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Effects of gold complexes on the assembly behavior of human islet amyloid polypeptide
Effects of gold complexes on the assembly behavior of human islet amyloid polypeptide Lei He, Dengsen Zhu, Cong Zhao, Xian Jia, Xuesong Wang, Weihong Du PII: DOI: Reference:
S0162-0134(15)30063-5 doi: 10.1016/j.jinorgbio.2015.08.020 JIB 9791
To appear in:
Journal of Inorganic Biochemistry
Received date: Revised date: Accepted date:
22 April 2015 9 August 2015 20 August 2015
Please cite this article as: Lei He, Dengsen Zhu, Cong Zhao, Xian Jia, Xuesong Wang, Weihong Du, Effects of gold complexes on the assembly behavior of human islet amyloid polypeptide, Journal of Inorganic Biochemistry (2015), doi: 10.1016/j.jinorgbio.2015.08.020
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Effects of gold complexes on the assembly behavior of human islet
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amyloid polypeptide
Lei He, Dengsen Zhu, Cong Zhao, Xian Jia, Xuesong Wang, Weihong Du*
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Department of Chemistry, Renmin University of China, Beijing, 100872
Keywords: hIAPP; assembly; gold complex; interaction; dimerization. *
Corresponding author. Tel/Fax: 8610-6251-2660/6444, Email:
[email protected] 1
ACCEPTED MANUSCRIPT Abstract Human islet amyloid polypeptide (hIAPP) is a well-known amyloid protein that
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is associated with type II diabetes. Inhibitors of this peptide include aromatic organic
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molecules, short peptides, and metal complexes, such as zinc, ruthenium and vanadium compounds. Various metal ions and their complexes affect the fibrillization of hIAPP in different action modes. However, the assembly mechanism of the peptide
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remains unclear. This study evaluated the inhibitory effects of three gold complexes
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with different nitrogen-containing aromatic ligands, namely, [Au(bipy)Cl2][PF6] (1), [Au(Ph2bpy)Cl2]Cl (2), and [Au(phen)Cl2]Cl (3), on the amyloid fibrillization of
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hIAPP. The complexes interacted with the peptide mainly through hydrophobic
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interaction and metal coordination. The concentration dependence of hIAPP
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aggregation on gold complex indicated that the assembly behavior of hIAPP is significantly affected by these compounds. The gold complexes inhibited peptide
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aggregation through dimerization and stabilized the peptide to monomers. Gold ion was found to be a key influencing factor of the binding mode and assembly behavior of hIAPP. The different effects of the complexes on peptide aggregation might be attributed to their special ligands. This study provided insights into the inhibitory mechanism of gold complexes against hIAPP fibrillization.
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ACCEPTED MANUSCRIPT Introduction Human islet amyloid polypeptide (hIAPP, amylin), a hormone comprising 37
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amino acids with strong amyloidogenic propensity, is synthesized by pancreatic
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β-cells and co-secreted with insulin [1]. Self-assembly of hIAPP into amyloid aggregates leads to the development of pancreatic β-cell dysfunction and death in the pathology of type II diabetes [2, 3]. The mechanism underlying hIAPP-induced β-cell
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toxicity and related processes, including receptor-mediated mechanism, localized
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inflammatory response, and IAPP-induced membrane damage, remain unclear [2, 4–7]. Thus, exploring the oligomeric structures and the conformational dynamics in
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therapeutic strategies.
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hIAPP fibrillization is crucial to understand the aggregation mechanism and develop
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Many previous studies elucidated the molecular mechanism underlying hIAPP aggregation. They reported that the native soluble form of hIAPP undergoes
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conformational transition from a random coil structure into the cytotoxic β-sheet secondary structure [8]. The amyloid formation of hIAPP generally occurs via a nucleated growth mechanism; once a nucleus forms, fibril growth rapidly proceeds through further association of monomers or oligomers [9–12]. Recent investigations have provided high-resolution structures of fibrils through solid-state NMR and X-ray crystallography [13, 14]. Real-time structural details have also been tracked using optical techniques and other devices, such as IMS–MS and 2D IR; these techniques and devices revealed the generation of a transient β-sheet intermediate in the amyloid fibrillization of hIAPP [15–17]. Bowers et al. [18] compared the dimeric structures of 3
ACCEPTED MANUSCRIPT hIAPP with those of nonamyloidogenic rat IAPP (rIAPP). They found that hIAPP dimers are more extended than rIAPP dimers, suggesting the production of high
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β-sheet content by extended β-hairpin-containing monomers. Radford monitored
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oligomer (up to hexamers) formation from IAPP and distinguished the action modes between different inhibitors and hIAPP self-assembly by using ESI–IMS–MS [19]. Much research focused on designing active molecules to inhibit amyloid
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aggregation. Metal complexes show advantages as potential inhibitors of amyloid
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fibrillization. For example, [Pt(phen)Cl2] (phen is 1,10-phenanthroline) plays a dual role in inhibiting Aβ aggregation through noncovalent interaction and platinum
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coordination [20]. Metal nanoparticles, such as gold nanoparticles (AuNPs), have
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been studied because of their action on protein amyloidogenesis [21–24]. AuNPs with
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different coatings of citrate, PAA, and poly (allylamine hydrochloride) (PAH) exhibit different inhibitory effects on Aβ1-40 aggregation [23]. By contrast, citrate-capped
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AuNPs display strong accelerating effects on α-synuclein aggregation [24]. Meanwhile, ruthenium and palladium complexes show distinct inhibitory effects on the aggregation of different amyloid proteins through metal coordination and hydrophobic or electrostatic interactions [25–28]. The sequence specificity of different proteins should be considered in designing metal inhibitors of amyloid protein fibrillization. hIAPP sequence contains one unique metal binding site, His18. Zinc binds to monomeric IAPP near the His18 residue and exerts a dual effect on hIAPP fibrillogenesis; that is, zinc increases lag-time for fibrillization at low concentrations and decreases it at high concentrations [29, 30]. 4
ACCEPTED MANUSCRIPT Vanadium complexes also exhibit strong inhibitory effects on hIAPP aggregation through hydrophobic and electrostatic interactions [31].
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Gold complexes are well-known pharmaceuticals that can be used as rheumatoid
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arthritis treatment and as potential anti-tumor reagents [32, 33]. The interaction of gold complexes with PrP106-126 indicates that these complexes display great binding affinity to histidine and methionine residues, showing a strong inhibitory effect on
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prion neuropeptide aggregation [34]. Metal complexes have advantages over their
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organic counterparts, wherein variation can be introduced by modifying the ligands or changing the metal center. Considering the aforementioned properties of gold
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complexes and the sequence specificity of hIAPP, we investigated the inhibitory
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effects of three gold complexes with different nitrogen-containing aromatic ligands,
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namely, [Au(bipy)Cl2][PF6] (1), [Au(Ph2bpy)Cl2]Cl (2), and [Au(phen)Cl2]Cl (3), on hIAPP fibrillization (Scheme S1). In each of three gold complexes, free chloride atom
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may easily leave for gold coordination with the peptide. Traditional hIAPP inhibitors are aromatic compounds such as congo red and rifampicin. The ligands were selected to supply effective hydrophobic and π-π interactions. Moreover, each selected ligand possesses its distinct size so that these gold complexes could exert different steric effect during their interaction with the peptide. Hence, the three gold complexes were used to compare and explore the ligand effect which contributes to amyloid peptide assembly. Thioflavin T (ThT) fluorescence assay, atomic force microscopy (AFM), and dynamic light scattering (DLS) experiments were used to display the concrete effects of the gold complexes on protein aggregation. Electrospray ionization–mass 5
ACCEPTED MANUSCRIPT spectrometry (ESI–MS) and intrinsic fluorescence method were performed to study the binding properties between the gold complexes and hIAPP. Moreover, the details
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of binding site were explored through NMR spectroscopy. This study intended to
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reveal the effects of selected gold complexes on the properties and assembly behavior of hIAPP, as well as provide a potential strategy for rational metallodrug design.
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Experimental section
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Materials
hIAPP was chemically synthesized by GL Biochem Co. Ltd. (Shanghai, China)
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and then separated and identified by HPLC and MS, respectively. The sample
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(>95% pure) was used directly without further purification. Metal complexes were
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prepared as previously described [35–37]. All other reagents were analytical grade. Lyophilized hIAPP was dissolved in hexafluoroisopropanol for 1 h to obtain a
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4 mg/mL stock solution. Aliquots of the peptide stock solution were lyophilized before use.
