Molecular Dynamics Simulations of Ibuprofen Binding to Aβ Peptides

Using replica exchange molecular dynamics simulations and the implicit solvent model we probed binding of ibuprofen to Aβ_10-40 monomers and amyloid fibrils. We found that the concave (CV) fibril edge has significantly higher binding affinity for ibuprofen than the convex edge. Furthermore, binding of ibuprofen to Aβ monomers, as compared to fibrils, results in a smaller free energy gain. The difference in binding free energies is likely to be related to the presence of the groove on the CV fibril edge, in which ibuprofen tends to accumulate. The confinement effect of the groove promotes the formation of large low-energy ibuprofen clusters, which rarely occur on the surface of Aβ monomers. These observations led us to suggest that the ibuprofen binding mechanism for Aβ fibrils is different from that for monomers. In general, ibuprofen shows a preference to bind to those regions of Aβ monomers (amino terminal) and fibrils (the CV edge) that are also the primary aggregation interfaces. Based on our findings and on available experimental data, we propose a rationale for the ibuprofen antiaggregation effect.     

Comparison of neurotoxicity of different aggregated forms of Aβ40, Aβ42 and Aβ43 in cell cultures

The abnormal deposition of amyloid‐β (Aβ) peptides in the brain is the main neuropathological hallmark of Alzheimer's disease (AD). Amyloid deposits are formed by a heterogeneous mixture of Aβ peptides, among which the most studied are Aβ40 and Aβ42. Aβ40 is abundantly produced in the human brain, but the level of Aβ42 is remarkably increased in the brain of AD patients. Aside from Aβ40 and Aβ42, recent data have raised the possibility that Aβ43 peptides may be instrumental in AD pathogenesis. Besides its length, whether the Aβ aggregated form accounts for the neurotoxicity is also particularly controversial. Aβ fibrils are generally considered as key pathogenic substances in AD pathogenesis. Nevertheless, recent data implicated soluble Aβ oligomers as the main cause of synaptic dysfunction and memory loss in AD. To further address this uncertainty, we analyzed the neurotoxicity of different Aβ species and Aβ forms at the cellular level. The results showed that Aβ42 could form oligomers significantly faster than Aβ40 and Aβ43 and Aβ42 oligomers showed the greatest level of neurotoxicity. Regardless of the length of Aβ peptides, Aβ oligomers induced significantly higher cytotoxicity compared with the other two Aβ forms. Surprisingly, the neurotoxicity of fibrils in PC12 cells was only marginally but not significantly stronger than monomers, contrary to previous reports. Altogether, our findings demonstrate the high pathogenicity of Aβ42 among the three Aβ species and support the idea that Aβ42 oligomers contribute to the pathological events leading to neurodegeneration in AD. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Antimicrobial protegrin-1 forms amyloid-like fibrils with rapid kinetics suggesting a functional link

Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich β-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a potent antibiotic agent. Earlier we reported that its cytotoxicity is mediated by its channel-forming ability. In this study, we have examined the amyloidogenic fibril formation properties of PG-1 in comparison with a well-defined amyloid, the amyloid-β (Aβ_1–42) peptide. We have used atomic force microscopy (AFM) and thioflavin-T staining to investigate the kinetics of PG-1 fibrils growth and molecular dynamics simulations to elucidate the underlying mechanism. AFM images of PG-1 on a highly hydrophilic surface (mica) show fibrils with morphological similarities to Aβ_1–42 fibrils. Real-time AFM imaging of fibril growth suggests that PG-1 fibril growth follows a relatively fast kinetics compared to the Aβ_1–42 fibrils. The AFM results are in close agreement with results from thioflavin-T staining data. Furthermore, the results indicate that PG-1 forms fibrils in solution. Significantly, in contrast, we do not detect fibrillar structures of PG-1 on an anionic lipid bilayer 2-dioleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; only small PG-1 oligomers can be observed. Molecular dynamics simulations are able to identify the presence of these small oligomers on the membrane bilayer. Thus, our current results show that cytotoxic AMP PG-1 is amyloidogenic and capable of forming fibrils. Overall, comparing β-rich AMPs and amyloids such as Aβ, in addition to cytotoxicity and amyloidogenicity, they share a common structural motif, and are channel forming. These combined properties support a functional relationship between amyloidogenic peptides and β-sheet-rich cytolytic AMPs, suggesting that amyloids channels may have an antimicrobial function.     

