- Email: [email protected]
Thermal effect on the degradation of hIAPP20–29 fibrils
Accepted Manuscript Thermal Effect on the Degradation of hIAPP20-29 Fibrils H.X. Zhang, Lei Liu, Jie Wang, Christian Bortolini, Mingdong Dong PII: DOI: Reference:
S0021-9797(17)31276-6 https://doi.org/10.1016/j.jcis.2017.10.107 YJCIS 22977
To appear in:
Journal of Colloid and Interface Science
Received Date: Revised Date: Accepted Date:
28 September 2017 24 October 2017 29 October 2017
Please cite this article as: H.X. Zhang, L. Liu, J. Wang, C. Bortolini, M. Dong, Thermal Effect on the Degradation of hIAPP20-29 Fibrils, Journal of Colloid and Interface Science (2017), doi: https://doi.org/10.1016/j.jcis. 2017.10.107
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.
Thermal Effect on the Degradation of hIAPP20-29 Fibrils H X Zhang1, Lei Liu1*, Jie Wang1,2, Christian Bortolini2, Mingdong Dong2. 1
Institute for Advanced Materials, Jiangsu Univeristy, Zhenjiang, 212013, China. Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C, Denmark.
2
Corresponding author at: Institute for Advanced Materials, Jiangsu University, China Email address: [email protected] Abstract Uncontrolled misfolding of proteins resulting in the formation of amyloid deposits is associated with over 40 types of diseases, for instance, type-2 diabetes. The human Islet amyloid polypeptide (hIAPP) amyloid formation is thought to be the cause of type-2 diabetes occurrence. A possible strategy to the current challenge of reducing the toxicity of its aggregates to pancreatic β-cell is the discovery of an efficient way to degrading amyloid deposits. In this work, hIAPP 2029, a core fibrillating fragment of hIAPP, was selected as model system to explore the thermal effect at different temperature on the degradation of hIAPP20-29 mature fibrils. Insights on the degradation mechanism are obtained by analyzing the morphologies, the mechanical properties, the interactions between the peptides, and the secondary structure of amyloid aggregates. In addition, thermal degradation displayed a possible way to breaking the interaction of peptides and further disassembling the amyloid fibrils. These findings may initiate a new avenue to degrade the amyloid peptide aggregates and enrich and update the current selection of nanostructure modulations.
1. Introduction Human islet amyloid polypeptide (hIAPP), is a 37residue peptide that is co-secreted with insulin by pancreatic β-cells from pre-amylin and is then stored with insulin in secretory granules.[1, 2, 3] One of the most striking hallmarks of type 2 diabetes (T2D) is the existence of highly aggregated amyloid deposits of hIAPP in the islets of pancreatic β-cells.[4, 5] The amyloid aggregates have been assumed to be the fibrillization of hIAPP which plays an important role in the pathogenic development of T2D.[4] Under abnormal condition, hIAPP monomers in the body will increase to aggregate into oligomers, further into amyloid fibrils with β-sheet secondary conformation in β-cells, which can finally lead to the injure and the death of pancreatic βcells and dysfunction in insulin secretion resulting in T2D.[6, 7, 8] To reduce the cytotoxicity of hIAPP, a great many of agents and inhibitors have been tried eg. natural products and derivatives,[9] synthetic short peptides,[10] metal complex (copper(II) ions and zinc coordination),[11, 12] polymeric nanoparticles,[13] etc, aiming for the inhibition of amyloid aggregation in the body. There is another promising way to inhibiting the amyloid peptide aggregation by nanomaterials such as graphene, and graphene oxide,[14] etc, which have been attracted lots of attention due to their small size, passing through the cell membrane and disturbing the selfassembly of amyloid protein.[15] In recent, one more promising strategy has been drawing great attention in this field via the degradation of amyloid peptide mature fibrils. In the previous research, it was explored that polyoxometalates as photocatalysts [16] and porphyrin derivatives [17] could degrade β-sheet amyloid fibrils by light-driving. Upon producing ROS by photo-excitation, polyoxometalates and porphyrin derivatives could break the hydrogen bond between peptides.[16, 17] It is a typical way to utilize to break the interaction of peptides and degrade the amyloid fibrillization. In addition, thermal effect is a common and fundamental way to break hydrogen bond. Generally,
hydrophobic interactions are essential for Aβ aggregation and be varied with temperature changing.[18] Therefore, it has been assumed that higher temperature could block the formation of toxic oligomers, or guide the monomeric peptide to the pathway of non-toxic intermediates, and also prevent the primary nucleation process by destabilizing oligomers, or destabilizing fibrils to form non-toxic oligomers or monomers. It has been reported that the thermal effect could modulate the amyloid peptide aggregation and change the pathway of amyloid nucleation.[19] In the near future, some photo-thermal agents can be designed to locally break the amyloid fibrils with the targets, which might be a promising way to reduce the cytotoxicity of amyloid aggregates. Therefore, we proposed that the thermal effect on the degradation of amyloid mature fibrils, which is a creative and fundamental study, and may be used in further practical application. Herein, we explored the thermal effect on the degradation of amyloid fibrils. hIAPP20-29 was selected to be the model system, which is the particular peptide region of hIAPP1-37 being responsible for the fibrillation.[19] Therefore, it is a simple and representative model system of amyloid fibrillation. We used high-resolution AFM and quantitative nanomechanical mapping to characterize the nanostructure and nanomechanical properties of amyloid fibrils and the ones degraded through the thermal treatments at different temperature. With regard to different thermal-degraded peptide aggregates, the morphologies of fibrils were changed from thick, rigid and twisted structures to be thin, straight and short filaments, and finally it was converted to be the amyloid film and short oligomers with the treatment at 80 °C for 24 hours. The nanomechnical properties of amyloid fibrils were also varied according to the different thermal treatments. Young’s modulus of amyloid fibrils was decreased after the thermal treatment, which implies that the molecular packing and interaction between the peptides in the fibrils become weak and the thermal treatment might break the hydrogen bond and hydrophobic interaction. Furthermore, we obtain the mechanistic insight of thermal degradation of amyloid
fibrils, and it is revealed that the changes of the secondary structure of peptides. The content of β-sheet conformation was decreased, which implied weak intermolecular interaction between peptides with less βsheet conformation in amyloid aggregates due to the thermal treatment. In sum up, the knowledge of thermal effect on degradation of hIAPP20-29 fibrils obtained in this work is a prerequisite for deciphering the retro-gradation mechanism of amyloid fibrillation, and it could have more practical applications in future.
