Evidence of the existence of micelles in the fibrillogenesis of beta-amyloid peptide

Alzheimer's disease (AD) is characterized by the deposition of fibrillar deposits formed by the amyloid beta (Abeta) peptide. The most widely accepted model of fibrillogenesis of Abeta affirms that fibrillogenesis occurs in two distinct stages, nucleation and elongation. A modification of the model includes the formation of micelles. We have demonstrated with accurate experimental determinations the existence of aggregates with micellar properties (namely, the critical micellar concentration, CMC, and aggregation number). Values of the CMC were obtained by analysis of surface tension (17.5 microM) and changes in the fluorescence of pyrene (17.6 microM), respectively. The average aggregation number determined by fluorescence quenching was 25, and it was independent of peptide concentration. The presence of micelles implies that above the CMC all excess peptide is incorporated into micelles, and consequently, the monomer concentration is kept almost constant. Thus, micelles act as a peptide reservoir. Micelles are located on-pathway, since they serves as nucleation centers. Experimental data support the model, since above 17.7 microM the time of half-aggregation is independent of peptide concentration, and the overall reaction of the conversion of monomer peptide into fibril can be treated as an apparent first-order reaction.     

Metal switch for amyloid formation: insight into the structure of the nucleus

The role of Zn2+ in pre-organizing Abeta(10-21) amyloid formation is shown to preferentially alter the relative rate of fibril nucleation and to have little influence on fibril propagation. Fibril morphology, as determined by small angle neutron scattering (SANS) and transmission electron microscopy (TEM), was unchanged in the presence and absence of Zn2+ in Abeta(10-21), as well as in a series of site-specifically altered variants. The metal-independence of the Abeta(10-21)H13Q peptide suggested that the increase in nucleation rate in Abeta(10-21) is due to Zn2+-mediated inter-sheet interactions, involving both histidine 13 and histidine 14.     

Structure and dynamics of the Abeta(21-30) peptide from the interplay of NMR experiments and molecular simulations

We combine molecular dynamics simulations and new high-field NMR experiments to describe the solution structure of the Abeta(21-30) peptide fragment that may be relevant for understanding structural mechanisms related to Alzheimer's disease. By using two different empirical force-field combinations, we provide predictions of the three-bond scalar coupling constants ((3)J(H(N)H(alpha))), chemical-shift values, (13)C relaxation parameters, and rotating-frame nuclear Overhauser effect spectroscopy (ROESY) crosspeaks that can then be compared directly to the same observables measured in the corresponding NMR experiment of Abeta(21-30). We find robust prediction of the (13)C relaxation parameters and medium-range ROESY crosspeaks by using new generation TIP4P-Ew water and Amber ff99SB protein force fields, in which the NMR validates that the simulation yields both a structurally and dynamically correct ensemble over the entire Abeta(21-30) peptide. Analysis of the simulated ensemble shows that all medium-range ROE restraints are not satisfied simultaneously and demonstrates the structural diversity of the Abeta(21-30) conformations more completely than when determined from the experimental medium-range ROE restraints alone. We find that the structural ensemble of the Abeta(21-30) peptide involves a majority population (approximately 60%) of unstructured conformers, lacking any secondary structure or persistent hydrogen-bonding networks. However, the remaining minority population contains a substantial percentage of conformers with a beta-turn centered at Val24 and Gly25, as well as evidence of the Asp23 to Lys28 salt bridge important to the fibril structure. This study sets the stage for robust theoretical work on Abeta(1-40) and Abeta(1-42), for which collection of detailed NMR data on the monomer will be more challenging because of aggregation and fibril formation on experimental timescales at physiological conditions. In addition, we believe that the interplay of modern molecular simulation and high-quality NMR experiments has reached a fruitful stage for characterizing structural ensembles of disordered peptides and proteins in general.     

Alzheimer amyloid beta(1-40) peptide: interactions with cationic gemini and single-chain surfactants

