Article pubs.acs.org/Langmuir
Cross-Sequence Interactions between Human and Rat Islet Amyloid Polypeptides Rundong Hu,† Mingzhen Zhang,† Kunal Patel,‡ Qiuming Wang,† Yung Chang,∥ Xiong Gong,§ Ge Zhang,‡ and Jie Zheng*,† †
Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, Ohio 44325, United States Department of Biomedical Engineering, The University of Akron, Akron, Ohio 44325, United States § College of Polymer Science and Polymer Engineering, The University of Akron, Akron, Ohio 44325, United States ∥ R&D Center for Membrane Technology and Department of Chemical Engineering, Chung Yuan University, Chung Li, Taoyuan 320, Taiwan ‡
ABSTRACT: Human islet amyloid polypeptide (hIAPP) can assemble into toxic oligomers and fibrils, which are associated with cell degeneration and the pathogenesis of type 2 diabetes. Cross-interaction of hIAPP with rat IAPP (rIAPP)a non-amyloidogenic peptide with high sequence similarity to hIAPPmight influence the aggregation and toxicity of hIAPP. However, the exact role of rIAPP in hIAPP aggregation and toxicity still remains unclear. In this work, we investigated the effect of cross-sequence interactions between fulllength hIAPP1−37 and rIAPP1−37 on hybrid amyloid structures, aggregation kinetics, and cell toxicity using combined computational and experimental approaches. Experimental results indicate a contrasting role of rIAPP in hIAPP aggregation, in which rIAPP initially inhibits the early aggregation and nuclei formation of hIAPP, but hIAPP seeds can also recruit both hIAPP and rIAPP to form more hybrid fibrils, thus promoting amyloid fibrillation ultimately. The coincubation of hIAPP and rIAPP also decreases cell viability, presumably due to the formation of more toxic hybrid oligomers at the prolonged lag phase. Comparative MD simulations confirm that the crosssequence interactions between hIAPP and rIAPP stabilize β-sheet structure and thus likely promote their fibrillization. This work provides valuable insights into a critical role of cross-amyloid interactions in protein aggregation.
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differs from hIAPP by only 6 residues and 3 of them are proline residues in the β-sheet region.16,17 The presence of 3 proline residues in the central region of 20−29 positions is considered as one of key factors for the inability of rIAPP to form fibrils, consistent with the general β-sheet blocker role of proline.18,19 Because of high sequence similarity to hIAPP and nonamyloidogenic nature of rIAPP, rIAPP should be used as an intuitive inhibitor. Surprisingly, only a few works20,21 explored the inhibitory ability of rIAPP on hIAPP aggregation and toxicity. Instead, significant efforts and progress have been reported to identify inhibitory sequences/fragments from fulllength hIAPP and to examine their inhibitory activities of these hIAPP fragments or modified fragments,22 including Nmethylated hIAPP segments,23 hIAPP fragments modified with α-aminoisobutyric acid,24 and N-methylated full length hIAPP.14 Cao et al.20 reported that full-length rIAPP was able to inhibit amyloid formation of hIAPP in bulk solution, lengthening both lag and growth phases and reducing fibril formation. They found that only when rIAPP was present in 5-
isfolding and self-aggregation of soluble native proteins into insoluble abnormal amyloid fibrils deposited in various human tissues are associated with more than 20 neurodegenerative diseases.1 In type 2 diabetes (T2D), human islet amyloid polypeptide (hIAPP, also known as amylin) has been identified as a major component of pancreatic amyloid deposits, a characteristic histopathological biomarker in more than 90% of T2D patients.2,3 hIAPP (amylin), a 37-residue hormone peptide, is synthesized in the islet β-cells and cosecreted with insulin. In the normal state, hIAPP realizes its metabolic functions in regulating hormonal activities and insulin release.4,5 In the disease state, increasing evidence has shown that when hIAPP aggregates into small oligomers, protofibrils, and mature fibrils, these hIAPP aggregates could all be toxic species and involved in amyloidosis, albeit through different mechanisms to kill β-cells.6−9 Moreover, during the aggregation process of hIAPP, conformation transition from random-coil monomers to β-sheet-rich aggregates and the increased propensity of β-sheet structures in hIAPP aggregates10,11 are also hypothetically linked to β-cell death.12 Therefore, inhibition of β-sheet and fibril formation of hIAPP is regarded as a potential therapeutic approach against T2D.13−15 Conversely, rat amylin (rIAPP) is well-known not to form amyloid fibrils and not cytotoxic to β-cells, although rIAPP © 2014 American Chemical Society
