiologic cause of AD1,2. During in this process, several types of soluble Aβ oligomers are formed3, 4, and they are among the most toxic species5, 6 because of their binding and disruption of the lipid cell membranes7-9. Based on this hypothesis, several modulating molecules,10 such as curcumin,11,12 EGCG,13-15 and peptides,16 have been used to suppress the aggregation of Aβ at its early stages to, ultimately, inhibit the formation of oligomers. Compared with other inhibitors, peptide inhibitor, because of their diverse structure, could specific bind to Aβ and inhibit the aggregation of Aβ, thereby preventing neuronal toxicity induced by the β-sheet structure. Among them, LPFFD and KLVFF were most widely studied and they were proved to inhibit Aβ-mediated neurotoxicity and Aβ deposition in vivo and improve behavioral deficiencies in animal models.17-20 Moreover, Ac-LVFFARK-NH2 (LK7)21 and N-methylated Aβ25-3522 have also described as adequate peptide inhibitors. Over the last decade, several investigations have demonstrated that the levels of some transition metal ions, such as Cu+/2+, Zn2+, and Fe2+/3+, are elevated in AD brain tissue and enriched in amyloid plaques.23,24 Among these ions, copper, due to its redox and catalysis potential, has been widely and deeply studied.25,26 Although the exact biochemical and


2

Page 3 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

pathophysiologic significance of the copper ion in Aβ aggregation and its toxicity are still disputed,27 there is general consensus that the copper ion participate in two processes related to AD pathology: accelerating Aβ aggregation by binding with Asp1, Ala2, His6, His13 or His14 in Aβ28 and catalyzing the production of neurotoxic reactive oxygen species (ROS), including oxygen free radicals and hydrogen peroxide (H2O2).29 Based on these findings, metal-ion chelators have been hypothesized as a potential method for AD therapy. Bush et al. first used clioquinol (CQ) and 8-hydroxy quinolone (PBT2) to chelate copper, and in turn, demetallated and dissolved the plaques and ameliorated its neurotoxicity both in vitro and in vivo.30,31 Qu et al. sensitively modified CQ on some inorganic nanoparticles and successfully prevented the aggregation of Aβ induced by Cu2+ and decreased ROS toxicity.32 However, even if the excess copper ion was chelated and the production of ROS was suppressed, the excess soluble Aβ that extracellularly accumulated still tend to aggregate to the most cytotoxic oligomers, protofibrils, and, ultimately, the fibrils.33 These findings suggest that chelation alone seems insufficient for AD treatment and the more effective modulating molecule should possess two functions: chelation of the copper ion and inhibition of Aβ aggregation. In 2012, Hureau and Faller et al. found that the Aβ12–20 and Aβ13–20, a combination of a peptide inhibitor and copper chelator, could inhibit Aβ aggregation, prevent H2O2 formation, and decrease the its toxic effects in neuronal cell culture. Within Aβ12–20 and Aβ13–20, Aβ1620(KLVFF) was the inhibitor sequence and both Aβ12-15 and Aβ13-15 was the Cu(II) binding site.34 Nevertheless, Aβ16-20(KLVFF), as the hydrophobic core for Aβ aggregation, has a strong tendency to self-assemble and form a β-sheet structure on its own,35 which would significantly interfere with the interaction between the peptide and Aβ monomer.


3

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 26

In our previous work, 36,37 an active decapeptide inhibitor RR (RYYAAFFARR) was designed, and it has been proved that RR can interact with Aβ through the hydrophobic interaction, electrostatic interactions and the presumably hydrogen bonding, which made it exhibit a 75% inhibition of Aβ40 fibrillation at an equimolar concentration and a near complete inhibition at a molar ratio of 1:4 (Aβ/RR), and it could be embedded into the β-sheet structure of Aβ fibril and disaggregate the fibril into nanorod-like fragments.38 More importantly, multiple arginine residues in both ends of RR increased the repulsion between each other, and effectively prevented self-assemble. In the present study, we designed a bifunctional Aβ aggregation inhibitor GGHRYYAAFFARR (GR), in which RYYAAFFARR (RR) acted as an aggregation inhibitor and GGH had chelator function for Cu(II) ions (Scheme 1). After evaluating the influence of the stoichiometric level of Cu(II) ions on Aβ aggregation and ROS production, the inhibitory effect of GR on Aβ aggregation and cytotoxicity with/without Cu(II) was assessed by the thioflavin T (ThT) fluorescence assay, transmission electron microscopy (TEM), coumarin-3carboxylic acid (CCA) fluorescence assay, and cell viability assay. Our findings may help guide the design of multi-functional inhibitors of Aβ aggregation and open doors to novel AD treatments.