ThT assay Fluorescence was monitored using an F-4500 fluorescence spectrometer (Hitachi, Japan) with a programmable temperature controller (PolyScience, USA). Metal compounds were added in equimolar amounts to hIAPP (5 μM) in a 10 mM phosphate buffer at pH 7.5. The samples were incubated at 310 K for 96 h and then added with 10 μM ThT for analysis. ThT signal was quantified by averaging the 6
ACCEPTED MANUSCRIPT fluorescence emission at 484 nm over 10 s when the sample was excited at 432 nm. The final spectrum was obtained from the mean of three repeated spectra. Moreover,
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the amyloid formation of hIAPP (5 μM) as a function of gold complex concentration
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was monitored using also ThT. Different concentrations (0, 1, 2, 3, 4, 5, 25, 50, 100, 150, 200, and 250 μM) of the gold complexes were used. For the time course experiments of hIAPP aggregation, 20 μM peptide was used in the absence and
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presence of a gold complex at a molar ratio of 0.2, 0.6, or 1.0. Other conditions were
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the same as mentioned above.
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AFM images
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Samples were prepared by adding a metal complex to hIAPP solution and
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incubating at 310 K for 4 days. The final peptide concentration of all samples was 5 μM. Images were obtained in tapping mode with a silicon tip under ambient
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conditions, a scanning rate of 1 Hz, and a scanning line of 512 by using a Veeco D3100 instrument (Veeco Instruments 151 Inc., USA). Height and radius distributions were obtained by analyzing 50 patterns of each sample.
DLS measurements DLS experiments were performed using a Zetasizer Nano instrument (Malvern Instruments, Worcestershire, UK). A 1 mL aliquot of 10 μM hIAPP solution with equimolar amounts of the gold complexes was incubated at 310 K for 96 h and then centrifuged at 12000 rpm for 10 min to remove large precipitates. The supernatants 7
ACCEPTED MANUSCRIPT were transferred to a fluorescence cuvette for measurement.
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Spectrofluorometric measurements
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A published fluorescence-based approach was adopted to measure the apparent dissociation constant (Kd) and determine the binding affinity between the gold complexes and hIAPP [38]. The peptide concentration used was 1 μM. A wavelength
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of 275 nm was selected to excite intrinsic tyrosine residue, and Kd was determined by
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the basic function relationship between the fluorescence intensity of peptide and the molar ratio of metal complex to peptide. Results were reported as the mean of three
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NMR spectroscopy
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repeated experiments.
NMR spectroscopy experiments were carried out on a Bruker Advance
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400/600 MHz spectrometer. Suppression of residual water signal was achieved using a watergate pulse program with gradients. Experiments involving 2D homonuclear total correlation spectroscopy (TOCSY) were conducted in D2O with a total spinlock time of 120 ms using a regular MLEV-17 mixing sequence. All samples were prepared with 10 mM phosphate buffer (pH 5.5). The peptide concentration used was 150 μM, and equimolar amounts of metal compound were added. Chemical shifts were referenced to the methyl resonance of 4,4-dimethyl-4-silapentane-1-sulfonic acid as an internal standard.
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ACCEPTED MANUSCRIPT ESI-MS ESI–MS spectra were recorded in a positive mode by directly introducing the
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samples at a flow rate of 3 μL min−1 in an APEX IV FT-ICR high-resolution MS
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(Bruker, USA) equipped with a conventional ESI source. The working conditions were as follows: end plate electrode voltage, −3500 V; capillary entrance voltage, −4000 V; skimmer, 1 and 30 V; and dry gas temperature, 473 K. The flow rates of the
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drying gas and the nebulizer gas were set at 12 and 6 L min−1, respectively. Data
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analysis 4.0 software (Bruker) was used to acquire data. Deconvoluted masses were determined using an integrated deconvolution tool. The peptide sample used in the
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ESI–MS experiments was kept constant at 50 μM. Different amounts of the gold
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complexes were added to the sample for assay.
Results
In
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Synthesis of gold complexes this
study,
the
three
gold
complexes
[Au(bipy)Cl2][PF6]
(1),
[Au(Ph2bpy)Cl2]Cl (2), and [Au(phen)Cl2]Cl (3) were synthesized and identified as previously reported. The products were in accordance with the literature and used for later investigation (Fig. S1). The ligands of the complexes were 2,2′-bipyridine (bpy) for 1, 4-4′-diphenyl-2,2’-bipyridyl (Ph2bpy) for 2, and 1,10-phenanthroline for 3.
Influence of gold complexes on hIAPP aggregation hIAPP may self-assemble to form amyloid deposits, which are closely related to 9
ACCEPTED MANUSCRIPT type II diabetes. ThT assay is a well-known method for detecting amyloid fibrillization in diseases associated with protein misfolding because a strong
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fluorescence is produced when ThT binds to any amyloid or amyloid-like fibril [39,
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40]. Thus, ThT assay was utilized to monitor the aggregation of hIAPP and the effects of the gold complexes on the fibrillization of hIAPP.
In this section, equivalent amounts of the interactive system were constructed
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and explored. Fig. 1 shows that the fluorescence spectrum emitted a strong signal
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accompanying the binding of ThT with aggregated hIAPP. By contrast, ThT fluorescence intensity noticeably decreased with the addition of equivalent amounts of
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gold complexes 1, 2, and 3, suggesting that hIAPP aggregation was reduced. The gold
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complexes displayed no ultraviolet-visible absorption near 484 nm (data not shown),
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suggesting that no interaction occurred between the complexes and ThT. These results indicated that the changes in fluorescence intensities were induced by interactions
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between the gold complexes and hIAPP, although ThT competition with the compound was not completely eliminated. AFM was performed without ThT disturbance to further identify the inhibitory effects of the gold complexes on hIAPP aggregation. This technique was used to determine the different states of amyloidogenic peptide. The average result of three experiments was described. The aggregates formed by hIAPP had a rigid fibrillar structure (Fig. 2A), suggesting a strong aggregation state after 96 h of incubation at 310 K. However, AFM micrographs of hIAPP in the presence of equivalent amounts of gold complexes showed that such aggregation was reversed, showing only several 10
ACCEPTED MANUSCRIPT granular/spherical structures on the silicon wafers (Fig. 2B–D). Complex 2 showed the best inhibition with a few oligomers, and the AFM images agreed with the results
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of the ThT assay.
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The size distribution of hIAPP aggregates was determined by dynamic light scattering (DLS) after hIAPP was incubated with or without the gold complexes at 310 K for 96 h (Fig. 3). The self-assembly of hIAPP alone produced large aggregates
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with a hydrodynamic diameter of approximately 450 nm. The hydrodynamic radius of
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hIAPP decreased when equivalent amounts of a gold complex were included in the hIAPP solution. These results agreed with ThT fluorescence and AFM results,
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providing strong support for the interaction of the gold complexes with hIAPP.
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Dependence of hIAPP aggregation on gold complex concentration ThT, AFM, and DLS studies illustrated the strong inhibitory effects of equivalent
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amounts of gold complexes on hIAPP aggregation. However, the inhibitory effects of low-concentration gold complexes on protein fibrillization were complicated. ThT assay results indicated that hIAPP aggregation decreased after hIAPP was incubated with 0.2 equivalent amounts of gold complex. When the concentrations of the gold complexes were enhanced to approximately 0.6 equivalents, the peptide aggregation rebounded and became weaker than that at 0.2 equivalents (Fig. 4). ThT fluorescence intensity decreased as the molar ratio of metal complex to peptide was increased. This result matched our aforementioned results at 1:1. For verification, we performed the ThT assay at a high concentration range. When the molar ratio was increased to 50, 11
ACCEPTED MANUSCRIPT complexes 2 and 3 showed strong inhibitory effects on hIAPP fibrillization, whereas complex 1 demonstrated fluctuating inhibitory effects on this process (Fig. S2).
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The AFM images provided further details about the morphology of hIAPP
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aggregates in the presence of the gold complexes. Compared with the observation at a 1:1, the morphology of hIAPP fibrillar aggregates changed into granular particles when hIAPP was treated with 0.2 equivalent amounts of the compounds (Fig. 5).
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hIAPP formed spherical particles with heights less than 30 nm and average radius at
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approximately 60 nm (Figs. S3 and S4). When the molar ratio of a gold complex to hIAPP was enhanced to 0.6, hIAPP formed many special flower-like patterns, with
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heights less than 40 nm and average radius at approximately 100 nm. The AFM
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results agreed with the ThT assay results, indicating that the gold complexes inhibited
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hIAPP aggregation at low concentrations. However, the effect rebounded within a certain concentration range. The concentration dependence relationship might
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disclose an extraordinary and interesting aggregation mechanism of hIAPP for such a powerful amyloid peptide.
hIAPP aggregation dynamics when treated with gold complexes To determine whether or not the gold complexes directly affect hIAPP amyloidogenesis, we monitored the time course of hIAPP fibrillization in the presence of different gold complex ratios, with ThT as a marker. Fig. 6 shows that the fibrillization of hIAPP at 20 μM was negatively affected by the three gold complexes. When only 0.2 equivalent amounts of the gold complexes were added to hIAPP 12
ACCEPTED MANUSCRIPT solution, the extent of amyloid formation reversed, the lag time increased, and the fluorescence intensity decreased. Increasing the molar ratio of the gold compound to
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0.6 weakened the final inhibitory effect compared with the previous one but evidently
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prolonged the lag phase of hIAPP aggregation. Similarly, the three gold complexes showed strong inhibitory effects on both fibrillization rate and aggregation extent when the ratio to hIAPP was 1:1. Although we could not completely eliminate the
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competition of ThT binding to the peptide, the time course experiments suggested that
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the gold complexes influenced the aggregation behavior of the peptide.