Received: February 18, 2014 Revised: April 3, 2014 Published: April 22, 2014 5193
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IAPP Purification and Preparation. Both IAPP peptides were obtained in a lyophilized form and stored at −20 °C as arrived. In order to prepare the monomeric IAPP solution, 1.0 mg of preaggregated IAPP was dissolved in HFIP for 2 h, sonicated for 30 min to remove any pre-existing aggregates or seeds, and centrifuged at 14 000 rpm for 30 min at 4 °C. 80% of the top IAPP solution was then extracted, subpackaged, frozen with liquid nitrogen, and then dried with a freeze-dryer. The dry IAPP powder was lyophilized at −80 °C and used within 1 week. A homogeneous monomer solution of IAPP is required for studying amyloid formation. 0.2 mg of purified hIAPP or rIAPP powder was aliquoted in 20 μL of DMSO and sonicated for 1 min to obtain homogeneous solution. The initiation of hIAPP (25 μM, containing 1% (v/v) DMSO) aggregation in solution was accomplished by adding that 20 μL of DMSO−hIAPP solution to 2 mL of 10 mM PBS buffer. This solution was then centrifuged with 14 000 rpm for 30 min at 4 °C to remove any existing oligomers, and 80% of the top solution was removed for further incubation. We used the same protocol to prepare mixed hIAPP:rIAPP solutions, with only difference in changing the initial amount and ratio of purified powder. All the solutions were incubated at 37 °C. Thioflavine T (ThT) Fluorescence Assay. ThT fluorescence assay is considered as a standard method to detect the formation of amyloid fibrils31 because ThT can specifically bind to the β-sheet structure of protein fibrils and gives a strong fluorescence emission. A ThT solution (2 mM) was prepared by adding 0.0324 g of ThT powder into 50 mL of DI water. The resulting 250 μL of the 2 mM ThT solution was further diluted into 50 mL of Tris-buffer (pH = 7.4) to a final concentration of 10 μM. 60 μL of IAPP solution was put into 3 mL of 10 μM ThT-Tris solution. Fluorescence spectra were obtained using a LS-55 fluorescence spectrometer (PerkinElmer Corp., Waltham, MA). All measurements were carried out in aqueous solution using a 1 cm × 1 cm quartz cuvette. ThT fluorescence emission wavelengths were recorded between 460 and 510 nm with an excitation wavelength of 450 nm. Each experiment was repeated by at least three times, and each sample was tested in quintuplicate. Tapping-Mode AFM. Morphology changes of IAPP during fibrillization were monitored by a tapping-mode AFM. A 20 μL sample used in the IAPP ThT fluorescence assay was taken for AFM measurement at different time points in order to correlate IAPP morphology change with IAPP growth kinetics. IAPP solution was deposited onto a freshly cleaved mica substrate for 1 min, rinsed three times with 50 mL of deionized water to remove salts and loosely bound IAPP, and dried with compressed air for 5 min before AFM imaging. Tapping mode AFM imaging was performed in air using a Nanoscope III multimode scanning probe microscope (Veeco Corp., Santa Barbara, CA) equipped with a 15 μm E scanner. Commercial Si cantilevers (NanoScience) with an elastic modulus of 40 N m−1 were used. All images were acquired as 512 × 512 pixel images at a typical scan rate of 1.0−2.0 Hz with a vertical tip oscillation frequency of 250−350 kHz. Representative AFM images were obtained by scanning at least six different locations of different samples. Cell Culture. Rat insulinoma (RIN-m5F) cells (ATCC, Manassas, VA) were used as model pancreatic β-cells, and cultured in 75 cm2 Tflasks in sterile-filtered RPMI-1640 medium (ATCC, Manassas, VA) containing 10% fetal bovine serum (ATCC, Manassas, VA) and 1% penicillin/streptomycin (ATCC, Manassas, VA). Flasks were incubated in a humidified incubator with 5% CO2 at 37 °C. Cells were then cultured to confluence and harvested using 0.25% Trypsin-EDTA (1×) solution (Lonza, Walkersville, MD). Cells were counted using a hemacytometer and plated in a 96-well tissue culture plate at 50 000 cells per well in 100 μL of medium, which allow them to attach inside the incubator for 24 h. MTT Toxicity Assay. MTT-based cell toxicity assays were performed to assess the cytotoxicity of hIAPP and rIAPP assemblies.32 A 96-well plate with cells were split into eight groups, with each group containing six replicates. The first group containing cells only in medium was used as a positive control. The second group containing 2% DMSO was used as an additional control for evaluating the effects