Sedimentation studies on human amylin fail to detect low-molecular-weight oligomers

Sedimentation velocity experiments show that only monomers coexist with amyloid fibrils of human islet amyloid-polypeptide. No oligomers containing <100 monomers could be detected, suggesting that the putative toxic oligomers are much larger than those found for the Alzheimer's peptide, Aβ(1-42).     

Naproxen Interferes with the Assembly of Aβ Oligomers Implicated in Alzheimer's Disease

Experimental and epidemiological studies have shown that the nonsteroidal antiinflammatory drug naproxen may be useful in the treatment of Alzheimer's disease. To investigate the interactions of naproxen with Aβ dimers, which are the smallest cytotoxic aggregated Aβ peptide species, we use united atom implicit solvent model and exhaustive replica exchange molecular dynamics. We show that naproxen ligands bind to Aβ dimer and penetrate its volume interfering with the interpeptide interactions. As a result naproxen induces a destabilizing effect on Aβ dimer. By comparing the free-energy landscapes of naproxen interactions with Aβ dimers and fibrils, we conclude that this ligand has stronger antiaggregation potential against Aβ fibrils rather than against dimers. The analysis of naproxen binding energetics shows that the location of ligand binding sites in Aβ dimer is dictated by the Aβ amino acid sequence. Comparison of the in silico findings with experimental observations reveals potential limitations of naproxen as an effective therapeutic agent in the treatment of Alzheimer's disease.     

Binding of the molecular chaperone αB-crystallin to Aβ amyloid fibrils inhibits fibril elongation

The molecular chaperone αB-crystallin is a small heat-shock protein that is upregulated in response to a multitude of stress stimuli, and is found colocalized with Aβ amyloid fibrils in the extracellular plaques that are characteristic of Alzheimer's disease. We investigated whether this archetypical small heat-shock protein has the ability to interact with Aβ fibrils in vitro. We find that αB-crystallin binds to wild-type Aβ₄₂ fibrils with micromolar affinity, and also binds to fibrils formed from the E22G Arctic mutation of Aβ₄₂. Immunoelectron microscopy confirms that binding occurs along the entire length and ends of the fibrils. Investigations into the effect of αB-crystallin on the seeded growth of Aβ fibrils, both in solution and on the surface of a quartz crystal microbalance biosensor, reveal that the binding of αB-crystallin to seed fibrils strongly inhibits their elongation. Because the lag phase in sigmoidal fibril assembly kinetics is dominated by elongation and fragmentation rates, the chaperone mechanism identified here represents a highly effective means to inhibit fibril proliferation. Together with previous observations of αB-crystallin interaction with α-synuclein and insulin fibrils, the results suggest that this mechanism is a generic means of providing molecular chaperone protection against amyloid fibril formation.     

Polymorphism of Alzheimer's Aβ17-42 (p3) Oligomers: The Importance of the Turn Location and Its Conformation

Aβ_17-42 (so-called p3) amyloid is detected in vivo in the brains of individuals with Alzheimer's disease or Down's syndrome. We investigated the polymorphism of Aβ_17-42 oligomers based on experimental data from steady-state NMR measurements, electron microscopy, two-dimensional hydrogen exchange, and mutational studies, using all-atom molecular-dynamics simulation with explicit solvent. We assessed the structural stability and the populations. Our results suggest that conformational differences in the U-turn of Aβ_17-42 lead to polymorphism in β-sheet registration and retention of an ordered β-strand organization at the termini. Further, although the parallel Aβ_17-42 oligomer organization is the most stable of the conformers investigated here, different antiparallel Aβ_17-42 organizations are also stable and compete with the parallel architectures, presenting a polymorphic population. In this study we propose that 1), the U-turn conformation is the primary factor leading to polymorphism in the assembly of Aβ_17-42 oligomers, and is also coupled to oligomer growth; and 2), both parallel Aβ_17-42 oligomers and an assembly of Aβ_17-42 oligomers that includes both parallel and antiparallel organizations contribute to amyloid fibril formation. Finally, since a U-turn motif generally appears in amyloids formed by full proteins or long fragments, and since to date these have been shown to exist only in parallel architectures, our results apply to a broad range of oligomers and fibrils.     

