Conformation-Dependent Manipulation of Human Islet Amyloid

Subscriber access provided by University of South Dakota

Biological and Medical Applications of Materials and Interfaces

Conformation-Dependent Manipulation of Human Islet Amyloid Polypeptide Fibrillation by Shiitake-Derived Lentinan Yanru Xin, Xiuxia Wang, Liang Luo, and Fanling Meng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11078 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018

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

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

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

ACS Applied Materials & Interfaces

Conformation-Dependent Manipulation of Human Islet Amyloid Polypeptide Fibrillation by Shiitake-Derived Lentinan Yanru Xina, Xiuxia Wanga, Liang Luoa, b*, Fanling Menga, b* a. National Engineering Research Center for Nanomedicine, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, China b. Wuhan Institute of Biotechnology, Wuhan, 430075, China

Key words: Diabetes; Islet amyloid Conformation-dependent; Inhibition

polypeptide; Lentinan; Triple-helical;

1

ACS Paragon Plus Environment

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

Page 2 of 32

ABSTRACT Misfolding and aggregation of human islet amyloid polypeptide (hIAPP) into fibrils is an important contribution to the pathology of type 2 diabetes. Developing effective inhibitors of protein aggregation and fibrillation has been considered a promising therapeutic approach to preventing and treating type 2 diabetes. Herein we report that Shiitake-derived polysaccharide lentinan manipulates in vitro hIAPP fibrillation and modulates IAPP-induced cytotoxicity, in a conformation dependent manner. In its triple-helical conformation, lentinan effectively inhibits hIAPP fibrillation, either in bulk solution or in the presence of lipid membrane, suppresses reactive oxygen species (ROS) generation, and attenuates hIAPP-induced cell toxicity. In contrast, lentinan accelerates hIAPP aggregation when it exists in a random-coil conformation, and shows no suppression on hIAPP-mediated ROS production. Further investigation shows that the interaction between triple-helical lentinan and monomeric hIAPP is more favorable than the inter-molecular binding of hIAPP, which redirects hIAPP aggregates to discrete nontoxic nanocomposites. To the best of our knowledge, this is the first time to report a conformation-dependent inhibition of hIAPP aggregation, which will provide new insights for our understanding of the manipulation mechanisms on hIAPP by natural polysaccharides, and open a new avenue for designing and screening potential amyloid inhibitors against type 2 diabetes.

2


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


INTRODUCTION Human islet amyloid polypeptide (hIAPP) is a 37-residue peptide containing a disulfide bond between Cys-2 and Cys-7 and an amidated C-terminus. It is synthesized and co-secreted with insulin in the pancreatic beta cells of both healthy people and diabetes patients. The misfolding and aggregation of monomeric hIAPP into β-sheet enriched oligomers and amyloid fibrils, which are toxic to cultured islet beta cells, is believed to contribute to the beta cell dysfunction and beta cell mass loss associated with the later stages of type 2 diabetes.1-7 Fibrillation of hIAPP has also been implicated as one of the potential causative agents to the failure of islet cell transplants.8-10 Although the molecular mechanisms of how peptide aggregates contribute to amyloid diseases has not been well understood, there is continuously increased interest in developing various inhibitors of hIAPP amyloid formation, including small molecules, peptides, polymer nanoparticles, and antibodies,11-24 as potential therapeutic approach to preventing and treating type 2 diabetes. However, most of the existing inhibitors are effective only at concentrations comparable to or higher than the total concentration of hIAPP, so that their further applications are greatly limited. Polysaccharides, as naturally occurring substances, have been proven to have strong correlations with amyloidogenic peptides,25-36 providing exciting new opportunities in the search for effective, non-toxic inhibitors of hIAPP fibrillation.37-40 Lentinan (LNT), isolated from Shiitake mushroom, is a β-1,3 beta-glucan with two β-1,6 glucosyl groups for every five glucose residues and has a comb-like branched 3



Page 4 of 32

primary structure (Figure 1A). Natively, LNT exists as a triple-helical structure that is stabilized by both inter and intramolecular hydrogen bonds. In dimethyl sulfoxide (DMSO) or concentrated NaOH solutions, the LNT triple-helixes are transformed into single random-coil chains, when the hydrogen bonds are disrupted.41-44 LNT exhibits multiple bioactivities in many in vitro and in vivo studies, such as anti-inflammation,45 antitumor activity,46-48 activating natural immunity,49-50 and protection pancreatic cells from streptozotocin-induced damage.51 However, the anti-amyloidogenic biological activity of LNT has not yet been reported. In addition, the biological activity of LNT is associated with its conformations. For instance, triple-helical LNT potentially inhibits the Sarcoma 18 solid tumor growth both in vivo and in vitro, while the random-coil LNT has no obvious effects.49 It is intriguing to investigate whether LNT can manipulate the fibrillation of amyloidogenic peptides, in a conformation dependent manner. In the work presented here, we have comprehensively characterized the effect of LNT on cytotoxic fibrillation of hIAPP, using a combination of instrumental methods, including thioflavin T (ThT) fluorescence kinetics assays, dynamic light scattering (DLS), circular dichroism (CD), and transmission electron microscopy (TEM). Strikingly, our results have revealed a conformation-dependent manipulation of hIAPP amyloid aggregation by LNT. When the conformation of LNT is triple-helical, the polysaccharide inhibits hIAPP fibrillation very effectively and attenuates hIAPP-mediated cytotoxicity simultaneously. On the contrary, LNT fails to inhibit hIAPP amyloid formation when existed as random coils. Instead, it accelerates hIAPP 4


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


nucleation and promotes fibrillation of the peptide. Our work has demonstrated that triple-helical LNT represents an interesting lead structure in designing inhibitors of hIAPP fibrillation, and also paved the pathway of investigating natural polysaccharides as potential pharmacological treatments of type 2 diabetes. EXPERIMENTAL SECTION Materials. Human islet amyloid polypeptide (hIAPP) was purchased from Science Peptide Biological Technology (Shanghai, China). Lentinan (LNT, average m. w. 300,000 Da) was purchased from Saccharidia Co., Ltd (Shanghai, China). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP, ≥ 99 %), Thioflavine-T (ThT), Trizma hydrochloride (Reagent grade, ≥ 99%), and Trizma base (≥ 99.9%) were purchased from Sigma-Aldrich (USA). Congo red (CR) was purchased from Energy Chemical (Shanghai,

China).

