Oligotyrosines Inhibit Amyloid Formation of Human Islet Amyloid

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Oligotyrosines Inhibit Amyloid Formation of Human Islet Amyloid Polypeptide in a Tyrosine-Number Dependent Manner Yanru Xin, Huazhi Zhang, Qigang Hu, Sidan Tian, Chenhui Wang, Liang Luo, and Fanling Meng ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01384 • Publication Date (Web): 08 Dec 2018 Downloaded from http://pubs.acs.org on December 14, 2018

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ACS Biomaterials Science & Engineering

Oligotyrosines Inhibit Amyloid Formation of Human Islet Amyloid Polypeptide in a Tyrosine-Number Dependent Manner

Yanru Xin†, Huazhi Zhang†, Qigang Hu†, Sidan Tian†, Chenhui Wang†, ‡, Liang Luo†, ‡*,

Fanling Meng†, ‡*

†.

National Engineering Research Center for Nanomedicine, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, China ‡. Wuhan Institute of Biotechnology, Wuhan, 430075, China *Corresponding authors E-mail address: [email protected] (F. Meng), [email protected] (L. Luo) Key words: Diabetes; Human islet amyloid polypeptide; Amyloid; Inhibition; Oligotyrosines

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ABSTRACT Misfolding and amyloid formation of human islet amyloid polypeptide (IAPP) is believed to be critical in the pathogenesis of type 2 diabetes. Inhibitors that can effectively prevent protein aggregation and fibrillation are considered as potential therapeutics for the prevention and treatment of type 2 diabetes. Here we report that oligotyrosines manipulate IAPP amyloid formation in vitro and modulate IAPPinduced cytotoxicity in a manner that is related to the number of tyrosine units. Tyr2 and Tyr3 can effectively inhibit the aggregation of IAPP, either in bulk solution or in the presence of lipid membranes, and alleviate IAPP-mediated cytotoxicity. On the contrary, Tyr, Tyr4, and Tyr6 do not show significant inhibitory effects on the IAPP aggregation at the same conditions. To the best of our knowledge, this is the first time to report a residue-number dependent inhibition of IAPP aggregation by oligotyrosines, and Tyr2 and Tyr3 are proved to be potent inhibitors of IAPP amyloid formation. The interactions between oligotyrosines and IAPP have been simulated through molecular docking, which provides us a new insight about the inhibition mechanism of IAPP amyloid formation that will be helpful for developing anti-diabetic drug candidates.

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INTRODUCTION Protein misfolding and amyloid formation are linked to many human diseases including Alzheimer’s disease, Parkinson’s disease and type 2 diabetes.1,2 Human islet amyloid polypeptide (amylin, or IAPP) is synthesized in the islet pancreatic β-cells and co-stored with insulin in the insulin secretory granule.3 IAPP is intrinsically soluble and unfolded in a monomer state under physiological conditions. It has strong propensity to aggregate and form amyloid fibrils,4-7 which are toxic to pancreatic β-cells and contributes to the pathology of type 2 diabetes.8-11 It was also reported that amyloid formation of IAPP plays a potentially deleterious role in islet transplantation.12 Although the molecular mechanisms of how IAPP amyloid formation contributes to type 2 diabetes has not been well clarified, many attempts have been applied to search for the therapeutic reagents which can potently inhibit IAPP amyloid formation and reduce IAPP-induced pancreatic β-cell toxicity.13-21 The driving forces for the formation and stabilization of amyloid fibrils include hydrogen bonding,22 π-π stacking,23 and hydrophobic interaction.24 For instance, both Phe15 and Phe23 in IAPP play an important role in the aggregation of the peptide, and mutating these 2 residues to Leu can significantly increase the time of IAPP amyloid formation.25-28 In addition, disrupting the hydrogen bonding formation in IAPP20-29 hydrophobic region can prevent IAPP aggregation, i.e. both I26P and G24P IAPP lose the ability of amyloid fibrils formation.29-31 Therefore, developing new inhibitors to interfere with these driving forces in amyloid aggregation has become an interesting attempt. Small molecule inhibitor (−)-Epigallocatechin 3-Gallate (EGCG) represents 3



