Amylin Monomer Research Articles

Multiscale Modeling of Macromolecular Interactions between Tau-Amylin Oligomers and Asymmetric Lipid Nanodomains That Link Alzheimer's and Diabetic Diseases.

The molecular events of protein misfolding and self-aggregation of tau and amylin are associated with the progression of Alzheimer's and diabetes, respectively. Recent studies suggest that tau and amylin can form hetero-tau-amylin oligomers. Those hetero-oligomers are more neurotoxic than homo-tau oligomers. So far, the detailed interactions between the hetero-oligomers and the neuronal membrane are unknown. Using multiscale MD simulations, the lipid binding and protein folding behaviors of hetero-oligomers on asymmetric lipid nanodomains or raft membranes were examined. Our raft membranes contain phase-separated phosphatidylcholine (PC), cholesterol, and anionic phosphatidylserine (PS) or ganglioside (GM1) in one leaflet of the lipid bilayer. The hetero-oligomers bound more strongly to the PS and GM1 than other lipids via the hydrophobic and hydrophilic interactions, respectively, in the raft membranes. The hetero-tetramer disrupted the acyl chain orders of both PC and PS in the PS-containing raft membrane, but only the GM1 in the GM1-containing raft membrane as effectively as the homo-tau-tetramer. We discovered that the alpha-helical content in the heterodimer was greater than the sum of alpha-helical contents from isolated tau and amylin monomers on both raft membranes, indicative of a synergetic effect of tau-amylin interactions in surface-induced protein folding. Our results provide new molecular insights into understanding the cross-talk between Alzheimer's and diabetes.

Molecular Mechanisms of Protein–Lipid Interactions and Protein Folding of Heterogeneous Amylin and Tau Oligomers on Lipid Nanodomains That Link to Alzheimer’s

The disruption of cell membranes by tau and amylin oligomers is linked to amyloid diseases such as Alzheimer’s and diabetes, respectively. The recent studies suggest that misfolded tau and amylin can form neurotoxic hetero-oligomers that are structurally different from homo-oligomers. However, the molecular interactions of these hetero-oligomers with the neuronal membranes remain unclear. Using MD simulations, we have investigated the binding behaviors, membrane disruption, and protein folding of hetero-oligomers on a raft membrane containing phase-separated lipid nanodomains like those found in neurons. We discovered that the hetero-oligomers bind to the liquid-order and liquid-disorder phase boundaries of the raft membrane. The major lipid-binding sites of these interactions include the L16 and I26 residues of amylin and the N-terminal of tau. Strong disruptions of the raft domain size by the hetero-tetramer were detected. Furthermore, the hetero-dimer disrupted the saturated phospholipid orientational order to a greater extent than the individual tau or amylin monomer. In addition, the constituent tau more strongly promoted the alpha-helix to the beta-sheet transition of the constituent amylin within the hetero-dimer when compared with the amylin monomer alone. Our results provide new molecular insights into understanding the neurotoxicity of the hetero-oligomers associated with the cross-talk between amyloid diseases.

Human Amylin in the Presence of SARS-COV-2 Protein Fragments.

COVID-19 can lead to the onset of type-II diabetes, which is associated with the aggregation of islet amyloid polypeptides, also called amylin. Using molecular dynamics simulations, we investigate how the equilibrium between amylin monomers in its functional form and fibrils associated with diabetes is altered in the presence of SARS-COV-2 protein fragments. For this purpose, we study the interaction between the fragment SFYVYSRVK of the envelope protein or the fragment FKNIDGYFKI of the spike protein with the monomer and two amylin fibril models. Our results are compared with earlier work studying such interactions for the two different proteins.

