Human Islet Amyloid Polypeptide Assembly: The Key Role of the 8â20

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Human Islet Amyloid Polypeptide Assembly: The Key Role of the 8-20 Fragment Li Wang, Alexandre Iourievich Ilitchev, Maxwell J. Giammona, Fei Li, Steven K. Buratto, and Michael T. Bowers J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b09475 • Publication Date (Web): 27 Oct 2016 Downloaded from http://pubs.acs.org on October 31, 2016

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The Journal of Physical Chemistry

Human Islet Amyloid Polypeptide Assembly: The Key Role of the 8-20 Fragment Li Wang†‡§, Alexandre I. Ilitchev†§, Maxwell J. Giammona†, Fei Li‡, Steven K. Buratto†, Michael T. Bowers†* † Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106, United States ‡ State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130012, P. R. China § These authors contributed equally to this work.

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ABSTRACT The aggregation of human islet amyloid polypeptide (hIAPP) has been closely associated with the pathogeny of type 2 diabetes mellitus (T2DM) and destruction of pancreatic islet βcells. Several amyloidogenic domains within the hIAPP sequence capable of self-association have been identified. Among them is the 8-20 region of hIAPP, which has formed β-sheet fibrils despite being contained within an α-helical region of full-length hIAPP. To further understand the propensity of this region for self-assembly, two peptide fragments were compared, one consisting of the residues 8-20 (WT8-20) and a mutant fragment with a His18Pro substitution (H18P8-20). The conformational distribution and aggregation propensity of these peptides was determined using a combination of ion mobility mass spectrometry and atomic force microscopy. Our results reveal that the two peptide fragments have vastly differing assembly pathways. WT820

produces a wide range of oligomers up to decamer whereas the H18P8-20 mutant produces only

low order oligomers. This study confirms the propensity of the 8-20 region to aggregate from its native α-helical structure into amyloid β-sheet oligomers and highlights the significance of the charged His18 in the aggregation process.

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INTRODUCTION Amyloid aggregation and fibril deposition contribute to the pathology of a variety of disorders including Alzheimer’s and Parkinson’s disease, as well as type 2 diabetes mellitus (T2DM).1-3 T2DM is characterized by aggregation and deposition of human islet amyloid polypeptide (hIAPP), a 37 amino acid co-secreted with insulin from β-cells within the islets of Langerhans of the pancreas.4-6 IAPP is over-expressed in patients with T2DM, aggregating into soluble oligomers and, subsequently, insoluble fibrils characteristic of amyloid disorders. The early oligomers of hIAPP have been characterized as toxic, contributing to islet β-cell dysfunction and death.7-8 Native hIAPP is disordered in its monomer form but is highly structured into stacked β-sheets once it forms insoluble fibrils.9-12 Understanding the aggregation mechanism from monomer to soluble oligomer to fibril requires consideration of aggregation prone domains within the IAPP sequence. Several of these domains have already been identified, with particular focus on the 2029 region S20NNFGAILSS29 (rat IAPP does not aggregate almost certainly due to proline residues at positions 25, 28 and 29).13-15 In addition to the 20-29 region of hIAPP, selfassociation has been observed for the fragments 1-8, 8-20, and 30-37, all of which form amyloidlike β-sheet fibrils.16-21 This is important because fragment amyloid formation has been computationally associated with the aggregation of full-length peptides.22-23 The 8-20 region has been found capable of self-association both within hIAPP and rIAPP. These differ only at position 18, which is histidine in hIAPP and arginine in rIAPP.21 Numerous studies have found that this region is α-helical within the disordered hIAPP monomer.24-25 There is also some indication that this region is important to molecular recognition leading to amyloid formation.26 Previous work has shown that the aggregation propensity of 8-20 is strongly affected by pH.27 3