fold excess or above, it showed strong inhibitory ability against hIAPP aggregation. Daniel et al.21 used ATR-FTIR spectroscopy to examine the interaction of hIAPP with anionic lipid bilayers in the absence and presence of peptide inhibitors including rIAPP. rIAPP was found to inhibit hIAPP aggregation and fibrillization on lipid bilayers. They suggest that the inhibitory effect of rIAPP on hIAPP fibrillogenesis at the membrane interface was due to the formation of nonamyloidogenic heterocomplexes of hIAPP and rIAPP. Two studies above consistently showed that rIAPP was able to prevent hIAPP amyloid formation at both lag and growth phases and also protect β-cells from apoptosis. Young et al.25 also observed the hetero-oligomer formation of hIAPP−rIAPP complexes using ionization-ion mobility spectrometry−mass spectrometry. However, Middleton et al. for the first time observed that rIAPP played a contrasting inhibitory role in hIAPP aggregation.26 When incubating with hIAPP, rIAPP initially inhibited hIAPP aggregation as evidenced by a lengthening of a lag phase, but as incubation time progressed, rIAPP could adopt β-sheet structures and grow with hIAPP templates via the “induced-fit and conformational selection” mechanism,27 thus eventually promoting amyloid fibril formation. Controversial data indicate that the cross-interaction between rIAPP and hIAPP is more complex than expected, and thus the inhibitory effect of rIAPP on hIAPP aggregation and underlying cross-interactions between rIAPP and hIAPP are not completely understood. More importantly, it is also a great interest to examine the cross-interaction between two different but similar amyloid sequences, which would provide a better fundamental understanding of the amyloid assembly mechanism.28−30 In this work, we examined the effect of cross-sequence interactions between hIAPP and rIAPP on hybrid amyloid structures, aggregation kinetics, and cell toxicity using combined computational and experimental approaches. Molar ratios of hIAPP:rIAPP were varied from 1:0.5 to 1:2. The fluorescent dye thioflavin-T (ThT) was used to probe the kinetic process of amyloid formation, and the corresponding fibril morphologies were monitored by atomic force microscopy (AFM). Cell viability assay was used to assess the inhibitory effect of rIAPP on β-cell viability. Molecular dynamics (MD) simulations were also performed to reveal the structural and energetic details of cross-interaction between hIAPP and rIAPP peptides at atomic level. Collective results showed that the presence of rIAPP extended both lag phase and growth phase of aggregation by hIAPP, but eventually it promoted amyloid fibril formation and induced the greater toxicity to cells. These results suggest that rIAPP is involved in the initial inhibition of hIAPP conformation transition and aggregation, but this is not effective to prevent hIAPP amyloid formation and hIAPPinduced cell toxicity. Once hIAPP seeds are formed, they can further recruit rIAPP to form more toxic amyloids. This work reveals a dual contrasting role of rIAPP as both an inhibitor and a coordinator when interacting with hIAPP.
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MATERIALS AND METHODS
Materials. 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP, ≥99.9%), dimethyl sulfoxide (DMSO, ≥99.9%), 10 mM PBS buffer (pH = 7.4), and thioflavin T (ThT, 98%) were purchased from Sigma-Aldrich (St. Louis, MO). Human IAPP (1−37) (≥95.0%) and rat IAPP (1− 37) (≥95.0%) were purchased from American Peptide Inc. (Sunnyvale, CA). All other chemicals were of the highest grade available. 5194
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of the DMSO on cells. DMSO−IAPP solutions diluted by cell medium were added to the groups of 3−8 to achieve the following final concentrations with different hIAPP:rIAPP molar ratios: 25 μM hIAPP, 50 μM hIAPP, 25 μM rIAPP, 25 μM hIAPP + 25 μM rIAPP, 25 μM hIAPP + 12.5 μM rIAPP, and 25 μM hIAPP + 50 μM rIAPP. The volume in each well was equalized to 137.5 μL by addition of cell medium. The cells were then incubated for another 24 h and assessed for cell toxicity using the Vybrant MTT Cell Proliferation assay kit (Life Technologies, Grand Island, NY). A 12 mM MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was prepared by dissolving 5 mg of MTT per 1 mL of sterile PBS (phosphate buffered saline). 