Biochemical and Biophysical Research Communications 533 (2020) 125e131

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Biochemical and Biophysical Research Communications

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Structural characterization and cryo-electron analysis of human islet amyloid polypeptide suggest a synchronous process of the hIAPP137 amyloid fibrillation * Xueli Zhang a, b, Dongyu Li a, c, Xushan Zhu a, b, Youwang Wang a, c, Ping Zhu a, b, c, a National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China b Sino-Danish College, University of Chinese Academy of Sciences, Beijing, 100049, China c College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China article info abstract

Article history: Revealing the aggregation and fibrillation process of variant amyloid is critical for understanding Received 22 August 2020 the molecular mechanism of related amyloidosis diseases. Here we characterized the fibrillation Accepted 25 August 2020 morphology and kinetics of type 2 diabetes (T2D) related human islet amyloid polypeptide (hIAPP1-37) Available online 14 September 2020 fibril formation process using negative staining transmission electron microscopy (NS-TEM), cryo- electron microscopy (cryo-EM) analysis, and 3D cryo-electron tomography (cryo-ET) reconstruction, Keywords: together with circular dichroism (CD) and Thioflavin-T (ThT) assays. Our results showed that various Human islet amyloid polypeptide (hIAPP) amyloid fibrils can be observed at different time points of hIAPP137 fibrillization process, while the Amylin fi Amyloid winding of proto brils presents in different growth stages, which suggests a synchronous process of fi Cryo-electron tomography hIAPP1-37 amyloid brillization. This work provides insights into the understanding of hIAPP1-37 amyloid Protofibril winding aggregation process and the pathogenesis of Type 2 diabetes disease. © 2020 Elsevier Inc. All rights reserved.