2. Experimental 2.1. Materials Human Islet amyloid polypeptide (hIAPP20-29) (amino acid sequence: NH2-SNNFGAILSS-COOH) was provided from Abbiochem Co., Ltd., (Shanghai, China). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Ethanol was supplied from Sinopharm Chemical Reagent Co., Ltd., (Shanghai, China). 2.2. Preparation of hIAPP20-29 mature fibrils and thermal degradation 1 mg hIAPP20-29 powder was dissolved in 1 mL 1,1,1,3,3,3-Hex -afluoro-2-propanol with 5 seconds ultrasonication, followed by 5 seconds of vortex mixing. Sonication and vortexing were repeated 3 times. The solution was put in a thermo-shaker (PHMT, Grant Instruments, UK) for 12 hours at 350 rpm min-1 at 25℃, to ensure peptide powder to be dissolved fully. 50 μL of hIAPP20-29 solution was transferred into a 1.5 mL centrifuge tube, and sealed with parafilm. The tube was put in a Vacuum Drying Oven for 2 hours at 25 ℃ in order to evaporate HFIP fully and the peptide thin film formed on the bottom of tube. 150 μL of MilliQ water was added to the tube, then the solution was sonicated for 5 seconds and vortexed for 5 seconds (repeated 3 times) until the solution became clear and homogeneous. Finally, the solution was incubated on a thermo-shaker at 350 rpm min-1 and 37 ℃ for 12 hours. The hIAPP 20-29 peptide aggregated into fibrils with a final concentration of about 330 μM. Furthermore, 150 μL of peptide fibril solution above was diluted into 250 μL with the final concentration of 200 μM, and then the diluted peptide fibril solution was placed in a thermo-shaker for 24 hours at 350 rpm min-1 at 50 ℃, 60 ℃ and 80 ℃, respectively. 2.3. Thioflavin T (ThT) fluorescence assay 50 μL of peptide fibril solution (200 μM) was mixed with 450 μL MilliQ water and 100 μL ThT (1 mM), then the mixed solution was measured by a fluorescence spectrophotometer (F-4500; Hitachi) with the excitation at 450 nm and the emission at 485 nm with a 1 cm quartz cells. Fluorescence spectra of amyloid peptide fibril was recorded. Measurements were carried out in quartz cell with sample volumes of 3 mL. 2.4. Circular dichroism spectrum (CD) Circular dichroism measurements were carried out at room temperature by a JASCO PTC-348W1 spectropolarimeter. CD spectra were performed between 250 and 190 nm with a 0.1 cm quartz cells. The slit-width
was set at 2 nm and scanning speed was set at 50 nm min1 . The signal of the Milli-Q water has been subtracted. Measurements were carried out in quartz cell with a sample volume of 250 μL. 2.5. Atomic force microscopy (AFM) 10 μL solution for all samples was deposited on the freshly cleaved mica surface, dried for 10 min in the air, and the residue liquid on the surface was removed. All AFM images were recorded by MultiMode VIII SPM (Bruker Company) in tapping mode with ultrasharp silicon cantilevers (OMCL-AC160TS-R3; Olympus) and a nominal spring constant of 26 N m-1 under ambient conditions. AFM imaging was carried out in tapping mode with optimized feedback parameters at a scan frequency of 1 Hz and 512 ×512-pixel resolution. 2.6. Quantitative nanomechanical (QNM) mapping The quantitative nanomechanical mapping is based on the measurements of the nanostructure and nanomechanical properties of biological samples. The measurements were performed using Multimode-VIII SPM and Nanoscope V controller (Bruker Company) with silicon cantilevers and a spring constant of 200 N m1 under the ambient conditions. The cantilever was calibrated by ramp and thermal tuning before mapping. Peak Force Tapping mode oscillates, but far below the cantilever resonant frequency, the vertical motion of the cantilever using the (main) Z piezo element and depends on feedback of peak force. Peak interaction force and nanoscale material property information is collected and recorded for each individual tap. The interactions between the tip and sample are mainly determined by the long-range electrostatic and van der Waals forces, and short-range mechanical restoration forces. The following formula derived for a spherical tip indenting a semi-infinite planar sample was used to estimate a local reduced elastic modulus in DMT model. Finteraction =(4/3)E*R1/2(d-d0)3/2+Fadh where Finteraction is the tip-sample force, E* is the reduced elastic modulus of the tip and the sample, R represents the tip radius, and d0 represents the surface rest position, and d-d0 is the depth of indentation and Fadh is the constant adhesion force during the contact.
3. Results and discussion 3.1. Thermal effect on the morphologies of the mature fibrils of hIAPP20-29 of Initially, we intuitively displayed the thermal effect on the morphology of hIAPP20-29 fibrils via the temperature control. Atomic force microscopy (AFM) was utilized to characterize the high resolutional nanostructure of hIAPP20-29 aggregates. As shown in Figure 1a, hIAPP20-29 tended to form long thick, rigid fibrils after 12 hour incubation at 37 °C. The height of mature fibrils of hIAPP20-29 was determined to be about 20 nm. However when the mature hIAPP20-29 fibrils were incubated at 50 ℃ for 24 hours, the fibrils became shorter amyloid species with height of 12 nm (Fig. 1c), that is distinct to the mature hIAPP20-29 ones. Then mature amyloid fibrils with the treatment at 60 ℃ for 24 hours, the fibrils disassembled into the filament with the height
of 9 nm (Fig. 1i). The fibrils were treated at 80 ℃ for 24 hours, the product is almost particles with the height of about 4 nm and a few film with the height of 1 nm (Fig. 1l). Compared to amyloid mature fibrils, the height of degraded amyloid fibril decreased and the morphology is different, which suggests that increasing temperature could degrade mature hIAPP20-29 fibrils and the
degradation effect become strong with the increment of temperature. The possible mechanism is in view that thermal incubation at the temperature larger than 50 °C might lead to the loose packing of peptide assembly, which will enable the amyloid fibril disassembling.
Figure 1. AFM images of hIAPP20–29 fibrils degradation. (a-b) hIAPP20–29 fibrils. (c-d, i-j) hIAPP20–29 fibril morphologies with the incubation at 50 ℃ and 60 ℃, respectively for 24 hours. (k-l) hIAPP20–29 fibril morphologies with the incubation at 80 ℃ for 24 hours. (e-f, g-h, m-n, o-p) Height lineprofile and distributions of amyloid fibrils and nanostructures according to a-b, c-d, i-j, and k-l, respectively.