Lipids

(dioleoylphosphatidylglycerol

(DOPG)

and

dioleoylphosphatidylcholine (DOPC)) were from Avanti Polar Lipids (AL, USA). Alamar Blue was purchased from Invitrogen (NY, USA). 2',7'-dichlorofluorescein diacetate (DCFH-DA) was purchased from Aladdin (Shanghai, China). RPMI 1640 was from Hyclone (South Logan, UT). Rat insulinoma cells were obtained from Shanghai Gefan Biotechnology Co., LTD (Shanghai, China). Preparation of hIAPP peptide solutions. For all experiments, hIAPP was freshly dissolved in HFIP and filtered through 0.22 µm filters before use. Aliquots of hIAPP were lyophilized under vacuum. The buffer was added into the dried hIAPP immediately before use. Thioflavin-T (ThT) fluorescence assay. Fluorescence measurements were 5



Page 6 of 32

recorded in 96-well black microplates (Corning Costar Corporation, USA) at 30 ºC by a fluorescence microplate reader (Varioskan LUX, Thermo Scientific). The excitation wavelength was set to 435 nm, and the emission was monitored at 485 nm. For the kinetics experiments to explore the effects of the triple helical LNT on hIAPP aggregation, LNT was dissolved in saline (0.9% NaCl solution) and all hIAPP solutions were prepared by adding saline to dried hIAPP peptide. Samples for the experiments to study the impact of random coils on hIAPP aggregation, LNT was dissolved in 100% DMSO, and then added to hIAPP solutions.

The final solution

condition for these hIAPP kinetic experiments was 4.7% DMSO in 20 mM Tris-HCl buffer (pH 7.4). For the kinetics experiments in the presence of lipid membranes, the concentration of lipid vesicles was adjusted to 630 µM. All samples contain 16 µM hIAPP and 40 µM ThT, and the final concentrations of LNT, when present, were 200 µg/ml, 100 µg/ml, and 50 µg/ml. Dynamic light scattering (DLS). DLS was conducted on a Malvern Nano ZS90 particle size analyzer. 16 µM hIAPP was incubated with or without LNT in a cuvette at room temperature with a scattering angle of 90°. The first measured time point was set as 0 h. The condition of each sample was the same as what was described in ThT assays (2.3). Each time point was measured 3 times. Far-UV circular dichroism (CD). CD spectra were recorded on a JASCO-810 circular dichroism spectrometer under a constant flow of nitrogen at 25 ºC. Wavelength scans were recorded from 190 nm to 260 nm at 1 nm intervals in a 0.2 cm path length quartz cuvette. Aliquots were removed from the ThT assays when the 6


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


amyloid kinetics experiments were completed, and submitted for CD spectroscopy measurement. The final spectra were the average of 5 scans. Background spectra were subtracted from the collected data. Transmission electronic microscopy (TEM). TEM experiments were performed on a Hitachi HT7700 Transmission Electron Microscope system with an accelerating voltage of 80 kV. Each sample was placed on a 300 mesh Formvar-carbon coated copper grid for 2 min and then negatively stained with saturated uranyl acetate for 2 min. Unilameller vesicle preparation. Unilameller vesicles used in this work were prepared from a 7:3 mixture of DOPC/DOPG. The chloroform was removed by rotary evaporation followed by drying under a stream of nitrogen. The dried film was hydrated in saline for 1 h. The hydration mixture was sonicated for 20 minutes. The size of the prepared vesicles was determined by a particle size analyzer (Nano ZS90, Malvern). The stock concentration of the prepared vesicles was 7.14 mM. The final concentration of the prepared vesicles in the kinetics experiments was 630 µM. Fluorescence imaging. hIAPP (16 µM) in the presence or absence of LNT was incubated at 37 ºC overnight. The concentration of LNT, when present, is 200 µg/ml, 100 µg/ml, and 50 µg/ml. CR (1 mM) and ThT (45 µM) were added into the sample solution respectively. The mixed solutions were placed onto a glass slide, covered with a cover glass and excited with a blue light under a fluorescence microscope to observe the ThT fluorescence signal (460-490 nm excitation wavelength). The samples used for CR fluorescence were dried in the air and then take the images under 7



Page 8 of 32

a fluorescence microscope. Cell toxicity assay. Rat insulinoma (INS-1) cells were cultured in RPMI-1640 media supplemented with 10% FBS, 1% sodium pyruvat, 1% penicillin-streptomycin, and 50 µM β-mecaptoethanol. Cells were incubated in 5% CO2 atmosphere and passaged at 80% cell density (0.25% trypsin-EDTA) for subsequent experiments. Cell viability was measured using the Alamar Blue reduction assay. Cells were plated at a density of 15,000 cells per well in 96-well plates and incubated at 37 ºC in 5% CO2 atmosphere overnight before addition of solutions. Samples of hIAPP (32 µM) in the presence or absence of LNT were prepared in saline. After an overnight incubation at 37 °C, samples were added directly to the cells (30% final media concentration) and incubated for another 5 h. Alamar Blue was diluted 10-fold in the media and co-incubated with cells for 3 h at 37 ºC. Fluorescence was measured with the microplate reader. The excitation wavelength was set to 530 nm, and emission was monitored at 590 nm. Measurement of reactive oxygen species (ROS) generation. ROS production was measured by incubating the cells with the fluorescent probe (DCFH-DA). INS-1 cells were treated with hIAPP in the presence or absence of LNT and then incubated at 37 °C for 5 h. After incubation, cells were treated with 10 µM DCFH-DA for 30 min at 37 °C. Cells were then washed twice with PBS before measurement. Fluorescence was measured at 485 nm excitation and 535 nm emission wavelengths by the fluorescence microplate reader. FTIR Measurement. FTIR spectra were collected at room temperature on a 8


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


BRUKER ATR-FTIR Alpha spectrometer. Each spectrum was collected with 40 scans at a resolution of 4 cm-1 and the collection range was 4000-600 cm-1. The samples (32 µM hIAPP, 1.34 µM LNT, and a mixture of 32 µM hIAPP and 1.34 µM LNT) were all dissolved in water to avoid the interference of saline. Each solution was added dropwise on the sample plate of the instrument and air-dried before measurement. Statistical analysis. Data were presented as mean ± standard deviation. Statistical significance was determined using one-way analysis of variance and two-tailed Student’s t-test. P values of p < 0.05 were considered to be statistically significant. RESULTS AND DISCUSSION Triple-helical and random-coil conformations of LNT. It has been reported that LNT can exist either as triple helixes or as random coils in different environments.43, 52

We have firstly examined the conformation of LNT upon addition into different

experiment conditions in our study. The TEM image of samples made by LNT in saline displayed entangled winding chains (Figure 1B), which is the typical morphology of LNT triple helixes in aqueous solutions. When LNT is dissolved in DMSO, its spherical morphology, as demonstrated by the TEM image (Figure 1C), suggests that LNT has a random-coil conformation in DMSO. The particle size of LNT in saline and in DMSO, as measured by DLS, is approximately 249.6 nm and 44.9 nm respectively, consistent with the corresponding conformation in each condition (Figure 1D).43 The conformations of LNT are further evidenced by the absorption spectroscopic change of Congo Red (CR), which is typically employed to 9



evaluate the conformational behaviors of polysaccharides in solution. When LNT and CR are mixed in saline, there is an apparent red shift of the maximum absorbance compared to the CR solution, as shown in Figure 1E, consistent with the formation of a complex between CR and triple-helical LNT.52 In contrast, the maximum absorbance wavelength of CR remains unchanged when it is mixed with LNT in DMSO (Figure 1F), suggesting that LNT exists as random coils under this condition. A

1

10

100

D / nm h

1000

CR in saline CR+LNT in saline

E

400

500

600

700

Wavelength(nm)

Absorbance (a. u.)

15

Absorbance (a. u.)