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Binding affinity of gold complexes to hIAPP determined by intrinsic fluorescence
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The gold complexes significantly inhibited hIAPP aggregation, although the
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inhibitory effect rebounded within a narrow concentration range. This result prompted us to further study the binding affinity between the gold complexes and hIAPP.
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Numerous studies utilized intrinsic fluorescence quenching to estimate Kd [38, 41, 42]. Fluorescence quenching of tyrosine residues can represent structural changes caused by a gold complex. The change in peptide fluorescence upon the addition of a gold complex may reflect the amount of binary complex produced. The fluorescence intensity of hIAPP at 303 nm in the presence of a gold complex was therefore used to estimate Kd by using a nonlinear least-squares regression analysis (Fig. S5). The Kd values were 7.9 ± 0.8 × 10−7, 1.1 ± 0.1 × 10−6, and 1.1 ± 0.2 × 10−6 M for complexes 1, 2, and 3, respectively. The strong binding ability of gold complex may stem from the hydrophobic interaction as determined by intrinsic fluorescence quenching. However, 13
ACCEPTED MANUSCRIPT the binding affinity may be not directly correlated with the action against hIAPP fibrillization for different ligands. In some cases, ligand spatial configuration
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contributes to inhibit fibrillization, even though a complex has relatively weak
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binding affinity.
Possible binding mode of gold complexes to hIAPP
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hIAPP contains only one His18 residue, which has been recognized as a critical
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binding target for metal ions in other amyloid peptide sequences [39, 43, 44]. The His111 from the PrP106-126 sequence plays an important role in peptide aggregation
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and binding affinity to [Au(dien)Cl]Cl2, a gold complex containing a tridentate ligand
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[34]. In the present study, NMR spectroscopy was performed to analyze the
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interaction of gold complexes with hIAPP and determine whether or not these complexes bind to hIAPP and His18 is probable binding site. H NMR spectra were acquired at pH 5.5 and 298 K as previously described
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[27]. As shown in Fig. 7, the 1H NMR spectrum of hIAPP was confirmed and consistent with the previous result. The signal of His18 CδHs at 7.24 ppm was not noticeably disturbed with the addition of equivalent complex 2 in peptide solution. Compared with the 1H NMR spectrum of independent gold complex, the resonance signals for Ha proton (at 8.90 ppm) and Hh proton (at 8.97 ppm), which came from the aromatic ring of the ligand (Scheme S1), obviously decreased after the addition of hIAPP. The change of other peaks from the complex could not be confirmed because they were overlapped with the amide resonances. The present result implied that 14
ACCEPTED MANUSCRIPT peptide–complex binding influenced the proton relaxation. Similar changes in NMR spectrum were also observed for complex 1 and 3 (Fig. S6 and S7). Subsequently, 2D
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NMR analysis was performed through TOCSY for the system of hIAPP and gold
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complexes. Taking complex 2 as an example, the cross peak from His18 side chain was clearly assigned (Fig. S8). When the gold complexes were incubated with hIAPP, the cross peak of His18 shifted to the down-field region, and the chemical shift of
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CεHs moved by approximately 0.1 ppm. This result revealed that the interaction of the
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metal complexes with the peptide and His18 might be a potential binding site. The NMR results revealed that the interaction of the three gold complexes with hIAPP
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for gold binding.
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may include metal coordination and π–π interaction, with histidine as a crucial residue
Dimer formation of hIAPP caused by gold complexes
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Dependence of peptide aggregation on gold complex concentration was confirmed through ESI–MS. The free hIAPP exhibited an intense peak near [3903(1+)], which corresponded to its expected monomer mass (Fig. S9). When 0.2 equivalent amounts of gold complexes were incubated with the peptide, a major peak with a large intensity appeared and corresponded to the peptide monomer. For complexes 1 and 2, a minor peak appeared near [7806(1+)], indicating a dimer state of the ionized peptide (Fig. S10). With the addition of a gold complex at 0.6 molar ratio, the dimer peak became a primary component in the MS spectra. The monomer peak returned to major status when the molar ratio was increased to 3.0 (Fig. 8). This result 15
ACCEPTED MANUSCRIPT indicated that peptide aggregation can be inhibited well at high concentration and that the metal complex can stabilize the peptide to monomers.
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A distinct adduct peak matching the formation of a [hIAPP+Au] complex was not
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observed, although it occasionally appeared in the system of complex 3 with the peptide (data not shown). However, the results of ThT assay, NMR, and AFM clearly showed that the gold complexes interacted with hIAPP. The phenomenon may be
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attributed to the specificity of the long peptide in ESI–MS analysis and the relatively
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weak metal coordination. Moreover, the mass difference for monomers or dimers in the given data possibly reflected the active difference that resulted from distinct
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ligands of these complexes. MS results demonstrated that the gold complexes
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inhibited hIAPP aggregation, undergoing a dimer process. This phenomenon may
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probably explain why the dependence of peptide aggregation on gold complex
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concentration rebounded within a narrow range.
Discussion
Influence of gold complexes on hIAPP fibrillization Metal complexes, such as ruthenium and vanadium compounds, inhibit hIAPP aggregation in a concentration-dependent manner. The gold complexes revealed a different action mode on hIAPP aggregation. The self-assembly of the peptide was influenced by the concentration of the gold complexes in solution. When the molar ratio of gold complexes to hIAPP was lower than 0.6, hIAPP showed a weak aggregation state, and fibril morphology was replaced by granular oligomers. The 16
ACCEPTED MANUSCRIPT inhibitory effect on hIAPP aggregation rebounded when the ratio was enhanced to approximately 0.6. The AFM images demonstrated that the presence of a gold
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complex caused the aggregates to differ from intrinsic hIAPP fibrils, which formed
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flower-like patterns with an expanded radius but with no changes in height. However, the strong inhibition on hIAPP aggregation reappeared when the molar ratio was increased to 1:1. Unexpectedly, ThT assay showed that complex 1 exerted fluctuating
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inhibitory effects on hIAPP aggregation at a high range (Fig. S2).
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As we know, gold complexes are good candidates as antitumor agents. Complex 3 and an analog of complex 1, [Au(bipy)(OH)2][PF6], have shown relevant cytotoxic
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effects toward human tumor cell lines [45,46]. In addition, it is reported that
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[Au(TPP)]Cl was more than 10-fold toxic to nasopharyngeal cancer cells when
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compared to normal peripheral blood mononuclear cells [47]. Moreover, our previous study has demonstrated that complexes 1 and 2 can well prevent the neurotoxicity
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induced by PrP106–126 [36]. However, the hIAPP induced INS-1 insulinoma cells toxicity was not remarkably restored by adding these complexes (data not shown). Nevertheless, this work centered around the distinct effects of gold complexes on the behavior of hIAPP assembly, and their positive effects on regulation of amyloid aggregation were valuable as mentioned above.
Interaction between gold complexes and hIAPP Many studies reported the profound effects of metal ions on amyloid aggregation. A well-known example is an Alzheimer’s disease-related Aβ protein that possesses 17
ACCEPTED MANUSCRIPT moderate affinity for copper and zinc [48-50]. The metal ions coordinated to the imidazole group of histidine and altered the aggregation and toxicity profiles of the
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peptide. Zinc ion exerts a dual effect on hIAPP fibrillogenesis; that is, zinc ion
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increases the lag time for fiber formation and decreases the rate of hIAPP aggregation at low concentrations but shows opposite effects at high concentrations [29]. Many studies identified metal binding sites with high affinity in amyloid proteins; however,
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a few studies elucidated the binding of metal ions to hIAPP through MS results [30,
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51]. Although previous MALDI–TOF/MS results provided direct evidence that nickel and copper combine with hIAPP, the binding peak was weak, and molar ratios were
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as high as 100:1 and 10:1, respectively [51].