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This pH dependence, and aggregation of the fragment in general, is negated when the histidine at position 18 is substituted by a less basic residue.27 Therefore it is thought that the protonated state of histidine plays some part in the aggregation process. Histidine has also been found to play a significant role in the aggregation of peptides responsible for other amyloid diseases, such as Alzheimer’s.28 Further, a study of the 8-16 fragment similarly concluded that this region of hIAPP aggregates into fibrils similar in composition and cytotoxicity to full-length hIAPP.29 Reconciling the α-helical state of this region in the monomer of the full peptide with the highly ordered β-sheet stacking of the aggregated fragment may give significant insight into the aggregation mechanism of hIAPP. In this study we use a combination of ion mobility mass spectrometry (IMS-MS) and atomic force microscopy (AFM) in order to probe the differences in the aggregation mechanisms of the 8-20 residue fragment (WT8-20) and a fragment containing a His18Pro substitution (H18P8-20). IMS-MS has been extensively used to characterize protein monomer and oligomer solution-like structures. This is done through the use of soft nano-electrospray ionization coupled with gentle ion mobility separation of conformational isomers and oligomers.30-31 AFM is used as a complimentary technique to ion mobility as it allows for the observation of larger macroscopic aggregates.32-34 In cases where oligomers observed with ion mobility are large enough, a direct comparison can be made between IMS-MS cross-section and the feature size seen in AFM measurements.35

MATERIALS AND METHODS Peptide Sample Preparation. Both peptide samples, ATQRLANFLVHSS (WT8-20) and its mutant, ATQRLANFLVPSS (H18P8-20), were purchased from Genscript (Piscataway, NJ) and 4


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used without further purification. Both peptides have unmodified free termini. Dry samples were dissolved in 100% hexafluoroisopropanol (HFIP) (Sigma-Aldrich, St. Louis, MO) to a concentration of 1 mg/mL and sonicated for 5 minutes in order to break up any pre-existing aggregates. Aliquots of the stock were dried under vacuum overnight and re-suspended in water for a final peptide concentration of 50 µM. Ion Mobility Experiments. Peptide samples were prepared at 50 µM in water and stored on ice until IMS-MS experiments were performed in order to slow the aggregation process. All experiments were done on a homebuilt ion mobility mass spectrometer which has been described in detail previously.36 In short, ions are generated using a nano-electrospray source and funneled into a 5-cm drift cell filled with ~3.5 Torr of helium buffer gas. The ions are pulled through the drift cell under the influence of a weak electric field and their motion is slowed by collisions with the buffer gas. Ions with different conformations and surface areas, but the same m/z, are therefore separated in arrival time due to differing collisions with the buffer gas. This allows for the separation of either conformers or oligomers that share the same m/z. Ions exit the drift cell and are mass-selected by a quadrupole before being detected by an electron multiplier detector. Additional experimental details are provided in the Supporting Information. Atomic Force Microscopy. The same peptide samples used for IMS-MS experiments were used directly for AFM experiments by depositing 20 µL of the 50 µM peptide solution onto V1grade mica (TedPella, Redding, CA) at indicated times and dried in a vacuum-desiccator overnight. Mica was freshly cleaved and pre-treated prior to deposition with 50 µL of 50 µM potassium chloride in order to prevent possible interactions between positively charged residues in the protein fragment and oxygen atoms in the mica.37 The KCl solution was left to sit on the mica in a covered sample dish for 30 minutes after which the mica was rinsed 3 times with 5



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deionized water. Tapping-mode AFM experiments were performed in air on an MFP-3D Atomic Force Microscope (Asylum Research, Goleta, CA) using a silicon probe with a cantilever spring constant of 7 N/m and a resonant frequency of 155 kHz (MikroMasch USA, Lady’s Island, SC). Aggregates were height analyzed using a mask starting at 1 nm which was adjusted to include all aggregates in the image. Images shown are 2 µM x 2 µM in size with a 512 x 512 pixel resolution in order to highlight the range of feature sizes. Perimeter and area of aggregates were extracted from 0.5 µM x 0.5 µM images with the same 512 x 512 pixel resolution to avoid convolution of the feature size with the pixel size for the smaller features. This allows for direct comparison of the smaller features observed in the AFM to the larger oligomers observed in the IMS-MS. Maximum height values were extracted from the 2 µM x 2 µM sized images to improve statistics; no height differences were observed when imaging at different length scales. Particle properties for calculating the characteristics for each masked aggregate are detailed in the Supporting Information. Thioflavin-T

Fluorescence

Assay.

Assays

were

performed

on

an

RF-5301PC

Spectrofluorophotometer (Shimadzu (China), P.R.C) at room temperature with λex = 440 nm and λem = 482 nm. All assay solutions were prepared by adding 20 µM thioflavin-T solution to 50 µM peptide solutions immediately before measurement.

