Biochemical and Biophysical Research Communications
Structural characterization and cryo-electron tomography analysis of human islet amyloid polypeptide suggest a synchronous process of the hIAPP1−37 amyloid fibrillation
Introduction
Amyloid aggregation is a common pathological feature among many unrelated diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Type 2 diabetes (T2D) and Mad cow disease [[1], [2], [3], [4]]. In these amyloid diseases, a particular protein, for example, Aβ for AD and α-synuclein for PD, was typically found to misfold and aggregate to form insoluble fibrils which are rich in β-sheet structures [5,6]. In type 2 diabetes, amyloid fibrils are formed by human Islet amyloid polypeptide (hIAPP) in the pancreatic islets of Langerhans [7,8]. Islet amyloid polypeptide (IAPP), also known as amylin, is a peptide hormone of 37 amino acid residues that is co-expressed and co-secreted with insulin by the pancreatic β-cell secretory granules [[9], [10], [11]]. As a member of the calcitonin family of hormones, IAPP has been found in all mammals studied to date, which can control the glucose homeostasis together with insulin by regulating glucose uptake and inhibiting insulin release and glucose elimination rate [10,[12], [13], [14]]. IAPP is normally soluble and naturally unfolded in monomeric state, but it was found to aggregate as islet amyloid in type 2 diabetes (T2D) and can be readily induced to form fibrils in vitro [8,10,12,15]. Islet amyloid formation can lead to islet β-cell dysfunction and death, and contribute to the failure of islet transplantation [10,[16], [17], [18], [19]]. The amyloid deposition of hIAPP presents in the pancreas in over 90 % of diabetic patients and the extent of amyloid deposition seems related to the severity of the disease [[20], [21], [22], [23], [24]]. Therefore, the hIAPP fibril formation plays an important role in the pathogenesis of T2D, and the study of the hIAPP amyloid aggregation and fibrillation kinetics will shed lights on the pathogenesis of T2D disease.
Although the aggregation mechanism of the Aβ or α-synuclein amyloid proteins had been extensively studied [[25], [26], [27], [28], [29]], the hIAPP 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 subsequent rapid growth phase [30,31]. Molecular dynamic simulation on Aβ1-40 revealed that amyloid monomers aggregate into crystalline oligomers, and the monomers sequentially assemble onto the preformed oligomers to form protofibrils with β-sheet structures [[32], [33], [34]]. The aggregation kinetic studies of Aβ or α-synuclein amyloid proteins in vitro suggested that the formation of amyloid fibrils is a multistage assembly process, which initially involves the soluble monomers oligomerize into a stable nucleus, and then the nucleus associates with either monomers or oligomers aggregate to form oligomers, protofibrils and fibrils. Finally, mature fibrils accumulate in plaques in the related organelles [[25], [26], [27]].
Protofibrils were thought the precursors of fibril assembly [35]. For Aβ, two kinds of protofibril growth modes, i.e., elongation by monomer deposition and protofibril-protofibril association, were proposed based on multi-angle light scattering and atomic force microscopy (AFM) analyses on Aβ1-40 [36]. For hIAPP, negative staining EM analysis suggested that the hIAPP fibrils are derived from annealing and winding of protofibrils, which were then spontaneously assemble into higher order fibril structures through coiling or side-by-side assembly [37]. However, the air-drying process and the 2D projection EM analysis hinder the observation of fibrillization process and the portofibril winding during fiber formation in a relatively native environment and in three-dimensional space.
In this study, we used negative staining transmission electron microscopy (NS-TEM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET), together with circular dichroism (CD) and Thioflavin-T (ThT) assays, to explore the hIAPP1−37 aggregation process and kinetics. We find that various fibril morphologies present in the whole stage of hIAPP1−37 aggregation, while the protofibril winding can be observed all over the time in different growth states. These results suggest that the hIAPP1−37 fibrillation is a synchronous process in which the protofibril elongation and the mature fibril formation are performed simultaneously.
Section snippets
Synthesis and preparation of hIAPP1-37
Human IAPP1-37 polypeptide (KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTYN-H2) was synthesized with purity of 95 % as determined by high performance liquid chromatography (HPLC) and mass spectrometry. Lyophilized hIAPP1-37 was first dissolved in hexafluoroisopropanol (HFIP) to a stock concentration of 2 mM. Aliquots of polypeptide stock solutions were pipetted into 1.5 mL centrifugal tubes, and HFIP was evaporated in the fume hood at room temperature for 10 min. The peptide was then diluted in Milli-Q
Various fibril morphologies were detected in different stages of hIAPP1−37 aggregation
The formation of amyloid fibrils is generally thought a multi-step process that involves converting the monomer into a β-sheet conformation [25,30]. To reveal the fibril morphology and fibrillation kinetics of the hIAPP1-37 aggregation process, we took samples from different time points and detected them by negative-stain transmission electron microscopy (NS-TEM), CD and ThT assays. Since the hIAPP1-37 fibrillization process is much faster in the presence of seed [30], the reactions in this
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This work was supported by grants from the Chinese Ministry of Science and Technology (2017YFA0504700), National Natural Science Foundation of China (31425007, 31730023), and the Strategic Priority Research Program from Chinese Academy of Sciences (XDB37010102). The cryo-EM data were collected at the Center for Biological Imaging (CBI), Institute of Biophysics, CAS.
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