3.2. Thermal effect on the mechanical properties of the mature fibrils of hIAPP20-29
Figure 2. Quantitative nanomechanical maps of hIAPP20-29 fibrils with the in-situ incubation at 37 ℃, 50 ℃, 60 ℃, respectively for 24 hours. (a)-(c) AFM morphology images of amyloid fibrils (d)-(f) The Young’s modulus maps of amyloid fibrils according to a-c, respectively; (g)-(j) Gaussian distribution of Young’s modulus according to map (d-f), respectively.
To understand thoroughly mechanism of thermal effect on degradation of hIAPP20-29 fibrils, quantitative nanomechanical (QNM) property was captured by observing the Young’s modulus of hIAPP 20-29 fibril. The deformation of analysed nanostructures was kept to 2 nm during the QNM mapping, to ensure that the moduli during measurements were correct and comparable. As shown in Figure. 2, the mapping of Young’s modulus of three amyloid fibrils were presented in three different conditions (37 ° C, 50 ° C, and 60 ° C), and the Young’s modulus of three fibrils were measured to be 4.5 ± 0.9 GPa (Fig. 4d), 3.2 ± 0.4 GPa (Fig. 4e) and 1.1 ± 0.1 GPa (Fig. 4f), respectively. The obtained modulus of amyloid mature fibrils in this measurement is consistent with the one of the amyloid fibrils in previous research.[20] After the identical fibrils with thermal incubation, the nanostructure and the mechanical properties of peptide assemblies were both modulated. The Young’s modulus of degraded peptide fibrils after the incubation at 50 ℃ and 60 ℃ for 24 hours decreased. It presented the thermal effect on the mechanical properties of amyloid fibrils. The thermal treatment eg. 50 ℃ and 60 ℃, might degrade the amyloid fibrils, and the molecular packing might loose and intermolecular interaction between the peptides might weaken. The secondary structure of amyloid peptide eg. the content of β-sheet conformation is likely to decease with the thermal treatment that can be explored Figure 4, which would facilitate the weak interaction between the peptides and result in the decreased stiffness of the assemblies.[20, 21] However hIAPP20-29 fibrils through the incubation with 80 ℃ for 24 hours became particles and membrane and the height of degraded nanostructure is about 4.1 nm, which clear displayed the thermal effect on the degradation of amyloid fibrils based the morphology variation of amyloid aggregates. The interaction and molecular packing in the degraded amyloid nanostructure is different from the one in the mature amyloid fibrils. 3.3. Thermal effect on the hydrophobic interactions of the mature fibrils of hIAPP20-29 To further verify the thermal effect on the degradation of amyloid fibrils, the adhesion force maps of amyloid fibrils were explored in three conditions (Figure 3a-3c). The adhesion force on the surface of amyloid mature fibril is determined to be 1.1 ± 0.2 nN (Figure 3d and 3e). The adhesion force on the surface of amyloid fibril decreased to be 1.1 ± 0.2 nN and 0.6 ± 0.3 nN, respectively, after the thermal treatment at 50 ℃ and 60 ℃ for 24 hours (Figure 3d-3h). The adhesion force is a function of interfacial energy. Johnson-Kendall-Roberts (JKR) theory relates the adhesive force (Fad) to the effective radius of an AFM tip (R) and the work of adhesion (Wad) as: Fad = 3/2πRWad , Wad is related to the excess free energy density (γ) of the interfaces of the system before and after separation as Wad = fibril-air + tipair - tip-fibril where fibril-air is the interfacial energy of the amyloid fibril in air, tip-air is the interfacial energy of the AFM tip in air, tip-fibril is the interfacial energy of the contact area formed between the AFM tip and amyloid fibril interface[22]. In air, tip-air is unchanged, and tip-fibril is changing not so much because the residues are same in the fibrils, therefore Wad is related to the fibril-air, and Fad relates tofibril-air. The decreasing of Fad, means fibril-air
decreases, and hydrophobicity becomes strong. Therefore, the adhesion force is considered as one parameter that can characterize the hydrophilicity of the material surface. Compared to the adhesion force of amyloid mature fibril, the ones of the degraded fibrils decreased after the thermal treatment, which implied the surface of amyloid fibril became less hydrophilic and more hydrophobic. In the assembly of amyloid peptide, hydrophobic interaction and hydrogen bond are dominant, which makes a great of contribution to the amyloid fibril formation, and involves in the molecular packing inside of fibril. The hydrophobic residues exposing outside would weaken the interaction of peptide assembly inside. It is consistent with the result of Young’s modulus obtained above. 3.4. Thermal effect on the secondary structure of the mature fibrils of hIAPP20-29 To obtain the molecular mechanical insight of the thermal effect on the degradation of amyloid fibrils, we investigated the secondary structure of amyloid peptides before and after thermal degradation of amyloid fibril. To assess the thermal effect on the peptide conformation conversion, CD experiments were carried out with an identical peptide solution, initially. hIAPP20-29 amyloid fibril presented the typical β-sheet secondary structure after the initial incubation, at 37 ℃ for 12 h. Subsequently, peptide fibril solution was characterized by CD spectra after the thermal treatment at 50 ℃, 60 ℃, 80 ℃, respectively, for 24 h. Compared to the spectra Figure 4d, CD signal at 208 nm decreased dramatically and also shift a lot with the treatment increasing temperature. It is revealed that high temperature treatment will leads to the decreasing of β-sheet content in the amyloid assemblies. Furthermore, we verified the thermal effect on the degradation hIAPP20-29 fibrils through thioflavin T (ThT) fluorescence assay. Thioflavin T (ThT) is the most commonly used dye to diagnose amyloid fibril formation and the intensity of ThT fluorescence could be proportional to the degree of amyloid fibril formation.[16] hIAPP20-29 monomers, were incubated at 37 ℃ for 12 hours in aqueous solution, and the ThT assay presented the strong signal which suggests that monomers converted into mature fibrils. Then the fibril solution was incubated at 50 ℃, 60 ℃ and 80 ℃ for 24 h respectively, and a drastic decrease in ThT fluorescence intensity indicates that thermal treatment had a great effect on hIAPP20-29 fibril degradation, which also suggests that βsheet structure converted into other secondary structure, and it is consistent with the previous studies demonstrating temperature-dependent β-sheet formation in β-amyloid Aβ1-40 peptide [18]. The result we obtained by ThT assay is consistent with the one by CD spectra above and both them illustrated β-sheet content reduction in the assemblies of hIAPP20-29 peptides after thermal treatment. Combined the thermal effect on the morphology variation of amyloid fibrils, mechanical properties and the molecular structure analysis, we proposed the scheme of thermal effect on the degradation of amyloid fibrillation. High temperature treatment could facilitate the amyloid fibril degradation by the reduction of β-sheet secondary structure and weaken the interaction between the peptides.