RESULTS AND DISCUSSION Distinct oligomerization pathways revealed for WT8-20 and H18P8-20 via IMS-MS. Mass spectra and ion mobility data were collected in positive-ion mode on a home-built ion mobility mass spectrometer at t = 0 days, 7 days, and 14 days. Mass spectra of both peptide samples at t = 0 days are shown in Figure 1. Both samples show peaks at m/z = ~701 and m/z = ~1403 6


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corresponding to oligomer-size to charge (n/z) values of 1/+2 and 1/+1 respectively. The WT8-20 mass spectrum also shows a significant additional 1/+3 peak (m/z = ~475), likely the result of protonation at His18 which does not occur when the amino acid is substituted to proline in H18P8-20. Mass-selected arrival time distributions (ATDs) were taken of all major features with data for the 1/+1 peak given in Figure 2. This peak was chosen for analysis as it represents the native solution-phase charge state of the peptides. The other, more prominent MS peaks, 1/+2 and 1/+3, each contain a single monomeric state in their ATD, most likely corresponding to a charged gas-phase structure (data not shown). Data taken at low injection energy (IE) are shown

Figure 1. Positive-mode mass spectra of (A) the 8-20 hIAPP fragment (WT8-20) and (B) the His18Pro mutant (H18P8-20) at t = 0 days. All peptide concentrations are 50 µM in deionized water. Peaks are annotated by oligomer size to charge ratio (n/z). in order to minimize instrumental dissociation of the oligomers. At t = 0 days (Figure 2A), WT8-20 shows a prominent monomer peak followed by a cluster of oligomers up to pentamer. After two weeks, very little monomer remains, with a broad range of higher order oligomers observed. These are broadly clustered into two groups; one contains dimer to pentamer and the 7



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other containing hexamer to 10-mer. H18P8-20, on the other hand, has only monomer, dimer and trimer peaks and remains largely unchanged over the span of two weeks, only adding a minor tetramer shoulder. These results indicate that substitution of His by Pro at position 18 dramatically reduces the oligomerization of the 8-20 hIAPP peptide fragment. We will address the implication of these results when we analyze the AFM data (Figure S3).

Figure 2. Representative ATDs of (A-C) the 8-20 hIAPP fragment (WT8-20) and (D-F) the His18Pro mutant (H18P8-20) of the n/z = 1/+1 mass spectra peaks at 0 days, 7 days, and 14 days. All distributions are annotated by oligomer size (n) and injection energy (IE). Each ATD is fit with multiple features using the procedure described in the Supporting Information section. The dashed lines represent the peak shape for a single conformation.

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Figure 3. Injection energy (IE) studies of WT8-20 (A) and H18P8-20 (B) hIAPP fragment at charge-to-oligomer ratio, n/z = 1/+1 and t = 14 days. All peaks are annotated by oligomer number (n). Lower injection energies favor higher order oligomers whereas higher injection energies break down higher order oligomers and shift the distribution to smaller order oligomers. The lowest injection energy (25V) represents an approximation of the in-solution oligomer distribution. Each peak is fit using the procedure described in the Supporting Information. The relative stability of these oligomers was probed using injection energy modulation. Increased injection energy provides the aggregates with sufficient internal energy to dissociate oligomers or to collapse solution-phase structures into more compact gas-phase conformations. As injection energy is increased for WT8-20, all peaks with low arrival times disappear, corroborating that they are indeed oligomeric aggregates (Figure 3A). This also occurs for the H18P8-20 sample, with both samples essentially in monomer form at IE = 100 eV (Figure 3B). No additional structures appear as injection energy is increased, indicating that the monomeric structure is relatively stable.

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Table 1: Collision Cross Section in Å² of IAPP Fragments in the n/z = 1/+1 ATDs

Sample WT8-20 H18P8-20

1

2

297 293

497 484

Oligomer Size (n) 3 4 5 6 Experimental Cross-Section (Å²) 660 841 1002 1099 631 761

8

10

1398

1659

A list of oligomer cross-sections is provided in Table 1 and plotted versus oligomer number in Figure 4. Oligomer sizes were compared to ideal isotropic growth defined by the equation σ = σmon • n2/3, where σmon is the monomer cross-section and n is the oligomer number.38 Oligomers of the mutant largely follow the isotropic growth line whereas the wildtype fragment cross sections deviate strongly above the isotropic line (Figure 4). The linear growth for the wildtype fragment from n = 1 to n = 5 suggests possible β-sheet stacking. Recent spectroscopic results have shown a strong correlation between the occurrence of β-sheet and positive deviation of cross section from the isotropic trendline for smaller peptide systems.39 Also of interest is a change in growth at n = 6 for the wildtype peptide. Previous work on 11 residue peptides has shown cylindrin/β-barrel structures occur at this size.40-41 The WT8-20 peptide is 13 residues in length and lacks a central glycine but nonetheless a transition from in-register β-sheet growth to cylindrin/β-barrel growth may be occurring here. While further work is needed to verify this possibility it might provide a clue regarding the mechanism of IAPP growth and cellular damage.