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Considering the difficulty of detecting hIAPP–metal complex adduct through
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MS, we used intrinsic fluorescence quenching to compare the binding affinity between gold complexes and hIAPP in the present study. The Kd values were 7.9 ± 0.8
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× 10−7 M, 1.1 ± 0.1 × 10−6, and 1.1 ± 0.2 × 10−6 M for complexes 1, 2, and 3, respectively. The high binding affinity may be attributed to hydrophobic interaction between the peptide and the complex. In addition, the NMR results provided plausible evidence that gold ion binds to histidine residue in the peptide. However, metal coordination may not be a major contributing factor in the inhibitory function of the complexes, and the role of the nitrogen-containing aromatic ligands cannot be ignored. This speculation agrees with the finding that aromatic organic compounds, such as Congo red, phenol red, and rifampicin, can inhibit the fibrillization and decrease 18
ACCEPTED MANUSCRIPT the cytotoxicity of hIAPP. In the present study, three ligand molecules (bpy, Ph2bpy, and phen) alone did not inhibit the fibrillization of the peptide as strongly as their gold
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complexes (Figure S11). Some studies report that the central gold ion has a strong
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tendency to coordinate with histidine residue during the inhibition of amyloid peptide fibrillization [34, 36, 52]. In this work, the [AuCl4]- itself showed feebler inhibitory effect on hIAPP aggregationwhen compared with the gold complex (Figure S12). This
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result backed up the significance of using metal complex to suppress hIAPP
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aggregation and far surpass the corresponding ligand and the metal ion. Furthermore, the MS data imply that the gold complexes elicit a unique dimer mechanism against
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hIAPP self-assembly and aggregation. These results verify that the peculiar functions
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of the gold complexes on the self-assembly of hIAPP may be attributed to multiple
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interactions, including metal coordination, ligand steric effects, and π-π stacking from the aromatic ligands and aromatic residues of the peptide.
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NMR results provided a plausible fact that the gold complexes can bind to His18 in the peptide. However, no direct binding adducts in the MS experiments were consistently observed. This result implied that the combined adducts of the gold complexes with hIAPP were few and sensitive to ionization, which complicated identification by MS. Different from the MS spectrum of hIAPP alone, the MS spectra of the peptide together with the metal compounds exhibited intense peaks of hIAPP monomers and dimers. Moreover, the peak intensity of monomers or dimers changed with the molar ratio of gold complex to peptide. This result corresponded with the concentration dependence observed in ThT assay, AFM, and DLS analysis. 19
ACCEPTED MANUSCRIPT A previous work reported that only monomeric states of hIAPP and fractional dimers can be observed through ESI–MS and that the fractional dimers disappear with the
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addition of zinc [30]. By contrast, the present results revealed that hIAPP showed
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different dependence levels on gold complex concentration. Gold complexes at an appropriate concentration range can restrain hIAPP fibrillization by inducing dimer formation and suppressing further oligomerization. Complexes 2 and 3 inhibited
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hIAPP aggregation at high concentrations, whereas complex 1 demonstrated
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fluctuating inhibitory effects at a high concentration range.
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Complicated assembly mechanism of hIAPP
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Early oligomeric states are key to protein self-assembly and subsequent amyloid
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disease. Several studies proposed hypothetical mechanisms for amyloid protein aggregation and their interaction with other molecules [19, 53–57]. Almost all the
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conjectures have referred to common on-pathway and off-pathway oligomers. Oligomers may be either on-pathway to help fibrillization or undergo conformational changes and allow off-pathway species to enter the aggregation pathway and form amyloid. Hence, most of the designed inhibitors, such as epigallocatechin gallate and silibinin, are aimed at binding and stabilizing monomers to prevent further protein polymerization [19]. In the present study, the gold complexes induced a concentration-dependent assembly of hIAPP dimerization within a narrow concentration range, and the dimerization can be dispelled by adding a large amount of gold complexes to hIAPP. Different from zinc ion on hIAPP fibrillization, 20
ACCEPTED MANUSCRIPT complexes 2 and 3 inhibited hIAPP aggregation at high concentrations. This study
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provided further insights into the behavior of hIAPP assembly.
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Conclusion
hIAPP is a vital amyloid peptide that correlates with type II diabetes. The present work demonstrated distinct effects of gold complexes on hIAPP assembly and
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aggregation. The self-assembly behavior of the peptide was affected by different
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molar ratios of gold complex to hIAPP. Complexes 2 and 3 showed strong inhibitory effects on peptide aggregation. By contrast, complex 1 displayed a fluctuated effect
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on peptide fibril formation at high concentration. The inhibitory effects were derived
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from multiple interactions, including possible gold–histidine coordination, ligand
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steric effects, and π–π stacking from the nitrogen-containing aromatic ligands and peptide aromatic residues. The results also suggested that gold complexes inhibited
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hIAPP aggregation through dimerization, stabilized hIAPP to monomers, and thus prevented further fibrillization of the peptide. Moreover, gold ion exerted a noninterchangeable effect on peptide assembly.
Abbreviations hIAPP, amylin rIAPP AuNPs PAH ThT H2TPP
Human islet amyloid polypeptide Rat IAPP gold nanoparticles poly (allylamine hydrochloride) Thioflavin T tetraphenylporphyrin
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ACCEPTED MANUSCRIPT Acknowledgements This work was supported by the National Nature Science Foundation of China
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(No. 21271185 & 21473251).
References
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ACCEPTED MANUSCRIPT Acad. Sci. USA 106(2009) 7828-7833. [57] R. Soong, J. R. Brender, P. M. Macdonald, A. Ramamoorthy, J. Am. Chem. Soc. 131(2009)
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ACCEPTED MANUSCRIPT Figure legends Scheme 1. Molecular structures of [Au(bipy)Cl2][PF6] (1), [Au(Ph2bpy)Cl2]Cl(2), and
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[Au(phen)Cl2]Cl (3).
Fig. 1. Evaluation of the ability of Au complexes to inhibit hIAPP aggregation as measured by ThT fluorescence. hIAPP was incubated with ThT in the absence (solid)
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and presence of [Au(bipy)Cl2][PF6] (dashed), [Au(Ph2bpy)Cl2]Cl (dash-dot), and
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[Au(phen)Cl2]Cl (dotted). Fluorescence curve of ThT alone was used for comparison
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(short dashed).
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Fig. 2. AFM images of hIAPP in the absence (A) and presence of equimolar amounts
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of [Au(bipy)Cl2][PF6] (B), [Au(Ph2bpy)Cl2]Cl (C), and [Au(phen)Cl2]Cl (D). The
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scale bar is 500 nm.
Fig. 3. DLS analysis of the multimodal size distribution of hIAPP aggregates in the absence (solid) and presence of [Au(bipy)Cl2][PF6] (dashed), [Au(Ph2bpy)Cl2]Cl (dash-dot), and [Au(phen)Cl2]Cl (dotted).
Fig. 4. The inhibitory effects of gold complexes on hIAPP aggregation at different molar
ratios.
(A)
[Au(bipy)Cl2][PF6],
(B)
[Au(Ph2bpy)Cl2]Cl,
and
(C)
[Au(phen)Cl2]Cl. Relative fluorescence intensity was detected at 484 nm. The concentration of hIAPP is 5 μM and the molar ratio of gold complex to hIAPP were 27
ACCEPTED MANUSCRIPT selected at 0, 0.2, 0.4, 0.6, 0.8 and 1.0, respectively.
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Fig. 5. AFM images for the inhibitory effects of gold complexes on hIAPP fibril
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formation. The molar ratio of gold complex to hIAPP were 0.2 (left) and 0.6 (right) respectively for [Au(bipy)Cl2][PF6] (A), [Au(Ph2bpy)Cl2]Cl(B), and [Au(phen)Cl2]Cl
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(C). The scale bar is 500nm.
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Fig. 6. ThT fluorescence monitored at 480 nm during hIAPP (20 μM) aggregation in the absence (solid) and presence of 0.2 (dashed), 0.6 (dotted) and 1.0 (dash-dot)
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and [Au(phen)Cl2]Cl (C).
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equimolar amounts of gold complex: [Au(bipy)Cl2][PF6] (A), [Au(Ph2bpy)Cl2]Cl (B),
Fig. 7. 1H NMR spectra of hIAPP (150 μM) and [Au(Ph2bpy)Cl2]Cl in H2O/DMSO at
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pH 5.5, 298 K. (A) [Au(Ph2bpy)Cl2]Cl; (B) hIAPP in the presence of equimolar amounts of [Au(Ph2bpy)Cl2]Cl; (C) hIAPP. The signals at 8.90 and 8.97 ppm (dot) of [Au(Ph2bpy)Cl2]Cl was obviously perturbed by the interaction with hIAPP.
Fig. 8. ESI-MS spectra of hIAPP in presence of three equimolar amounts of [Au(bipy)Cl2][PF6] (A), [Au(Ph2bpy)Cl2]Cl (B), and [Au(phen)Cl2]Cl (C).
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Synopsis for the Graphic Abstract
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Gold complexes inhibit the fibril formation of human islet amyloid
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polypeptide through dimerization.
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Au complexes could bind to hIAPP and inhibit its aggregation behavior.
Different assembly behaviors of hIAPP were dependent on gold complex
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concentration.
The binding modes were included hydrophobic interaction and metal coordination.