Stoichiometry and Affinity of the Human Serum Albumin-Alzheimer's Aβ Peptide Interactions

A promising strategy to control the aggregation of the Alzheimer's Aβ peptide in the brain is the clearance of Aβ from the central nervous system into the peripheral blood plasma. Among plasma proteins, human serum albumin plays a critical role in the Aβ clearance to the peripheral sink by binding to Aβ oligomers and preventing further growth into fibrils. However, the stoichiometry and the affinities of the albumin-Aβ oligomer interactions are still to be fully characterized. For this purpose, here we investigate the Aβ oligomer-albumin complexes through a novel and generally applicable experimental strategy combining saturation transfer and off-resonance relaxation NMR experiments with ultrafiltration, domain deletions, and dynamic light scattering. Our results show that the Aβ oligomers are recognized by albumin through sites that are evenly partitioned across the three albumin domains and that bind the Aβ oligomers with similar dissociation constants in the 1–100 nM range, as assessed based on a Scatchard-like model of the albumin inhibition isotherms. Our data not only explain why albumin is able to inhibit amyloid formation at physiological nM Aβ concentrations, but are also consistent with the presence of a single high affinity albumin-binding site per Aβ protofibril, which avoids the formation of extended insoluble aggregates.     

Association Thermodynamics and Conformational Stability of β-Sheet Amyloid β(17-42) Oligomers: Effects of E22Q (Dutch) Mutation and Charge Neutralization

Amyloid fibrils are associated with many neurodegenerative diseases. It was found that amyloidogenic oligomers, not mature fibrils, are neurotoxic agents related to these diseases. Molecular mechanisms of infectivity, pathways of aggregation, and molecular structure of these oligomers remain elusive. Here, we use all-atom molecular dynamics, molecular mechanics combined with solvation analysis by statistical-mechanical, three-dimensional molecular theory of solvation (also known as 3D-RISM-KH) in a new MM-3D-RISM-KH method to study conformational stability, and association thermodynamics of small wild-type Aβ_17-42 oligomers with different protonation states of Glu²², as well the E22Q (Dutch) mutants. The association free energy of small β-sheet oligomers shows near-linear trend with the dimers being thermodynamically more stable relative to the larger constructs. The linear (within statistical uncertainty) dependence of the association free energy on complex size is a consequence of the unilateral stacking of monomers in the β-sheet oligomers. The charge reduction of the wild-type Aβ_17-42 oligomers upon protonation of the solvent-exposed Glu²² at acidic conditions results in lowering the association free energy compared to the wild-type oligomers at neutral pH and the E22Q mutants. The neutralization of the peptides because of the E22Q mutation only marginally affects the association free energy, with the reduction of the direct electrostatic interactions mostly compensated by the unfavorable electrostatic solvation effects. For the wild-type oligomers at acidic conditions such compensation is not complete, and the electrostatic interactions, along with the gas-phase nonpolar energetic and the overall entropic effects, contribute to the lowering of the association free energy. The differences in the association thermodynamics between the wild-type Aβ_17-42 oligomers at neutral pH and the Dutch mutants, on the one hand, and the Aβ_17-42 oligomers with protonated Glu²², on the other, may be explained by destabilization of the inter- and intrapeptide salt bridges between Asp²³ and Lys²⁸. Peculiarities in the conformational stability and the association thermodynamics for the different models of the Aβ_17-42 oligomers are rationalized based on the analysis of the local physical interactions and the microscopic solvation structure.     