4

Page 5 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Scheme 1. The amino acids sequences of GR and the possible binding sites to Aβ and Cu(II) ions

Materials and Methods Materials. 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP, 99%) and thioflavin T (ThT, 75%) were purchased from Heowns (China). Aβ40(95%), RR (Ac-RYYAAFFARR-NH2, 95%), GGH (95%) and GR (GGHRYYAAFFARR-NH2, 95%) were obtained from GL Biochem Ltd. (Shanghai, China). Coumarin-3-carboxylic acid (CCA) and copper chloride (CuCl2) were purchased from Alfa Aesar. 2-(4-(2-hydroxyethyl)-1-piperazinyl) ethanesulfonic acid (HEPES), N-(2-Acetamido)-2aminoethanesulfonic acid (ACES) and L-Ascorbic acid were purchased from DingGuo Biotech Ltd. (Beijing, China). The cell lines PC12 were obtained from Cell Bank of Chinese Academy of Science. All the cell culture reagents were purchased from GIBCO (Grand land, NY) unless otherwise indicated. The other reagents were commercially available and used without purification. Aβ Pretreatment and Sample Solution Preparation. Aβ40 was pretreated by HFIP as described in the literature.39 First, Aβ40 was dissolved in HFIP to 1.0 mg/ml at room temperature for at least 2 h for disaggregation. Then, HFIP was evaporated off under N2 and the remaining white film was lyophilized overnight. Finally, the peptide was stored in refrigerator at -20 ºC. To prepare the fresh Aβ monomer solution, Aβ was dissolved with 10 mM NaOH solution and sonicated for 1 min, and then diluted in 10 mM HEPES (pH 7.4) for different concentrations.


5

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 26

In this study, all the aqueous solutions were made by Milli-Elix water filtered through 0.22 µm filters. For ThT assay, TEM, turbidimetric analysis and CD measurements, we incubated Aβ (20 µM) with 0, 5, 10, 20, 40 and 80 µM of CuCl2 in 10 mM HEPES (pH 7.4) at 37 ºC with continuously shaking at 120 rpm for 3 days. For each peptide inhibitor (GGH, RR, GR), Aβ (20 µM), CuCl2 (5µM) and inhibitor (20µM) were co-incubated in HEPES (10mM, pH 7.4) at the same conditions. To prepare the sample for MTT assay, the solution of Aβ with/without different concentration of CuCl2 and peptide inhibitor (GGH, RR, GR) was diluted in DMEM containing 10% FBS and incubated at 37ºC for 1 day with 120rpm shaking before added to cells. Thioflavin T Fluorescence Assay. ThT fluorescence assay, used to detect the β-sheet content of Aβ aggregates, was carried out by an F-7000 Fluorescent spectrophotometer (Hitachi, Japan). For most ThT experiments, 1 mL incubated sample solution was treated with 10 µL ThT solutions with the concentration of 1 mg/ml and shaken for 1 min in the dark. For experiments of Aβ co-incubated with GGH, RR or GR, the protocols were applied as shown in pervious paper.37 Briefly, a volume of 100 µL sample liquids were mixed with 900 µL ThT buffer solutions of 10 µM. All measurements were operated with excitation at 440 nm (slit width=5.0 nm) and emission at 485nm (slit width=5.0 nm) in 1×1 cm2quartz cuvette. Three measurements were performed and the data were averaged. Turbidimetric analysis. Aβ aggregation was observed by turbidimetric measurement which was described by Huang et al.40 The solution samples above were added to a flat bottom 96-well plate (Greiner bio-one, Germany) with 200 µL for each. Then, turbidity absorbance intensity at 400 nm was recorded


6

Page 7 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

using an Enspire microplate reader (PerkinElmer LLC, USA) at 25 ºC. The absorbance intensity measured in an average of six wells was adopted for further analysis. Transmission Electron Microscopy. All experiments were performed by using TEM (FEI, Tecnai G2F20, USA) with the accelerating voltage of 200 kV. To prepare samples, 20 µL droplet of incubated solutions was dropped onto carbon-coated copper grids (200 mesh) and dried for 2 min. The excess fluid was removed by filtered paper on the opposite side of the film. Then, 10 µL phosphotungstic acid (1%, w/w) was added and kept for another 2 min for negatively staining and the excess solution was blotted up. The grids were stored in a vacuum drier before use. Circular Dichroism Measurements. Circular dichroism (CD) spectra were carried out using a MOS-450 CD Spectrometer (BioLogic, France). The spectra were recorded in a 0.1 cm quartz cuvette with 10 mm path length at room temperature. Points in a range between 190 and 260 nm were taken at 0.5 nm, with an integration time of 1.0 s. The spectra were smoothed using the noise reducing option in the software supplied by the vendor. The background signal from the HEPES buffer has been subtracted and the data were averaged after being measured three times. Measurement of HO˙ in vitro. Coumarin-3-carboxylic acid (CCA) was used for assessment of hydroxyl radicals produced in Cu-Aβ solutions containing L-Ascorbic acid (Asc). The reaction generates 7-OH-CCA, which could be detected by fluorescence, could be used as an indicator of ROS level. The solution of 2 mM CCA and the solution of 400 mM Asc dissolved in 10 mM HEPES (pH=7.4) were prepared just before use. The samples for fluorescent tests, made up of 0.5 mL protein solutions, 0.25 mL CCA and 0.25 mL Asc solution, were incubated at 37 ºC with shaking rate of 120 rpm for 1 h