Au complexes inhibited hIAPP assembly by dimerization and stabilized it to
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This study provided further insights into the behavior of hIAPP assembly.
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monomers.
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S0162-0134(15)30063-5 doi: 10.1016/j.jinorgbio.2015.08.020 JIB 9791
To appear in:
Journal of Inorganic Biochemistry
Received date: Revised date: Accepted date:
22 April 2015 9 August 2015 20 August 2015
Please cite this article as: Lei He, Dengsen Zhu, Cong Zhao, Xian Jia, Xuesong Wang, Weihong Du, Effects of gold complexes on the assembly behavior of human islet amyloid polypeptide, Journal of Inorganic Biochemistry (2015), doi: 10.1016/j.jinorgbio.2015.08.020
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effects of gold complexes on the assembly behavior of human islet
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amyloid polypeptide
Lei He, Dengsen Zhu, Cong Zhao, Xian Jia, Xuesong Wang, Weihong Du*
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Department of Chemistry, Renmin University of China, Beijing, 100872
Keywords: hIAPP; assembly; gold complex; interaction; dimerization. *
Corresponding author. Tel/Fax: 8610-6251-2660/6444, Email:
[email protected] 1
ACCEPTED MANUSCRIPT Abstract Human islet amyloid polypeptide (hIAPP) is a well-known amyloid protein that
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is associated with type II diabetes. Inhibitors of this peptide include aromatic organic
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molecules, short peptides, and metal complexes, such as zinc, ruthenium and vanadium compounds. Various metal ions and their complexes affect the fibrillization of hIAPP in different action modes. However, the assembly mechanism of the peptide
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remains unclear. This study evaluated the inhibitory effects of three gold complexes
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with different nitrogen-containing aromatic ligands, namely, [Au(bipy)Cl2][PF6] (1), [Au(Ph2bpy)Cl2]Cl (2), and [Au(phen)Cl2]Cl (3), on the amyloid fibrillization of
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hIAPP. The complexes interacted with the peptide mainly through hydrophobic
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interaction and metal coordination. The concentration dependence of hIAPP
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aggregation on gold complex indicated that the assembly behavior of hIAPP is significantly affected by these compounds. The gold complexes inhibited peptide
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aggregation through dimerization and stabilized the peptide to monomers. Gold ion was found to be a key influencing factor of the binding mode and assembly behavior of hIAPP. The different effects of the complexes on peptide aggregation might be attributed to their special ligands. This study provided insights into the inhibitory mechanism of gold complexes against hIAPP fibrillization.
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ACCEPTED MANUSCRIPT Introduction Human islet amyloid polypeptide (hIAPP, amylin), a hormone comprising 37
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amino acids with strong amyloidogenic propensity, is synthesized by pancreatic
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β-cells and co-secreted with insulin [1]. Self-assembly of hIAPP into amyloid aggregates leads to the development of pancreatic β-cell dysfunction and death in the pathology of type II diabetes [2, 3]. The mechanism underlying hIAPP-induced β-cell
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toxicity and related processes, including receptor-mediated mechanism, localized
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inflammatory response, and IAPP-induced membrane damage, remain unclear [2, 4–7]. Thus, exploring the oligomeric structures and the conformational dynamics in
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therapeutic strategies.
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hIAPP fibrillization is crucial to understand the aggregation mechanism and develop
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Many previous studies elucidated the molecular mechanism underlying hIAPP aggregation. They reported that the native soluble form of hIAPP undergoes
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conformational transition from a random coil structure into the cytotoxic β-sheet secondary structure [8]. The amyloid formation of hIAPP generally occurs via a nucleated growth mechanism; once a nucleus forms, fibril growth rapidly proceeds through further association of monomers or oligomers [9–12]. Recent investigations have provided high-resolution structures of fibrils through solid-state NMR and X-ray crystallography [13, 14]. Real-time structural details have also been tracked using optical techniques and other devices, such as IMS–MS and 2D IR; these techniques and devices revealed the generation of a transient β-sheet intermediate in the amyloid fibrillization of hIAPP [15–17]. Bowers et al. [18] compared the dimeric structures of 3
ACCEPTED MANUSCRIPT hIAPP with those of nonamyloidogenic rat IAPP (rIAPP). They found that hIAPP dimers are more extended than rIAPP dimers, suggesting the production of high
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β-sheet content by extended β-hairpin-containing monomers. Radford monitored
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oligomer (up to hexamers) formation from IAPP and distinguished the action modes between different inhibitors and hIAPP self-assembly by using ESI–IMS–MS [19]. Much research focused on designing active molecules to inhibit amyloid
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aggregation. Metal complexes show advantages as potential inhibitors of amyloid
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fibrillization. For example, [Pt(phen)Cl2] (phen is 1,10-phenanthroline) plays a dual role in inhibiting Aβ aggregation through noncovalent interaction and platinum
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coordination [20]. Metal nanoparticles, such as gold nanoparticles (AuNPs), have
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been studied because of their action on protein amyloidogenesis [21–24]. AuNPs with
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different coatings of citrate, PAA, and poly (allylamine hydrochloride) (PAH) exhibit different inhibitory effects on Aβ1-40 aggregation [23]. By contrast, citrate-capped
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AuNPs display strong accelerating effects on α-synuclein aggregation [24]. Meanwhile, ruthenium and palladium complexes show distinct inhibitory effects on the aggregation of different amyloid proteins through metal coordination and hydrophobic or electrostatic interactions [25–28]. The sequence specificity of different proteins should be considered in designing metal inhibitors of amyloid protein fibrillization. hIAPP sequence contains one unique metal binding site, His18. Zinc binds to monomeric IAPP near the His18 residue and exerts a dual effect on hIAPP fibrillogenesis; that is, zinc increases lag-time for fibrillization at low concentrations and decreases it at high concentrations [29, 30]. 4
ACCEPTED MANUSCRIPT Vanadium complexes also exhibit strong inhibitory effects on hIAPP aggregation through hydrophobic and electrostatic interactions [31].
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Gold complexes are well-known pharmaceuticals that can be used as rheumatoid
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arthritis treatment and as potential anti-tumor reagents [32, 33]. The interaction of gold complexes with PrP106-126 indicates that these complexes display great binding affinity to histidine and methionine residues, showing a strong inhibitory effect on
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prion neuropeptide aggregation [34]. Metal complexes have advantages over their
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organic counterparts, wherein variation can be introduced by modifying the ligands or changing the metal center. Considering the aforementioned properties of gold
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complexes and the sequence specificity of hIAPP, we investigated the inhibitory
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effects of three gold complexes with different nitrogen-containing aromatic ligands,
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namely, [Au(bipy)Cl2][PF6] (1), [Au(Ph2bpy)Cl2]Cl (2), and [Au(phen)Cl2]Cl (3), on hIAPP fibrillization (Scheme S1). In each of three gold complexes, free chloride atom
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may easily leave for gold coordination with the peptide. Traditional hIAPP inhibitors are aromatic compounds such as congo red and rifampicin. The ligands were selected to supply effective hydrophobic and π-π interactions. Moreover, each selected ligand possesses its distinct size so that these gold complexes could exert different steric effect during their interaction with the peptide. Hence, the three gold complexes were used to compare and explore the ligand effect which contributes to amyloid peptide assembly. Thioflavin T (ThT) fluorescence assay, atomic force microscopy (AFM), and dynamic light scattering (DLS) experiments were used to display the concrete effects of the gold complexes on protein aggregation. Electrospray ionization–mass 5
ACCEPTED MANUSCRIPT spectrometry (ESI–MS) and intrinsic fluorescence method were performed to study the binding properties between the gold complexes and hIAPP. Moreover, the details
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of binding site were explored through NMR spectroscopy. This study intended to
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reveal the effects of selected gold complexes on the properties and assembly behavior of hIAPP, as well as provide a potential strategy for rational metallodrug design.
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Experimental section
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Materials
hIAPP was chemically synthesized by GL Biochem Co. Ltd. (Shanghai, China)
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and then separated and identified by HPLC and MS, respectively. The sample
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(>95% pure) was used directly without further purification. Metal complexes were
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prepared as previously described [35–37]. All other reagents were analytical grade. Lyophilized hIAPP was dissolved in hexafluoroisopropanol for 1 h to obtain a
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4 mg/mL stock solution. Aliquots of the peptide stock solution were lyophilized before use.