Structural Differences between Aβ(1-40) Intermediate Oligomers and Fibrils Elucidated by Proteolytic Fragmentation and Hydrogen/Deuterium Exchange

The aggregation of amyloid-β protein (Aβ) in vivo is a critical pathological event in Alzheimer's disease. Although more and more evidence shows that the intermediate oligomers are the primary neurotoxic species in Alzheimer's disease, the particular structural features responsible for the toxicity of these intermediates are poorly understood. We measured the peptide level solvent accessibility of multiple Aβ(1-40) aggregated states using hydrogen exchange detected by mass spectrometry. A gradual reduction in solvent accessibility, spreading from the C-terminal region to the N-terminal region was observed with ever more aggregated states of Aβ peptide. The observed hydrogen exchange protection begins with reporter peptides 20-34 and 35-40 in low molecular weight oligomers found in fresh samples and culminates with increasing solvent protection of reporter peptide 1-16 in long time aged fibrillar species. The more solvent exposed structure of intermediate oligomers in the N-termini relative to well-developed fibrils provides a novel explanation for the structure-dependent neurotoxicity of soluble oligomers reported previously.     

Size distribution of amyloid nanofibrils

We consider the size distribution of amyloid nanofibrils (protofilaments) in nucleating protein solutions when the nucleation process occurs by the mechanism of direct polymerization of β-strands (extended peptides or protein segments) into β-sheets. Employing the atomistic nucleation theory, we derive a general expression for the stationary size distribution of amyloid nanofibrils constituted of successively layered β-sheets. The application of this expression to amyloid β_1-40 (Aβ₄₀) fibrils allows us to determine the nanofibril size distribution as a function of the protein concentration and temperature. The distribution is most remarkable with its exhibiting a series of peaks positioned at “magic” nanofibril sizes (or lengths), which are due to deep local minima in the work for fibril formation. This finding of magic sizes or lengths is consistent with experimental results for the size distribution of aggregates in solutions of Aβ₄₀ proteins. Also, our approach makes it possible to gain insight into the effect of point mutations on the nanofibril size distribution, an effect that may play a role in experimentally observed substantial differences in the fibrillation lag-time of wild-type and point-mutated amyloid-β proteins.     

Structural Determination of Aβ25-35 Micelles by Molecular Dynamics Simulations

Amyloid-β (Aβ) peptides and other amyloidogenic proteins can form a wide range of soluble oligomers of varied morphologies at the early aggregation stage, and some of these oligomers are biologically relevant to the pathogenesis of Alzheimer's disease. Spherical micelle-like oligomers have been often observed for many different types of amyloids. Here, we report a hybrid computational approach to systematically construct, search, optimize, and rank soluble micelle-like Aβ_25-35 structures with different side-chain packings at the atomic level. Simulations reveal for the first time, to our knowledge, that two Aβ micelles with antiparallel peptide organization and distinct surface hydrophobicity display high structural stability. Stable micelles experience a slow secondary structural transition from turn to α-helix. Energetic analysis coupled with computational mutagenesis reveals that van der Waals and solvation energies play a more pronounced role in stabilizing the micelles, whereas the electrostatic energies present a stable but minor energetic contribution to peptide assemblies. Modeled Aβ micelles with shapes and dimensions similar to those of experimentally derived spherical structures also provide detailed information about the roles of structural dynamics and transition in the formation of amyloid fibrils. The strong binding affinity of our micelles to antibodies implies that micelles may be a biologically relevant species.     