one such inhibitor. Recent works have demonstrated that EGCG can prevent IAPP amyloid formation and disassemble the formed protein aggregates through hydrogen bonding, aromatic interaction, and hydrophobic interaction.32,33 Inspired by the polyphenolic molecular structure of EGCG, we have attempted to evaluate inhibitory effect of peptides possessing polyphenol features on IAPP amyloid formation. Oligotyrosines (Figure 1) which contain multi-phenyl groups have emerged as appealing candidates. They are bioactive peptides that contributes to the regulation of blood pressure,34 and the supplement of tyrosine is important for the treatment of phenylketonuria, Parkinson’s disease, and acute stress.35 More interestingly, due to the phenol group on tyrosine, the oligotyrosine peptides have the potential to interfere with IAPP through multiple interactions (hydrogen bonding, hydrophobic effect, and π-π interaction) and inhibit the aggregation of IAPP. In this work, we have evaluated the inhibition effect of a series of oligotyrosines on IAPP fibrillation, and explored the influence of the number of tyrosine in these peptides on the inhibitory effect. We have demonstrated that oligotyrosines Tyr2 and Tyr3 are potent inhibitors of amyloid aggregation both in the bulk solution and in the presence of membrane by using kinetic assay and transmission electron microscopy. Tyr2 and Tyr3 can effectively protect INS-1 cells from IAPP-induced cell toxicity. Molecular docking simulations have been employed to elucidate the molecular mechanism of oligotyrosines inhibiting IAPP aggregation. Typically, the amyloid formation peptide inhibitors are truncated, modified IAPP fragments15 or mimics of α-helix.18 The peptide inhibitors we have developed here are rationalized directly from the molecular structure. 4


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In addition, this is the first time to report that oligotyrosines have the ability to inhibit amyloid formation, which provides a new paradigm for developing anti-diabetic agents. A

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Figure 1: (A) The primary sequence of human IAPP. The peptide contains a disulfide bridge between Cys-2 and Cys-7 and has an amidated C-terminus. (B) The molecular structure of designed oligotyrosines. MATERIALS AND METHODS Materials Human islet amyloid polypeptide (IAPP) was purchased from Science Peptide Biological Technology and GL Biochem (Shanghai, China). L-Tyrosine was purchased from Shanghai Regal Biology Technology Co., Ltd (Shanghai, China). Tyr2, Tyr4 and

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Tyr6 were purchased from Science Peptide Biological Technology (Shanghai, China). Tyr3 was purchased from Aladdin (Shanghai, China). 1,1,1,3,3,3-Hexafluoro-2propanol (HFIP, ≥ 99 %), Thioflavine-T (ThT), Trizma hydrochloride (Reagent grade, ≥ 99%) and Trizma base (≥99.9%) were purchased from Sigma-Aldrich (USA). Lipids (dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC)) were from Avanti Polar Lipids (AL, USA). MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5Diphenyltetrazolium Bromide) was purchased from Energy Chemical (Shanghai, China). RPMI 1640 was from Hyclone (South Logan, UT). Rat insulinoma cells were obtained from Shanghai Gefan Biotechnology (Shanghai, China). Thioflavin-T (ThT) fluorescence assay For all experiments, IAPP was freshly dissolved in HFIP and filtered through 0.22 µm filters before use. The concentration of IAPP stock solution is 1.6 mM. The IAPP solution for ThT kinetics was prepared by diluting IAPP stock solution into 20 mM trisHCl buffer with ThT. The final solution condition is 32 μM IAPP, 25 μM ThT in 2% HFIP. The inhibitor stock solutions were made by dissolving in 1 M HCl (Tyr, Tyr2 and Tyr3) or 0.2 M NaOH (Tyr4 and Tyr6) solution and then diluting in deionized water to a final concentration of 1.6 mM. The final concentrations of inhibitors, when present in the kinetics assay, were 32 μM, 80 μM, and 160 μM respectively for IAPP:inhibitor 1:1, 1:2.5 and 1:5 molar ratio. The pH values for solutions in all kinetic assays were kept at 7.4. For the experiments of IAPP in the presence of lipid vesicles, the concentration of lipid membrane was 630 µM. Samples were added to 96-well black microplates (Corning Costar Corporation, USA) and fluorescence was measured at 30 ºC with a 6