ThT assay Fluorescence was monitored using an F-4500 fluorescence spectrometer (Hitachi, Japan) with a programmable temperature controller (PolyScience, USA). Metal compounds were added in equimolar amounts to hIAPP (5 μM) in a 10 mM phosphate buffer at pH 7.5. The samples were incubated at 310 K for 96 h and then added with 10 μM ThT for analysis. ThT signal was quantified by averaging the 6
ACCEPTED MANUSCRIPT fluorescence emission at 484 nm over 10 s when the sample was excited at 432 nm. The final spectrum was obtained from the mean of three repeated spectra. Moreover,
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the amyloid formation of hIAPP (5 μM) as a function of gold complex concentration
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was monitored using also ThT. Different concentrations (0, 1, 2, 3, 4, 5, 25, 50, 100, 150, 200, and 250 μM) of the gold complexes were used. For the time course experiments of hIAPP aggregation, 20 μM peptide was used in the absence and
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presence of a gold complex at a molar ratio of 0.2, 0.6, or 1.0. Other conditions were
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the same as mentioned above.
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AFM images
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Samples were prepared by adding a metal complex to hIAPP solution and
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incubating at 310 K for 4 days. The final peptide concentration of all samples was 5 μM. Images were obtained in tapping mode with a silicon tip under ambient
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conditions, a scanning rate of 1 Hz, and a scanning line of 512 by using a Veeco D3100 instrument (Veeco Instruments 151 Inc., USA). Height and radius distributions were obtained by analyzing 50 patterns of each sample.
DLS measurements DLS experiments were performed using a Zetasizer Nano instrument (Malvern Instruments, Worcestershire, UK). A 1 mL aliquot of 10 μM hIAPP solution with equimolar amounts of the gold complexes was incubated at 310 K for 96 h and then centrifuged at 12000 rpm for 10 min to remove large precipitates. The supernatants 7
ACCEPTED MANUSCRIPT were transferred to a fluorescence cuvette for measurement.
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Spectrofluorometric measurements
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A published fluorescence-based approach was adopted to measure the apparent dissociation constant (Kd) and determine the binding affinity between the gold complexes and hIAPP [38]. The peptide concentration used was 1 μM. A wavelength
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of 275 nm was selected to excite intrinsic tyrosine residue, and Kd was determined by
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the basic function relationship between the fluorescence intensity of peptide and the molar ratio of metal complex to peptide. Results were reported as the mean of three
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NMR spectroscopy
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repeated experiments.
NMR spectroscopy experiments were carried out on a Bruker Advance
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400/600 MHz spectrometer. Suppression of residual water signal was achieved using a watergate pulse program with gradients. Experiments involving 2D homonuclear total correlation spectroscopy (TOCSY) were conducted in D2O with a total spinlock time of 120 ms using a regular MLEV-17 mixing sequence. All samples were prepared with 10 mM phosphate buffer (pH 5.5). The peptide concentration used was 150 μM, and equimolar amounts of metal compound were added. Chemical shifts were referenced to the methyl resonance of 4,4-dimethyl-4-silapentane-1-sulfonic acid as an internal standard.
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ACCEPTED MANUSCRIPT ESI-MS ESI–MS spectra were recorded in a positive mode by directly introducing the
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samples at a flow rate of 3 μL min−1 in an APEX IV FT-ICR high-resolution MS
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(Bruker, USA) equipped with a conventional ESI source. The working conditions were as follows: end plate electrode voltage, −3500 V; capillary entrance voltage, −4000 V; skimmer, 1 and 30 V; and dry gas temperature, 473 K. The flow rates of the
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drying gas and the nebulizer gas were set at 12 and 6 L min−1, respectively. Data
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analysis 4.0 software (Bruker) was used to acquire data. Deconvoluted masses were determined using an integrated deconvolution tool. The peptide sample used in the
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ESI–MS experiments was kept constant at 50 μM. Different amounts of the gold
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Results
In
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Synthesis of gold complexes this
study,
the
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complexes
[Au(bipy)Cl2][PF6]
(1),
[Au(Ph2bpy)Cl2]Cl (2), and [Au(phen)Cl2]Cl (3) were synthesized and identified as previously reported. The products were in accordance with the literature and used for later investigation (Fig. S1). The ligands of the complexes were 2,2′-bipyridine (bpy) for 1, 4-4′-diphenyl-2,2’-bipyridyl (Ph2bpy) for 2, and 1,10-phenanthroline for 3.
Influence of gold complexes on hIAPP aggregation hIAPP may self-assemble to form amyloid deposits, which are closely related to 9
ACCEPTED MANUSCRIPT type II diabetes. ThT assay is a well-known method for detecting amyloid fibrillization in diseases associated with protein misfolding because a strong
IP
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fluorescence is produced when ThT binds to any amyloid or amyloid-like fibril [39,
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40]. Thus, ThT assay was utilized to monitor the aggregation of hIAPP and the effects of the gold complexes on the fibrillization of hIAPP.
In this section, equivalent amounts of the interactive system were constructed
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and explored. Fig. 1 shows that the fluorescence spectrum emitted a strong signal
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accompanying the binding of ThT with aggregated hIAPP. By contrast, ThT fluorescence intensity noticeably decreased with the addition of equivalent amounts of
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gold complexes 1, 2, and 3, suggesting that hIAPP aggregation was reduced. The gold
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complexes displayed no ultraviolet-visible absorption near 484 nm (data not shown),
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suggesting that no interaction occurred between the complexes and ThT. These results indicated that the changes in fluorescence intensities were induced by interactions
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between the gold complexes and hIAPP, although ThT competition with the compound was not completely eliminated. AFM was performed without ThT disturbance to further identify the inhibitory effects of the gold complexes on hIAPP aggregation. This technique was used to determine the different states of amyloidogenic peptide. The average result of three experiments was described. The aggregates formed by hIAPP had a rigid fibrillar structure (Fig. 2A), suggesting a strong aggregation state after 96 h of incubation at 310 K. However, AFM micrographs of hIAPP in the presence of equivalent amounts of gold complexes showed that such aggregation was reversed, showing only several 10
ACCEPTED MANUSCRIPT granular/spherical structures on the silicon wafers (Fig. 2B–D). Complex 2 showed the best inhibition with a few oligomers, and the AFM images agreed with the results
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of the ThT assay.
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The size distribution of hIAPP aggregates was determined by dynamic light scattering (DLS) after hIAPP was incubated with or without the gold complexes at 310 K for 96 h (Fig. 3). The self-assembly of hIAPP alone produced large aggregates
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with a hydrodynamic diameter of approximately 450 nm. The hydrodynamic radius of
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hIAPP decreased when equivalent amounts of a gold complex were included in the hIAPP solution. These results agreed with ThT fluorescence and AFM results,
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providing strong support for the interaction of the gold complexes with hIAPP.
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Dependence of hIAPP aggregation on gold complex concentration ThT, AFM, and DLS studies illustrated the strong inhibitory effects of equivalent
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amounts of gold complexes on hIAPP aggregation. However, the inhibitory effects of low-concentration gold complexes on protein fibrillization were complicated. ThT assay results indicated that hIAPP aggregation decreased after hIAPP was incubated with 0.2 equivalent amounts of gold complex. When the concentrations of the gold complexes were enhanced to approximately 0.6 equivalents, the peptide aggregation rebounded and became weaker than that at 0.2 equivalents (Fig. 4). ThT fluorescence intensity decreased as the molar ratio of metal complex to peptide was increased. This result matched our aforementioned results at 1:1. For verification, we performed the ThT assay at a high concentration range. When the molar ratio was increased to 50, 11
ACCEPTED MANUSCRIPT complexes 2 and 3 showed strong inhibitory effects on hIAPP fibrillization, whereas complex 1 demonstrated fluctuating inhibitory effects on this process (Fig. S2).
IP
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The AFM images provided further details about the morphology of hIAPP
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aggregates in the presence of the gold complexes. Compared with the observation at a 1:1, the morphology of hIAPP fibrillar aggregates changed into granular particles when hIAPP was treated with 0.2 equivalent amounts of the compounds (Fig. 5).
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hIAPP formed spherical particles with heights less than 30 nm and average radius at
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approximately 60 nm (Figs. S3 and S4). When the molar ratio of a gold complex to hIAPP was enhanced to 0.6, hIAPP formed many special flower-like patterns, with
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heights less than 40 nm and average radius at approximately 100 nm. The AFM
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results agreed with the ThT assay results, indicating that the gold complexes inhibited
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hIAPP aggregation at low concentrations. However, the effect rebounded within a certain concentration range. The concentration dependence relationship might
AC
disclose an extraordinary and interesting aggregation mechanism of hIAPP for such a powerful amyloid peptide.
hIAPP aggregation dynamics when treated with gold complexes To determine whether or not the gold complexes directly affect hIAPP amyloidogenesis, we monitored the time course of hIAPP fibrillization in the presence of different gold complex ratios, with ThT as a marker. Fig. 6 shows that the fibrillization of hIAPP at 20 μM was negatively affected by the three gold complexes. When only 0.2 equivalent amounts of the gold complexes were added to hIAPP 12
ACCEPTED MANUSCRIPT solution, the extent of amyloid formation reversed, the lag time increased, and the fluorescence intensity decreased. Increasing the molar ratio of the gold compound to
IP
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0.6 weakened the final inhibitory effect compared with the previous one but evidently
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prolonged the lag phase of hIAPP aggregation. Similarly, the three gold complexes showed strong inhibitory effects on both fibrillization rate and aggregation extent when the ratio to hIAPP was 1:1. Although we could not completely eliminate the
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competition of ThT binding to the peptide, the time course experiments suggested that
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the gold complexes influenced the aggregation behavior of the peptide.