Analysis of Baboon IAPP Provides Insight into Amyloidogenicity and Cytotoxicity of Human IAPP

The polypeptide hormone islet amyloid polypeptide (IAPP) forms islet amyloid in type 2 diabetes, a process which contributes to pancreatic β-cell dysfunction and death. Not all species form islet amyloid, and the ability to do so correlates with the primary sequence. Humans form islet amyloid, but baboon IAPP has not been studied. The baboon peptide differs from human IAPP at three positions containing K1I, H18R, and A25T substitutions. The K1I substitution is a rare example of a replacement in the N-terminal region of amylin. The effect of this mutation on amyloid formation has not been studied, but it reduces the net charge, and amyloid prediction programs suggest that it should increase amyloidogenicity. The A25T replacement involves a nonconservative substitution in a region of IAPP that is believed to be important for aggregation, but the effects of this replacement have not been examined. The H18R point mutant has been previously shown to reduce aggregation in vitro. Baboon amylin forms amyloid on the same timescale as human amylin in vitro and exhibits similar toxicity toward cultured β-cells. The K1I replacement in human amylin slightly reduces toxicity, whereas the A25T substitution accelerates amyloid formation and enhances toxicity. Photochemical cross-linking reveals that the baboon amylin, like human amylin, forms low-order oligomers in the lag phase of amyloid formation. Ion-mobility mass spectrometry reveals broadly similar gas phase collisional cross sections for human and baboon amylin monomers and dimers, with some differences in the arrival time distributions. Preamyloid oligomers formed by baboon amylin, but not baboon amylin fibers, are toxic to cultured β-cells. The toxicity of baboon oligomers and lack of significantly detectable toxicity with exogenously added amyloid fibers is consistent with the hypothesis that preamyloid oligomers are the most toxic species produced during IAPP amyloid formation.

Monomer-targeting affinity peptide inhibitors of amyloid with no self-fibrillation and low cytotoxicity.

We utilized solution-phase biopanning to obtain a de novo peptide (LA12) that specifically bound to the core region of the human amylin monomer. LA12 stabilized the random coil conformation of amylin to suppress aggregation in a dose-dependent manner with the highest suppression effect of 78% and reduced the cytotoxicity of amylin.

Interplay Between Membrane Composition and Structural Stability of Membrane-Bound hIAPP.

Amyloid aggregates are characteristic of many serious diseases such as Alzheimer's disease, Parkinson's, and type 2 diabetes and commonly involve intrinsically disordered proteins (IDPs), those that populate an ensemble of conformations rather than a single folded structure. Human islet amyloid polypeptide (hIAPP or amylin) is an amyloidogenic IDP implicated in pancreatic β-cell death during the pathogenesis of type 2 diabetes. The target of amylin's toxic activity is thought to be the cell's lipid membrane, which may also act as a catalyst for aggregation. Since amylin is intrinsically disordered, differing environments can have a large impact on its equilibrium conformational ensemble. We apply atomistic molecular dynamics simulations on multiple systems containing a full-length amylin monomer and a lipid bilayer to study the changes induced by the membrane. We observe stabilized helical conformations structurally similar to those determined by NMR experiments conducted in similar environments. We also find that bilayers of different compositions result in greatly different equilibrium ensembles of amylin. Finally, we discuss how a mixed bilayer containing zwitterionic and anionic lipid headgroups can allow for greater preference toward conformations which are adsorbed below the membrane surface through rearrangement of lipids for more favorable protein-lipid interactions.

Insight into a New Binding Site of Zinc Ions in Fibrillar Amylin.

Amylin peptides are secreted together with insulin and zinc ions from pancreatic β-cells. Under unknown conditions, the amylin peptides aggregate to produce oligomers and fibrils, and in some cases Zn2+ ions can bind to amylin peptides to form Zn2+-aggregate complexes. Consequently, these aggregates lead to the death of the β-cells and a decrease in insulin, which is one of the symptoms of type-2 diabetes (T2D). Therefore, it is crucial to investigate the binding sites of the Zn2+ ions in fibrillary amylin. It was previously found by in vitro and simulation studies that Zn2+ ion binds to two or four His residues in the turn domain of fibrillary amylin. In the current study, we present a new Zn2+ binding site in the N-terminus of fibrillary amylin with three different coordination modes. Our simulations showed that Zn2+ ions bind to polymorphic amylin fibrils with a preference to bind to four Cys residues rather than two Cys residues of two neighboring amylin monomers. The new binding site leads to conformational changes, increases the number of polymorphic states, and demonstrates the existence of competition between various binding sites. Our study provides insight into the molecular mechanisms through which Zn2+ ions that play a critical role in amylin aggregation can bind to amylin and promote amylin aggregation in T2D.