7

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 26

and kept in dark. After that, the fluorescent intensity at 450 nm (slit width=5.0 nm) upon excitation at 388 nm (slit width=5.0 nm) was recorded by an F-7000 Fluorescent spectrophotometer (Hitachi, Japan). Results are the average of three independent experiments each following normalization (n = 3). Isothermal Titration Calorimetry. ITC titrations were carried out on a MicroCal VP-ITC (GE, USA) at 37 ºC. Copper ions and peptides (GGH, RR or GR) were dissolved in the same 20 mM ACES buffer (pH 7.4, 100 mM NaCl). All solutions were degassed for at least 15 minutes to remove air bubbles before titration. The CuCl2 buffer solution was in the syringe while peptides solution was loaded in the cell. Stirrer speed was 307 rpm. An initial delay of 120 s and an initial injection of 2 mL were adopted. The interval between two adjacent injections was 210 s. The heat of dilution was determined and subtracted from titration data. Then, Origin 7.0 software package supplied by manufacturer was applied to analyze the data using one-site binding model. For titration of CuCl2 to GGH, CuCl2 of 5 mM was injected to 0.5 mM of GGH with each droplet of 10 mL for 28 times. For other experiments, CuCl2 of 2.5 mM was added to 0.5 mM of RR or GR with each one of 5 mL for 34 droplets. MALDI-TOF mass spectrometry. The peptide of GR was dissolved in 10 mM HEPES (pH 7.4) to reach the concentration of 20 mM and cultured at 37 ºC with shaking for 24 hours. Then, the measurement was conducted using MALDI-TOF mass spectrometer (Bruker, Autoflex III LRF200-CID, Germany) to test whether the GR would self-assemble. Cell Toxicity Assays.


8

Page 9 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Highly differentiated rat pheochromocytoma (PC-12) cells were cultured in a medium of DMEM with 10% fetal calf serum at 37 °C in an atmosphere of 5% CO2. For MTT assays, cells were plated in 96-well plates at a density of 8×103 cells per well for 24 h. Then different kinds of pre-incubated sample solution were introduced to the plates and co-cultured for 48h. Afterwards, the cells were treated with 20 mL MTT（5 mg/mL in PBS）for 4 h at 37 ºC, and then were lysed in DMSO at room temperature in the dark. Absorbance intensity at 490 nm was measured with an ELISA reader. All the experiments were carried out with five replicates. Results and Discussion The effects of Cu(II) ions on Aβ aggregation With the development of the metal ion hypothesis, more and more research have focused on the role of copper ions in AD pathogenesis as well as the identification of a suitable metal chelator as a possible therapeutic reagent.41-43 However, the mechanistic role of copper ions in Aβ aggregation and Aβ-Cu(II) cytotoxicity has yet to be elucidated.40,44-47 First, we studied the effect of Cu(II) levels on Aβ aggregation and cytotoxicity of the Aβ-Cu(II) complex. Turbidimetric analysis was first used to study the process of Cu(II)-induced Aβ aggregation. The effects of Cu(II) ions at different stoichiometric levels on the turbidity are shown in Fig.1A. During the observation period, the solution of Aβ alone sample (20 µM) only developed a small increase in turbidity. In contrast, in the presence of Cu(II) ions, the turbidity of the Aβ solution significantly increased with the increasing concentration of Cu(II) ions, indicating the promotion effect of Cu(II) ions on Aβ aggregation. Subsequently, the Thioflavin T (ThT) assay was used to investigate the influence of the stoichiometric level of Cu(II) ions on Aβ aggregation. Fig.1B exhibits that the fluorescence intensity increases quickly at the early stage with a sub-equimolar Cu(II) ions (Cu(II)/Aβ = 0.25:1), indicating that the Cu(II) ions accelerated the aggregation of


9

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 26

Aβ and induced the formation of the β-sheet structure. However, with the increasing addition of Cu(II) ion, the fluorescence intensity and increasing rate rapidly decreased to the extent that the fluorescence signal was undetectable at a molar ratio of 4:1(Cu(II)/Aβ). In the CD spectra, it was also easy to see the reduction of the strength of peak in 210 nm, which indicated the decrease of β-sheet structure (Fig.S1). Combined with the turbidimetric analysis results, we hypothesized that the aggregates induced by Cu(II) ions was not the β-sheet structure, so TEM was used to directly study its morphology. As shown in Fig.1C, when Cu(II) was added at a molar ratio of 0.25:1, the fibril formed that was similar with Aβ in shape but low in amount. With increasing concentration of Cu(II) ions, fibril structure was obviously reduced and spherical aggregates appeared. When the molar ratio of Cu(II)/Aβ was 4:1, fibrils completely disappeared and only spherical aggregates were observed. These results indicated that Cu(II) ions could not only accelerate the aggregation of Aβ forming β-sheet structure with a sub-stoichiometric level but also result in spherical aggregates at a supra-stoichiometric balance. These phenomena were reported in Sarell and Chen’s work as well.44,48 It is generally conjectured that these may result from the interaction of copper ions with Aβ, which is likely due to chelating and electrostatic interactions. At low concentrations, the copper ions can bind to a small amount of monomeric Aβ to accelerate the nucleation process, and, in turn, accelerate the formation of fibrils with βsheet structure. While, at a supra-equimolar quantity, numerous Aβ-Cu(II) complex can be formed and Aβ monomers gather together quickly through electrostatic interaction. This aggregation process was too fast to translate conformation of Aβ for forming the fibrils with regular β-sheet structure. Thus, the amorphous aggregates were subsequently generated.49 Another thing should be noticed that, besides of Pedersen’s work 27, the Aβ-Cu(II) comlex could form fribril at equimolar Cu(II)/Aβ in most of other researches, which was higher than our work


10

Page 11 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(1:0.25). This results indicated that the self-assembling process of Aβ-Cu(II) complex was very complicated and maybe influenced by lots of factors, such as, purity and concentration of Aβ, buffer species and some unknown factors.