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fluorescence microplate reader (Varioskan LUX, Thermo Scientific) at 10-minute intervals. The excitation wavelength was set to 435 nm, and emission was monitored at 485 nm. Transmission electronic microscopy (TEM) 10 µL samples of IAPP (32 µM) with or without oligotyrosines (80 µM or 160 µM) were removed from the ThT assays at the end of the reactions and placed on a 300 mesh Formvar-carbon coated copper grid for 2 min. It was negatively stained with 1% uranyl acetate for 2 minutes. After the grid was dried, TEM experiments were performed on a Hitachi HT7700 transmission electron microscope system with an accelerating voltage of 80 kV. Unilameller vesicle preparation The unilameller vesicles used in this work was prepared by mixing DOPC and DOPG at a 7:3 equivalent molar ratio. The chloroform was removed by rotary evaporation. The obtained dry film was hydrated in 20 mM tris-HCl (pH 7.4) for 1 hour. The hydrated mixture was finally sonicated for 20 minutes. The size of the prepared vesicles was measured with a particle size analyzer (Nano ZS90, Malvern). The final concentration of vesicles in the kinetic system was 630 µM. Cell toxicity assay Rat insulinoma (INS-1) cells were maintained in RPMI-1640 medium (Hyclone) containing 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvat, 100 U/ml penicillin 100 mg/ml streptomycin solution, and 50 µM β-mecaptoethanol in a humidified 5% CO2 atmosphere at 37 ºC. Prior to adding the sample solution, cells were seeded in 96 7



well plates at a density of 15,000 cells per well and incubated overnight at 37 ºC in 5% CO2 atmosphere. Stock samples of IAPP (50 µM) in the presence or absence of Tyr2 or Tyr3 at different molar ratios were prepared in 20 mM tris-HCl. After pre-incubation at 37 ºC for 2 hours, the stock sample solution was mixed with the culture medium at a ratio of 4:6 (v/v) and added to the cells (100 µL/well). After the cells were incubated with the sample solution for 5 hours, the sample solution was aspirated. The 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/ml in PBS) was diluted 5-fold with culture medium and added to the cells for an additional 4 hours. The medium was aspirated and 150 µL DMSO was added to dissolve the formazan salt. The absorbance of the formazan solution was measured using a microplate reader (Varioskan LUX, Thermo Scientific) at 570 nm to reflect the viability of the cells. Molecular docking simulation All oligotyrosines were built by ChemDraw software and optimized to achieve stable formation conformations in model dynamics. The PDB file of IAPP was downloaded from the Protein Data Bank (PDB ID: 5mgq). The program suite AutoDock 4.2.0 (http://www.scripps.edu/mb/olson/doc/autodock) and AutoDock Vina was used for docking modeling of the interaction between IAPP and oligotyrosine based on Lamarckian Genetic Algorithm (LGA). In the docking process, IAPP was considered to be a receptor and the oligotyrosines were set as ligands. The docking models of IAPP and oligotyrosines were displayed using Discovery Studio. Statistical analysis 8


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Statistical significance was determined using two-tailed Student’s t-test and Oneway analysis of variance. Differences were considered statistically significant when the p < 0.05. RESULTS AND DISCUSSION Tyrosine-number-dependent manipulation of IAPP aggregation by oligotyrosines We first investigated the manipulation effect of a series of oligotyrosines with different tyrosine unit numbers on IAPP fibrillation by monitoring the kinetics of IAPP amyloid formation using Thioflavin T (ThT) assays.36 With the prolongation of the incubation time, the ThT fluorescence showed a typical sigmoidal curve, which started with a lag phase, followed by an amyloid-forming growth phase and finally a plateau region where monomers and fibrils reached an equilibrium. The lag phase time of amyloid formation for IAPP alone is about 260 min (black curve, Figure 2A). The presence of amino acid L-tyrosine (Tyr) in the IAPP kinetic assay (IAPP:Tyr 1:5 molar ratio) almost had negligible effect on the kinetics of IAPP aggregation, with a lag phase time of around 250 min. We next examined the inhibition effect of oligotyrosines with increased tyrosine units, including Tyr2 ，Tyr3 ，Tyr4 and Tyr6, on the IAPP amyloid formation. The oligotyrosines exhibited an interesting tyrosine-number-dependent manipulation effects on IAPP kinetics at the same IAPP:oligotyrosine molar ratio (1:5). For IAPP with Tyr2 or Tyr3, there was no obvious fluorescence intensity detected during the entire experiment period, suggesting that both Tyr2 and Tyr3 were highly effective in preventing IAPP amyloid formation (Figure 2A), and the lag phase times in these cases were extended to over 800 min (Figure 2B). Whereas in the presence of Tyr4, the 9