15.3 μL of this MTT solution was added to each well. The cells were incubated for 4 h at 37 °C to convert MTT to formazan crystals. Cells were then removed from the incubator, and all but 50 μL of medium was removed from each well. Fomazan crystals formed at the bottom of each well were dissolved by adding 100 μL of DMSO per well and thoroughly mixed. Cells were incubated for an additional 10 min at 37 °C and mixed again to ensure formazan was fully dissolved. Plates were placed in a Synergy H1 microplate reader (BioTek, Winooski, VT), and absorbance was read at 540 nm to determine formazan content. The absorbance of positive control wells were averaged and subtracted from all other samples to eliminate background effect. Sample absorbance was then compared to the control groups to determine cell viability. hIAPP−rIAPP Simulation System. By averaging 10 solid-state NMR structures of full-length hIAPP fibrils kindly provided by the Tycko lab,10 the initial monomer coordinate of hIAPP1−37 peptide was constructed with a β-strand (Lys1-Val17)−turn (His18-Leu27)−βstrand (Ser28-Tyr37), U-bend fold. At the N-termini, the intramolecular disulfide bond between Cys2 and Cys7 was formed to stabilize the structure. The N- and C-termini were constructed by NH3+ and COO− groups, giving rise to a net charge of +3e at a pH of 7.4. Consistent with experimental molar ratios of hIAPP and rIAPP mixtures, the hIAPP1−37 oligomers (from dimer to pentamer) were built by stacking hIAPP1−37 monomers on top of each other in a parallel and register manner, where the peptide−peptide separation distance was set to 4.7 Å. The starting structure of the rIAPP1−37 monomer was obtained by mutating six amino acids of hIAPP monomer to His18Arg, Phe23Leu, Ala25Pro, Ile26Val, Ser28Pro, and Ser29Pro. The initial rIAPP monomer was first optimized in energy using 500 steps of the steepest decent algorithm with peptide backbone being restrained, followed by additional 500 steps of the conjugate gradient minimization with all atoms being relaxed. We applied similar procedure to construct hybrid hIAPP−rIAPP assemblies.33,34 Briefly, our working hypothesis of “induce-and-fit” suggests that hIAPP should recruit conformationally similar rIAPP to form hIAPP-rIAPP assemblies. Thus, we used the NMR-derived βstrand−turn−β-strand motif as a building block to construct rIAPP1−37 peptides, which then was packed on the top of the hIAPP1−37 oligomers in the same registered way as the hIAPP oligomers being constructed, allowing to maximize side chain contacts at the hIAPP− rIAPP interface. MD Simulation Protocol. All MD simulations of heteroassemblies in the explicit water molecules were performed using the NAMD software package35 with the CHARMM27 force field for peptides and the modified TIP3P model for water.36 The solvent box for each hIAPP−rIAPP assembly was constructed with a margin of ∼15 Å from any edge of the water box to any peptide atom. All the systems were neutralized by Cl− and Na+ ions to mimic ∼150 mM ionic strength. The 5000 steps of steepest decent minimization with peptide backbone atoms harmonically constrained and additional 5000 steps of conjugate gradient minimization without any constraint were performed to remove any unreasonable contact. After the minimization, the short 1 ns MD simulations were carried out to gradually heat system from 0 to 310 K by constraining the backbones of oligomers. Then, two independent 80 ns simulations for each hIAPP−rIAPP complex were conducted to study the structural and binding characteristics. All systems were subjected to an isothermal− isochoric ensemble (NPT, T = 310 K and P = 1 atm) under periodic
boundary conditions throughout the simulations. The Langevin piston method with a decay period of 100 fs and a damping time of 50 fs was employed to achieve a constant pressure of 1 atm, while the Langevin thermostat method with a damping coefficient of 1 ps−1 was used to maintain the temperature at 310 K. The hydrogen-involved covalent bonds were constrained using the RATTLE method,37 and the 2 fs time step was used in the velocity Verlet integration. The switch function with a cutoff at 12 and 14 Å was employed for van der Waals (VDW) interactions, while the force-shifted method with a 14 Å cutoff was used to calculate long-range electrostatic interactions. Trajectories were saved every 2 ps for analysis. All data analyses were performed using tools within the CHARMM, VMD,38 and codes developed inhouse.
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RESULTS AND DISCUSSION Effect of hIAPP:rIAPP Ratios on Amyloid Fibril Formation. We first examined the effect of cross-sequence interaction between hIAPP and rIAPP peptides on selfassembly and inhibition of amyloid fibrils using thioflavine T (ThT) fluorescence assay. Figure 1 showed the aggregation
Figure 1. Time-dependent ThT fluorescence curves for pure hIAPP, pure rIAPP, and mixed hIAPP/rIAPP at different ratios of 1:0.5, 1:1, and 1:2. Error bars represent the average of five replicate experiments.