LNT in DMSO LNT in saline

30

0

C

B

D Number(%)

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

Page 10 of 32

CR in DMSO CR + LNT in DMSO

F

500

600

700

Wavelength(nm)

Figure 1. (A) The structure of LNT. (B) TEM image of 0.1 mg/ml LNT in saline. (C) TEM image of 0.1 mg/ml LNT in DMSO. (D) Size distribution of LNT in DMSO and in saline characterized by DLS. (E) Absorbance spectrum of CR only and a mixture of CR and LNT in saline. (F) Absorbance spectrum of CR only and a mixture of CR and LNT in DMSO. Scale bar of the TEM images: 100 nm. Inhibition of hIAPP Fibril formation by triple-helical LNT. Thioflavin (ThT) fluorescence assays monitored the fibrillation kinetics of hIAPP in the absence and presence of LNT as a function of time. To investigate the effect of triple-helical LNT 10


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


on hIAPP fibrillation, the kinetics experiments were performed in saline, where LNT existed dominantly as triple helixes. With prolonged incubation time, kinetics of hIAPP in the absence of LNT exhibited a typical sigmoidal curve (Black curve, Figure 2A), which consisted of a lag phase during which peptide oligomers were produced, a growth phase when amyloid fibrils were generated, and a final plateau region where the fibrils and soluble peptide reached an equilibrium. The average time needed to reach the half of the plateau fluorescence intensity, or t50, is about 520 min for hIAPP alone. However, when LNT was present with a concentration of 200 µg/ml, there was no detectable change in ThT fluorescence intensity observed (red curve, Figure 2A), suggesting an ultra-effective extension of the lag phase and prevention of amyloid fibril formation by triple-helical LNT. The molar ratio of hIAPP:LNT was calculated to be 24:1 under this condition, on basis of the average LNT molecular weight of 300,000 Da. Complete inhibition of hIAPP fibrillation was still observed at reduced concentration of LNT (100 µg/ml, or 0.33 µM) during the time course of the whole experiment (> 800 min). Under this condition, the molar ratio of hIAPP:LNT was calculated to be 48:1, more potent than most of the existing inhibitors of hIAPP fibrillation reported to date. Triple-helical LNT could effectively inhibit hIAPP amyloid fibril assembly even when the concentration of LNT was decreased to 50

µg/ml (0.17 µM, hIAPP:LNT molar ratio of 96:1), with an apparent increase in the lag time and a reduction in the final fluorescence intensity of 85% when compared to in the absence of LNT. Far-UV circular dichroism (CD) spectra of samples at the end of the kinetics 11



Page 12 of 32

experiments were used to determine the impact of triple-helical LNT on hIAPP secondary structure, as shown in Figure 2B. The CD spectrum of hIAPP alone displayed a typical amyloid β-sheet structure with a characteristic negative peak at 222 nm. On the contrary, CD spectra of hIAPP treated with different doses of triple-helical LNT were all consistent with largely unstructured peptides, the negative peak wavelength shifting from 205 nm to 200 nm with increased LNT concentrations. The CD spectroscopic results suggested that triple-helical LNT effectively inhibits the hIAPP conformation transition from the unfolded structure to the β-sheet structure, on a dose dependent basis. The inhibition of triple-helical LNT on hIAPP amyloid fibril formation was further confirmed by TEM images recorded at the end of the kinetics experiments. A dense mat of fibrils were observed for the sample of hIAPP alone, which was typically found for in vitro hIAPP amyloid deposits (Figure 2C). As a comparison, the samples of hIAPP added with 200 and 100 µg/ml of triple-helical LNT displayed a majority of discrete nanocomposites on the TEM images (Figure 2D and 2E). In addition, a few fibril-like structures, different from amyloid fibrils but consistent with the morphology of LNT triple helixes shown in Figure 1B, were visible in the TEM images. When smaller amount of LNT was presented (50 µg/ml), some hIAPP fibrils, with much thicker and longer appearance, started to emerge on the TEM images (Figure 2F), consistent with the less effective inhibition effect on hIAPP fibrillation at reduced LNT concentration shown in Figure 2A.

12


100

Normalized Fluorescence

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


A

LNT concentration (µg/ml) 0 50 100 200

50

0 0

200

D

400 600 Time (min)

800

B

4

Ellipticity (mdeg)

Page 13 of 32

2

LNT concentration (µg/ml)

C

0 50 100 200

0 -2 -4 200

220 240 Wavelength (nm)

260

E

F

Figure 2. Effects of triple-helical LNT on hIAPP fibrillation monitored by ThT assays, CD, and TEM. (A) hIAPP fibrillation kinetics monitored by ThT assays, in the presence of LNT saline solutions at different concentrations. (B) CD spectra of samples at the end of the kinetics experiments. (C-F) TEM images of samples at the end of the kinetics studies. The corresponding LNT concentration in each sample was (C) 0 µg/ml, (D) 200 µg/ml, (E) 100 µg/ml, (F) 50 µg/ml. To further confirm that the observed fibrillar structure in Figure 2C and 2D is mainly formed by LNT but not hIAPP, we employed ThT and CR to individually stain the hIAPP samples that had been co-incubated overnight with different concentrations of LNT, taking advantage of the different binding properties of these two fluorophores: ThT only binds with amyloid fibrils but CR can bind with both amyloid fibrils and polysaccharide triple helixes. The fluorescence images (Figure 3) of ThT-stained samples showed strong signals when no LNT was presented, indicating the formation of a large quantity of hIAPP fibrils. For the samples co-incubated with LNT (200 µg/ml and 100 µg/ml), very weak fluorescence of ThT was shown under the 13



microscope, suggesting that hIAPP fibrillation was well inhibited in each case. Some fluorescence signals showed up in samples treated with 50 µg/ml LNT, suggesting the formation of very small amounts of hIAPP fibrils. On the contrary, intensive fluorescence signals of CR were observed for all samples, either with or without LNT, clearly evidencing that triple-helical LNT was existed in the kinetics experiments. These results further confirmed that the observed fibrils in the kinetic experiment

ThT

were majorly from triple-helical LNT.

CR

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

Page 14 of 32

0 µg/ml

200 µg/ml

100 µg/ml

50 µg/ml

Figure 3. Fluorescence images of ThT- or CR-stained hIAPP samples which had been co-incubated in the presence of different concentrations (0, 200, 100, and 50 µg/ml) of LNT overnight (Magnification 200 ×). In addition, dynamic light scattering (DLS) experiments have also been employed to determine the influence of triple-helical LNT on hIAPP aggregation, by measuring the particle sizes in the individual system as a function of time. The average size of hIAPP without LNT gradually increased to 2,000 nm after incubating for 8 h (Figure 4A), indicating the formation of large aggregates during the period. Incubation the 14


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


hIAPP solution for longer time resulted in the formation of precipitates that were not suitable to measure by DLS. However, incubating hIAPP solutions in the presence of triple-helical LNT showed very different results. The average particle sizes of all solutions remained almost unchanged (less than 200 nm) after being incubated for 8 h, as shown in Figure 4B-4D, which suggested that triple-helical LNT could effectively prevent the formation of large aggregates of hIAPP. The DLS measurement results, together with the results from ThT assays, CD, and TEM studies, unambiguously demonstrate that LNT can effectively inhibit hIAPP amyloid fibril formation when it is in triple-helical conformation. A