lag phase time increased to 340 min, and the final fluorescence intensity was similar to that for IAPP alone, suggesting that Tyr4 was still able to inhibit the amyloid formation of IAPP, but it was less effective than Tyr2 and Tyr3. Keeping on increasing the number of tyrosine in the oligotyrosines continued to vanish the inhibition effect. The presence of Tyr6 led to a lag phase time of 310 min and the final fluorescence intensity is almost identical to that of IAPP alone. In addition, to test if the efficient inhibition by Tyr2 and Tyr3 was simply due to the increase of the total amounts of tyrosine, we had examined the inhibitory effect of Tyr at higher concentrations. The results showed that increasing the concentration of Tyr to an IAPP:Tyr molar ratio of 1:10 and 1:20 had no obvious change on the kinetics of IAPP amyloid formation as monitored by ThT assay (Figure S1), suggesting that the potent inhibition of Tyr2 and Tyr3 was not a simple sum of individual tyrosine inhibitory effect. To further validate the effect of different oligotyrosines on IAPP fibril formation, TEM images had been recorded at the end of kinetic experiments. For the sample with IAPP alone, the TEM images showed that a large quantity of fibrils have been formed, with a typical morphology of IAPP-derived amyloid (Figure 2C). The samples of IAPP with Tyr and Tyr6, each with an IAPP:oligotyrosine molar ratio of 1:5, displayed amyloid fibrils that were similar to the IAPP amyloids (Figure 2D, H). As a comparison, both IAPP samples with Tyr2 and Tyr3 exhibited spherical, discrete nanostructures in the TEM images and no fibers had been generated (Figure 2E and 2F). As for IAPP in the presence of Tyr4, the TEM image showed amyloid fibrils formed (Figure 2G). The ThT assays, together with the TEM results, have demonstrated that the inhibitory effect 10


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of oligotyrosines are directly related to the number of tyrosine units in them. When an oligotyrosine is present at an IAPP:oligotyrosine molar ratio of 1:5, it can completely inhibit the amyloid formation of IAPP when it contains two or three tyrosine residues. It becomes less effective when the tyrosine residue number increases to four, while it has no effect on the kinetics of IAPP fibrillation when the tyrosine residue number is one or six (Figure 2B).

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Figure 2：Effects of oligotyrosines on amyloid formation of IAPP in vitro. (A) IAPP fibrillation kinetics monitored by ThT fluorescence with or without oligotyrosines. The molar ratio of IAPP:oligotyrosine was set as 1:5 in each experiment. (B) The lag phase time of IAPP kinetics experiments in the presence of different oligotyrosines. (C-H) TEM images of samples at the end of the kinetics studies. (C) IAPP alone; (D) IAPP 12