Figure 1. (A) Monitoring the effects of Cu(II) ions on Aβ aggregation by turbidimetric analysis (B) β-sheet structure content in samples of Aβ-Cu(II) complex was monitored using the ThT fluorescence assay. The samples A and B were incubated with 20 µM Aβ monomer and 0, 5, 20, 80 µM CuCl2 for 3 days, respectively (n = 3). (C) Negative-stained TEM images of Aβ and AβCu(II) complex after 3 days incubation. Scale bars = 500 nm. It was also reported that ROS may be another important cause that induces cytotoxicity of the Aβ-Cu(II) complex.50 Thus, the quantity of ROS produced by Cu(II) ions and the cytotoxicity with different stoichiometric level of Cu(II) ions was measured by CCA fluorescence and MTT assay. For the CCA fluorescence (Fig.2A), the ROS quantity steadily increased with increasing concentrations of Cu(II) ions, and the Cu(II)-Aβ complex group was lower than the Cu(II) ions alone in every molar ratio. However, the MTT assay displayed a more complicated behavior. As shown in Fig.2B, the toxicity of Cu(II) ions alone was increased with increasing levels of Cu(II),


11

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 26

which was consistent with the CCA assay. The viability of PC12 cells treated with Aβ-Cu(II) complex increased at first but subsequently declined with increasing Cu(II) levels. When Aβ and Cu(II) reached the equimolar ratio (Cu(II) ions concentration was 20µM), the toxicity of AβCu(II) complex greatly increased and continued with additional Cu(II) ions. Our findings indicate that the cytotoxicity of the Aβ-Cu(II) complex is influenced by the content of the β-sheet structure and ROS at the same time. When Cu(II) ion was in a low concentration, such as 5 µM, the ROS level catalyzed by the complex was low as well in the system (Fig.2A), so its toxicity mainly attributed to the β-sheet aggregates (Fig.2B). As the Cu(II) ions slightly increased, the βsheet structure in the aggregates was greatly reduced (Fig.1B) and the amorphous product showed a low toxicity, and this conclusion was consist with Sarell’s research 44. When the Cu(II) ion concentration was increased to more than equimolar quantities, its cytotoxicity suddenly increased, which likely came from the ROS catalyzed by it. To our knowledge, there was few study focus on the effect of aggregation morphology and ROS production of Aβ-Cu(II) complex on cell toxicity synthetically before.

Figure.2 (A)ROS production was quantified by measurements of CCA fluorescence for Cu(II) alone and Aβ-Cu(II) complex solutions (n = 3).(B) Cytotoxicity of Cu(II) and Aβ-Cu(II) complex to PC12 cells. The concentration of Aβ in all samples was 20 µM. Cell viability was


12

Page 13 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

determined using the MTT assay and is shown as a percentage of the untreated cells (n = 5). Statistically significant differences are indicated with asterisks: *p < 0.05. To further explore the cytotoxicity of the amorphous aggregates, we introduced nickel ions (Ni(II)) as a research model in place of copper. It was previously reported in Li’s work that nickel could reproduce copper’s effects on Aβ40 conformation and aggregation.51 However, because it cannot catalyze the production of ROS in experimental conditions, the nickel model could serve as a negative control for ROS-mediated cytotoxicity. Indeed, Aβ formation was inducible by Ni(II) to form amorphous aggregates with a high molar ratio of Ni(II)/Aβ (Fig.S2), and these aggregates exhibited low toxicity on PC12 cells (Fig.S3). These results were consistent with our above hypothesis and Li’s work. The effects of GR on Aβ aggregation in the absence of Cu(II) ions Although metal chelation as a possible therapeutic strategy for AD has been intensively studied, Aβ itself has great potential to self-assemble and form the toxic aggregates even in the absence of copper ions. Therefore, metal chelation alone is likely insufficient to modulate the aggregation of Aβ and reduce if not inhibit its cytotoxicity. Therefore, we designed a bifunctional peptide inhibitor GR, which was expected offering both anti-amyloid effect and the chelation of metal ions. The decapeptide RR, which was designed in our previous study32 and shown to have high efficiency in inhibiting the aggregation of Aβ and its cytotoxicity, was put into the bifunctional inhibitor GR to act as an inhibitor. For chelating, a tripeptide GGH was chosen, which was reported in previous studies to chelate Cu(II) ion with -NH2 of Gly1, -NHfrom the Gly1-Gly2 and Gly2-His3 peptide bond and Nδ atoms of the imidazole ring of His3 (Scheme 1).52