Amyloid Aggregation of Amylin: Gain of Function along Aggregation Pathway?

Abstract: Aggregation of human amylin, a 37 amino acid residue neuropeptide of pancreatic origin, into amyloid aggregates is implicated in the etiology of diabetes mellitus type II. Despite its clinical significance, details of progression of nontoxic amylin monomers into cytotoxic oligomers are not very clear. Studies based on Amyloid-β (Aβ), a peptide with similar size, have suggested that a conformational transition along the aggregation pathway makes the oligomers of Aβ membrane-binding competent (1). We have explored the possibility of amylin undergoing a similar transition during aggregation as it could shed light on potential commonalities in the conformation and function of different toxic amyloid species. Using fluorescence correlation spectroscopy, we identify two distinct oligomers of amylin along the aggregation pathway having hydrodynamic radius of 0.90 nm and 1.6 nm respectively. The membrane affinity of amylin increases remarkably from ∼15 % in smaller species to ∼ 85% in the larger one as assessed by an in vitro membrane-binding assay developed in our lab (2). We observe similar difference in the cell membrane attachment ability of these two species in RIN5mf cell lines using confocal microscopy. A preliminary conformational study in artificial lipid bilayers using a SERS based methodology (3) suggests a temporal conformational reorganization in the peptide backbone. Our data suggest that amylin might acquire toxic function by a mechanism which depends on similar conformational features as Aβ in presence of membranes. Further studies aimed at obtaining high-resolution structural details of amylin oligomers in solution and in membranes are currently in progress.

Rapid assessment of human amylin aggregation and its inhibition by copper(II) ions by laser ablation electrospray ionization mass spectrometry with ion mobility separation.

Native electrospray ionization (ESI) mass spectrometry (MS) is often used to monitor noncovalent complex formation between peptides and ligands. The relatively low throughput of this technique, however, is not compatible with extensive screening. Laser ablation electrospray ionization (LAESI) MS combined with ion mobility separation (IMS) can analyze complex formation and provide conformation information within a matter of seconds. Islet amyloid polypeptide (IAPP) or amylin, a 37-amino acid residue peptide, is produced in pancreatic beta-cells through proteolytic cleavage of its prohormone. Both amylin and its precursor can aggregate and produce toxic oligomers and fibrils leading to cell death in the pancreas that can eventually contribute to the development of type 2 diabetes mellitus. The inhibitory effect of the copper(II) ion on amylin aggregation has been recently discovered, but details of the interaction remain unknown. Finding other more physiologically tolerated approaches requires large scale screening of potential inhibitors. Here, we demonstrate that LAESI-IMS-MS can reveal the binding stoichiometry, copper oxidation state, and the dissociation constant of human amylin-copper(II) complex. The conformations of hIAPP in the presence of copper(II) ions were also analyzed by IMS, and preferential association between the β-hairpin amylin monomer and the metal ion was found. The copper(II) ion exhibited strong association with the -HSSNN- residues of the amylin. In the absence of copper(II), amylin dimers were detected with collision cross sections consistent with monomers of β-hairpin conformation. When copper(II) was present in the solution, no dimers were detected. Thus, the copper(II) ions disrupt the association pathway to the formation of β-sheet rich amylin fibrils. Using LAESI-IMS-MS for the assessment of amylin-copper(II) interactions demonstrates the utility of this technique for the high-throughput screening of potential inhibitors of amylin oligomerization and fibril formation. More generally, this rapid technique opens the door for high-throughput screening of potential inhibitors of amyloid protein aggregation.

Free energy simulations of amylin I26P mutation in a lipid bilayer.