pH-Dependent Binding of Synthetic β-Amyloid Peptides to Glycosaminoglycans

The seinile plaques found within the cerebral cortex and hippocampus of the Alzheimer disease brain contain β-amyloid peptide (Aβ) fibrils that are associated with a variety of macromolecular species, including dermatan sulfate proteoglycan and heparan sulfate proteoglycan. The latter has been shown recently to bind tightly to both amyloid precursor protein and A/β, and this binding has been attributed largely to the interaction of the core protein of heparan sulfate proteoglycan with Aβ and its precursor. Here we have examined the ability of synthetic Aβ s to bind to and interact with the glycosaminoglycan moieties of proteoglycans. Aβ(1–28) associates with heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate. The interaction of these sulfated polysaccharides with the amyloid peptide results in the formation of large aggregates that are readily sedimented by centrifugation. The ability of both Aβ(1–28) and Aβ(1–40) to bind glycosaminoglycans is pH-dependent, with increasing interaction as the pH values fall below neutrality and very little binding at pH 8.0. The pH profile of heparin-induced aggregation of Aβ(1–28) has a midpoint pH of approximately 6.5, suggesting that one or more histidine residues must be protonated for binding to occur. Analysis of the Aβ sequence reveals a consensus heparin-binding domain at residues 12–17, and this motif contains histidines at positions 13 and 14 that may be involved in the interaction with glycosaminoglycans. This hypothesis is supported by the following observations: (a) Aβ(13–17) binds tightly to a heparin affinity column at pH 4.0, but not at pH 8.0; and (b) an Aβ(13–17) in which histidine residues 13 and 14 have been replaced with serines does not bind to a heparin column at either pH 8.0 or 4.0. Together, the data indicate that Aβ is capable of binding to the glycosaminoglycan chains of proteoglycans, and such an interaction may be relevant to the etiology and pathology of Alzheimer's disease.     

Nanomedicine: Action of Metal Nanoparticles on Neuronal Nitric Oxide Synthase—Fluorimetric Analysis on the Mechanism for Fibrillogenesis

The incubation of neuronal nitric oxide synthase with the five amyloid peptide fragments [Aβ_17–21; Aβ_25–29; Aβ_29–33; Aβ_33–37; Aβ_25–37] catalyzed the formation of fibrils. The role of neuronal isomer (nNOS) involved the entrapment of free monomers and seed aggregates to initiate the events of nucleation and elongation, critical for the formation of fibrils. It was evident that the hydrophobic nature of Aβ_17–21, the three glycine zipper peptides [Aβ_25–29; Aβ_29–33; Aβ_33–37] and Aβ_25–37 was a trigger in the formation of fibrils and was a force critical in the association of the peptides with the enzyme. Gold and silver nanoparticles (average 4.0 nm) inhibited fibril formation when added to the induced fibrils from nNOS-Aβ incubation. The addition of nNOS and/or Aβ to co-incubated solutions of nanoparticle-Aβ or nanoparticle-nNOS respectively did not prevent fibril formation but reversed it. Three mechanisms for this reversal were proposed: (1) depletion of free Aβ monomer in solution and blocking potential aggregation sites on the nNOS molecule due to large surface area of the nanoparticle (2) hydrophobic interaction between the Aβ peptide and nanoparticle (3) disruption of binary adducts between Aβ-peptides and nNOS by nanoparticles.     

Mapping Conformational Ensembles of Aβ Oligomers in Molecular Dynamics Simulations

Although the oligomers formed by Aβ peptides appear to be the primary cytotoxic species in Alzheimer's disease, detailed information about their structures appears to be lacking. In this article, we use exhaustive replica exchange molecular dynamics and an implicit solvent united-atom model to study the structural properties of Aβ monomers, dimers, and tetramers. Our analysis suggests that the conformational ensembles of Aβ dimers and tetramers are very similar, but sharply distinct from those sampled by the monomers. The key conformational difference between monomers and oligomers is the formation of β-structure in the oligomers occurring together with the loss of intrapeptide interactions and helix structure. Our simulations indicate that, independent of oligomer order, the Aβ aggregation interface is largely confined to the sequence region 10-23, which forms the bulk of interpeptide interactions. We show that the fractions of β structure computed in our simulations and measured experimentally are in good agreement.     