1

8h

10

100

1000

B

8h

6h

6h

4h

4h

2h

2h

0h

0h

10000

1

10

C

1

100

/ nm

1000

D

8h

10

100

10000

/ nm

/ nm

1000

8h

6h

6h

4h

4h

2h

2h

0h

0h

10000

1

10

100

1000

10000

/ nm

Figure 4. Temporal evolution of the size distribution of hIAPP in the absence (A) and presence of LNT saline solutions co-incubated at 30 ºC, as determined by dynamic light scattering. The LNT concentrations were 200 µg/ml (B); 100 µg/ml (C); and 50 15



Page 16 of 32

µg/ml (D). The effect of LNT random coils on hIAPP fibrillation. While triple-helical LNT can super-effectively inhibit hIAPP amyloid formation, the situation for LNT random coils is completely different. We examined the effect of LNT on hIAPP fibrillation by conducting the kinetics experiment where LNT was added as random coils. In this experiment, the LNT stock solution in DMSO was added to the hIAPP solution in 20 mM Tris-HCl (pH 7.4). Under this DMSO-containing buffer (DCB) condition, the data obtained from ThT fluorescence assays of hIAPP alone still showed a typical sigmoidal curve (Black curve Figure 5A). The t50 was measured to be 650 min, suggesting that hIAPP had a longer lag phase in DCB than in saline (t50 of 520 min) (Figure 5B). Interestingly, adding LNT into the DCB-mediated system decreased the lag time of the kinetics and lowered the final fluorescence intensity (Figure 5A). The t50 decreased with the increase of the concentration of LNT, as shown in Figure 5B, suggesting that the presence of LNT random coils could promote the formation of hIAPP amyloid fibrils. As a comparison, the t50 of the kinetics in the presence of triple-helical LNT increases with the increase of LNT concentration, in consistent with the effective inhibition of hIAPP fibrillation by triple-helical LNT. The TEM images of the samples at the end of the kinetics experiments in DCB media all displayed large quantities of hIAPP amyloid fibrils, either in the absence or in the presence of LNT (Figure 5C-5F), further confirming that LNT failed to inhibit the fibrillation of hIAPP as random coils.

16


Page 17 of 32

B 800

A 100

LNT concentration (µg/ml) 0 50 100 200

50

700 t50 (min)


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


LNT conformation Triple helixes Random coils

600 500 400

0 0

200

400 600 Time(min)

0

800

C

D

E

F

50 100 150 200 LNT concentration (µg/ml)

Figure 5. The effect of LNT random coils on hIAPP fibrillation characterized by ThT assays and TEM. (A) hIAPP aggregation kinetics monitored by ThT assays in the presence of different concentrations of LNT. Experiments conducted in DCB solutions. hIAPP concentration in each experiment: 16 µM. (B) t50 of hIAPP kinetics experiments in the presence of different concentrations of triple-helical LNT and random-coil LNT. For LNT triple helixes at 100 and 200 µg/ml, t50 is represented by the whole experiment time of 800 min. (C-F) TEM images of samples at the end of the kinetics studies. LNT concentration: (C) 0 µM; (D) 50 µg/ml; (E) 100 µg/ml; (F) 200 µg/ml. Scale bar: 200 nm. The effect of LNT on hIAPP fibrillation at membrane. We have also examined the inhibitory effect of LNT under phospholipid membrane conditions, because many 17



Page 18 of 32

inhibitors get deactivated in the existence of lipids 53. In addition, this study is of great importance in that the interaction between membrane and misfolded peptide is involved in the hIAPP-induced cell toxicity in type 2 diabetes. We use DOPC:DOPG (7:3) as a membrane model system, which is close to the anionic contents of cell membrane phospholipids. The characteristic sigmoidal curve was observed for hIAPP in the presence of lipid vesicles, with a very short t50 of less than 50 min (Figure 6A), indicating the accelerating effect of lipid membrane on IAPP amyloid formation which has been well established.54 The TEM image of the sample after the kinetics study demonstrated numerous amyloid fibrils (Figure 6C). In the presence of triple-helical LNT (200 µg/ml), significant inhibition of hIAPP aggregation was observed, where the t50 increased by a factor of 3. The TEM image for the samples at the end of the kinetics studies displayed significant less amount of amyloid fibrils than in the absence of LNT, as shown in Figure 6D. Inhibition was still observed when lower concentrations of LNT is present (50 and 100 µg/ml), where the t50 increased to 120 and 80 minutes respectively. The final fluorescence intensities of all samples have reduced to approximately 70% of that observed for hIAPP alone. On the other hand, hIAPP exhibited very different kinetics behaviors in the presence of lipid vesicles when random-coil LNT was added. Negligible effect on the hIAPP fibrillation was observed, and a dense mat of hIAPP fibrils formed at the end of the kinetics studies for hIAPP with or without random-coil LNT (Figure S1). The inhibition activity of triple-helical LNT under cell membrane conditions was further confirmed by CD spectroscopy. The CD spectrum of hIAPP alone in the 18


Page 19 of 32

lipid-containing solution showed a negative absorbance band at 222 nm, which suggests that hIAPP formed characteristic amyloid β-sheet structures. In the presence of triple-helical LNT, the CD spectra of hIAPP in the lipid-containing solutions have much less β-sheet structures formed (Figure 6B), and the content of β-sheet structures gradually increased as the concentration of LNT decreased. The above experiments have strongly proved that triple-helical LNT is so effective that it can inhibit amyloid formation at membrane surface. A

B

100

0

50

Ellipcity (mdeg)


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


0 50 100 200

0 0

100 200 Time(min)

300

-1

0 50 100 200

-2

200

C

D

E

F

220 240 Wavelength (nm)

260

Figure 6. Triple-helical LNT inhibits amyloid formation of hIAPP in the presence of 630 µM of lipid vesicles. (A) Kinetics of hIAPP amyloid formation in the presence of different concentration (0, 50, 100, and 200 µg/ml) of LNT monitored by ThT 19



Page 20 of 32

fluorescence. (B) CD spectra of hIAPP in the presence of different concentration (0, 50, 100, and 200 µg/ml) of LNT. Aliquots were removed for CD experiment at the end of the kinetics reactions. (C-F) TEM images of hIAPP at the end of each kinetics experiment. (C) IAPP alone, (D) IAPP with 200 µg/ml LNT, (E) IAPP with 100 µg/ml LNT and (F) IAPP with 50 µg/ml LNT. The concentration of hIAPP in all experiment is 16 µM in saline. Scale bar: 500 nm. Manipulation of hIAPP-mediated ROS generation and cytotoxicity by LNT. hIAPP fibrillation is considered critical in the islet beta cell death, as well as in the pathogenesis of type 2 diabetes. Oxidative stress has been implicated as a potential contributing factor in hIAPP-induced cell toxicity in vitro and in human type 2 diabetes patients.55-60 To gain an in-depth understanding of the biochemical impact of LNT with different conformations, the production of reactive oxygen species (ROS) in rat insulinoma (INS-1) β-cells has been examined by fluorescein-labeled dye DCFH-DA (Figure 7A and 7B). When the experiment was conducted in saline, hIAPP upregulated the intracellular ROS production to 185% relative to the control cells treated with saline only, as determined by the DCFH fluorescence intensity. Treating cells with hIAPP in the presence of triple-helical LNT reduces ROS generation in a dose-dependent manner. Higher concentration of LNT triple helixes resulted in lower production of ROS in INS-1 cells. The findings were graphically validated by fluorescence microscopy (Figure 7B). The fluorescence microscope images were taken immediately prior to the assessment of ROS production. The cells treated with hIAPP alone displayed bright fluorescence, while in the presence of triple-helical LNT, 20