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with Tyr; (E) IAPP with Tyr2; (F) IAPP with Tyr3; (G) IAPP with Tyr4; (H) IAPP with Tyr6. The concentrations of IAPP and polytyrosines were 32 and 160 μM respectively and the experiments were performed in 20 mM Tris-HCl, pH 7.4 at 30 °C. Scale bar: 500 nm. Tyr3 is a more efficient inhibitor of IAPP amyloid formation than Tyr2 To further compare the inhibition effects between Tyr2 and Tyr3, as well as to explore the inhibition potency of the two inhibitors, we investigated the effects of Tyr2 and Tyr3 on the kinetics of IAPP at lower stoichiometric ratios. The results of ThT kinetics assays suggested that decreasing the concentration of Tyr2 led to reduced inhibitory effects (Figure 3A). When the molar ratio of IAPP:Tyr2 decreased to 1:2.5 or 1:1, only moderate inhibitory effect was achieved. On the contrary, when IAPP and Tyr3 was coincubated at an IAPP: Tyr3 molar ratio of 1:2.5, the kinetics experiment showed no ThT fluorescence intensity change during the experiment time, suggesting that Tyr3 was very effective at this concentration. When the concentration of Tyr3 continued to decrease to an IAPP:Tyr3 molar ratio of 1:1, it failed to inhibit IAPP amyloid formation (Figure 3B). The effect of Tyr2 and Tyr3 on the aggregated state of IAPP at the end of the kinetics was further confirmed by TEM images. After 12 hours of incubation, the sample of IAPP with Tyr2 at 1:2.5 molar ratio showed the presence of many mature fibers (Figure 3C). When the molar ratio of IAPP:Tyr3 were 1:2.5, the discrete oligomers were observed in the TEM images and there was no generation of mature fibers, confirming that Tyr3 could completely inhibit the fiber production of IAPP at this concentration 13



(Figure 3E). The ThT-monitored kinetics experiments and TEM images confirmed that Tyr3 is the most potent inhibitor among the examined oligotyrosines, which can effectively inhibit IAPP amyloid formation at as low as 1:2.5 molar ratio. A

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Figure 3: The inhibition ability of Tyr2 and Tyr3 at different IAPP:inhibitor molar ratios. (A) IAPP aggregation kinetics monitored by ThT assays in the presence of different concentrations of Tyr3. (B) IAPP aggregation kinetics monitored by ThT assays in the presence of different concentrations of Tyr3. (C-D) TEM images of samples at the end of the kinetics studies. (C) IAPP with Tyr2 at a molar ratio of 1:2.5 (D) IAPP with Tyr3 at a molar ratio of 1:2.5. The IAPP concentration in each experiment was 32 μM and the experiments were conducted in 20 mM tris-HCl, pH 7.4 at 30 °C. Scale bar: 500 nm. Tyr2 and Tyr3 inhibit IAPP amyloid formation at the membrane

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The interaction between membrane and misfolded peptide plays a vital role in IAPPinduced cell death in type 2 diabetes.37,38 In addition, many inhibitors become inactive for vesicle-bound amyloid aggregation.39,40 Here we have examined whether Tyr2 or Tyr3 have the ability to regulate IAPP aggregation in the presence of lipid vesicles. We used vesicles from a 7:3 mixture of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG) to mimic the anionic contents of cell membrane phospholipids. In the presence of lipid vesicles, ThT-monitored IAPP aggregation kinetics showed a typical sigmoidal curve with a lag phase time of about 100 min, faster than in the absence of membrane (around 260 min) (Figure 4A and 4B). At the end of the kinetics experiment, aliquots of the IAPP sample were removed for TEM characterization. A large amount of mature fibers had been observed (Figure 4C), consistent with the kinetics results. For IAPP with Tyr2 at an IAPP:inhibitor molar ratio of 1:5, it was interesting to observe that the kinetics assay showed no obvious fluorescence signal during the entire experiment time, indicating the complete inhibition of IAPP amyloid formation by Tyr2 in the presence of lipid membrane (Figure 4A). Accordingly, the TEM image of the corresponding sample showed discrete nanostructure (Figure 4D). As for Tyr3, when it was co-incubated with IAPP in the presence of lipid membrane at an IAPP:Tyr3 molar ratio of 1:5, it could completely prevent IAPP amyloid formation, yielding a similar kinetics curve and discrete nanostructures (Figure 4F) as Tyr2. When the concentration of Tyr3 was decreased to an IAPP:inhibitor molar ratio of 1:2.5, the fluorescence intensity of sample increased to about 30% of IAPP alone, with a lag phase time of around 220 min. The 15



corresponding TEM image showed that there still had limited amount of amyloid fibrils formed (Figure 4E). The above experiment results proved that Tyr2 and Tyr3 can potently inhibit IAPP aggregation, even in the presence of lipid vesicles.