Figure 3. Quantitative nanomechanical maps (adhesion maps) of hIAPP 20-29 fibrils with the in-situ incubation at 37 ℃, 50 ℃, 60 ℃, respectively for 24 hours. (a-c) The Adhesion force maps of amyloid fibril at 37 ℃, and the degraded ones at 50 ℃, 60 ℃, respectively. (d) The line-profile of adhesion force of amyloid fibrils in three conditions. (e-g) Gaussian distribution of Adhesion force according to maps (a-c), respectively. (h) The comparison of adhesion force of amyloid fibrils in three conditions.
Figure 4. Overview of the thermal effect on the secondary structure of peptide in the degradation of hIAPP 20–29 fibrils. (a) Circular dichroism (CD) spectra of hIAPP20–29 fibrils representing the peptide secondary structure and the ones with the in-situ incubation at 50 ℃, 60 ℃, 80 ℃, respectively for 24 hours; (b) ThT assay of hIAPP20–29 fibrils and the ones with the in-situ incubation at 50 ℃, 60 ℃, 80 ℃, respectively for 24 hours; (c) Analysis of CD spectra at 208 nm of a; (d) Analysis of CD spectra shift at 208 nm of a. (e) Analysis of ThT intensity of b.
Figure 5. The scheme of thermal effect on the degradation of amyloid fibrillation. (a) Schematic model of hIAPP 20–29. (b) Schematic illustration of thermal modulating hIAPP20–29 peptide fibril structure. (1) Mature fibrils were degraded at 50 ℃ for 24 hours. (2) Mature fibrils were degraded at 60 ℃ for 24 hours. (3) Mature fibrils were degraded at 80 ℃ for 24 hours.
4. Conclusion To sum up, we have explored the thermal effect on the degradation of amyloid fibrils, and the obtained results clearly indicated that amyloid fibrils would be degraded by the thermal treatment by temperature augmentation. The morphology of amyloid fibril varied
by the thermal treatment, and finally it converted into nanofilm and particles. The thermal degradation of amyloid fibrils was ascribed to β-sheet secondary structure reduction in amyloid peptide fibrils, and the molecular packing and hydrophobic interaction became weak which is responsible for the amyloid fibril formation. This finding may open up a new facile method
to degrade and modulate the amyloid peptide aggregates, which is also a possible strategy for treating various amyloid related diseases in the future.
Acknowledgement The authors thank the National Nature Science Foundation of China (Grant no. 21573097, 51503087), the Foundation of Jiangsu Province (BK20150490, BK20140528, BK20140013), the Foundation of Jiangsu Distinguished Professor. The authors owe their groups thanks for help. This paper was also subsidized by Jiangsu Planned Projects for Postdoctoral Research Funds (1401068B) and Jiangsu University Funds (14JDG061 and 11JDG098). Reference 1. Wang, L., et al., 2DIR Spectroscopy of Human Amylin Fibrils Reflects Stable β-Sheet Structure, Journal of the American Chemical Society, 40 (2011) 16062-16071. 2. Profit, A. A., et al., Aromaticity and amyloid formation: Effect of π-electron distribution and aryl substituent geometry on the selfassembly of peptides derived from hIAPP 22–29, Archives of biochemistry and biophysics, (2015) 46-58. 3. Rigacci, S., et al., Oleuropein aglycon prevents cytotoxic amyloid aggregation of human amylin, The Journal of nutritional biochemistry, 8 (2010) 726-735. 4. Brender, J. R., et al., Amyloid Fiber Formation and Membrane Disruption are Separate Processes Localized in Two Distinct Regions of IAPP, the Type-2-Diabetes-Related Peptide, Journal of the American Chemical Society, 20 (2008) 6424-6429. 5. L pe , C. L., et al., Benzbromarone, Quercetin, and Folic Acid Inhibit Amylin Aggregation, International Journal of Molecular Sciences, 6 (2016). 6. Haataja, L., et al., Islet amyloid in type 2 diabetes, and the toxic oligomer hypothesis, Endocrine reviews, 3 (2008) 303-316. 7. Leckström, A., et al., Renal elimination of islet amyloid polypeptide, Biochemical and biophysical research communications, 1 (1997) 265-268. 8. Brender, J. R., et al., Role of zinc in human islet amyloid polypeptide aggregation, Journal of the American Chemical Society, 26 (2010) 8973-8983. 9. Pithadia, A., et al., Inhibition of IAPP aggregation and toxicity by natural products and derivatives, Journal of diabetes research, (2015).