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


very weak fluorescence signals were observed, suggesting that triple-helical LNT was very effective in suppressing hIAPP-mediated ROS generation. In contrast, when the ROS tests were conducted in DCB, hIAPP only upregulated the ROS production to 130% of the control cells treated with DCB alone, attributed to the different experiment conditions. However, adding LNT under this condition showed no difference in ROS production than bare hIAPP (Figure 7C), indicating that random-coil LNT had no suppression effect on hIAPP-mediated ROS generation. To further confirm the difference in ROS production between hIAPP treated with triple-helical and with random-coil LNT, as well as to eliminate the possible effect of DMSO on the cells, we have also carried out ROS generation experiment of hIAPP with triple-helical LNT where DMSO was added into the saline solution of LNT (the final v% of DMSO is 4.7%). The results showed that hIAPP upregulated the ROS production to 130% of the control cells treated with DMSO-containing saline. However, triple-helical LNT still alleviated the generation of ROS under this condition in a dose-dependent manner, as shown in Figure S2, evidencing that the suppression effect of LNT on hIAPP-mediated ROS generation is conformation dependent. We next evaluated the ability of triple-helical LNT to protect against the toxicity of hIAPP fibrillation to the INS-1 cells. Incubation of hIAPP with INS-1 cells for 5 hours leads to 70% cell viability relative to control cells, as determined by Alamar Blue assays (Figure 7D), suggesting that the process of hIAPP fibrillation serves as one of the key factors for the cell damage. Co-incubating hIAPP and triple-helical 21



LNT (50 µg/ml) alleviated hIAPP-induced cytotoxicity significantly, leading to a loss of less than 15% cell viability compared to control cells. Increasing the concentration of triple-helical LNT to 200 µg/ml can completely protect cells from hIAPP-induced toxicity. These results implicated that triple-helical LNT could effectively downregulate the generation of ROS and rescue the cells from hIAPP-mediated damage. A DCF fluorescence intensity (% of control)

B

*** ***

250

0 µg/ml

50 µg/ml

100 µg/ml

200 µg/ml

*

200 150 100 50 0

hIAPP LNT 0 (µg/ml)

+

-

-

-

+

+

0

50 100 200 50 100 200

+

D

C

NS

200

*

NS

**

**

NS

Cell viability (%)

DCF fluorescence intensity (% of control)

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

Page 22 of 32

150

100

100

50

50 0

0

hIAPP LNT (µg/ml) 0

+

+

0

50

+

+

-

-

-

100 200 50 100 200

hIAPP LNT (µg/ml)

-

+

+

+

0

0

50

100

+

-

-

-

200 50 100 200

Figure 7. (A) ROS generated by hIAPP in the absence and presence of different concentrations of triple-helical LNT in saline, determined by measuring the fluorescence intensity of an oxidation-sensitive fluorescein DCFH-DA using ex/em of 495/525 nm. (B) ROS generation in the absence and presence of different 22


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


concentrations of triple-helical LNT examined by fluorescence microscopy (magnification 200 ×). After treatment, INS-1 cells were stained with DCFH-DA and imaged under a fluorescence microscope. (C) ROS generated by hIAPP in the absence and presence of different concentrations of random-coil LNT. (D) Cell toxicity of hIAPP in the absence and presence of triple-helical LNT evaluated by Alamar Blue assays. All data were obtained from three independent experiments and presented as the means ± SD. NS: no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001. Understanding the interaction between LNT and hIAPP. To understand the different inhibitory effects of LNT with different conformations, FTIR was employed to study the interaction between hIAPP and LNT. The FTIR spectra of a mixed sample of hIAPP (32 µM) and triple-helical LNT (400 µg/ml), a sample of hIAPP alone, and a sample of free triple-helical LNT, were recorded as a function of time to illustrate the structural change of hIAPP, as shown in Figure 8. The freshly prepared monomeric hIAPP has a characteristic peak of disordered structure at 1655 cm-1 (C=O bond stretching) at the beginning of the experiment (black spectrum, Figure 8), while a new absorption peak at 1627 cm-1 appeared after prolonged time (20 h), suggesting that C=O bonds within the hIAPP molecule were involved in the intermolecular hydrogen bonding that ultimately led to the formation of amyloid β-structure.61 When triple-helical LNT was added, hIAPP still showed the characteristic peak of 1655 cm-1 after immediately mixing with LNT (red spectrum). However, after co-incubating LNT and hIAPP for just 10 minutes at room temperature, the peak at 1627 cm-1 appeared on the FTIR spectrum. The fast appearance of the 1627 cm-1 peak could be 23



Page 24 of 32

originated from the intermolecular interactions between hIAPP, or from the interaction between hIAPP and LNT triple helixes. The fact that no fibrils (by TEM) or β-structure (by CD) were observed at this stage clearly demonstrated that the 1627 cm-1 peak was not attributed to the intermolecular hydrogen bonding between hIAPP molecules. Therefore the interaction between hIAPP and triple-helical LNT should account for the emergence of the 1627 cm-1 peak. For IAPP with random-coil LNT (Figure S3), there was only one absorption peak of 1655 cm-1 shown, suggesting that there is no such interaction between hIAPP and LNT. We conclude that the strong interaction between hIAPP and the conformation-specific LNT triple helixes is more favorable than the intermolecular interactions of hIAPP, which represents the major factor that contributes to its excellent inhibition capability on hIAPP fibrillation (Figure 8B).