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Figure 4: Effects of Tyr2 and Tyr3 on IAPP amyloid formation in the presence of lipid vesicles. (A) ThT monitored kinetics of IAPP fibrillation in the presence and in the absence of inhibitors at membrane surface at different IAPP/inhibitor molar ratios in the presence of lipid vesicles. (B) The lag phase time of IAPP kinetics experiments with or without inhibitors at membrane surface at different IAPP/inhibitor molar ratios. (C16


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F) TEM images of the samples taken at the end of the kinetics studies while lipid vesicles were present. (C) IAPP alone; (D) IAPP: Tyr2 1:5; (E) IAPP: Tyr3 1:2.5, and (F) IAPP: Tyr3 1:5. The IAPP and lipid vesicle concentrations in each experiment were 32 μM and 630 μM, respectively, and the experiments were conducted in 20 mM trisHCl, pH 7.4 at 30 °C. Scale bar: 500 nm. Tyr2 and Tyr3 protect rat insulinoma (INS-1) cells against the toxic effects of IAPP The misfolding and fibrillation of IAPP are considered important pathological factors of type 2 diabetes.6 There is increasing evidence suggesting that oligomers and mature fibers of IAPP can disrupt cell membranes, release intracellular contents, and cause death of β-cells.41,42 On basis of the fact that Tyr2 and Tyr3 can effectively inhibit the process of IAPP misfolding, we have further investigated the regulation effect of these two inhibitors (Tyr2 and Tyr3) on IAPP-induced cytotoxicity to INS-1 cells. As shown in Figure 5, incubation of 20 µM IAPP for 5 hours with cells triggered 30% toxicity relative to the untreated control as determined by MTT assays. When Tyr2 or Tyr3 was present, the cell viability had been significantly improved. The percentage of viable cells increased to 98% when Tyr2 had been co-incubated with IAPP at 1:5 IAPP:inhibitor molar ratio. On the other hand, when Tyr3 was present with IAPP at an IAPP:inhibitor molar ratio of 1:2.5, the cell viability was recovered to 97%. The cell viability could only increase to 85% at an IAPP:Tyr3 molar ratio of 1:5. This was partially due to the intrinsic cytotoxicity of Tyr3 at this concentration. Overall, the MTT results have unambiguously evidenced that both Tyr2 and Tyr3 can effectively alleviate IAPP-mediated β-cell toxicity. 17



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Figure 5: Tyr2 and Tyr3 protect rat INS-1 cells against the toxic effects of IAPP. Cell viability was determined by MTT assays. Data were obtained from three independent experiments and presented as the means ± SD. The concentration of IAPP, when present, was 20 µM. *p < 0.05, **p < 0.01. The mechanism of Tyr2 and Tyr3 to inhibit IAPP aggregation To explore the interaction between the inhibitors and IAPP, we have performed molecular docking simulations, using the AutoDock Vina software, based on the previously reported solution NMR structure of IAPP43. The structures of IAPP and inhibitors employed to perform docking studies are shown in Figure S2, and IAPP exists as an amphiphilic helix in the N-terminal region (8-19 residues). From the docking result of Tyr2 and IAPP (Figure 6A and 6B), Tyr2 interacts with IAPP through binding to the hydrophobic face of IAPP 8-19 helix. Previous studies have shown that the IAPP fragment (residues 1-19) plays a very important role in IAPP-IAPP association, IAPP-membrane binding, and membrane damage.44-48 The docking results