10. Mao, Y., et al., New peptide inhibitors modulate the selfassembly of islet amyloid polypeptide residues 11–20 in vitro, European journal of pharmacology, (2017) 102-110. 11. Li, H., et al., Rapid assessment of human amylin aggregation and its inhibition by copper (II) ions by laser ablation electrospray ionization mass spectrometry with ion mobility separation, Analytical chemistry, 19 (2015) 9829-9837. 12. Nedumpully-Govindan, P., et al., Promotion or inhibition of islet amyloid polypeptide aggregation by zinc coordination depends on its relative concentration, Biochemistry, 50 (2015) 7335-7344. 13. Cabaleiro-Lago, C., et al., Inhibition of IAPP and IAPP (20− 29) fibrillation by polymeric nanoparticles, Langmuir, 5 (2009) 34533461. 14. Nedumpully-Govindan, P., et al., Graphene oxide inhibits hIAPP amyloid fibrillation and toxicity in insulin-producing NIT-1 cells, Physical Chemistry Chemical Physics, 1 (2016) 94-100. 15. Guo, J., et al., Exploring the influence of carbon nanoparticles on the formation of β-sheet-rich oligomers of IAPP22–28 peptide by molecular dynamics simulation, PLoS One, 6 (2013) e65579. 16. Li, M., et al., Photodegradation of β-sheet amyloid fibrils associated with Alzheimer's disease by using polyoxometalates as photocatalysts, Chemical Communications, 97 (2013) 11394-11396. 17. Hirabayashi, A., et al., Photodegradation of amyloid β and reduction of its cytotoxicity to PC12 cells using porphyrin derivatives, Chemical Communications, 67 (2014) 9543-9546. 18. Gursky, O.,Aleshkov, S., Temperature-dependent β-sheet formation in β-amyloid Aβ 1–40 peptide in water: uncoupling β-structure folding from aggregation, Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1 (2000) 93-102. 19. Bennett, R. G., et al., An insulin-degrading enzyme inhibitor decreases amylin degradation, increases amylin-induced cytotoxicity, and increases amyloid formation in insulinoma cell cultures, Diabetes, 9 (2003) 2315-2320. 20. Yang, G., et al., Light-driven porphyrin modulating fibrillation of hIAPP 20–29 peptide, Journal of Colloid and Interface Science, (2017) 37-43. 21. Krotee, P., et al., Atomic structures of fibrillar segments of hIAPP suggest tightly mated β-sheets are important for cytotoxicity, eLife, (2017) e19273. 22. Alsteens, D., et al., Direct measurement of hydrophobic forces on cell surfaces using AFM, Langmuir, 24 (2007) 11977-11979.
Graphical abstract
S0021-9797(17)31276-6 https://doi.org/10.1016/j.jcis.2017.10.107 YJCIS 22977
To appear in:
Journal of Colloid and Interface Science
Received Date: Revised Date: Accepted Date:
28 September 2017 24 October 2017 29 October 2017
Please cite this article as: H.X. Zhang, L. Liu, J. Wang, C. Bortolini, M. Dong, Thermal Effect on the Degradation of hIAPP20-29 Fibrils, Journal of Colloid and Interface Science (2017), doi: https://doi.org/10.1016/j.jcis. 2017.10.107
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.
Thermal Effect on the Degradation of hIAPP20-29 Fibrils H X Zhang1, Lei Liu1*, Jie Wang1,2, Christian Bortolini2, Mingdong Dong2. 1
Institute for Advanced Materials, Jiangsu Univeristy, Zhenjiang, 212013, China. Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C, Denmark.
2
Corresponding author at: Institute for Advanced Materials, Jiangsu University, China Email address: [email protected] Abstract Uncontrolled misfolding of proteins resulting in the formation of amyloid deposits is associated with over 40 types of diseases, for instance, type-2 diabetes. The human Islet amyloid polypeptide (hIAPP) amyloid formation is thought to be the cause of type-2 diabetes occurrence. A possible strategy to the current challenge of reducing the toxicity of its aggregates to pancreatic β-cell is the discovery of an efficient way to degrading amyloid deposits. In this work, hIAPP 2029, a core fibrillating fragment of hIAPP, was selected as model system to explore the thermal effect at different temperature on the degradation of hIAPP20-29 mature fibrils. Insights on the degradation mechanism are obtained by analyzing the morphologies, the mechanical properties, the interactions between the peptides, and the secondary structure of amyloid aggregates. In addition, thermal degradation displayed a possible way to breaking the interaction of peptides and further disassembling the amyloid fibrils. These findings may initiate a new avenue to degrade the amyloid peptide aggregates and enrich and update the current selection of nanostructure modulations.
1. Introduction Human islet amyloid polypeptide (hIAPP), is a 37residue peptide that is co-secreted with insulin by pancreatic β-cells from pre-amylin and is then stored with insulin in secretory granules.[1, 2, 3] One of the most striking hallmarks of type 2 diabetes (T2D) is the existence of highly aggregated amyloid deposits of hIAPP in the islets of pancreatic β-cells.[4, 5] The amyloid aggregates have been assumed to be the fibrillization of hIAPP which plays an important role in the pathogenic development of T2D.[4] Under abnormal condition, hIAPP monomers in the body will increase to aggregate into oligomers, further into amyloid fibrils with β-sheet secondary conformation in β-cells, which can finally lead to the injure and the death of pancreatic βcells and dysfunction in insulin secretion resulting in T2D.[6, 7, 8] To reduce the cytotoxicity of hIAPP, a great many of agents and inhibitors have been tried eg. natural products and derivatives,[9] synthetic short peptides,[10] metal complex (copper(II) ions and zinc coordination),[11, 12] polymeric nanoparticles,[13] etc, aiming for the inhibition of amyloid aggregation in the body. There is another promising way to inhibiting the amyloid peptide aggregation by nanomaterials such as graphene, and graphene oxide,[14] etc, which have been attracted lots of attention due to their small size, passing through the cell membrane and disturbing the selfassembly of amyloid protein.[15] In recent, one more promising strategy has been drawing great attention in this field via the degradation of amyloid peptide mature fibrils. In the previous research, it was explored that polyoxometalates as photocatalysts [16] and porphyrin derivatives [17] could degrade β-sheet amyloid fibrils by light-driving. Upon producing ROS by photo-excitation, polyoxometalates and porphyrin derivatives could break the hydrogen bond between peptides.[16, 17] It is a typical way to utilize to break the interaction of peptides and degrade the amyloid fibrillization. In addition, thermal effect is a common and fundamental way to break hydrogen bond. Generally,
hydrophobic interactions are essential for Aβ aggregation and be varied with temperature changing.[18] Therefore, it has been assumed that higher temperature could block the formation of toxic oligomers, or guide the monomeric peptide to the pathway of non-toxic intermediates, and also prevent the primary nucleation process by destabilizing oligomers, or destabilizing fibrils to form non-toxic oligomers or monomers. It has been reported that the thermal effect could modulate the amyloid peptide aggregation and change the pathway of amyloid nucleation.[19] In the near future, some photo-thermal agents can be designed to locally break the amyloid fibrils with the targets, which might be a promising way to reduce the cytotoxicity of amyloid aggregates. Therefore, we proposed that the thermal effect on the degradation of amyloid mature fibrils, which is a creative and fundamental study, and may be used in further practical application. Herein, we explored the thermal effect on the degradation of amyloid fibrils. hIAPP20-29 was selected to be the model system, which is the particular peptide region of hIAPP1-37 being responsible for the fibrillation.[19] Therefore, it is a simple and representative model system of amyloid fibrillation. We used high-resolution AFM and quantitative nanomechanical mapping to characterize the nanostructure and nanomechanical properties of amyloid fibrils and the ones degraded through the thermal treatments at different temperature. With regard to different thermal-degraded peptide aggregates, the morphologies of fibrils were changed from thick, rigid and twisted structures to be thin, straight and short filaments, and finally it was converted to be the amyloid film and short oligomers with the treatment at 80 °C for 24 hours. The nanomechnical properties of amyloid fibrils were also varied according to the different thermal treatments. Young’s modulus of amyloid fibrils was decreased after the thermal treatment, which implies that the molecular packing and interaction between the peptides in the fibrils become weak and the thermal treatment might break the hydrogen bond and hydrophobic interaction. Furthermore, we obtain the mechanistic insight of thermal degradation of amyloid
fibrils, and it is revealed that the changes of the secondary structure of peptides. The content of β-sheet conformation was decreased, which implied weak intermolecular interaction between peptides with less βsheet conformation in amyloid aggregates due to the thermal treatment. In sum up, the knowledge of thermal effect on degradation of hIAPP20-29 fibrils obtained in this work is a prerequisite for deciphering the retro-gradation mechanism of amyloid fibrillation, and it could have more practical applications in future.