24


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


A

0 min

10 min

20 h

2.5 h

LNT

hIAPP + LNT

hIAPP

2000

1600

2000

1600

2000

1600

2000

1600

-1

Wavenumber (cm )

B

Native hIAPP monomers

Small

Elongated

oligomers

oligomers

Fibrils

Triple-helical LNT Complex of triple-helical and IAPP monomers

Figure 8. (A) FTIR spectra of LNT, hIAPP, and a mixture of hIAPP (32 µM) and triple-helical LNT (400 µg/ml) at different time points. (B) Schematic illustration of hypothetic inhibition mechanism of triple-helical LNT on hIAPP fibrillation process. To examine if all triple-helical polysaccharide can inhibit amyloid formation of hIAPP, we have investigated another triple-helical polysaccharide curdlan. The results demonstrate that curdlan has no obvious inhibition effects on hIAPP amyloid formation (Figure S4). Thus, the potent inhibitory effect of triple-helical LNT on hIAPP amyloid formation is not simply because of the conformation of triple helixes. 25



Page 26 of 32

The unique interaction between the polysaccharide and peptide should also be related to the structure of the polysaccharide, in addition to the triple-helical conformation. CONCLUSIONS We have demonstrated that Shiitake-derived LNT can manipulate the amyloid fibril formation of hIAPP in a conformation-dependent manner. The triple-helical LNT is a potent inhibitor of hIAPP fibrillation, which can effectively redirect hIAPP into discrete nanoparticles, reduce the generation of ROS, and alleviate hIAPP-induced cytotoxicity. The random-coil LNT cannot inhibit hIAPP amyloid fibril formation, but accelerate IAPP aggregation to some extent. This natural polysaccharide can effectively prevent amyloid formation, even in the presence of cell membrane. To the best of our knowledge, this is the first report on the conformation selectivity of a natural polysaccharides on inhibiting fibrillation of amyloidogenic peptides. The excellent inhibition capability of triple-helical LNT also provides a new structural class of potential amyloid inhibitors to explore. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the website. Thioflavin-T (ThT) fluorescence assay and TEM of hIAPP amyloid formation in DCB in the presence of lipid vesicles, measurement of ROS generation by hIAPP in the absence and presence of triple-helical LNT where DMSO was added into the saline solution of LNT, FTIR spectra of LNT in 0.3M NaOH, 26


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


hIAPP only, and the mixture of IAPP and LNT, Thioflavin-T (ThT) fluorescence assay, and TEM images of hIAPP amyloid formation in the presence and in the absence of curdlan. AUTHOR INFORMATION Corresponding Author *E-mail address: [email protected] (F. Meng); [email protected] (L. Luo). Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS We thank Dr. Chenhui Wang for the help in cell toxicity experiments. We also thank Ms. Han Luo and Dr. Jianyong Sheng for the help in lentinan solution preparation. 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. REFERENCES (1) Cooper, G. J. S.; Willis, A. C.; Clark, A.; Turner, R. C.; Sim, R. B.; Reid, K. B. M. Purification and Characterization of a Peptide from Amyloid-Rich Pancreases of Type-2 Diabetic-Patients. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 8628-8632. (2) Kahn, S. E.; Andrikopoulos, S.; Verchere, C. B. Islet Amyloid: A Long-Recognized but Underappreciated Pathological Feature of Type 2 Diabetes. Diabetes 1999, 48, 241-253. (3) Westermark, P.; Wernstedt, C.; Wilander, E.; Hayden, D. W.; Obrien, T. D.; Johnson, K. H. Amyloid Fibrils in Human Insulinoma and Islets of Langerhans of the Diabetic Cat Are Derived from a Neuropeptide-Like Protein Also Present in Normal Islet Cells. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 3881-3885. (4) Westermark, P.; Wernstedt, C.; Wilander, E.; Sletten, K. A Novel Peptide in the Calcitonin Gene Related Peptide Family as an Amyloid Fibril Protein in the 27



Page 28 of 32

Endocrine Pancreas. Biochem. Bioph. Res. Co. 1986, 140, 827-831. (5) Hull, R. L.; Westermark, G. T.; Westermark, P.; Kahn, S. E. Islet Amyloid: A Critical Entity in the Pathogenesis of Type 2 Diabetes. J. Clin. Endocrinol. Metab. 2004, 89, 3629-3643. (6) Jaikaran, E. T. A. S.; Higham, C. E.; Serpell, L. C.; Zurdo, J.; Gross, M.; Clark, A.; Fraser, P. E. Identification of a Novel Human Islet Amyloid Polypeptide beta-Sheet Domain and Factors Influencing Fibrillogenesis. J. Mol. Biol. 2001, 308, 515-525. (7) Marzban, L.; Park, K.; Verchere, C. B. Islet Amyloid Polypeptide and Type 2 Diabetes. Exp. Gerontol. 2003, 38, 347-351. (8) Udayasankar, J.; Kodama, K.; Hull, R. L.; Zraika, S.; Aston-Mourney, K.; Subramanian, S. L.; Tong, J.; Faulenbach, M. V.; Vidal, J.; Kahn, S. E. Amyloid Formation Results in Recurrence of Hyperglycaemia Following Transplantation of Human IAPP Transgenic Mouse Islets. Diabetologia 2009, 52, 145-153. (9) Westermark, G. T.; Westermark, P.; Berne, C.; Korsgren, O.; Transpla, N. N. C. I. Widespread Amyloid Deposition in Transplanted Human Pancreatic Islets. New. Engl. J. Med. 2008, 359, 977-979. (10) Westermark, G. T.; Westermark, P.; Nordin, A.; Tornelius, E.; Andersson, A. Formation of Amyloid in Human Pancreatic Islets Transplanted to the Liver and Spleen of Nude Mice. Upsala. J. Med. Sci. 2003, 108, 193-203. (11) Meng, F. L.; Abedini, A.; Plesner, A.; Middleton, C. T.; Potter, K. J.; Zanni, M. T.; Verchere, C. B.; Raleigh, D. P. The Sulfated Triphenyl Methane Derivative Acid Fuchsin Is a Potent Inhibitor of Amyloid Formation by Human Islet Amyloid Polypeptide and Protects against the Toxic Effects of Amyloid Formation. J. Mol. Biol. 2010, 400, 555-566. (12) Meng, F. L.; Abedini, A.; Plesner, A.; Verchere, C. B.; Raleigh, D. P. The Flavanol (-)-Epigallocatechin 3-Gallate Inhibits Amyloid Formation by Islet Amyloid Polypeptide, Disaggregates Amyloid Fibrils, and Protects Cultured Cells against IAPP-Induced Toxicity. Biochemistry 2010, 49, 8127-8133. (13) Abedini, A.; Meng, F. L.; Raleigh, D. P. A Single-Point Mutation Converts the Highly Amyloidogenic Human Islet Amyloid Polypeptide into a Potent Fibrillization Inhibitor. J. Am. Chem. Soc. 2007, 129, 11300-1. (14) Ladiwala, A. R. A.; Bhattacharya, M.; Perchiacca, J. M.; Cao, P.; Raleigh, D. P.; Abedini, A.; Schmidt, A. M.; Varkey, J.; Langen, R.; Tessier, P. M. Rational Design of Potent Domain Antibody Inhibitors of Amyloid Fibril Assembly. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 19965-19970. (15) Kumar, S.; Birol, M.; Schlamadinger, D. E.; Wojcik, S. P.; Rhoades, E.; Miranker, A. D. Foldamer-Mediated Manipulation of a Pre-Amyloid Toxin. Nat. Commun. 2016, 7, 1412 (16) Gurzov, E. N.; Wang, B.; Pilkington, E. H.; Chen, P. Y.; Kakinen, A.; Stanley, W. J.; Litwak, S. A.; Hanssen, E. G.; Davis, T. P.; Ding, F.; Ke, P. C. Inhibition of hIAPP Amyloid Aggregation and Pancreatic beta-Cell Toxicity by OH-Terminated PAMAM Dendrimer. Small 2016, 12, 1615-1626. (17) Yan, L. M.; Tatarek-Nossol, M.; Velkova, A.; Kazantzis, A.; Kapurniotu, A. 28