1. Introduction regulating glucose uptake and inhibiting insulin release and glucose elimination rate [10,12e14]. IAPP is normally soluble and Amyloid aggregation is a common pathological feature among naturally unfolded in monomeric state, but it was found to aggre- many unrelated diseases, such as Alzheimer’s disease (AD), Par- gate as islet amyloid in type 2 diabetes (T2D) and can be readily kinson’s disease (PD), Type 2 diabetes (T2D) and Mad cow disease induced to form fibrils in vitro [8,10,12,15]. Islet amyloid formation [1e4]. In these amyloid diseases, a particular , for example, can lead to islet b- dysfunction and death, and contribute to the Ab for AD and a-synuclein for PD, was typically found to misfold failure of islet transplantation [10,16e19]. The amyloid deposition and aggregate to form insoluble fibrils which are rich in b-sheet of hIAPP presents in the pancreas in over 90 % of diabetic patients structures [5,6]. In type 2 diabetes, amyloid fibrils are formed by and the extent of amyloid deposition seems related to the severity human Islet amyloid polypeptide (hIAPP) in the pancreatic islets of of the disease [20e24]. Therefore, the hIAPP fibril formation plays Langerhans [7,8]. Islet amyloid polypeptide (IAPP), also known as an important role in the pathogenesis of T2D, and the study of the amylin, is a peptide hormone of 37 amino acid residues that is co- hIAPP amyloid aggregation and fibrillation kinetics will shed lights expressed and co-secreted with insulin by the pancreatic b-cell on the pathogenesis of T2D disease. secretory granules [9e11]. As a member of the calcitonin family of Although the aggregation mechanism of the Ab or a-synuclein hormones, IAPP has been found in all mammals studied to date, amyloid proteins had been extensively studied [25e29], the hIAPP which can control the glucose homeostasis together with insulin by amyloid aggregation and hIAPP fibrillation process still remain poorly understood. The kinetic profile of amyloid fibril formation is generally believed to be a nucleation-dependent polymerization process, which is characterized by a slow nucleation phase and a * Corresponding author. National Laboratory of Biomacromolecules, CAS Center subsequent rapid growth phase [30,31]. Molecular dynamic simu- for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of lation on Ab revealed that amyloid monomers aggregate into Sciences, Beijing, 100101, China. 1-40 E-mail address: [email protected] (P. Zhu). crystalline oligomers, and the monomers sequentially assemble https://doi.org/10.1016/j.bbrc.2020.08.088 0006-291X/© 2020 Elsevier Inc. All rights reserved. 126 X. Zhang et al. / Biochemical and Biophysical Research Communications 533 (2020) 125e131 onto the preformed oligomers to form protofibrils with b-sheet preparation, respectively. Three microliters of each fibril solution structures [32e34]. The aggregation kinetic studies of Ab or a- was dropped on a freshly glow-discharged holey carbon grid synuclein amyloid proteins in vitro suggested that the formation of (Quantifoil Cu R2/1, 300 mesh), blotted with filter paper for 3 s to amyloid fibrils is a multistage assembly process, which initially remove excess sample, and vitrified by plunging into liquid involves the soluble monomers oligomerize into a stable nucleus, nitrogen-cooled liquid ethane using Vitrobot Mark IV (Thermo and then the nucleus associates with either monomers or oligo- Fisher) at 4 C and with 100 % humidity. Grids were transferred to a mers aggregate to form oligomers, protofibrils and fibrils. Finally, Titan Krios (Thermo Fisher) operating at mature fibrils accumulate in plaques in the related organelles 300 kV. Micrographs were recorded using a Gatan-K2 Summit [25e27]. counting camera (Gatan Company) in super-resolution mode with a Protofibrils were thought the precursors of fibril assembly [35]. nominal magnification of 130,000 , resulting in a calibrated pixel For Ab, two kinds of protofibril growth modes, i.e., elongation by size of 1.04 Å. monomer deposition and protofibril-protofibril association, were For Cryo-electron tomography (Cryo-ET), 3 mLoffibril solution proposed based on multi-angle light scattering and atomic force incubated for 6 h with agitation was applied to a freshly glow- microscopy (AFM) analyses on Ab1-40 [36]. For hIAPP, negative discharged holey carbon grid (Quantifoil Cu R2/2, 200 mesh), staining EM analysis suggested that the hIAPP fibrils are derived blotted for 3 s, and then plunged into liquid ethane using a Vitrobot from annealing and winding of protofibrils, which were then Mark IV (Thermo Fisher). Cryo-electron tomograms were acquired spontaneously assemble into higher order fibril structures through with a Talos Arctica microscope (Thermo Fisher) operating at coiling or side-by-side assembly [37]. However, the air-drying 200 kV. Tilt-series were collected automatically from 54 to þ54 process and the 2D projection EM analysis hinder the observation with 3 increments at 53,000 of magnification (pixel of fibrillization process and the portofibril winding during fiber size ¼ 2.73 Å), using a defocus 3e5 mm and dose of 1.5 e /Å2 per formation in a relatively native environment and in three- image. Digital images were drift corrected and the cryo-electron dimensional space. tomograms were aligned by patch tracking with bin 5 in IMOD In this study, we used negative staining transmission electron [38]. The last round of reconstruction was done in ICON [39] which microscopy (NS-TEM), cryo-electron microscopy (cryo-EM), and uses a compressed sensing arithmetic. cryo-electron tomography (cryo-ET), together with circular di- To improve the volume’s signal-to-noise ratio (SNR) and to chroism (CD) and Thioflavin-T (ThT) assays, to explore the hIA- make the characteristics more visible, the volumes in the tomo- PP137 aggregation process and kinetics. We find that various fibril gram including the interested fibrils area were selected, segmented morphologies present in the whole stage of hIAPP137 aggregation, in eman 2 [40] with a tool of machine learning, and displayed in while the protofibril winding can be observed all over the time in Chimera [41] with different colors in the map. different growth states. These results suggest that the hIAPP137 fibrillation is a synchronous process in which the protofibril elon- 2.3. Circular dichroism (CD) spectroscopy gation and the mature fibril formation are performed simultaneously. CD spectra (average of three scans) were obtained using a Chira scan Plus spectropolarimeter and 1 mm path-length quartz cuvette. 