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indicated that Tyr2 inhibits IAPP amyloid formation by preventing IAPP intermolecular interaction and association. On the other hand, the binding model of IAPP with Tyr3 are shown in Figure 6C and 6D. According to the docking results, Tyr3 binds to the central hydrophobic core fragment of IAPP (20-29) through hydrogen bonding, hydrophobic interactions, and interacts with Phe15 through π-π stacking. It has been evidenced that hydrophobic interaction and π-π stacking are important driving forces for IAPP amyloid formation,23,24 and the interaction between Tyr3 and IAPP can interfere the aggregation of IAPP by blocking IAPP intermolecular hydrophobic interactions and interrupting intramolecular π-π stacking. In addition, several studies have indicated that EGCG interacts with Phe23 (through π-π interaction) and Ile26 (through hydrophobic interaction) of IAPP, which are critical for the efficient inhibition effect of EGCG.33,49,50 The inhibition mechanism of Tyr3 on fibrillation of IAPP is similar to that of EGCG, and they both inhibit the aggregation of IAPP via hydrogen bonding, hydrophobic interaction and π-π stacking. On the other hand, we also performed the molecular docking simulation of IAPP interacts with Tyr4 and Tyr6. According to the docking results shown in Figure S3, the binding modes of both Tyr4 and Tyr6 are similar to that of Tyr3. They interact with IAPP through hydrogen bonding, hydrophobic interaction and π-π stacking. However, our biophysical studies suggest that Tyr4 and Tyr6 are not effective inhibitors of IAPP amyloid formation. The possible reason for this inconsistency is that the hydrophobicity of the oligotyrosines raise with the increase of the tyrosine number, leading to strong self-association and aggregation propensity of the oligopeptides, and in consequence 19



interrupting the interactions between the oligopeptides and IAPP.

A

B

D

C

Phe15

Figure 6: Docking studies of IAPP with Tyr2 and with Tyr3. (A-B) Low energy conformation from docking study of IAPP in the presence of Tyr2 shown in cartoon representation (A) and in surface representation (B). Low energy conformation from docking study of IAPP interacts with Tyr3 shown in cartoon representation (C) and in surface representation (D). The π-π interactions between Phe15 and Tyr3 are shown in red. Both Tyr2 and Tyr3 are shown in sticks (green). CONCLUSION 20


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The data presented here have clearly demonstrated that oligotyrosines can regulate the formation of IAPP amyloid in a tyrosine-number dependent manner. When the oligotyrosine contains three tyrosine units or less, its inhibition effect increases with the increase of the tyrosine unit number, while the inhibition effect decreases gradually when the oligotyrosine contains more than three tyrosine units. Two oligotyrosines, Tyr2 and Tyr3, have been identified to effectively inhibit IAPP amyloid fibrillation, even in the presence of lipid membrane, and alleviate IAPP-induced cytotoxicity to INS-1 cells. Molecular docking studies suggest that Tyr2 inhibits IAPP amyloid formation by binding to N-terminal region and interrupting IAPP-IAPP association, whereas Tyr3 can interact with hydrophobic core region and aromatic residue, leading to an effective prevention of IAPP aggregation. These results provide us useful information for understanding the mechanism of inhibition of IAPP amyloid formation and developing candidates for anti-diabetic drugs. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the website. Thioflavin-T (ThT) fluorescence assays of hIAPP amyloid formation in the presence of different concentrations of Tyr, the structures of IAPP and oligotyrosines employed to perform docking studies, and the docking simulation results of IAPP with Tyr4 and with Tyr6. Notes The authors declare no competing financial interests. 21



ACKNOWLEDGEMENTS We thank our group members for the helpful discussions. This work is supported by National Basic Research Program of China (2018YFA0208900) and the innovation programs (0118170135 and 3004170118) of Huazhong University of Science and Technology. We also thank the HUST Analytical and Testing Center for allowing us to use its facilities. REFERENCES (1) Eisenberg, D.; Jucker, M. The amyloid state of proteins in human diseases. Cell 2012, 148, 1188-1203. DOI: 10.1016/j.cell.2012.02.022 (2) Chiti, F.; Dobson, C. M. Protein Misfolding, Functional Amyloid, and Human Disease. Annu. Rev. Biochem. 2006, 75, 333-366. DOI: 10.1146/annurev.biochem.75.101304.123901

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For Table of Contents Use Only Oligotyrosines Inhibit Amyloid Formation of Human Islet Amyloid Polypeptide in a Tyrosine-Number Dependent Manner Yanru Xin†, Qigang Hu†, Sidan Tian†, Huazhi Zhang†, Chenhui Wang†, ‡, Liang Luo†, ‡*,

Fanling Meng†, ‡*

26


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Oligotyrosines Inhibit Amyloid Formation of Human Islet Amyloid

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