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Binding affinity of gold complexes to hIAPP determined by intrinsic fluorescence
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The gold complexes significantly inhibited hIAPP aggregation, although the
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inhibitory effect rebounded within a narrow concentration range. This result prompted us to further study the binding affinity between the gold complexes and hIAPP.
AC
Numerous studies utilized intrinsic fluorescence quenching to estimate Kd [38, 41, 42]. Fluorescence quenching of tyrosine residues can represent structural changes caused by a gold complex. The change in peptide fluorescence upon the addition of a gold complex may reflect the amount of binary complex produced. The fluorescence intensity of hIAPP at 303 nm in the presence of a gold complex was therefore used to estimate Kd by using a nonlinear least-squares regression analysis (Fig. S5). The Kd values were 7.9 ± 0.8 × 10−7, 1.1 ± 0.1 × 10−6, and 1.1 ± 0.2 × 10−6 M for complexes 1, 2, and 3, respectively. The strong binding ability of gold complex may stem from the hydrophobic interaction as determined by intrinsic fluorescence quenching. However, 13
ACCEPTED MANUSCRIPT the binding affinity may be not directly correlated with the action against hIAPP fibrillization for different ligands. In some cases, ligand spatial configuration
IP
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contributes to inhibit fibrillization, even though a complex has relatively weak
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binding affinity.
Possible binding mode of gold complexes to hIAPP
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hIAPP contains only one His18 residue, which has been recognized as a critical
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binding target for metal ions in other amyloid peptide sequences [39, 43, 44]. The His111 from the PrP106-126 sequence plays an important role in peptide aggregation
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and binding affinity to [Au(dien)Cl]Cl2, a gold complex containing a tridentate ligand
TE
[34]. In the present study, NMR spectroscopy was performed to analyze the
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interaction of gold complexes with hIAPP and determine whether or not these complexes bind to hIAPP and His18 is probable binding site. H NMR spectra were acquired at pH 5.5 and 298 K as previously described
AC
1
[27]. As shown in Fig. 7, the 1H NMR spectrum of hIAPP was confirmed and consistent with the previous result. The signal of His18 CδHs at 7.24 ppm was not noticeably disturbed with the addition of equivalent complex 2 in peptide solution. Compared with the 1H NMR spectrum of independent gold complex, the resonance signals for Ha proton (at 8.90 ppm) and Hh proton (at 8.97 ppm), which came from the aromatic ring of the ligand (Scheme S1), obviously decreased after the addition of hIAPP. The change of other peaks from the complex could not be confirmed because they were overlapped with the amide resonances. The present result implied that 14
ACCEPTED MANUSCRIPT peptide–complex binding influenced the proton relaxation. Similar changes in NMR spectrum were also observed for complex 1 and 3 (Fig. S6 and S7). Subsequently, 2D
IP
T
NMR analysis was performed through TOCSY for the system of hIAPP and gold
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complexes. Taking complex 2 as an example, the cross peak from His18 side chain was clearly assigned (Fig. S8). When the gold complexes were incubated with hIAPP, the cross peak of His18 shifted to the down-field region, and the chemical shift of
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CεHs moved by approximately 0.1 ppm. This result revealed that the interaction of the
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metal complexes with the peptide and His18 might be a potential binding site. The NMR results revealed that the interaction of the three gold complexes with hIAPP
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for gold binding.
D
may include metal coordination and π–π interaction, with histidine as a crucial residue
Dimer formation of hIAPP caused by gold complexes
AC
Dependence of peptide aggregation on gold complex concentration was confirmed through ESI–MS. The free hIAPP exhibited an intense peak near [3903(1+)], which corresponded to its expected monomer mass (Fig. S9). When 0.2 equivalent amounts of gold complexes were incubated with the peptide, a major peak with a large intensity appeared and corresponded to the peptide monomer. For complexes 1 and 2, a minor peak appeared near [7806(1+)], indicating a dimer state of the ionized peptide (Fig. S10). With the addition of a gold complex at 0.6 molar ratio, the dimer peak became a primary component in the MS spectra. The monomer peak returned to major status when the molar ratio was increased to 3.0 (Fig. 8). This result 15
ACCEPTED MANUSCRIPT indicated that peptide aggregation can be inhibited well at high concentration and that the metal complex can stabilize the peptide to monomers.
IP
T
A distinct adduct peak matching the formation of a [hIAPP+Au] complex was not
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observed, although it occasionally appeared in the system of complex 3 with the peptide (data not shown). However, the results of ThT assay, NMR, and AFM clearly showed that the gold complexes interacted with hIAPP. The phenomenon may be
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attributed to the specificity of the long peptide in ESI–MS analysis and the relatively
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weak metal coordination. Moreover, the mass difference for monomers or dimers in the given data possibly reflected the active difference that resulted from distinct
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ligands of these complexes. MS results demonstrated that the gold complexes
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inhibited hIAPP aggregation, undergoing a dimer process. This phenomenon may
CE P
probably explain why the dependence of peptide aggregation on gold complex
AC
concentration rebounded within a narrow range.
Discussion
Influence of gold complexes on hIAPP fibrillization Metal complexes, such as ruthenium and vanadium compounds, inhibit hIAPP aggregation in a concentration-dependent manner. The gold complexes revealed a different action mode on hIAPP aggregation. The self-assembly of the peptide was influenced by the concentration of the gold complexes in solution. When the molar ratio of gold complexes to hIAPP was lower than 0.6, hIAPP showed a weak aggregation state, and fibril morphology was replaced by granular oligomers. The 16
ACCEPTED MANUSCRIPT inhibitory effect on hIAPP aggregation rebounded when the ratio was enhanced to approximately 0.6. The AFM images demonstrated that the presence of a gold
IP
T
complex caused the aggregates to differ from intrinsic hIAPP fibrils, which formed
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flower-like patterns with an expanded radius but with no changes in height. However, the strong inhibition on hIAPP aggregation reappeared when the molar ratio was increased to 1:1. Unexpectedly, ThT assay showed that complex 1 exerted fluctuating
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inhibitory effects on hIAPP aggregation at a high range (Fig. S2).
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As we know, gold complexes are good candidates as antitumor agents. Complex 3 and an analog of complex 1, [Au(bipy)(OH)2][PF6], have shown relevant cytotoxic
D
effects toward human tumor cell lines [45,46]. In addition, it is reported that
TE
[Au(TPP)]Cl was more than 10-fold toxic to nasopharyngeal cancer cells when
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compared to normal peripheral blood mononuclear cells [47]. Moreover, our previous study has demonstrated that complexes 1 and 2 can well prevent the neurotoxicity
AC
induced by PrP106–126 [36]. However, the hIAPP induced INS-1 insulinoma cells toxicity was not remarkably restored by adding these complexes (data not shown). Nevertheless, this work centered around the distinct effects of gold complexes on the behavior of hIAPP assembly, and their positive effects on regulation of amyloid aggregation were valuable as mentioned above.
Interaction between gold complexes and hIAPP Many studies reported the profound effects of metal ions on amyloid aggregation. A well-known example is an Alzheimer’s disease-related Aβ protein that possesses 17
ACCEPTED MANUSCRIPT moderate affinity for copper and zinc [48-50]. The metal ions coordinated to the imidazole group of histidine and altered the aggregation and toxicity profiles of the
IP
T
peptide. Zinc ion exerts a dual effect on hIAPP fibrillogenesis; that is, zinc ion
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increases the lag time for fiber formation and decreases the rate of hIAPP aggregation at low concentrations but shows opposite effects at high concentrations [29]. Many studies identified metal binding sites with high affinity in amyloid proteins; however,
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a few studies elucidated the binding of metal ions to hIAPP through MS results [30,
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51]. Although previous MALDI–TOF/MS results provided direct evidence that nickel and copper combine with hIAPP, the binding peak was weak, and molar ratios were
D
as high as 100:1 and 10:1, respectively [51].