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


Design of a Mimic of Nonamyloidogenic and Bioactive Human Islet Amyloid Polypeptide (IAPP) as Nanomolar Affinity Inhibitor of IAPP Cytotoxic Fibrillogenesis. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 2046-2051. (18) Meng, F. L.; Raleigh, D. P.; Abedini, A. Combination of Kinetically Selected Inhibitors in Trans Leads to Highly Effective Inhibition of Amyloid Formation. J. Am. Chem. Soc. 2010, 132, 14340-14342. (19) Cabaleiro-Lago, C.; Lynch, I.; Dawson, K. A.; Linse, S. Inhibition of IAPP and IAPP((20-29)) Fibrillation by Polymeric Nanoparticles. Langmuir 2010, 26, 3453-3461. (20) Cao, P.; Meng, F.; Abedini, A.; Raleigh, D. P. The Ability of Rodent Islet Amyloid Polypeptide To Inhibit Amyloid Formation by Human Islet Amyloid Polypeptide Has Important Implications for the Mechanism of Amyloid Formation and the Design of Inhibitors. Biochemistry 2010, 49, 872-881. (21) Kumar, S.; Miranker, A. D., A Foldamer Approach to Targeting Membrane Bound Helical States of Islet Amyloid Polypeptide. Chem. Commun. 2013, 49, 4749-4751. (22) Nedumpully-Govindan, P.; Kakinen, A.; Pilkington, E. H.; Davis, T. P.; Ke, P. C.; Ding, F. Stabilizing Off-pathway Oligomers by Polyphenol Nanoassemblies for IAPP Aggregation Inhibition. Sci. Rep. 2016, 6, 19463 (23) Sormanni, P.; Aprile, F. A.; Vendruscolo, M. Rational Design of Antibodies Targeting Specific Epitopes within Intrinsically Disordered Proteins. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 9902-9907. (24) Zhao, D. S.; Chen, Y. X.; Li, Y. M. Rational Design of an Orthosteric Regulator of hIAPP Aggregation. Chem. Commun. 2015, 51, 2095-2098. (25) Abedini, A.; Tracz, S. M.; Cho, J. H.; Raleigh, D. P. Characterization of the Heparin Binding Site in the N-Terminus of Human Pro-Islet Amyloid Polypeptide: Implications for Amyloid Formation. Biochemistry 2006, 45, 9228-9237. (26) Marzban, L.; Rhodes, C. J.; Steiner, D. F.; Haataja, L.; Halban, P. A.; Verchere, C. B. Impaired NH2-terminal Processing of Human Proislet Amyloid Polypeptide by the Prohormone Convertase PC2 Leads to Amyloid Formation and Cell Death. Diabetes 2006, 55, 2192-2201. (27) Meng, F.; Abedini, A.; Song, B.; Raleigh, D. P. Amyloid Formation by Pro-Islet Amyloid Polypeptide Processing Intermediates: Examination of the Role of Protein Heparan Sulfate Interactions and Implications for Islet Amyloid Formation in Type 2 Diabetes. Biochemistry 2007, 46, 12091-12099. (28) Vieira, T.; Cordeiro, Y.; Caughey, B.; Silva, J. L. Heparin Binding Confers Prion Stability and Impairs Its Aggregation. FASEB J. 2014, 28, 2667-2676. (29) Hull, R. L.; Peters, M. J.; Perigo, S. P.; Chan, C. K.; Wight, T. N.; Kinsella, M. G. Overall Sulfation of Heparan Sulfate from Pancreatic Islet beta-TC3 Cells Increases Maximal Fibril Formation but Does Not Determine Binding to the Amyloidogenic Peptide Islet Amyloid Polypeptide. J. Biol. Chem. 2012, 287, 37154-37164. (30) Papy-Garcia, D.; Morin, C.; Huynh, M. B.; Sineriz, F.; Sissoeff, L.; Sepuveda-Diaz, J. E.; Raisman-Vozari, R. Glycosaminoglycans, Protein Aggregation 29



Page 30 of 32

and Neurodegeneration. Curr. Protein Peptide Sci. 2011, 12, 258-268. (31) Valle-Delgado, J. J.; Alfonso-Prieto, M.; de Groot, N. S.; Ventura, S.; Samitier, J.; Rovira, C.; Fernandez-Busquets, X. Modulation of A beta(42) Fibrillogenesis by Glycosaminoglycan Structure. FASEB J. 2010, 24, 4250-4261. (32) Bravo, R.; Arimon, M.; Valle-Delgado, J. J.; Garcia, R.; Durany, N.; Castel, S.; Cruz, M.; Ventura, S.; Fernandez-Busquets, X., Sulfated Polysaccharides Promote the Assembly of Amyloid beta(1-42) Peptide into Stable Fibrils of Reduced Cytotoxicity. J. Biol. Chem. 2008, 283, 32471-32483. (33) Castellani, R. J.; Siedlak, S. L.; Fortino, A. E.; Perry, G.; Ghetti, B.; Smith, M. A. Chitin-Like Polysaccharides in Alzheimer's Disease Brains. Curr. Alzheimer Res. 2005, 2, 419-423. (34) Furukawa, K.; Sopher, B. L.; Rydel, R. E.; Begley, J. G.; Pham, D. G.; Martin, G. M.; Fox, M.; Mattson, M. P. Increased Activity-Regulating and Neuroprotective Efficacy of alpha-Secretase-Derived Secreted Amyloid Precursor Protein Conferred by a C-Terminal Heparin-Binding Domain. J. Neurochem. 1996, 67, 1882-1896. (35) Oskarsson, M. E.; Singh, K.; Wang, J.; Vlodavsky, I.; Li, J. P.; Westermark, G. T. Heparan Sulfate Proteoglycans Are Important for Islet Amyloid Formation and Islet Amyloid Polypeptide-Induced Apoptosis. J. Biol. Chem. 2015, 290, 15121-15132. (36) Park, K.; Verchere, C. B. Identification of a Heparin Binding Domain in the N-Terminal Cleavage Site of Pro-Islet Amyloid Polypeptide-Implications for Islet Amyloid Formation. J. Biol. Chem. 2001, 276, 16611-16616. (37) Jiang, T.; Yu, W. B.; Yao, T.; Zhi, X. L.; Pan, L. F.; Wang, J.; Zhou, P. Trehalose Inhibits Wild-Type alpha-Synuclein Fibrillation and Overexpression and Protects against the Protein Neurotoxicity in Transduced PC12 Cells. Rsc. Adv. 2013, 3, 9500-9508. (38) Oliveri, V.; Bellia, F.; Grasso, G. I.; Pietropaolo, A.; Vecchio, G. Trehalose-8-Hydroxyquinoline Conjugates as Antioxidant Modulators of A beta Aggregation. Rsc. Adv. 2016, 6, 47229-47236. (39) Qi, W.; Zhang, A. M.; Good, T. A.; Fernandez, E. J. Two Disaccharides and Trimethylamine N-Oxide Affect A beta Aggregation Differently, but All Attenuate Oligomer-Induced Membrane Permeability. Biochemistry 2009, 48, 8908-8919. (40) Wada, M.; Miyazawa, Y.; Miura, Y. A Specific Inhibitory Effect of Multivalent Trehalose Toward A beta(1-40) Aggregation. Polym. Chem. 2011, 2, 1822-1829. (41) Zhang, X. F.; Zhang, L. N.; Xu, X. J. Morphologies and Conformation Transition of Lentinan in Aqueous NaOH Solution. Biopolymers 2004, 75, 187-195. (42) Zhang, Y. Y.; Xu, X. J.; Zhang, L. Gel Formation and Low-Temperature Intramolecular Conformation Transition of a Triple-Helical Polysaccharide Lentinan in Water. Biopolymers 2008, 89, 852-861. (43) Wang, X. H.; Xu, X. J.; Zhang, L. Thermally Induced Conformation Transition of Triple-Helical Lentinan in NaCl Aqueous Solution. J. Phys. Chem. B 2008, 112, 10343-10351. (44) Xu, X. J.; Zhang, X. F.; Zhang, L. N.; Wu, C. Collapse and Association of Denatured Lentinan in Water/Dimethlysulfoxide Solutions. Biomacromolecules 2004, 5, 1893-1898. 30