13

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 26

It had been shown in our previous work that multiple arginines moieties in RR could increase the repulsion between each other and thereby prevent its self-aggregation.37 Thus, we studied the self-assembling behavior of GR by MALDI-TOF spectra in the present study. As shown in Fig.S4, a single peak appeared at 1570.8 m/z after 24 hours incubation, indicating that no oligomer was formed by GR itself. In addition, the MTT assay demonstrated that GR was not cytotoxic (Fig.S5). This feature was very important for GR in the successive application as an AD treatment reagent. To test the influence of GR on Aβ aggregation without Cu(II) ions, the thioflavin-T (ThT) fluorescence assay was first used. As shown in Fig.3A, in contrast to GGH, GR and RR could diminish the fluorescence intensity of Aβ to about 30% after 3 days of co-incubation and both showed prominent inhibitory activity at 68% and 73%, respectively. These results were supported by TEM imaging (Fig.3C). The GGH treated group displayed mature fibrils as Aβ, and the RR treated group only developed short fibrils (about 500-nanometer-long), which was in line with our previous findings. For the GR treated group, few short fibrils and some amorphous aggregates were detected. Subsequently, the MTT assay was used to explore their cytotoxicity. As shown in Fig.3B, the GGH treated group had similar cell viability with Aβ, but the addition of RR and GR greatly improved the viability of PC12 cells to over 90%. Taken together, these results indicated that GR could efficiently inhibit the aggregation of Aβ alone and greatly reduce its cytotoxicity.


14

Page 15 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure.3 (A) Monitoring the effects of GGH, RR, and GR on Aβ aggregation using a ThT fluorescence assay for 3 days (n = 3). (B) Viability of PC12 cells treated with Aβ and other peptides for 48 h. Cell viability was measured with the MTT assay and viability is shown as a percentage of the untreated cells (n = 5; *p< 0.05). All samples of A and B were incubated with 20 µM Aβ and 20 µM GGH, RR, or GR, respectively. (C) Negative-stained TEM images of Aβ with GGH, RR, or GR after 3 days incubation. Scale bars = 500 nm. The effects of GR on Cu(II)-induced Aβ aggregation Since Aβ is predominantly as a fibril type in amyloid plaques in AD brains, based on above studies, we chose a molar ratio of 0.25:1(Cu(II)/Aβ) at which the aggregation of Cu-Aβ complex exhibited fibrils that were most similar to that found in AD brain to evaluate the influence of GR on Cu(II)-induced Aβ aggregation and cytotoxicity. In the turbidimetric analysis (Fig.4A), GGH greatly decreased the turbidity of the Aβ-Cu(II) complex to make it approach that of Aβ alone, and this illustrated that equimolar GGH could


15

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 26

inhibit the inducing effect of Cu(II) ions on Aβ aggregation. RR and GR groups displayed a great increase in turbidity, which was in line with the control experiments lacking Cu(II) (See Fig.S6). We hypothesized that the addition of GR or RR inhibited the fibrillar aggregation of Aβ and formed some amorphous aggregates, which exhibited strong light scattering. In the ThT fluorescence assay (Fig.4B), the GR treated group exhibited the lowest β-sheet content, which could achieve an inhibition of approximately 68%, in keeping with the inhibition efficiency of GR in the Aβ only system. This favorable result indicated that GR could chelate the Cu(II) ions and inhibit the Cu(II)-induced aggregation of Aβ in the early stage, which was confirmed by ITC (Table.S1, Fig.S7). More importantly, GR could also inhibit Aβ self-assembly, resulting in the prominent inhibitory effect on the formation of the β-sheet structure, which was also confirmed by CD measurement (Fig.S8). Distinct from GR, GGH addition could reduce the increasing rate of fluorescence intensity over the first 12 hours. However, the final β-sheet structure in the system was not inhibited. This demonstrated that GGH could inhibit the Cu(II)induced aggregation by chelating copper ions (Table.S1, Fig.S9), but it could not inhibit the fibril formation of Aβ itself (Fig.3A). The final fluorescence intensity was lower than Aβ fibrils alone and higher than the Aβ-Cu(II) complex. This may be due to the decline of Cu(II)-induced amorphous aggregation and, consequently, the increase in β-sheet content from Aβ self-assembly. The similar phenomenon was also observed in Sharma’s work. In their research, the presence of ion-chelator had a significant effect on the Cu2+-mediated oligomerization of Aβ42 and promoted the formation of larger Aβ42 aggregates.53 The addition of RR could inhibit the aggregation by approximately 44%, which was lower than that by GR (68%) and lower than the Aβ only system (73%, Fig.3A) due to the present of Cu(II) ions. Moreover, it is remarkable that the interaction between RR and Aβ may also be influenced


16

Page 17 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

by the nucleation induced by Cu(II) ions, so the initial rate of Aβ aggregation was slightly lower than that of the Aβ-Cu(II) complex. On the other hand, ITC examination revealed that RR had no interaction with Cu(II) (Fig.S10), so its aggregating rate in the early stage was faster than in the GGH treated group. In Jensen’s work, Aβ12-20 and Aβ13-20 were also used as bifunctional peptides for inhibiting aggregation of Aβ-Cu(II) complex. Although Aβ12-20 showed the protecting effect on SH-SY5Y cells in the present of Aβ42-Cu(II) complex, it was not able to inhibit the aggregation of Aβ40 alone and Aβ40-Cu(II) complex.34 TEM was used to directly observe the production of different groups. As shown in Fig.4C, the GGH group could form good fibrils that were similar to Aβ alone and exhibited a low turbidity. However, the RR group exhibited some amorphous aggregates and short fibrils. The GR group displayed some spherical aggregates with a diameter of ≈ 100 nm and a few short fibrils. These were consistent with the turbidimetric results that the amorphous and spherical aggregates had strong light scattering.