TE
Considering the difficulty of detecting hIAPP–metal complex adduct through
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MS, we used intrinsic fluorescence quenching to compare the binding affinity between gold complexes and hIAPP in the present study. The Kd values were 7.9 ± 0.8
AC
× 10−7 M, 1.1 ± 0.1 × 10−6, and 1.1 ± 0.2 × 10−6 M for complexes 1, 2, and 3, respectively. The high binding affinity may be attributed to hydrophobic interaction between the peptide and the complex. In addition, the NMR results provided plausible evidence that gold ion binds to histidine residue in the peptide. However, metal coordination may not be a major contributing factor in the inhibitory function of the complexes, and the role of the nitrogen-containing aromatic ligands cannot be ignored. This speculation agrees with the finding that aromatic organic compounds, such as Congo red, phenol red, and rifampicin, can inhibit the fibrillization and decrease 18
ACCEPTED MANUSCRIPT the cytotoxicity of hIAPP. In the present study, three ligand molecules (bpy, Ph2bpy, and phen) alone did not inhibit the fibrillization of the peptide as strongly as their gold
IP
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complexes (Figure S11). Some studies report that the central gold ion has a strong
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tendency to coordinate with histidine residue during the inhibition of amyloid peptide fibrillization [34, 36, 52]. In this work, the [AuCl4]- itself showed feebler inhibitory effect on hIAPP aggregationwhen compared with the gold complex (Figure S12). This
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result backed up the significance of using metal complex to suppress hIAPP
MA
aggregation and far surpass the corresponding ligand and the metal ion. Furthermore, the MS data imply that the gold complexes elicit a unique dimer mechanism against
D
hIAPP self-assembly and aggregation. These results verify that the peculiar functions
TE
of the gold complexes on the self-assembly of hIAPP may be attributed to multiple
CE P
interactions, including metal coordination, ligand steric effects, and π-π stacking from the aromatic ligands and aromatic residues of the peptide.
AC
NMR results provided a plausible fact that the gold complexes can bind to His18 in the peptide. However, no direct binding adducts in the MS experiments were consistently observed. This result implied that the combined adducts of the gold complexes with hIAPP were few and sensitive to ionization, which complicated identification by MS. Different from the MS spectrum of hIAPP alone, the MS spectra of the peptide together with the metal compounds exhibited intense peaks of hIAPP monomers and dimers. Moreover, the peak intensity of monomers or dimers changed with the molar ratio of gold complex to peptide. This result corresponded with the concentration dependence observed in ThT assay, AFM, and DLS analysis. 19
ACCEPTED MANUSCRIPT A previous work reported that only monomeric states of hIAPP and fractional dimers can be observed through ESI–MS and that the fractional dimers disappear with the
IP
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addition of zinc [30]. By contrast, the present results revealed that hIAPP showed
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different dependence levels on gold complex concentration. Gold complexes at an appropriate concentration range can restrain hIAPP fibrillization by inducing dimer formation and suppressing further oligomerization. Complexes 2 and 3 inhibited
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hIAPP aggregation at high concentrations, whereas complex 1 demonstrated
MA
fluctuating inhibitory effects at a high concentration range.
D
Complicated assembly mechanism of hIAPP
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Early oligomeric states are key to protein self-assembly and subsequent amyloid
CE P
disease. Several studies proposed hypothetical mechanisms for amyloid protein aggregation and their interaction with other molecules [19, 53–57]. Almost all the
AC
conjectures have referred to common on-pathway and off-pathway oligomers. Oligomers may be either on-pathway to help fibrillization or undergo conformational changes and allow off-pathway species to enter the aggregation pathway and form amyloid. Hence, most of the designed inhibitors, such as epigallocatechin gallate and silibinin, are aimed at binding and stabilizing monomers to prevent further protein polymerization [19]. In the present study, the gold complexes induced a concentration-dependent assembly of hIAPP dimerization within a narrow concentration range, and the dimerization can be dispelled by adding a large amount of gold complexes to hIAPP. Different from zinc ion on hIAPP fibrillization, 20
ACCEPTED MANUSCRIPT complexes 2 and 3 inhibited hIAPP aggregation at high concentrations. This study
IP
T
provided further insights into the behavior of hIAPP assembly.
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Conclusion
hIAPP is a vital amyloid peptide that correlates with type II diabetes. The present work demonstrated distinct effects of gold complexes on hIAPP assembly and
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aggregation. The self-assembly behavior of the peptide was affected by different
MA
molar ratios of gold complex to hIAPP. Complexes 2 and 3 showed strong inhibitory effects on peptide aggregation. By contrast, complex 1 displayed a fluctuated effect
D
on peptide fibril formation at high concentration. The inhibitory effects were derived
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from multiple interactions, including possible gold–histidine coordination, ligand
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steric effects, and π–π stacking from the nitrogen-containing aromatic ligands and peptide aromatic residues. The results also suggested that gold complexes inhibited
AC
hIAPP aggregation through dimerization, stabilized hIAPP to monomers, and thus prevented further fibrillization of the peptide. Moreover, gold ion exerted a noninterchangeable effect on peptide assembly.
Abbreviations hIAPP, amylin rIAPP AuNPs PAH ThT H2TPP
Human islet amyloid polypeptide Rat IAPP gold nanoparticles poly (allylamine hydrochloride) Thioflavin T tetraphenylporphyrin
21
ACCEPTED MANUSCRIPT Acknowledgements This work was supported by the National Nature Science Foundation of China
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IP
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(No. 21271185 & 21473251).
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26
ACCEPTED MANUSCRIPT Figure legends Scheme 1. Molecular structures of [Au(bipy)Cl2][PF6] (1), [Au(Ph2bpy)Cl2]Cl(2), and
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IP
T
[Au(phen)Cl2]Cl (3).
Fig. 1. Evaluation of the ability of Au complexes to inhibit hIAPP aggregation as measured by ThT fluorescence. hIAPP was incubated with ThT in the absence (solid)
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and presence of [Au(bipy)Cl2][PF6] (dashed), [Au(Ph2bpy)Cl2]Cl (dash-dot), and
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[Au(phen)Cl2]Cl (dotted). Fluorescence curve of ThT alone was used for comparison
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(short dashed).
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Fig. 2. AFM images of hIAPP in the absence (A) and presence of equimolar amounts
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of [Au(bipy)Cl2][PF6] (B), [Au(Ph2bpy)Cl2]Cl (C), and [Au(phen)Cl2]Cl (D). The
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scale bar is 500 nm.
Fig. 3. DLS analysis of the multimodal size distribution of hIAPP aggregates in the absence (solid) and presence of [Au(bipy)Cl2][PF6] (dashed), [Au(Ph2bpy)Cl2]Cl (dash-dot), and [Au(phen)Cl2]Cl (dotted).
Fig. 4. The inhibitory effects of gold complexes on hIAPP aggregation at different molar
ratios.
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[Au(bipy)Cl2][PF6],
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[Au(Ph2bpy)Cl2]Cl,
and
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[Au(phen)Cl2]Cl. Relative fluorescence intensity was detected at 484 nm. The concentration of hIAPP is 5 μM and the molar ratio of gold complex to hIAPP were 27
ACCEPTED MANUSCRIPT selected at 0, 0.2, 0.4, 0.6, 0.8 and 1.0, respectively.
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Fig. 5. AFM images for the inhibitory effects of gold complexes on hIAPP fibril
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formation. The molar ratio of gold complex to hIAPP were 0.2 (left) and 0.6 (right) respectively for [Au(bipy)Cl2][PF6] (A), [Au(Ph2bpy)Cl2]Cl(B), and [Au(phen)Cl2]Cl
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(C). The scale bar is 500nm.
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Fig. 6. ThT fluorescence monitored at 480 nm during hIAPP (20 μM) aggregation in the absence (solid) and presence of 0.2 (dashed), 0.6 (dotted) and 1.0 (dash-dot)
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and [Au(phen)Cl2]Cl (C).
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equimolar amounts of gold complex: [Au(bipy)Cl2][PF6] (A), [Au(Ph2bpy)Cl2]Cl (B),
Fig. 7. 1H NMR spectra of hIAPP (150 μM) and [Au(Ph2bpy)Cl2]Cl in H2O/DMSO at
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pH 5.5, 298 K. (A) [Au(Ph2bpy)Cl2]Cl; (B) hIAPP in the presence of equimolar amounts of [Au(Ph2bpy)Cl2]Cl; (C) hIAPP. The signals at 8.90 and 8.97 ppm (dot) of [Au(Ph2bpy)Cl2]Cl was obviously perturbed by the interaction with hIAPP.
Fig. 8. ESI-MS spectra of hIAPP in presence of three equimolar amounts of [Au(bipy)Cl2][PF6] (A), [Au(Ph2bpy)Cl2]Cl (B), and [Au(phen)Cl2]Cl (C).
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Synopsis for the Graphic Abstract
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Gold complexes inhibit the fibril formation of human islet amyloid
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polypeptide through dimerization.
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ACCEPTED MANUSCRIPT Highlights
Au complexes could bind to hIAPP and inhibit its aggregation behavior.
Different assembly behaviors of hIAPP were dependent on gold complex
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concentration.
The binding modes were included hydrophobic interaction and metal coordination.
Au complexes inhibited hIAPP assembly by dimerization and stabilized it to
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This study provided further insights into the behavior of hIAPP assembly.
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monomers.
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