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Figure 4. Plot of oligomer size (n) versus experimental cross-section (σ, Å²) for WT8-20 (A) and H18P8-20 (B) hIAPP fragments. Experimental results are plotted against an isotropic model of aggregation (σmon • n2/3) where σmon is the monomer cross section for the peptide.

AFM shows substantial aggregation of WT8-20 and minimal aggregation for H18P8-20. Atomic force microscopy images were taken in parallel to ATD acquisition at t = 0 days and 14 days (Figure 5). WT8-20 shows significant globular aggregates at 0 days. H18P8-20, meanwhile, shows a much smaller quantity of structured features overall, including sparse, short fibril-like structures. After two weeks, WT8-20 displays pervasive amyloid fibrils with minor globular features throughout. The mutant fragment, meanwhile, appears largely unchanged, with slightly larger globular aggregates and minor contributions from the fibril-like structures. Overall these results correlate well with the IMS-MS results, with WT8-20 showing much more amyloid-like growth than the mutant H18P8-20 fragment.

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Figure 5. Time-lapse incubation of WT8-20 (A-B) and H18P8-20 (C-D) hIAPP fragments at 50µM over the course of two weeks. WT8-20 shows rapid oligomerization with gradual fibrilgrowth over the course of the experiment. H18P8-20 meanwhile appears to have minimal aggregation at 0 days with minor fibril-like features. After two weeks the mutant fragment shows larger globular aggregates with similar fibril-like features as seen at 0 days.

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Figure 6. Plots of particle perimeter versus area for the wildtype 8-20 hIAPP fragment (A) and H18P 8-20 mutant fragment (B). The area versus perimeter curve for a circle, which would be expected for isotropic growth, is plotted in orange as a reference. The WT8-20 fragment shows several elongated aggregation trajectories whereas the H18P8-20 mutant shows predominantly isotropic growth. AFM feature distribution parallels IMS-MS data. A direct comparison between microscopy imaging and arrival time distributions is often illusive as observed AFM features are typically significantly larger than those seen in IMS-MS while smaller features of the AFM image are convoluted with the background. In this paper we present a more direct connection between ATD and AFM by a particle analysis. The size progressions of feature perimeter versus area generated from 500 nm by 500 nm AFM images are shown in Figure 6. It should be noted that the area and perimeter of features are susceptible to tip convolution which makes particles in the AFM appear larger than they are in actuality. If a direct comparison of observed feature size was to be made, tip deconvolution would be required. Even without tip deconvolution, however, this analysis allows for a direct comparison of the growth trends observed in the ion-mobility experiments at small size scales and provides a continuation of those trends into larger size 13



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scales, highlighting how the two techniques are complimentary. Based on this analysis it is possible to see 2-3 district growth trajectories for the wild type fragment relative to the isotropic growth of an expanding circle given by the orange curve. The green line shows a trajectory that is only slightly elongated relative to a circle, and the yellow and red lines showing growth trajectories which are significantly elongated, indicating fibrillar growth. In contrast, only a single trajectory which trends very closely with isotropic growth curve is present for the H18P mutant. These data agree well with the IMS-MS data in Figure 4 and show the trend observed for small oligomers continues for much larger aggregates. Histograms used to generate these plots are available in the Supporting Information (Figure S4 and S5). This type of presentation presents an exciting correlation between AFM imaging and ion mobility, showing that the distribution of oligomers seen in IMS-MS and those observed in AFM appear to closely represent the general solution-phase distribution of structures.