2. Experimental 2.1. Materials Human Islet amyloid polypeptide (hIAPP20-29) (amino acid sequence: NH2-SNNFGAILSS-COOH) was provided from Abbiochem Co., Ltd., (Shanghai, China). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Ethanol was supplied from Sinopharm Chemical Reagent Co., Ltd., (Shanghai, China). 2.2. Preparation of hIAPP20-29 mature fibrils and thermal degradation 1 mg hIAPP20-29 powder was dissolved in 1 mL 1,1,1,3,3,3-Hex -afluoro-2-propanol with 5 seconds ultrasonication, followed by 5 seconds of vortex mixing. Sonication and vortexing were repeated 3 times. The solution was put in a thermo-shaker (PHMT, Grant Instruments, UK) for 12 hours at 350 rpm min-1 at 25℃, to ensure peptide powder to be dissolved fully. 50 μL of hIAPP20-29 solution was transferred into a 1.5 mL centrifuge tube, and sealed with parafilm. The tube was put in a Vacuum Drying Oven for 2 hours at 25 ℃ in order to evaporate HFIP fully and the peptide thin film formed on the bottom of tube. 150 μL of MilliQ water was added to the tube, then the solution was sonicated for 5 seconds and vortexed for 5 seconds (repeated 3 times) until the solution became clear and homogeneous. Finally, the solution was incubated on a thermo-shaker at 350 rpm min-1 and 37 ℃ for 12 hours. The hIAPP 20-29 peptide aggregated into fibrils with a final concentration of about 330 μM. Furthermore, 150 μL of peptide fibril solution above was diluted into 250 μL with the final concentration of 200 μM, and then the diluted peptide fibril solution was placed in a thermo-shaker for 24 hours at 350 rpm min-1 at 50 ℃, 60 ℃ and 80 ℃, respectively. 2.3. Thioflavin T (ThT) fluorescence assay 50 μL of peptide fibril solution (200 μM) was mixed with 450 μL MilliQ water and 100 μL ThT (1 mM), then the mixed solution was measured by a fluorescence spectrophotometer (F-4500; Hitachi) with the excitation at 450 nm and the emission at 485 nm with a 1 cm quartz cells. Fluorescence spectra of amyloid peptide fibril was recorded. Measurements were carried out in quartz cell with sample volumes of 3 mL. 2.4. Circular dichroism spectrum (CD) Circular dichroism measurements were carried out at room temperature by a JASCO PTC-348W1 spectropolarimeter. CD spectra were performed between 250 and 190 nm with a 0.1 cm quartz cells. The slit-width
was set at 2 nm and scanning speed was set at 50 nm min1 . The signal of the Milli-Q water has been subtracted. Measurements were carried out in quartz cell with a sample volume of 250 μL. 2.5. Atomic force microscopy (AFM) 10 μL solution for all samples was deposited on the freshly cleaved mica surface, dried for 10 min in the air, and the residue liquid on the surface was removed. All AFM images were recorded by MultiMode VIII SPM (Bruker Company) in tapping mode with ultrasharp silicon cantilevers (OMCL-AC160TS-R3; Olympus) and a nominal spring constant of 26 N m-1 under ambient conditions. AFM imaging was carried out in tapping mode with optimized feedback parameters at a scan frequency of 1 Hz and 512 ×512-pixel resolution. 2.6. Quantitative nanomechanical (QNM) mapping The quantitative nanomechanical mapping is based on the measurements of the nanostructure and nanomechanical properties of biological samples. The measurements were performed using Multimode-VIII SPM and Nanoscope V controller (Bruker Company) with silicon cantilevers and a spring constant of 200 N m1 under the ambient conditions. The cantilever was calibrated by ramp and thermal tuning before mapping. Peak Force Tapping mode oscillates, but far below the cantilever resonant frequency, the vertical motion of the cantilever using the (main) Z piezo element and depends on feedback of peak force. Peak interaction force and nanoscale material property information is collected and recorded for each individual tap. The interactions between the tip and sample are mainly determined by the long-range electrostatic and van der Waals forces, and short-range mechanical restoration forces. The following formula derived for a spherical tip indenting a semi-infinite planar sample was used to estimate a local reduced elastic modulus in DMT model. Finteraction =(4/3)E*R1/2(d-d0)3/2+Fadh where Finteraction is the tip-sample force, E* is the reduced elastic modulus of the tip and the sample, R represents the tip radius, and d0 represents the surface rest position, and d-d0 is the depth of indentation and Fadh is the constant adhesion force during the contact.