2. Materials and methods Samples at a concentration of 100 mM hIAPP1-37 picked at different growth time points were measured at 1 nm intervals between wave 2.1. Synthesis and preparation of hIAPP1-37 lengths of 190 nm and 260 nm, respectively. Background values were subtracted from all collected spectra data. Human IAPP1-37 polypeptide (KCNTATCATQRLANFLVHSSNNF- GAILSSTNVGSNTYN-H2) was synthesized with purity of 95 % as 2.4. Thioflavin-T (ThT) fluorescence spectroscopy determined by high performance liquid chromatography (HPLC) and mass spectrometry. Lyophilized hIAPP1-37 was first dissolved in Each sample prepared for CD was also examined using ThT hexafluoroisopropanol (HFIP) to a stock concentration of 2 mM. fluorescence spectroscopy. Aliquots (20 mL) were pipetted into a Aliquots of polypeptide stock solutions were pipetted into 1.5 mL black 96-well plate (Thermo) and then mixed with 10 mM ThT centrifugal tubes, and HFIP was evaporated in the fume hood at (180 mL). The fluorescence signal was recorded with an excitation room temperature for 10 min. The peptide was then diluted in wavelength of 450 nm and an emission wavelength of 482 nm Milli-Q water to a concentration of 100 mM with ~2 % HFIP for fibril using a Perkin Elmer EnSight Multifunctional Microplate Reader. formation experiments. Samples were sonicated and incubated for Experiments were performed in triplicate and the average values designated time at 37 C in the presence and in the absence of were reported with the corresponding standard deviation. constant agitation (200 rpm) to form fibrils. 3. Results and discussion 2.2. Transmission electron microscopy (TEM) 3.1. Various fibril morphologies were detected in different stages of For negative-stain electron microscopy, 100 mM hIAPP1-37 hIAPP137 aggregation monomer solutions were incubated at 37 C both in the presence and in the absence of stirring. At the scheduled time, the solutions The formation of amyloid fibrils is generally thought a multi- were taken out and applied on freshly glow-discharged carbon- step process that involves converting the monomer into a b-sheet coated copper grids for 1min and the excess samples were removed conformation [25,30]. To reveal the fibril morphology and fibrilla- by blotting on filter paper. Grids were stained with 1 % (w/v) uranyl tion kinetics of the hIAPP1-37 aggregation process, we took samples acetate for 1 min and the excess uranyl acetate was blotted off. from different time points and detected them by negative-stain Grids were examined by a Tecnai Spirit transmission electron mi- transmission electron microscopy (NS-TEM), CD and ThT assays. croscope (Thermo Fisher) at an accelerating voltage of 120 kV. Since the hIAPP1-37 fibrillization process is much faster in the For cryo-electron microscopy (cryo-EM), 100 mM hIAPP1-37 so- presence of seed [30], the reactions in this work were not seeded. lutions that were incubated for 6 h in the presence of agitation or Negative staining micrographs showed that various amyloid fibril 24 h in the absence of agitation were selected for frozen sample structures can be observed in different stages both with and X. Zhang et al. / Biochemical and Biophysical Research Communications 533 (2020) 125e131 127 without stirring during the fibril growth process. Interestingly, CD investigation on the sample in the process of fibril growth noticeable long amyloid fibrils co-existed with protofibrils at the showed that typical random coil conformation (negative peak at very early time point, e.g., 5 min, while the protofibrils can be ~202 nm) appeared at the early sampling time point, i.e., 5 min with readily detected at the very late incubation time, e.g., 288 h (Fig. 1A agitation or 24 h without agitation, while it completely converted and B). This is in contrast with the previous report of Ab1-40 amyloid to b-sheet (negative peak at ~217 nm) after 9 h in the presence of fibril formation, which showed that fibrils became visible after 3 stirring or 192 h in the absence of stirring, respectively (Fig. 1C and days in the presence of seed, or after 15 days without the initial D). The ThT fluorescence spectrum also reflected that the b-sheet seeding process, while protofibrils were not observed in the late content of the sample reached the highest peak at 9 h in the stage [35], suggesting different fibrillation kinetics in the hIAPP1-37 presence of agitation, which is consistent with the CD results and Ab1-40 amyloid fibril formation processes. Previous FastScan (Fig. 1E). These results suggest that the fibrils start to appear in the atomic force microscopy study on hIAPP1-37 formation showed that very early stage of hIAPP137 aggregation growth and protofibrils the fibril elongation speed was not directly related to peptide remain visible at late time points. In addition, the mixture of syn- concentration [42]. These results implied that unlike the fibrilli- thesized hIAPP1-37 will eventually self-assemble to form a b-sheet zation process of Ab amyloid fibrils, the hIAPP1-37 protofibril ag- structure both with or without agitation, while the fibril aggrega- gregation and fibril elongation are performed simultaneously. Our tion process can be significantly accelerated by agitation, possibly