kinetics of amyloid fibril formation at different molar ratios of hIAPP:rIAPP from 1:0.5 to 1:2. When incubating hIAPP of 25 μM alone at 37 °C for 20 h, ThT fluorescence profile showed a typical nucleation−polymerization kinetic of amyloid fibrillation, including a 4 h lag phase (nuclei formation), followed by an exponential growth phase (protofibril formation) between 4 and 6 h and a plateau phase where mature fibrils were formed after 6 h. As expected, pure rIAPP did not form amyloid fibrils itself under the same incubation conditions as pure hIAPP, since hIAPP and rIAPP displayed completely different aggregation and fibrillazation behaviors, despite their high sequence similarity. This fact indicates that both peptides may preserve some molecular recognition on one hand and may prevent amyloid assembly on the other. To examine this idea, we further studied the kinetics of fibril formation when coincubating hIAPP and rIAPP at different ratios of 1:0.5, 1:1, and 1:2 using the time-dependent ThT fluorescence. In the presence of an equimolar ratio of hIAPP (25 μM) and rIAPP (25 μM), the addition of rIAPP clearly showed an increase of lag time by an additional 2 h and a reduced growth rate, as compared to those values obtained 5195
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from pure hIAPP (25 μM) alone. This suggests that addition of rIAPP slows down the nucleation and early aggregation of hIAPP. However, after 20 h coincubation of hIAPP−rIAPP mixture (1:1), the maximum intensity of ThT fluorescence measured for a total of formation of amyloid fibrils approached to a stable plateau of ∼66, and this value was in between ∼55 and ∼100 for incubation of pure hIAPP at 25 and 50 μM, respectively. Compared to pure hIAPP (25 μM), increased maximum ThT intensity for hIAPP:rIAPP (1:1) mixture suggests that some rIAPP peptides are recruited by and grown onto the existing fibrils, leading to more fibrils being formed, despite the non-amyloidogenic nature of rIAPP itself. On the other hand, the hIAPP:rIAPP (1:1) mixture did not yield more fibrils as pure hIAPP at the same total concentration of 50 μM, suggesting that cross-interaction between rIAPP and hIAPP is not as equally effective as homogeneous interaction between hIAPP. Some cross-sequence barriers could exist and render different sequences difficult to specifically and selectively recognize and pack with each other due to the sequenceinduced polymorphic structures of peptides. Consistently, the presence of the other two hIAPP:rIAPP ratios of 1:0.5 and 1:2 also led to the changes in both lag time and the maximum fluorescence intensity, as comparing with the ThT profile of pure hIAPP (25 μM). When a 0.5 molar ratio of rIAPP was added, although the lag time was not significantly changed, the maximum ThT intensity was nevertheless increased by 16% at 20 h. When the hIAPP:rIAPP ratio was further increased to 1:2, a stronger inhibition on amyloid nucleation was observed with a lag time increased to ∼6 h. We should note that when hIAPP was incubated in the presence of rIAPP at different molar ratios, the maximum ThT intensities were only slightly increased as rIAPP:hIAPP ratios. The results suggest a saturate concentration of the rIAPP required for efficient fibril growth of hIAPP. Overall, the fibrillation kinetics monitored by ThT fluorescence exhibited concentration-dependent nucleation and growth (Figure 1), consistent with observations for the amyloid formation of many proteins and peptides. But, the most striking observation is that rIAPP appears to play a dual contrasting role in initial nucleation and final fibril growth. The introduction of rIAPP retards hIAPP nuclei formation, thus lengthening its lag phase. Once template amyloid seeds are formed, they can recruit conformationally similar rIAPP to promote more hybrid fibrils being formed. We should note that in all tested hIAPP−rIAPP mixtures containing the same amount of hIAPP (25 μM), any increase in final ThT intensity does not result from hIAPP fibrils alone, instead from new hybrid hIAPP−rIAPP fibrils. Young et al.25 found that the hetero-oligomers exhibited the less amyloid propensity than pure hIAPP oligomers of the same mass. This result is consistent with our results and explanation that cross-sequence interaction between hIAPP and rIAPP has less efficiency than homosequence interaction between hIAPP. Fibrils provide a catalytic surface for growth from either hIAPP or rIAPP peptides, but the availability of binding sites on the fibrillar surface is limited, which explains the less efficiency of interfacial interaction and subsequent fibril growth by rIAPP than by hIAPP. Structural Morphologies of Amyloid Fibrils. Parallel to ThT measurement, atomic force microscopy (AFM) is another way to monitor the structural changes of amyloids at different hIAPP:rIAPP ratios. All AFM samples of different time points were prepared at the same conditions as the corresponding
ThT experiments. Figure 2 shows a series of representative AFM images of hIAPP coincubated with and without rIAPP of
Figure 2. Representative AFM images for pure hIAPP, pure rIAPP, and mixed hIAPP/rIAPP aggregates after incubation with 0, 4, 6, 8, and 20 h.