3. Results and discussion 3.1. Thermal effect on the morphologies of the mature fibrils of hIAPP20-29 of Initially, we intuitively displayed the thermal effect on the morphology of hIAPP20-29 fibrils via the temperature control. Atomic force microscopy (AFM) was utilized to characterize the high resolutional nanostructure of hIAPP20-29 aggregates. As shown in Figure 1a, hIAPP20-29 tended to form long thick, rigid fibrils after 12 hour incubation at 37 °C. The height of mature fibrils of hIAPP20-29 was determined to be about 20 nm. However when the mature hIAPP20-29 fibrils were incubated at 50 ℃ for 24 hours, the fibrils became shorter amyloid species with height of 12 nm (Fig. 1c), that is distinct to the mature hIAPP20-29 ones. Then mature amyloid fibrils with the treatment at 60 ℃ for 24 hours, the fibrils disassembled into the filament with the height
of 9 nm (Fig. 1i). The fibrils were treated at 80 ℃ for 24 hours, the product is almost particles with the height of about 4 nm and a few film with the height of 1 nm (Fig. 1l). Compared to amyloid mature fibrils, the height of degraded amyloid fibril decreased and the morphology is different, which suggests that increasing temperature could degrade mature hIAPP20-29 fibrils and the
degradation effect become strong with the increment of temperature. The possible mechanism is in view that thermal incubation at the temperature larger than 50 °C might lead to the loose packing of peptide assembly, which will enable the amyloid fibril disassembling.
Figure 1. AFM images of hIAPP20–29 fibrils degradation. (a-b) hIAPP20–29 fibrils. (c-d, i-j) hIAPP20–29 fibril morphologies with the incubation at 50 ℃ and 60 ℃, respectively for 24 hours. (k-l) hIAPP20–29 fibril morphologies with the incubation at 80 ℃ for 24 hours. (e-f, g-h, m-n, o-p) Height lineprofile and distributions of amyloid fibrils and nanostructures according to a-b, c-d, i-j, and k-l, respectively.
3.2. Thermal effect on the mechanical properties of the mature fibrils of hIAPP20-29
Figure 2. Quantitative nanomechanical maps of hIAPP20-29 fibrils with the in-situ incubation at 37 ℃, 50 ℃, 60 ℃, respectively for 24 hours. (a)-(c) AFM morphology images of amyloid fibrils (d)-(f) The Young’s modulus maps of amyloid fibrils according to a-c, respectively; (g)-(j) Gaussian distribution of Young’s modulus according to map (d-f), respectively.
To understand thoroughly mechanism of thermal effect on degradation of hIAPP20-29 fibrils, quantitative nanomechanical (QNM) property was captured by observing the Young’s modulus of hIAPP 20-29 fibril. The deformation of analysed nanostructures was kept to 2 nm during the QNM mapping, to ensure that the moduli during measurements were correct and comparable. As shown in Figure. 2, the mapping of Young’s modulus of three amyloid fibrils were presented in three different conditions (37 ° C, 50 ° C, and 60 ° C), and the Young’s modulus of three fibrils were measured to be 4.5 ± 0.9 GPa (Fig. 4d), 3.2 ± 0.4 GPa (Fig. 4e) and 1.1 ± 0.1 GPa (Fig. 4f), respectively. The obtained modulus of amyloid mature fibrils in this measurement is consistent with the one of the amyloid fibrils in previous research.[20] After the identical fibrils with thermal incubation, the nanostructure and the mechanical properties of peptide assemblies were both modulated. The Young’s modulus of degraded peptide fibrils after the incubation at 50 ℃ and 60 ℃ for 24 hours decreased. It presented the thermal effect on the mechanical properties of amyloid fibrils. The thermal treatment eg. 50 ℃ and 60 ℃, might degrade the amyloid fibrils, and the molecular packing might loose and intermolecular interaction between the peptides might weaken. The secondary structure of amyloid peptide eg. the content of β-sheet conformation is likely to decease with the thermal treatment that can be explored Figure 4, which would facilitate the weak interaction between the peptides and result in the decreased stiffness of the assemblies.[20, 21] However hIAPP20-29 fibrils through the incubation with 80 ℃ for 24 hours became particles and membrane and the height of degraded nanostructure is about 4.1 nm, which clear displayed the thermal effect on the degradation of amyloid fibrils based the morphology variation of amyloid aggregates. The interaction and molecular packing in the degraded amyloid nanostructure is different from the one in the mature amyloid fibrils. 3.3. Thermal effect on the hydrophobic interactions of the mature fibrils of hIAPP20-29 To further verify the thermal effect on the degradation of amyloid fibrils, the adhesion force maps of amyloid fibrils were explored in three conditions (Figure 3a-3c). The adhesion force on the surface of amyloid mature fibril is determined to be 1.1 ± 0.2 nN (Figure 3d and 3e). The adhesion force on the surface of amyloid fibril decreased to be 1.1 ± 0.2 nN and 0.6 ± 0.3 nN, respectively, after the thermal treatment at 50 ℃ and 60 ℃ for 24 hours (Figure 3d-3h). The adhesion force is a function of interfacial energy. Johnson-Kendall-Roberts (JKR) theory relates the adhesive force (Fad) to the effective radius of an AFM tip (R) and the work of adhesion (Wad) as: Fad = 3/2πRWad , Wad is related to the excess free energy density (γ) of the interfaces of the system before and after separation as Wad = fibril-air + tipair - tip-fibril where fibril-air is the interfacial energy of the amyloid fibril in air, tip-air is the interfacial energy of the AFM tip in air, tip-fibril is the interfacial energy of the contact area formed between the AFM tip and amyloid fibril interface[22]. In air, tip-air is unchanged, and tip-fibril is changing not so much because the residues are same in the fibrils, therefore Wad is related to the fibril-air, and Fad relates tofibril-air. The decreasing of Fad, means fibril-air
decreases, and hydrophobicity becomes strong. Therefore, the adhesion force is considered as one parameter that can characterize the hydrophilicity of the material surface. Compared to the adhesion force of amyloid mature fibril, the ones of the degraded fibrils decreased after the thermal treatment, which implied the surface of amyloid fibril became less hydrophilic and more hydrophobic. In the assembly of amyloid peptide, hydrophobic interaction and hydrogen bond are dominant, which makes a great of contribution to the amyloid fibril formation, and involves in the molecular packing inside of fibril. The hydrophobic residues exposing outside would weaken the interaction of peptide assembly inside. It is consistent with the result of Young’s modulus obtained above. 3.4. Thermal effect on the secondary structure of the mature fibrils of hIAPP20-29 To obtain the molecular mechanical insight of the thermal effect on the degradation of amyloid fibrils, we investigated the secondary structure of amyloid peptides before and after thermal degradation of amyloid fibril. To assess the thermal effect on the peptide conformation conversion, CD experiments were carried out with an identical peptide solution, initially. hIAPP20-29 amyloid fibril presented the typical β-sheet secondary structure after the initial incubation, at 37 ℃ for 12 h. Subsequently, peptide fibril solution was characterized by CD spectra after the thermal treatment at 50 ℃, 60 ℃, 80 ℃, respectively, for 24 h. Compared to the spectra Figure 4d, CD signal at 208 nm decreased dramatically and also shift a lot with the treatment increasing temperature. It is revealed that high temperature treatment will leads to the decreasing of β-sheet content in the amyloid assemblies. Furthermore, we verified the thermal effect on the degradation hIAPP20-29 fibrils through thioflavin T (ThT) fluorescence assay. Thioflavin T (ThT) is the most commonly used dye to diagnose amyloid fibril formation and the intensity of ThT fluorescence could be proportional to the degree of amyloid fibril formation.[16] hIAPP20-29 monomers, were incubated at 37 ℃ for 12 hours in aqueous solution, and the ThT assay presented the strong signal which suggests that monomers converted into mature fibrils. Then the fibril solution was incubated at 50 ℃, 60 ℃ and 80 ℃ for 24 h respectively, and a drastic decrease in ThT fluorescence intensity indicates that thermal treatment had a great effect on hIAPP20-29 fibril degradation, which also suggests that βsheet structure converted into other secondary structure, and it is consistent with the previous studies demonstrating temperature-dependent β-sheet formation in β-amyloid Aβ1-40 peptide [18]. The result we obtained by ThT assay is consistent with the one by CD spectra above and both them illustrated β-sheet content reduction in the assemblies of hIAPP20-29 peptides after thermal treatment. Combined the thermal effect on the morphology variation of amyloid fibrils, mechanical properties and the molecular structure analysis, we proposed the scheme of thermal effect on the degradation of amyloid fibrillation. High temperature treatment could facilitate the amyloid fibril degradation by the reduction of β-sheet secondary structure and weaken the interaction between the peptides.