17

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 26

Figure.4 (A) Monitoring the effects of peptides on Aβ-Cu(II) complex aggregation using turbidimetric analysis (B) β-sheet structure content in the samples of Aβ-Cu(II) complex incubated with different peptides was monitored by ThT fluorescence assay. The samples of A and B were incubated with 20 µM Aβ, 5µM CuCl2, and 20 µM peptides for 3 days (n=3). (C) Negative-stained TEM images of Aβ-Cu(II) complex incubated with different peptides for 3 days. Scale bars = 500 nm. Finally, the MTT assay was used to study the effects of GR on inhibiting the cytotoxicity of Aβ-Cu(II) complex. The results of MTT assay are shown in Fig.5. The cell viability of the Aβ group was 62% and the addition of Cu(II) ions greatly reduced cell viability to 35%. Combined with the results displayed in Fig.2A, the ROS production of this molar ratio Aβ-Cu(II) complex (Cu(II)/Aβ = 0.25:1) showed a similarly low level to that of Aβ alone. Thus, we hypothesized that ROS was not the primary reason for the increasing toxicity of the Aβ-Cu(II) complex at this molar ratio and that it may come from two components. On the one hand, the redox ability of copper ions may be altered after the formation of the Aβ-Cu(II) complex; on the other hand, the Aβ itself may reduce the ROS level catalyzed by Cu(II), a finding that has been reported in Mayes and Jiang’s previous work.29,54 These conjectures will be tested in our future studies. Thus, the cytotoxicity of the Aβ-Cu(II) complex is still mostly attributed to the β-sheet structure induced by Cu(II) ions. Therefore, when GGH was added, the cell viability could be significantly improved to 49% because of its ability to chelate Cu(II) ions and, in turn, inhibit Aβ aggregation. When GR, which has the ability to chelate Cu(II) ions and have anti-amyloid effect, was added, the cell viability improved to 88%, significantly better than that of the RR group(68%). This improvement illustrated the wonderful properties of the bifunctional inhibitor in reducing the


18

Page 19 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

cytotoxicity of Aβ-Cu(II) complex and its advantage over the single functional inhibitor GGH and RR.

Figure.5 Viability of PC12 cells treated with Aβ alone, Aβ-Cu(II) complex or Aβ-Cu(II) complex co-incubated with different peptides for 48 h. Cell viability was determined using the MTT assay and is shown as a percentage of the untreated cells (n = 5). Statistical significance level is expressed by asterisks (control as Aβ group, *p < 0.05) and pound (control as Aβ-Cu(II) group, #p< 0.05). Conclusions In the present study, a bifunctional Aβ aggregation inhibitor peptide GR, which can both chelate the Cu(II) ions and inhibited the β-sheet structure formation, was designed, and the influence of stoichiometric levels of Cu(II) ions on Aβ aggregation, ROS production, and cytotoxicity was evaluated. Our results indicate that Cu(II) ions can induce the formation of toxic β-sheet structures at a sub-stoichiometric level, but result in a low-toxic spherical aggregation at a supra-stoichiometric one. GR could slow the aggregation of Aβ with/without Cu(II) ions, and, in turn, inhibit the formation of mature fibrils. More importantly, GR reduced the toxicity of the Aβ-Cu(II) complex on PC12 cells, and the cell viability improved to 88%,


19

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 26

which was higher than the single functional peptide GGH and RR, which yielded cell viabilities of 49% and 68%, respectively. Further structural studies at high resolution, such as NMR and docking calculations,14,55 are still needed to fully understand the role of GR in this process, and the relative work will be done in the future work . Overall, the bifunctional Aβ aggregation inhibitor may be an effective and promising therapeutic reagent for AD.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXX CD spectrometry of Aβ and Aβ-Cu(II) complex, MALDI-TOF mass spectrometry of fresh GR monomer and incubated GR, the turbidimetric analysis of GR/RR and Aβ solution, TEM images of Aβ-Ni(II) complex, cytotoxicity of Ni(II) and Aβ-Ni(II) complex to PC12 cells, and the calorimetric titration of Cu(II) to GGH, Cu(II) to RR and Cu(II) to GR. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Notes The authors declare no competing financial interest.


20

Page 21 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

ACKNOWLEDGMENT We gratefully acknowledge the National Natural Science Foundation of China (51273094) and PCSIRT (IRT1257) for support of this work. We also thank Prof. Deling Kong and Jun Yang for their help on cell-related experiments.

REFERENCES (1)

Mattson, M. P. Nature 2004, 430, 631-639.

(2)

Karran, E.; Mercken, M.; De Strooper, B. Nat. Rev. Drug Discov. 2011, 10, 698-712.

(3)

Suzuki, Y.; Brender, J. R.; Soper, M. T.; Krishnamoorthy, J.; Zhou, Y. L.; Ruotolo, B. T.;

Kotov, N. A.; Ramamoorthy, A.; Marsh, E. N. G. Biochemistry 2013, 52, 1903-1912. (4)

Vivekanandan, S.; Brender, J. R.; Lee, S. Y.; Ramamoorthy, A. Biochem. Biophys. Res.

Commun. 2011, 411, 312-316. (5)

Haass, C.; Selkoe, D. J. Nat. Rev. Mol. Cell Biol. 2007, 8, 101-112.

(6)

Kayed, R.; Head, E.; Thompson, J. L.; McIntire, T. M.; Milton, S. C.; Cotman, C. W.;

Glabe, C. G. Science 2003, 300, 486-489. (7)

Korshavn, K. J.; Bhunia, A.; Lim, M. H.; Ramamoorthy, A. Chem Commun. 2016. ASAP.