Thioflavin-T assay shows WT8-20 has more amyloid-like β-sheet character than H18P8-20. While IMS-MS and AFM show the oligomerization and aggregation of peptide fragments, there is little information to be obtained about the specific secondary structure of the oligomers from these techniques. A hallmark trait of amyloid aggregates is their high concentration of β-sheet assemblies. Thioflavin-T is a small fluorescent dye which intercalates between protein β-sheet structures and is therefore used as a bulk kinetic technique to determine the presence and rate of aggregation of amyloid peptides.42-43 Both WT8-20 and H18P8-20 aggregation kinetics were monitored over the span of two weeks, as seen in Figure 7. Within the first 6 days both WT8-20 and H18P8-20 show an increase in fluorescence, representative of increased β-sheet concentration in solution. The proline mutant, however, shows much lower maximum fluorescence than the 14


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WT8-20 peptide. Additionally, there is a second major increase in the total fluorescence of WT8-20 between 10 and 14 days not seen in the H18P8-20 sample, suggesting that there is some structural rearrangement that occurs after approximately 10 days which leads to higher order oligomer formation. A range of new higher-order oligomers appears in the IMS-MS for the WT8-20 after two weeks, which corresponds well to the structural rearrangement seen in the ThT assay. Overall the ThT results are consistent with both the IMS-MS data and the AFM data which indicate a major fraction of β-sheet formation for WT8-20 and a minor fraction of β-sheet formation for H18P8-20.

Figure 7. Time dependence of Thioflavin-T fluorescence intensity of 50 µM WT8-20 (black) and H18P8-20 (red). WT8-20 shows an additional increase in fluorescence after one week which could correspond to the formation of additional higher-order oligomers seen at two weeks in the ion mobility and the observation of fibrils in AFM. CONCLUSIONS In this work we examine the aggregation propensity of the 8-20 region of hIAPP through the study of two peptide fragments, the wildtype 8-20 (WT8-20) and a His18Pro mutant of the same 15



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fragment (H18P8-20). A combination of ion mobility mass spectrometry, atomic force microscopy, and Thioflavin-T fluorescence revealed disparate aggregation pathways for the two fragments. H18P8-20 forms low order isotropic oligomers with only a small amount of fibril growth observed in either the AFM or ThT assay. Ion mobility studies show WT8-20 forms numerous higher-order oligomers up to 10-mer, with fibril growth observed via AFM and amyloid-like β-sheet aggregation detected with ThT. General aggregation trends observed in IMS-MS correlate well with particle distributions seen in AFM particle analysis, with WT8-20 forming assorted fibrillar assemblies while H18P8-20 generally favors isotropic aggregates. As has been previously determined this 8-20 fragment is capable of extensive oligomerization, albeit at a slower pace than the full-length peptide (hIAPP1-37). It also comprises the membrane associating α-helix region of hIAPP1-37, indicating that it must fundamentally change its secondary structure before associating into stacked β-sheets. This could mean that the 8-20 region serves as an association site, but only contributes relatively late in the aggregation process. Of particular note is the formation of hexamer and octamer aggregates for WT8-20 which appear to dominate in solution after two weeks. Recent experiments with fragments of Alzheimer’s disease protein Aβ42 have shown that cylindrin and β-barrel aggregates are formed composed of 6 or 8 peptides.41 Additionally, full-length Aβ42 has been observed to form dodecamers via hexamer stacking, which is similar to the stacking aggregation mechanism observed for the 8-20 hIAPP fragment after 1-2 weeks.35 Given that the cross-section and oligomer number of the WT8-20 fragment is similar to those observed for the C-terminal fragment aggregates of Aβ42 a future trajectory of work should focus on determining the structure of these WT8-20 hexamer and octamer aggregates to see if they are indeed cylindrin/β-barrel structures.

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ASSOCIATED CONTENT Supporting Information. Ion mobility experimental details, atomic force microscopy particle mask calculation details, injection energy studies of the WT8-20 and H18P8-20, and AFM particle analysis of both peptides. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Michael T. Bowers, Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106, USA. E-mail: [email protected]. Phone: 805-893-2893. Fax: 805-893-8703. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. § These authors contributed equally to this work.

ACKNOWLEDGMENT The authors thank Nicholas J. Economou for his assistance in the development of analytical methods for interpreting the AFM data. Support from the National Science Foundation under grants CHE-1301032 and CHE-1565941 (M.T.B.) are gratefully acknowledged. L.W. was supported in part by a grant from the Chinese Scholarship Council (No. 201406170165). 17



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Human Islet Amyloid Polypeptide Assembly: The Key Role of the 8â20

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