The amylin peptide in a dioleoylphosphatidylcholine (DOPC) bilayer is studied using united atom molecular dynamics (MD) simulations. Dynamics and transport properties of the peptide and the phospholipid bilayer are investigated. The lateral diffusion of DOPC is in the order of 10(-8) cm(2) s(-1), which is in agreement with the experimental results. The order parameter and density profile for phospholipid molecules in the bilayer are calculated. The secondary structure of amylin peptide shows that the amino acids in two terminals are structureless and two α-helical segments in the peptide are connected through an unstructured link. This structure is similar to the experimental structure in the membrane-mimicking media. Free energy calculations of the Ile26→Pro mutation in the amylin peptide are performed in the bilayer and in aqueous solution using molecular dynamics simulations and a thermodynamic cycle. It is shown that in the mutated peptide in aqueous solution, the α-helix structure changes to a 5-helix, whereas this configuration is preserved in the bilayer environment. It is interesting that the accessible surface area increases for hydrophobic residues in the bilayer and for hydrophilic residues in aqueous solution as the coupling parameter changes from 0 to 1. These results are significant to understanding the aggregation mechanism of human amylin monomers in membranes to the dimers, trimers, oligomers, and fibrils associated with the type 2 diabetes at the atomic level.

Distinct Internalization Pathways of Human Amylin Monomers and Its Cytotoxic Oligomers in Pancreatic Cells

Toxic human amylin oligomers and aggregates are implicated in the pathogenesis of type 2 diabetes mellitus (TTDM). Although recent studies have shown that pancreatic cells can recycle amylin monomers and toxic oligomers, the exact uptake mechanism and trafficking routes of these molecular forms and their significance for amylin toxicity are yet to be determined. Using pancreatic rat insulinoma (RIN-m5F) beta (β)-cells and human islets as model systems we show that monomers and oligomers cross the plasma membrane (PM) through both endocytotic and non-endocytotic (translocation) mechanisms, the predominance of which is dependent on amylin concentrations and incubation times. At low (≤100 nM) concentrations, internalization of amylin monomers in pancreatic cells is completely blocked by the selective amylin-receptor (AM-R) antagonist, AC-187, indicating an AM-R dependent mechanism. In contrast at cytotoxic (µM) concentrations monomers initially (1 hour) enter pancreatic cells by two distinct mechanisms: translocation and macropinocytosis. However, during the late stage (24 hours) monomers internalize by a clathrin-dependent but AM-R and macropinocytotic independent pathway. Like monomers a small fraction of the oligomers initially enter cells by a non-endocytotic mechanism. In contrast a majority of the oligomers at both early (1 hour) and late times (24 hours) traffic with a fluid-phase marker, dextran, to the same endocytotic compartments, the uptake of which is blocked by potent macropinocytotic inhibitors. This led to a significant increase in extra-cellular PM accumulation, in turn potentiating amylin toxicity in pancreatic cells. Our studies suggest that macropinocytosis is a major but not the only clearance mechanism for both amylin’s molecular forms, thereby serving a cyto-protective role in these cells.

α-helix to β-hairpin transition of human amylin monomer.

The human islet amylin polypeptide is produced along with insulin by pancreatic islets. Under some circumstances, amylin can aggregate to form amyloid fibrils, whose presence in pancreatic cells is a common pathological feature of Type II diabetes. A growing body of evidence indicates that small, early stage aggregates of amylin are cytotoxic. A better understanding of the early stages of the amylin aggregation process and, in particular, of the nucleation events leading to fibril growth could help identify therapeutic strategies. Recent studies have shown that, in dilute solution, human amylin can adopt an α-helical conformation, a β-hairpin conformation, or an unstructured coil conformation. While such states have comparable free energies, the β-hairpin state exhibits a large propensity towards aggregation. In this work, we present a detailed computational analysis of the folding pathways that arise between the various conformational states of human amylin in water. A free energy surface for amylin in explicit water is first constructed by resorting to advanced sampling techniques. Extensive transition path sampling simulations are then employed to identify the preferred folding mechanisms between distinct minima on that surface. Our results reveal that the α-helical conformer of amylin undergoes a transformation into the β-hairpin monomer through one of two mechanisms. In the first, misfolding begins through formation of specific contacts near the turn region, and proceeds via a zipping mechanism. In the second, misfolding occurs through an unstructured coil intermediate. The transition states for these processes are identified. Taken together, the findings presented in this work suggest that the inter-conversion of amylin between an α-helix and a β-hairpin is an activated process and could constitute the nucleation event for fibril growth.