DOI: 10.1039/C5CC08634E. (8)

Sciacca, M. F. M.; Kotler, S. A.; Brender, J. R.; Chen, J.; Lee, D. K.; Ramamoorthy, A.

Biophys. J. 2012, 103, 702-710.


21

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(9)

Page 22 of 26

Kotler, S. A.; Walsh, P.; Brender, J. R.; Ramamoorthy, A. Chem. Soc. Rev. 2014, 43,

6692-6700. (10) Liu, L.; Zhang, L.; Niu, L.; Xu, M.; Mao, X.; Yang, Y.; Wang, C. ACS Nano 2011, 5, 6001-6007. (11) Reinke, A. A.; Gestwicki, J. E. Chem. Biol. Drug Des. 2007, 70, 206-215. (12) Yang, F.; Lim, G. P.; Begum, A. N.; Ubeda, O. J.; Simmons, M. R.; Ambegaokar, S. S.; Chen, P. P.; Kayed, R.; Glabe, C. G.; Frautschy, S. A.; Cole, G. M. J. Biol. Chem. 2005, 280, 5892-5901. (13) Liu, F.-F.; Dong, X.-Y.; He, L.; Middelberg, A. P. J.; Sun, Y. J. Phys. Chem. B. 2011, 115, 11879-11887. (14) Hyung, S. J.; DeToma, A. S.; Brender, J. R.; Lee, S.; Vivekanandan, S.; Kochi, A.; Choi, J. S.; Ramamoorthy, A.; Ruotolo, B. T.; Lim, M. H. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 3743-3748. (15) Rezai-Zadeh, K.; Shytle, D.; Sun, N.; Mori, T.; Hou, H.; Jeanniton, D.; Ehrhart, J.; Townsend, K.; Zeng, J.; Morgan, D.; Hardy, J.; Town, T.; Tan, J. J. Neurosci. 2005, 25, 88078814. (16) Richman, M.; Wilk, S.; Chemerovski, M.; Warmlander, S.; Wahlstrom, A.; Graslund, A.; Rahimipour, S. J. Am. Chem. Soc. 2013, 135, 3474-3484. (17) Soto, C.; Sigurdsson, E. M.; Morelli, L.; Kumar, R. A.; Castano, E. M.; Frangione, B. Nat. Med. 1998, 4, 822-826.


22

Page 23 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(18) Adessi, C.; Frossard, M. J.; Boissard, C.; Fraga, S.; Bieler, S.; Ruckle, T.; Vilbois, F.; Robinson, S. M.; Mutter, M.; Banks, W. A.; Soto, C. J. Biol. Chem. 2003, 278, 13905-13911. (19) Permanne, B.; Adessi, C.; Saborio, G. P.; Fraga, S.; Frossard, M. J.; Van Dorpe, J.; Dewachter, I.; Banks, W. A.; Van Leuven, F.; Soto, C. Faseb J. 2002, 16, 860-862. (20) Lowe, T. L.; Strzelec, A.; Kiessling, L. L.; Murphy, R. M. Biochemistry 2001, 40, 78827889. (21) Xiong, N.; Dong, X. Y.; Zheng, J.; Liu, F. F.; Sun, Y. ACS Appl Mater Interfaces 2015, 7, 5650-5662. (22) Hughes, E.; Burke, R. M.; Doig, A. J. J. Biol. Chem. 2000, 275, 25109-25115. (23) Kepp, K. P. Chem. Rev. 2012, 112, 5193-5239. (24) Faller, P.; Hureau, C.; Berthoumieu, O. Inorg. Chem. 2013, 52, 12193-12206. (25) Eskici, G.; Axelsen, P. H. Biochemistry 2012, 51, 6289-6311. (26) Savelieff, M. G.; Liu, Y.; Senthamarai, R. R. P.; Korshavn, K. J.; Lee, H. J.; Ramamoorthy, A.; Lim, M. H. Chem Commun. 2014, 50, 5301-5303. (27) Pedersen, J. T.; Ostergaard, J.; Rozlosnik, N.; Gammelgaard, B.; Heegaard, N. H. H. J. Biol. Chem. 2011, 286, 26952-26963. (28) Hureau, C.; Dorlet, P. Coord. Chem. Rev. 2012, 256, 2175-2187. (29) Mayes, J.; Tinker-Mill, C.; Kolosov, O.; Zhang, H.; Tabner, B. J.; Allsop, D. J. Biol. Chem. 2014, 289, 12052-12062.