different concentrations. Specifically, when incubating pure hIAPP (25 μM) alone, at the first 4 h, only a few amorphous aggregates, but no trace of fibrils was observed; at 6 h, some very short and unbranched protofibrils of 2−3 nm were observed; after 8 h, a significant amount of densely branched fibrils was formed, confirming the observation by strong ThT emission. Further increase of hIAPP concentration to 50 μM significantly promoted the formation of amyloid nuclei and fibrils. As expected, AFM images for incubation of pure rIAPP (25 μM) showed no fiber-like aggregates, but a few amorphous aggregates, confirming that rIAPP itself cannot form amyloid fibrils. For comparison, the morphologies of hIAPP aggregates in the presence of freshly prepared rIAPP of different molar ratios were examined. When a 0.5 molar ratio of rIAPP was added with hIAPP, no obvious changes in the morphology of hIAPP amyloids were observed. When the molar ratios of rIAPP:hIAPP were further increased to 1:1 and 2:1, a stronger inhibition on hIAPP amyloid formation was observed, with only a small amount of short fibrils and amorphous aggregates being formed at 6 h, respectively. Although dose-dependent morphologies were observed at a lag phase, coincubation of hIAPP with rIAPP of different concentrations eventually led to extensive branched fibrils with typical morphologies similar to pure hIAPP deposits. Taken together, the structural changes by AFM images, despite nonquantitative, were generally consistent with ThT data, confirming the acquired kinetics of amyloid fibrillation. Both ThT and AFM data clearly demonstrate that rIAPP indeed exerts inhibitory effect on hIAPP aggregation at 5196
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dynamics (MD) simulations to study the interactions of hIAPP dimer with rIAPP monomer, dimer, and tetramer at different molar ratios of hIAPP:rIAPP from 1:0.5, 1:1, to 1:2 used in our experiments. Considering that a hIAPP dimer is the most common building block39,40 and the atomic structures of hybrid hIAPP−rIAPP assemblies are not yet available, we used the U-shaped, cross-β-sheet structural models of hIAPP amyloid fibrils to construct a hIAPP dimer as the potentially smallest seed to interact with rIAPP monomer, dimer, and tetramer with similar U-shaped conformations. As revealed in Figure 4 throughout 80 ns MD simulations, both hIAPP and
the very early stage, as evidenced by prolonged lag time. But, once hIAPP forms cross-β structures, they can recruit rIAPP peptides and associate with them to further grow into hybrid amyloid fibrils similar to hIAPP fibrils. The kinetic and structural characteristics of rIAPP−hIAPP aggregation are generally consistent with the results by Middleton et al.,26 in which rIAPP initially inhibits hIAPP aggregation at 8 h but ultimately promotes hybrid hIAPP-rIAPP fibril formation at 24 h. Modulation of the hIAPP Cytotoxic Effect on β-Cells by rIAPP. To further determine the ability of the rIAPP to modulate the cytotoxicity of hIAPP assemblies, we conducted cell viability experiments to compare the toxic effects of hIAPP, rIAPP, and mixed hIAPP/rIAPP at different molar ratios (1:0.5, 1:1, and 1:2) on the RIN-m5F cells using the MTT assay (Figure 3). As expected, hIAPP exhibited strong cytotoxicity to
Figure 4. Representative top and side views of hIAPP (cyan) and rIAPP (pink) heterocomplex structures for hIAPP2−rIAPP1, hIAPP2− rIAPP2, and hIAPP2−rIAPP4 averaged from the last 20 ns MD simulations.
rIAPP peptides in three hybrid hIAPP−rIAPP assemblies were able to associate with each other, with a certain degree of twisting between adjacent peptides. The hIAPP peptides in the hybrid assemblies retained well for their two β-sheets and Uturn conformations, and the difference between their conformations was small. In contrast, the rIAPP peptides showed a noticeable tendency for a disturbed conformation at C-terminal β-sheet region, but the N-terminal β-sheet of rIAPP was relatively well preserved. Side-by-side structural comparison of hIAPP and rIAPP peptides in both pure and hybrid forms in our previous and present studies41 clearly showed that pure forms of hIAPP dimer or rIAPP oligomers displayed much larger structural instability than the corresponding hIAPP or rIAPP in hybrid forms. This indicates that the presence of cross-sequence interactions between hIAPP and rIAPP can mutually enhance structural stability of both hIAPP and rIAPP peptides. Additionally, we also compared the structural stability of hIAPP dimer extracted from both pure hIAPP trimer and hIAPP2−rIAPP1 trimer. RMSD results further confirmed that hIAPP dimer extracted from pure hIAPP trimer (RMSD = 5.4 Å) had the higher structural stability than hIAPP dimer from mixed hIAPP2−rIAPP1 trimer (RMSD = 6.5 Å). High sequence similarity and conformational flexibility of both hIAPP and rIAPP would enable such interactions. It appears that the introduction of rIAPP peptides into hIAPP dimer imposes little influence on interfacial interactions involving hydrophobic contacts and hydrogen bonds between adjacent hIAPP and rIAPP. Figure 5 shows quantitative analysis of geometric and structural characterization of the hybrid assemblies. Overall conformation changes of hybrid assemblies were measured by root-mean-square deviation (RMSD, Figure 5a) and radius of gyration (Rg, Figure 5b). It can be seen that relative to their initial structures of three hybrid hIAPP−rIAPP assemblies,
Figure 3. RIN-m5F cell viabilities in the presence of pure hIAPP, pure rIAPP, and mixed hIAPP/rIAPP as determined by the MTT assay. Error bars represent the average of six replicate experiments.