Fig. 1. Time-dependent aggregation of hIAPP1-37 in different growth states. (A and B) Negative staining EM results of hIAPP1-37 aggregates in the presence (A) and in the absence of agitation (B). Fibrils appear at the earliest sampling time point, i.e., 5 min with or without agitation, while protofibrils co-exist with fibrils at late time points, i.e., 21 h with or 288 h without agitation. The red arrows indicate long fibrils, and the blue arrows indicate protofibrils. Scale bar represents 200 nm. (C and D) CD spectra for hIAPP1-37 in the presence (C) and in the absence of agitation (D). The negative band at ~202 nm indicating of a random coil structure, and the negative band at ~217 nm indicating of a b-sheet backbone structure.

(E) ThT spectra for hIAPP1-37 in water exhibited S-shaped growth curve. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 128 X. Zhang et al. / Biochemical and Biophysical Research Communications 533 (2020) 125e131 because agitation accelerates the initial stage of the amyloid pro- supplemental material). By fitting the recently resolved hIAPP14-37 tein self-assembly process [43]. fibril structure [46] into our protofibril cryo-ET density map, we found that the wound fibril could accommodate two protofibrils (Fig. 3D). In contrast to Ab fibril, in which the winding was only 3.2. Cryo-EM and ET analysis revealed the winding of protofibrils in 1-40 observed at late stage of protofibril-to-fibril transition [35], the the fibril assembly process hIAPP fibrils were proposed to be derived from annealing and winding of protofibrils [37]. It is generally believed that mature Our negative staining EM images of hIAPP1-37 amyloid fibrils fibrils are the final assembly form after the amyloid aggregation showed that Y-shaped fibrils, which suggest winding of protofibrils, process which undergoes monomers, oligomers and protofibrils, can be observed at every sampling time point of amyloid fibril respectively [25]. Our 3-D cryo-ET analysis of hIAPP fibrillization growth periods (Fig. 2). To rule out the possibility that the observed 1-37 suggested that two or more intertwined protofibrils parallel to the protofibril windings were artefacts introduced during the negative fibril axis interact with each other to form a ribbon or twisted staining EM sample preparation, e.g., the staining solution used or mature fibril. the air-drying process, we used cryo-electron microscopy (cryo- Supplementary video related to this article can be found at EM), which can significantly preserve the native structures of https://doi.org/10.1016/j.bbrc.2020.08.088 proteins [44], to further study the hIAPP1-37 fibril structure and fibrillation process. Remarkably, the cryo-EM images showed that the protofibril winding was apparently detectable (Fig. 3A and B), 3.3. hIAPP1-37 amyloid aggregation is a synchronous process comparable to those in the negative staining EM. Interestingly, characterized by different widths and crossover lengths, the As shown in Figs. 1B and 2B, in the absence of agitation, proto- aggregated hIAPP1-37 amyloid fibrils formed by winding displayed fibrils, fibrils and protofibril winding can be visualized at as early as variable size and geometries, for example, the fibrils picked at the the time point of 5 min. Strikingly, the protofibrils and winding of time point of 6 h aggregated in the presence of agitation and those protofibrils can also be detected even after the time point of 288 h. picked at 24 h in the absence of agitation showed different pitches, These results suggested that various amyloid fibril morphologies indicating the variable polymorphism of hIAPP1-37 assembly pro- including protofibril winding can be observed all over the time in cess (Fig. 3A and B). the hIAPP1-37 fibril assembly process. Our results, combined with To rule out the possibility that the observed winding of proto- inferences from the previous experiments, suggest that the hIAPP1- fibrils, i.e., Y-shaped fibrils, in two-dimensional (2-D) EM obser- 37 amyloid aggregation is a synchronous process, which includes vation were the overlap illusions caused by different projection protofibril elongation, winding of protofibrils and mature fibril angles, we performed cryo-electron tomography (cryo-ET) analysis, formation at different time points (Fig. 4). Although various fibril which provides the most direct way to visualize the three- morphologies such as protofibrils, winding of protofibrils, fibrils dimensional (3-D) morphology of heterogeneous protein assem- and higher order fibrils formed by protofibrils winding can be bly samples [45], to further determinate the 3-D hIAPP1-37 amyloid observed at the different stage of hIAPP137 aggregation, it remains fibril structures in their native conditions. The 3-D reconstruction to be determined whether the higher order products are formed by results showed that two protofibrils intertwined with each other to protofibril winding or directly formed by protofibril elongation form a higher order fibril (Fig. 3C; see also Movie S1 in the (Fig. 4).

Fig. 2. Negative-staining EM examination of protofibril winding in the hIAPP1-37 fibrillation. Protofibril winding can be observed at every time point of hIAPP1-37 fibrillation in the presence (A) and in the absence of agitation (B). Two or more portofibrils intertwine to each other to form a larger width fibril. The red arrows indicate the wound fibrils. Scale bar represents 200 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) X. Zhang et al. / Biochemical and Biophysical Research Communications 533 (2020) 125e131 129

Fig. 3. hIAPP1-37 amyloid fibrils were examined by Cryo-EM and Cryo-ET. (A and B) Cryo-EM results of hIAPP1-37 amyloid fibrils at 6 h in the presence of agitation (A) and 24 h in the absence of agitation (B). The red arrows indicate the wound fibrils. (C) The winding of hIAPP1-37 fibril was analyzed by Cryo-ET. Protofibrils are colored by cyan (diameter ~6 nm, boxed as 1 and 2) and fibril is colored by red (diameter ~12 nm, boxed as 3), two protofibrils are intertwined (colored by yellow) to form a fibril. (D) Magnified views of different regions of Cryo-ET results corresponding to boxes in Fig. C. Previously resolved structure of hIAPP (EMDB: 21,410 [46]) are docked into Cryo-ET results. One model is fitted into protofibrils (panel 1 and 2), while two models are fitted into fibrils (panel 3). Scale bar represents 50 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4. Schematic diagram of the seeded hIAPP1-37 amyloid fibril growth. Various fibrils morphologies including protofibrils, winding of protofibrils and mature fibrils can be observed at different time points. 130 X. Zhang et al. / Biochemical and Biophysical Research Communications 533 (2020) 125e131

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