Figure 3. Quantitative nanomechanical maps (adhesion maps) of hIAPP 20-29 fibrils with the in-situ incubation at 37 ℃, 50 ℃, 60 ℃, respectively for 24 hours. (a-c) The Adhesion force maps of amyloid fibril at 37 ℃, and the degraded ones at 50 ℃, 60 ℃, respectively. (d) The line-profile of adhesion force of amyloid fibrils in three conditions. (e-g) Gaussian distribution of Adhesion force according to maps (a-c), respectively. (h) The comparison of adhesion force of amyloid fibrils in three conditions.
Figure 4. Overview of the thermal effect on the secondary structure of peptide in the degradation of hIAPP 20–29 fibrils. (a) Circular dichroism (CD) spectra of hIAPP20–29 fibrils representing the peptide secondary structure and the ones with the in-situ incubation at 50 ℃, 60 ℃, 80 ℃, respectively for 24 hours; (b) ThT assay of hIAPP20–29 fibrils and the ones with the in-situ incubation at 50 ℃, 60 ℃, 80 ℃, respectively for 24 hours; (c) Analysis of CD spectra at 208 nm of a; (d) Analysis of CD spectra shift at 208 nm of a. (e) Analysis of ThT intensity of b.
Figure 5. The scheme of thermal effect on the degradation of amyloid fibrillation. (a) Schematic model of hIAPP 20–29. (b) Schematic illustration of thermal modulating hIAPP20–29 peptide fibril structure. (1) Mature fibrils were degraded at 50 ℃ for 24 hours. (2) Mature fibrils were degraded at 60 ℃ for 24 hours. (3) Mature fibrils were degraded at 80 ℃ for 24 hours.
4. Conclusion To sum up, we have explored the thermal effect on the degradation of amyloid fibrils, and the obtained results clearly indicated that amyloid fibrils would be degraded by the thermal treatment by temperature augmentation. The morphology of amyloid fibril varied
by the thermal treatment, and finally it converted into nanofilm and particles. The thermal degradation of amyloid fibrils was ascribed to β-sheet secondary structure reduction in amyloid peptide fibrils, and the molecular packing and hydrophobic interaction became weak which is responsible for the amyloid fibril formation. This finding may open up a new facile method
to degrade and modulate the amyloid peptide aggregates, which is also a possible strategy for treating various amyloid related diseases in the future.
Acknowledgement The authors thank the National Nature Science Foundation of China (Grant no. 21573097, 51503087), the Foundation of Jiangsu Province (BK20150490, BK20140528, BK20140013), the Foundation of Jiangsu Distinguished Professor. The authors owe their groups thanks for help. This paper was also subsidized by Jiangsu Planned Projects for Postdoctoral Research Funds (1401068B) and Jiangsu University Funds (14JDG061 and 11JDG098). Reference 1. Wang, L., et al., 2DIR Spectroscopy of Human Amylin Fibrils Reflects Stable β-Sheet Structure, Journal of the American Chemical Society, 40 (2011) 16062-16071. 2. Profit, A. A., et al., Aromaticity and amyloid formation: Effect of π-electron distribution and aryl substituent geometry on the selfassembly of peptides derived from hIAPP 22–29, Archives of biochemistry and biophysics, (2015) 46-58. 3. Rigacci, S., et al., Oleuropein aglycon prevents cytotoxic amyloid aggregation of human amylin, The Journal of nutritional biochemistry, 8 (2010) 726-735. 4. Brender, J. R., et al., Amyloid Fiber Formation and Membrane Disruption are Separate Processes Localized in Two Distinct Regions of IAPP, the Type-2-Diabetes-Related Peptide, Journal of the American Chemical Society, 20 (2008) 6424-6429. 5. L pe , C. L., et al., Benzbromarone, Quercetin, and Folic Acid Inhibit Amylin Aggregation, International Journal of Molecular Sciences, 6 (2016). 6. Haataja, L., et al., Islet amyloid in type 2 diabetes, and the toxic oligomer hypothesis, Endocrine reviews, 3 (2008) 303-316. 7. Leckström, A., et al., Renal elimination of islet amyloid polypeptide, Biochemical and biophysical research communications, 1 (1997) 265-268. 8. Brender, J. R., et al., Role of zinc in human islet amyloid polypeptide aggregation, Journal of the American Chemical Society, 26 (2010) 8973-8983. 9. Pithadia, A., et al., Inhibition of IAPP aggregation and toxicity by natural products and derivatives, Journal of diabetes research, (2015).
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Graphical abstract