Amide Proton Solvent Protection in Amylin Fibrils Probed by Quenched Hydrogen Exchange NMR

Amylin is an endocrine hormone that accumulates in amyloid plaques in patients with advanced type 2 diabetes. The amyloid plaques have been implicated in the destruction of pancreatic β-cells, which synthesize amylin and insulin. To better characterize the secondary structure of amylin in amyloid fibrils we assigned the NMR spectrum of the unfolded state in 95% DMSO and used a quenched hydrogen-deuterium exchange technique to look at amide proton solvent protection in the fibrils. In this technique, partially exchanged fibrils are dissolved in 95% DMSO and information about amide proton occupancy in the fibrils is determined from DMSO-denatured monomers. Hydrogen exchange lifetimes at pH 7.6 and 37°C vary between ∼5 h for the unstructured N-terminus to 600 h for amide protons in the two β-strands that form inter-molecular hydrogen bonds between amylin monomers along the length of the fibril. Based on the protection data we conclude that residues A8-H18 and I26-Y37 comprise the two β-strands in amylin fibrils. There is variation in protection within the β-strands, particularly for strand β1 where only residues F15-H18 are strongly protected. Differences in protection appear to be due to restrictions on backbone dynamics imposed by the packing of two-layers of C2-symmetry-related β-hairpins in the protofilament structure, with strand β1 positioned on the surface and β2 in the interior.

Conformational dynamics of human IAPP monomers

We study the conformational dynamics of the human Islet Amyloid Polypeptide (hIAPP) molecule – a 37 residue-long peptide associated to type 2 diabetes – using molecular dynamics (MD) simulations. We identify partially structured conformational states of the hIAPP monomer, categorized by both end-to-end distance and secondary structure, as suggested by previous experimental and computational studies. The MD trajectories of hIAPP are analyzed using data-driven methods, in particular principal component analysis, in order to identify preferred conformational states of the amylin monomer and to discuss their relative stability as compared to corresponding states in the amylin dimer. These potential hIAPP conformational states could be further tested and described experimentally, or in conjunction with modern computational analysis tools such as Markov state-based methods for extracting kinetics and thermodynamics from atomistic MD trajectories.

Clustering and Internalization of Toxic Amylin Oligomers in Pancreatic Cells Require Plasma Membrane Cholesterol

Self-assembly of the human pancreatic hormone amylin into toxic oligomers and aggregates is linked to dysfunction of islet β-cells and pathogenesis of type 2 diabetes mellitus. Recent evidence suggests that cholesterol, an essential component of eukaryotic cells membranes, controls amylin aggregation on model membranes. However, the pathophysiological consequence of cholesterol-regulated amylin polymerization on membranes and biochemical mechanisms that protect β-cells from amylin toxicity are poorly understood. Here, we report that plasma membrane (PM) cholesterol plays a key role in molecular recognition, sorting, and internalization of toxic amylin oligomers but not monomers in pancreatic rat insulinoma and human islet cells. Depletion of PM cholesterol or the disruption of the cytoskeleton network inhibits internalization of amylin oligomers, which in turn enhances extracellular oligomer accumulation and potentiates amylin toxicity. Confocal microscopy reveals an increased nucleation of amylin oligomers across the plasma membrane in cholesterol-depleted cells, with a 2-fold increase in cell surface coverage and a 3-fold increase in their number on the PM. Biochemical studies confirm accumulation of amylin oligomers in the medium after depletion of PM cholesterol. Replenishment of PM cholesterol from intracellular cholesterol stores or by the addition of water-soluble cholesterol restores amylin oligomer clustering at the PM and internalization, which consequently diminishes cell surface coverage and toxicity of amylin oligomers. In contrast to oligomers, amylin monomers followed clathrin-dependent endocytosis, which is not sensitive to cholesterol depletion. Our studies identify an actin-mediated and cholesterol-dependent mechanism for selective uptake and clearance of amylin oligomers, impairment of which greatly potentiates amylin toxicity.