23

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 26

(30) Ritchie, C. W.; Bush, A. I.; Mackinnon, A.; Macfarlane, S.; Mastwyk, M.; MacGregor, L.; Kiers, L.; Cherny, R.; Li, Q. X.; Tammer, A.; Carrington, D.; Mavros, C.; Volitakis, I.; Xilinas, M.; Ames, D.; Davis, S.; Volitakis, I.; Xilinas, M.; Ames, D.; Davis, S.; Beyreuther, K.; Tanzi, R. E.; Masters, C. L. Arch. Neurol. 2003, 60, 1685-1691. (31) Lannfelt, L.; Blennow, K.; Zetterberg, H.; Batsman, S.; Ames, D.; Hrrison, J.; Masters, C. L.; Targum, S.; Bush, A. I.; Murdoch, R.; Wilson, J.; Ritchie, C. W.; Grp, P. E. S. Lancet Neurol. 2008, 7, 779-786. (32) Shi, P.; Li, M.; Ren, J.; Qu, X. Adv. Funct. Mater. 2013, 23, 5412-5419. (33) Jan, A.; Adolfsson, O.; Allaman, I.; Buccarello, A. L.; Magistretti, P. J.; Pfeifer, A.; Muhs, A.; Lashuel, H. A. J. Biol. Chem. 2011, 286, 8585-8596. (34) Jensen, M.; Canning, A.; Chiha, S.; Bouquerel, P.; Pedersen, J. T.; Ostergaard, J.; Cuvillier, O.; Sasaki, I.; Hureau, C.; Faller, P. Chem.-Eur. J. 2012, 18, 4836-4839. (35) Krysmann, M. J.; Castelletto, V.; Kelarakis, A.; Hamley, I. W.; Hule, R. A.; Pochan, D. J. Biochemistry 2008, 47, 4597-4605. (36) Liu, J.; Yuan, Z. Chin. J. Polym. Sci. 2015, 33, 1009-1017. (37) Liu, J.; Wang, W.; Zhang, Q.; Zhang, S.; Yuan, Z. Biomacromolecules 2014, 15, 931939. (38) Zhang, Q.; Liu, J.; Hu, X.; Wang, W.; Yuan, Z. ACS Macro Lett. 2015, 339-342. (39) Cao, M.; Han, Y.; Wang, J.; Wang, Y. J. Phys. Chem. B. 2007, 111, 13436-13443.


24

Page 25 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(40) Huang, X. D.; Atwood, C. S.; Moir, R. D.; Hartshorn, M. A.; Tanzi, R. E.; Bush, A. I. J. Biol. Inorg. Chem. 2004, 9, 954-960. (41) Braymer, J. J.; Choi, J.-S.; DeToma, A. S.; Wang, C.; Nam, K.; Kampf, J. W.; Ramamoorthy, A.; Lim, M. H. Inorg. Chem. 2011, 50, 10724-10734. (42) DeToma, A. S.; Krishnamoorthy, J.; Nam, Y.; Lee, H. J.; Brender, J. R.; Kochi, A.; Lee, D.; Onnis, V.; Congiu, C.; Manfredini, S.; Vertuani, S.; Balboni, G.; Ramamoorthy, A.; Lim, M. H. Chem. Sci. 2014, 5, 4851-4862. (43) Jones, M. R.; Service, E. L.; Thompson, J. R.; Wang, M. C. P.; Kimsey, I. J.; DeToma, A. S.; Ramamoorthy, A.; Lim, M. H.; Storr, T. Metallomics 2012, 4, 910-920. (44) Sarell, C. J.; Wilkinson, S. R.; Viles, J. H. J. Biol. Chem. 2010, 285, 41533-41540. (45) Tougu, V.; Karafin, A.; Zovo, K.; Chung, R. S.; Howells, C.; West, A. K.; Palumaa, P. J. Neurochem. 2009, 110, 1784-1795. (46) Ha, C.; Ryu, J.; Park, C. B. Biochemistry 2007, 46, 6118-6125. (47) Mold, M.; Ouro-Gnao, L.; Wieckowski, B. M.; Exley, C. Sci. Rep. 2013, 3, 1256. (48) Chen, W. T.; Liao, Y. H.; Yu, H. M.; Cheng, I. H.; Chen, Y. R. J. Biol. Chem. 2011, 286, 9646-9656. (49) Yoshiike, Y.; Tanemura, K.; Murayama, O.; Akagi, T.; Murayama, M.; Sato, S.; Sun, X.; Tanaka, N.; Takashima, A. J. Biol. Chem. 2001, 276, 32293-32299. (50) Li, M.; Liu, Z.; Ren, J. S.; Qu, X. G. Chem. Sci. 2012, 3, 868-873.


25

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 26

(51) Jin, L.; Wu, W. H.; Li, Q. Y.; Zhao, Y. F.; Li, Y. M. Nanoscale 2011, 3, 4746-4751. (52) Yang, W. R.; Jaramillo, D.; Gooding, J. J.; Hibbert, D. B.; Zhang, R.; Willett, G. D.; Fisher, K. J. Chem Commun. 2001, 1982-1983. (53) Sharma, A. K.; Kim, J.; Prior, J. T.; Hawco, N. J.; Rath, N. P.; Kim, J.; Mirica, L. M. Inorg Chem. 2014, 53, 11367-11376. (54) Jiang, D.; Li, X.; Liu, L.; Yagnik, G. B.; Zhou, F. J. Phys. Chem. B. 2010, 114, 48964903. (55) Kotler, S. A.; Brender, J. R.; Vivekanandan, S.; Suzuki, Y.; Yamamoto, K.; Monette, M.; Krishnamoorthy, J.; Walsh, P.; Cauble, M.; Holl, M. M. B.; Marsh, E. N. G.; Ramamoorthy, A. Sci Rep 2015, 5, 12.

Insert Table of Contents Graphic and Synopsis Here


26

Study of a Bifunctional AÎ² Aggregation Inhibitor ... - ACS Publications

Recommend Documents