cells, with cell viability decreased to 65.1% in the presence of 25 μM hIAPP and 48.6% in the presence of 50 μM hIAPP. In contrast, rIAPP (25 μM) alone exhibited almost no intrinsic cytotoxicity to cells, with cell viability of 97.5%. Interestingly, when coincubating hIAPP (25 μM) with nontoxic rIAPP at different concentrations (12.5, 25, and 50 μM), cell viability decreased to 55.5%, 54.2%, and 50.5%, respectively, as compared to 65.1% for pure hIAPP (25 μM). It appears that the introduction of rIAPP induces, not prevents, cell death as the increase of rIAPP:hIAPP ratios. The gain-of-toxic activity of both IAPP peptides is directly linked to their ability to misfold and self-assemble into β-sheet aggregates and fibrils. Various aggregated species during the aggregation process could induce the toxicity to β-cells. Among them, oligomeric intermediates are widely considered as the most toxic species. As described by our AFM and ThT data, addition of rIAPP to hIAPP generally prolongs the lag time to some extent, which increases the possibilities to produce more toxic oligomeric species and thus to induce more cell death. Cell viability experiments also provide additional evidence that the new hybrid species produced by cross-sequence interaction are toxic to cells. MD Simulations of Cross-Sequence Interactions between rIAPP and hIAPP. To better understand atomic details of the cross-sequence interactions between rIAPP and hIAPP, we performed all-atom explicit-water molecular 5197
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Figure 5. Geometric and structural characterization of the hybrid hIAPP−rIAPP assemblies. Time evolution of (a) RMSD and (b) Rg of hIAPP2− rIAPP1, hIAPP2−rIAPP2, and hIAPP2−rIAPP4 heteroassemblies. (c) Respective RMSF of hIAPP and rIAPP in heteroassemblies. (d) β-Sheet percentage of N-terminal and C-terminal regions in respective hIAPP and rIAPP of heteroassemblies.
conformation drifts were 8.0 Å for hIAPP2−rIAPP1, 8.2 Å for hIAPP2−rIAPP2, and 6.6 Å for hIAPP2−rIAPP4. Interestingly, upon relaxation in aqueous solution, Rg of three hybrid assemblies stayed leveled or even became smaller as the trajectories progressed, and eventually overall sizes of the heteroassemblies were well conserved. This indicates that the strand-to-strand association at the hIAPP−rIAPP interface is quite stable for all heteroassemblies. To further characterize the origin of conformational changes of the heteroassemblies, residue-based root-mean-square fluctuation (RMSF) was calculated separately for both hIAPP and rIAPP peptides. The average RMSF values for hIAPP and rIAPP give a more quantitative description of the contribution of the structural component to the overall stability of the heteroassemblies. When the fluctuations between hIAPP and rIAPP were compared, Figure 5c shows that rIAPP had relatively larger RMSF values, i.e., larger structural fluctuation, than hIAPP. The residue-based RMSFs of rIAPP in three heteroassemblies also showed that the residues from N-terminal β-strands were consistently stable, while the residues from C-terminal βstrands and turns were in general much more flexible. We also performed the RMSF comparison between edge hIAPP and rIAPP peptides in all three heteroassemblies. RMSF data showed that the edge rIAPP had the relatively larger RMSF (the larger structural fluctuation) than the edge hIAPP (data not shown). Considering that initial structures of hIAPP and rIAPP are almost identical to each other, such a difference in structural fluctuation (i.e., residue movement) could be partially contributed to the difference in overall structural stability of the heteroassemblies. To further quantify whether different flexibilities of N- and C-terminal residues actually result in secondary structure changes, secondary structures derived from C-terminal residues (position 20−37) and N-terminal residues (position 1−19) of
rIAPP and hIAPP were analyzed and compared using the DSSP algorithm. Specifically, β-structure contents were compared between hIAPP and rIAPP and between C-terminal residues and N-terminal residues; as Figure 5d shows, we observed several interesting differences and similarities for secondary structure distributions. First, rIAPP exhibited a much lower percentage of β-structure than hIAPP, in which hIAPP and rIAPP adopted 46.95−53.32% and 30.63−35.36% β-structure, respectively. Consistent with our results, Middleton et al.26 also reported that upon formation of hIAPP−rIAPP complexes Nterminal residues of both hIAPP and rIAPP peptides remained in a relatively stable β-sheet conformation, which provides a potential template for further β-sheet formation and association. Second, for hIAPP peptides in all heteroassemblies, βstructure contents from C-terminal and N-terminal regions were comparable to each other, with slight 4.01−8.89% of higher β-structure from N-terminal regions. Conversely, for rIAPP peptides, we observed a dramatic difference in βstructure propensities between C-terminal and N-terminal residues, which adopted 7.67−11.59% and 22.78−25.45% βstructure, respectively. This finding is, in part, consistent with previous studies of pure hIAPP and rIAPP assemblies in aqueous solution.17,39,42,43 These results confirm that despite structural difference in the C-terminal region of the heteroassemblies, hIAPP and rIAPP can still cross-seed to form amyloid-like structures. Possible Mechanistic Model for Cross-Interaction between rIAPP and hIAPP. Our recent simulations41 showed that hIAPP trimer to pentamer, but not monomer or dimer, exhibited high structural stability with well-preserved in-register parallel β-sheet and the U-bend conformation, suggesting that hIAPP trimer appears to be the smallest minimal seed in solution. In contrast, all of rIAPP aggregates from monomer to pentamer exhibited less structural stability, particularly with 5198
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Figure 6. Schematic model for cross-assembly and interaction between hIAPP (blue) and rIAPP (orange).