Stable and Metastable States of Human Amylin in Solution

Patients with type II diabetes exhibit fibrillar deposits of human amylin protein in the pancreas. It has been proposed that amylin oligomers arising along the aggregation or fibril-formation pathways are important in the genesis of the disease. In a step toward understanding these aggregation pathways, in this work we report the conformational preferences of human amylin monomer in solution using molecular simulations and infrared experiments. In particular, we identify a stable conformer that could play a key role in aggregation. We find that amylin adopts three stable conformations: one with an α-helical segment comprising residues 9–17 and a short antiparallel β-sheet comprising residues 24–28 and 31–35; one with an extended antiparallel β-hairpin with the turn region comprising residues 20–23; and one with no particular structure. Using detailed calculations, we determine the relative stability of these various conformations, finding that the β-hairpin conformation is the most stable, followed by the α-helical conformation, and then the unstructured coil. To test our predicted structure, we calculate its infrared spectrum in the amide I stretch regime, which is sensitive to secondary structure through vibrational couplings and linewidths, and compare it to experiment. We find that theoretically predicted spectra are in good agreement with the experimental line shapes presented herein. The implications of the monomer secondary structures on its aggregation pathway and on its interaction with cell membranes are discussed.

Evidence for a Partially Structured State of the Amylin Monomer

Islet amyloid polypeptide (amylin) is the main component in amyloid deposits formed in type II diabetes. We used triplet quenching to probe the dynamics of contact formation between the N-terminal disulfide loop and a C-terminal tryptophan in monomeric amylins from human and rat. Quenching rates measured in the absence of denaturant are four times larger than those in 6 M guanidinium chloride, indicating a decrease in the average end-to-end distance (collapse) at low denaturant concentrations. We were surprised to find an even greater (sevenfold) increase in quenching rates on removal of denaturant for a hydrophilic control peptide containing the disulfide loop compared to the same peptide without the loop (twofold change). These results suggest that collapse is driven by backbone-backbone and backbone-side chain interactions involving the disulfide loop portion of the chain rather than by the formation of side-chain hydrophobic contacts. Molecular dynamics simulations of the control peptide show that the collapse results from hydrogen-bonding interactions between the central residues of the chain and the disulfide loop. The quenching experiments also indicate that the monomer of the human, amyloidogenic form of amylin is more compact than the rat form, which does not form amyloid. We discuss these newly observed differences between human and rat amylin in solution and their possible relation to aggregation and to the physiological function of amylin binding to the calcitonin receptor.

Insulin is a kinetic but not a thermodynamic inhibitor of amylin aggregation

One of the most important pathological features of type 2 diabetes is the formation of islet amyloid, of which the major component is amylin peptide. However, the presence of a natural inhibitor such as insulin may keep amylin stable and physiologically functional in healthy individuals. Some previous studies demonstrated that insulin was a potent inhibitor of amylin fibril formation in vitro, but others obtained contradictory results. Hence, it is necessary to elucidate the effects of insulin on amylin aggregation. Here we report that insulin is a kinetic inhibitor of amylin aggregation, only keeping its inhibitory effect for a limited time period. Actually, insulin promotes amylin aggregation after long-term incubation. Furthermore, we found that this promotional effect could be attributed to the copolymerization of insulin and amylin. We also found that insulin copolymerized with amylin monomer or oligomer rather than preformed amylin fibrils. These results suggest that the interaction between insulin and amylin may contribute not only to the inhibition of amylin aggregation but also to the coaggregation of both peptides in type 2 diabetes.