On the Origin of the Stronger Binding of PIB over Thioflavin T to Protofibrils of the Alzheimer Amyloid-β Peptide: A Molecular Dynamics Study

Pittsburgh compound B (PIB) is a neutral derivative of the fluorescent dye Thioflavin T (ThT), which displays enhanced hydrophobicity and binding affinity to amyloid fibrils. We present molecular dynamics simulations of binding of PIB and ThT to a common cross-β-subunit of the Alzheimer Amyloid-β peptide (Aβ). Our simulations of binding to Aβ_9-40 protofibrils show that PIB, like ThT, selectively binds to the hydrophobic or aromatic surface grooves on the β-sheet surface along the fibril axis. The lack of two methyl groups and charge in PIB not only improves its hydrophobicity but also leads to a deeper insertion of PIB compared to ThT into the surface grooves. This significantly increases the steric, aromatic, and hydrophobic interactions, and hence leads to stronger binding. Simulations on protofibrils consisting of the more-toxic Aβ_17-42 revealed an additional binding mode in which PIB and ThT insert into the channel that forms in the loop region of the protofibril, sandwiched between two sheet layers. Our simulations indicate that the rotation between the two ring parts of the dyes is significantly more restricted when the dyes are bound to the surface of the cross-β-subunits or to the channel inside the Aβ_17-42 cross-β-subunit, compared with free solution. The specific conformations of the dyes are influenced by small chemical modifications (ThT versus PIB) and by the environment in which the dye is placed.     

Spontaneous formation of twisted Aβ(16-22) fibrils in large-scale molecular-dynamics simulations

Protein aggregation is associated with fatal neurodegenerative diseases, including Alzheimer's and Parkinson's. Mapping out kinetics along the aggregation pathway could provide valuable insights into the mechanisms that drive oligomerization and fibrillization, but that is beyond the current scope of computational research. Here we trace out the full kinetics of the spontaneous formation of fibrils by 48 Aβ_16-22 peptides, following the trajectories in molecular detail from an initial random configuration to a final configuration of twisted protofilaments with cross-β-structure. We accomplish this by performing large-scale molecular-dynamics simulations based on an implicit-solvent, intermediate-resolution protein model, PRIME20. Structural details such as the intersheet distance, perfectly antiparallel β-strands, and interdigitating side chains analogous to a steric zipper interface are explained by and in agreement with experiment. Two characteristic fibrillization mechanisms—nucleation/templated growth and oligomeric merging/structural rearrangement—emerge depending on the temperature.     

Structure and dynamics of oligomeric intermediates in β2-microglobulin self-assembly

β₂-Microglobulin is a 99-residue protein with a propensity to form amyloid-like fibrils in vitro which exhibit distinct morphologies dependent on the solution conditions employed. Here we have used ion mobility spectrometry-mass spectrometry to characterize the oligomeric species detected during the formation of worm-like fibrils of β₂-microglobulin at pH 3.6. Immediately upon sample dissolution, β₂-microglobulin monomer and oligomers—the latter ranging in size from dimer to hexamer—are present as a pool of rapidly interconverting species. Increasing the ionic strength of the solution initiates fibril formation without a lag-phase whereupon these oligomers become more stable and higher-order species (7-mer to >14-mer) are observed. The oligomers detected have collision cross-sectional areas consistent with a linearly stacked assembly comprising subunits of native-like volume. The results provide insights into the identity and properties of the transient, oligomeric intermediates formed during assembly of worm-like fibrils and identify species that differ significantly from the oligomers previously characterized during the nucleated assembly of long, straight fibrils. The data presented demonstrate the interrelationship between different fibril-forming pathways and identify their points of divergence.     

Temperature-induced dissociation of Abeta monomers from amyloid fibril

Using all-atom molecular dynamics, we study the temperature-induced dissociation of Aβ monomers from the fibril protofilament. To accelerate conformational sampling, simulations are performed at elevated temperatures and peptide concentrations. By computing free energy disconnectivity graphs we mapped the free energy landscape of monomers on the surface of Aβ fibril. We found that Aβ monomers sample diverse sets of low free energy states with different degrees of association with the fibril. Some of these states have residual amounts of fibril interactions, whereas others lack fibril-like content. Generally, Aβ monomers with partially formed fibril-like interactions have the lowest free energies, but their backbone conformations may differ considerably from those in the fibril interior. Overall, Aβ amyloid protofilaments seem to be highly resistant to thermal dissociation. Monomer dissociation from the fibril edge proceeds via multiple stages and pathways. Our simulation findings are discussed in the context of recent experimental results.