disturbed C-terminal β-sheet and turn conformation. Interestingly, the N-terminal β-sheet of rIAPP oligomers was largely preserved. On the basis of our computational observation, we hypothesized that the relatively conserved N-terminal β-sheet of rIAPP peptides, which is similar to that of hIAPP peptides, offers basic structural features and possibilities to be associated with and grown on the exteriors of the hIAPP oligomers and fibrils. To explore this hypothesis, further MD simulations in this work showed that hIAPP and rIAPP can form stable complexes with no apparent tendency for peptide disassociation within 80 ns. Although the structures of hIAPP−rIAPP complexes were not as perfect as those of pure hIAPP oligomers with well-preserved in-register parallel β-sheet and the U-bend conformation, both N-terminal residues of both peptides retained a stable β-sheet conformation, which could serve as a template to stabilize peptide association. Meanwhile, simulation results also demonstrate that although the presence of mismatch sequences between hIAPP and rIAPP increases energy barriers for efficient cross-interactions, similar conformations of both peptides, even partially, can “repair” the effect of the mismatch. MD simulations also provide structural basis for better understanding experimental observations. As shown in Figure 6, hIAPP peptides can aggregate into β-sheet-rich oligomers, protofibrils, and fibrils. Once stable hIAPP β-sheet aggregates are formed, the N-terminal β-sheet of hIAPP aggregates could serve as a template interface either to recruit and accommodate rIAPP with conformational similar N-terminal β-sheet or to facilitate the structural transition of rIAPP to partially fold into compatible β-sheet structures, or both. The cross-interactions of different sequences would increase the free energy barrier for peptide unfolding and subsequent elongation, thus requiring a longer time to form heteroassemblies particularly at the initial lag phase. This also explains the less efficiency of crossinteraction than homointeraction, as evidenced by that the final ThT intensities of hIAPP−rIAPP mixtures were always lower than those of pure hIAPP of the same or even lower total peptide concentrations. To a broader extent, according to the selective molecular recognition mechanism, cross-sequence interaction of amyloid species could be governed by conformational selection of compatible states. If the dominant conformations of two species are similar, they can interact with each other.27,44−48
hIAPP-induced neurotoxicity but also develop new therapies for T2D. In this work, considering that rIAPP has nonamyloidogenic properties and high sequence similarity to hIAPP, we study the effects of the cross- and self-interaction of hIAPP and rIAPP on the amyloidogenicity of both peptides using ThT fluorescence, AFM, MTT cell viability assay, and MD simulations. ThT and AFM results show that at different molar ratios of hIAPP:rIAPP (1:0.5, 1:1, and 1:2), hIAPP− rIAPP interactions suppress initial nucleation but promote final fibrillization. The cross-amyloid aggregates gain the higher toxic activity to cultured cells as compared to pure hIAPP aggregates. Thus, the cross-sequence interaction between amyloidogenic hIAPP and non-amyloidogenic rIAPP has been shown to promote amyloid formation and induce cell neurotoxicity. MD simulations further confirm that rIAPP can bind strongly to hIAPP to form heterogeneous rIAPP−hIAPP assemblies, which may serve as precursors to further self-aggregate into hybrid fibrils. The cross-sequence interaction between hIAPP and rIAPP has the less efficiency than homosequence interaction between hIAPP. These findings suggest that molecular recognition between hIAPP, rIAPP, and likely other amyloid peptides could mediate cross-amyloid interactions and protein self-assembly, which may assist in design of inhibitors to prevent these processes.
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AUTHOR INFORMATION
Corresponding Author
*Phone 330-972-2096; Fax 330-972-5856; e-mail zhengj@ uakron.edu (J.Z.). Author Contributions
R.H. and M.Z. contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the National Science Foundation grants (CAREER Award CBET-0952624 and CBET-1158447) for support of this research.
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REFERENCES
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