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


(45) Ahn, H.; Jeon, E.; Kim, J. C.; Kang, S. G.; Yoon, S.; Ko, H. J.; Kim, P. H.; Lee, G. S. Lentinan from Shiitake Selectively Attenuates AIM2 and Non-Canonical Inflammasome Activation While Inducing Pro-Inflammatory Cytokine Production. Sci. Rep. 2017, 7, 1314. (46) Wang, H.; Cai, Y.; Zheng, Y.; Bai, Q. X.; Xie, D. L.; Yu, J. F. Efficacy of Biological Response Modifier Lentinan with Chemotherapy for Advanced Cancer: A Meta-Analysis. Cancer Med. 2017, 6, 2222-2233. (47) Zhang, Y.; Li, Q.; Wang, J. F.; Cheng, F.; Huang, X.; Cheng, Y.; Wang, K. P. Polysaccharide from Lentinus Edodes Combined with Oxaliplatin Possesses the Synergy and Attenuation Effect in Hepatocellular Carcinoma. Cancer Lett. 2016, 377, 117-125. (48) Jia, X. W.; Liu, Q. Y.; Zou, S. W.; Xu, X. J.; Zhang, L. N. Construction of Selenium Nanoparticles/beta-Glucan Composites for Enhancement of the Antitumor Activity. Carbohyd. Polym. 2015, 117, 434-442. (49) Zhang, L.; Li, X. L.; Xu, X. J.; Zeng, F. B. Correlation Between Antitumor Activity, Molecular Weight, and Conformation of Lentinan. Carbohyd. Res. 2005, 340, 1515-1521. (50) Xu, X. F.; Yan, H. D.; Tang, J.; Chen, J.; Zhang, X. W. Polysaccharides in Lentinus edodes: Isolation, Structure, Immunomodulating Activity and Future Prospective. Crit. Rev. Food Sci. Nutr. 2014, 54, 474-487. (51) Zhang, Y. Q.; Mei, H. L.; Shan, W.; Shi, L.; Chang, X. A.; Zhu, Y. X.; Chen, F.; Han, X. Lentinan Protects Pancreatic Cells from STZ-Induced Damage. J. Cell. Mol. Med. 2016, 20, 1803-1812. (52) Xu, X. J.; Wang, X. H.; Cai, F.; Zhang, L. N. Renaturation of Triple Helical Polysaccharide Lentinan in Water-Diluted Dimethylsulfoxide Solution. Carbohyd. Res. 2010, 345, 419-424. (53) Engel, M. F. M.; VandenAkker, C. C.; Schleeger, M.; Velikov, K. P.; Koenderink, G. H.; Bonn, M. The Polyphenol EGCG Inhibits Amyloid Formation Less Efficiently at Phospholipid Interfaces than in Bulk Solution. J. Am. Chem. Soc. 2012, 134, 14781-14788. (54) Hebda, J. A.; Miranker, A. D. The Interplay of Catalysis and Toxicity by Amyloid Intermediates on Lipid Bilayers: Insights from Type II Diabetes. Annu. Rev. Biophys. 2009, 38, 125-152. (55) Sakuraba, H.; Mizukami, H.; Yagihashi, N.; Wada, R.; Hanyu, C.; Yagihashi, S. Reduced beta-Cell Mass and Expression of Oxidative Stress-Related DNA Damage in the Islet of Japanese Type II Diabetic Patients. Diabetologia 2002, 45, 85-96. (56) Konarkowska, B.; Aitken, J. F.; Kistler, J.; Zhang, S. P.; Cooper, G. J. S. Thiol Reducing Compounds Prevent Human Amylin-Evoked Cytotoxicity. FEBS J. 2005, 272, 4949-4959. (57) Westermark, P.; Andersson, A.; Westermark, G. T. Islet Amyloid Polypeptide, Islet Amyloid, and Diabetes Mellitus. Physiol. Rev. 2011, 91, 795-826. (58) Abedini, A.; Plesner, A.; Cao, P.; Ridgway, Z.; Zhang, J. H.; Tu, L. H.; Middleton, C. T.; Chao, B.; Sartori, D. J.; Meng, F. L.; Wang, H.; Wong, A. G.; Zanni, M. T.; Verchere, C. B.; Raleigh, D. P.; Schmidt, A. M. Time-Resolved Studies Define 31



Page 32 of 32

the Nature of Toxic IAPP Intermediates, Providing Insight for Anti-Amyloidosis Therapeutics. Elife 2016, 5, e12977. (59) Masters, S. L.; Dunne, A.; Subramanian, S. L.; Hull, R. L.; Tannahill, G. M.; Sharp, F. A.; Becker, C.; Franchi, L.; Yoshihara, E.; Chen, Z.; Mullooly, N.; Mielke, L. A.; Harris, J.; Coll, R. C.; Mills, K. H. G.; Mok, K. H.; Newsholme, P.; Nunez, G.; Yodoi, J.; Kahn, S. E.; Lavelle, E. C.; O'Neill, L. A. J. Activation of the NLRP3 Inflammasome by Islet Amyloid Polypeptide Provides a Mechanism for Enhanced IL-1 beta in Type 2 Diabetes. Nat. Immunol. 2010, 11, 897-904. (60) Zraika, S.; Hull, R. L.; Udayasankar, J.; Aston-Mourney, K.; Subramanian, S. L.; Kisilevsky, R.; Szarek, W. A.; Kahn, S. E. Oxidative Stress is Induced by Islet Amyloid Formation and Time-Dependently Mediates Amyloid-Induced beta Cell Apoptosis. Diabetologia 2009, 52, 626-635. (61) Nilsson, M. R.; Raleigh, D. P. Analysis of Amylin Cleavage Products Provides New Insights into the Amyloidogenic Region of Human Amylin. J. Mol. Biol. 1999, 294, 1375-1385.

Table of Contents (TOC)

32


Conformation-Dependent Manipulation of Human Islet Amyloid

Recommend Documents