The aggregation of amyloid beta-peptide [Abeta(1-40)] into fibril is a key pathological process associated with Alzheimer's disease. The effect of cationic gemini surfactant hexamethylene-1,6-bis-(dodecyldimethylammonium bromide) [C(12)H(25)(CH(3))(2)N(CH(2))(6)N(CH(3))(2)C(12)H(25)]Br(2) (designated as C(12)C(6)C(12)Br(2)) and single-chain cationic surfactant dodecyltrimethylammonium bromide (DTAB) on the Alzheimer amyloid beta-peptide Abeta(1-40) aggregation behavior was studied by microcalorimetry, circular dichroism (CD), and atomic force microscopy (AFM) measurements at pH 7.4. Without addition of surfactant, 0.5 g/L Abeta(1-40) mainly exists in dimeric state. It is found that the addition of the monomers of C(12)C(6)C(12)Br(2) and DTAB may cause the rapid aggregation of Abeta(1-40) and the fibrillar structures are observed by CD spectra and the AFM images. Due to the repulsive interaction among the head groups of surfactants and the formation of a small hydrophobic cluster of surfactant molecules, the fibrillar structure is disrupted again as the surfactant monomer concentration is increased, whereas globular species are observed in the presence of micellar solution. Different from single-chain surfactant, C(12)C(6)C(12)Br(2) has a much stronger interaction with Abeta(1-40) to generate larger endothermic energy at much lower surfactant concentration and has much stronger ability to induce the aggregation of Abeta(1-40).     

Understanding Amyloid‐β Oligomerization at the Molecular Level: The Role of the Fibril Surface

The aggregation of the amyloid β‐peptide into fibrils is a complex process that involves mechanisms such as primary and secondary nucleation, fibril elongation and fibril fragmentation. Some of these processes generate neurotoxic Aβ oligomers, which are involved in the development of Alzheimer's disease. Recent experimental studies have emphasized the role of the fibril as a catalytic surface for the production of highly toxic oligomers during secondary nucleation. By using molecular dynamics simulations, we show that it is the hydrophobic fibril region that causes the structural changes required for catalyzing the formation of β‐sheet‐rich Aβ1‐42 oligomers on the fibril surface. These results reveal, for the first time, the molecular basis of the secondary nucleation pathway.     

Amyloid-like formation by self-assembly of peptidolipids in two dimensions

The accumulation of beta-amyloid peptide (Abeta) in the human brain is known to be the major cause that drives Alzheimer's disease pathogenesis. Abeta, a 39-42 amino acid peptide, is the cleavage product of amyloid precursor protein in the hydrophobic transmembrane region. The present study employs a two-dimensional (2D) approach. Two synthetic peptidolipids, C18-IIGLM-OH and C18-IIGLM-NH2, are selected based on the fragment 31-35 of Abeta which is recognized as one of the determining segments that induces formation of amyloid fibril plaques. The aliphatic hydrocarbon chain C18 is attached to the N-terminal of the fragment 31-35 to facilitate the 2D study at the air-water interface. The aggregation process is observed by two measurements: (1) surface pressure-area and surface dipole moment-area isotherms and (2) epifluorescence microscopy of the Langmuir films to investigate the topography of the amyloid-like formation.     

A Competition of Secondary and Primary Nucleation Controls Amyloid Fibril Formation of the Parathyroid Hormone

Functional amyloids belong to an increasing class of non-toxic biologic material, in contrast to the prominent disease-related amyloids. Herein, this work reports on the fibril formation of the parathyroid hormone PTH₈₄ as a representative candidate following the same generic principles of primary and secondary nucleation. Employing Thioflavin T monitored kinetics analyses and negative-staining transmission electron microscopy, an intricate, concentration dependent behavior of time dependent generation and morphologies of PTH₈₄ fibrils are found. While at low peptide concentrations, fibril formation is driven by surface catalyzed secondary nucleation, an increased amount of peptides cause a negative feedback on fibril elongation and secondary nucleation. Moreover, the source of primary nuclei is found to regulate the overall macroscopic fibrillation. As a consequence, the concentration dependent competition of primary versus secondary nucleation pathways is found to dominate the mechanism of fibril generation. This work is able to hypothesize an underlying monomer-oligomer equilibrium providing high-order species for primary nucleation and, additionally, negatively affecting the available monomer pool.     

Micellization of surfactin and its effect on the aggregate conformation of amyloid beta(1-40)

The aggregation of amyloid beta-peptide (Abeta(1-40)) into fibrils is a key pathological process associated with Alzheimer's disease. This work has investigated the micellization process of biosurfactant surfactin and its effect on the aggregation behavior of Abeta(1-40). The results show that surfactin has strong self-assembly ability to form micelles and the micelles tend to form larger aggregates. Surfactin adopts a beta-turn conformation at low micelle concentration but a beta-sheet conformation at high micelle concentration. The effect of surfactin on the Abeta(1-40) aggregation behavior exhibits a strong concentration-dependent fashion. Below the critical micelle concentration of surfactin, the electrostatic binding of surfactin monomers on Abeta(1-40) causes Abeta(1-40) molecules to unfold. Assisted by the hydrophobic interaction among surfactin monomers on the Abeta(1-40) chain, the conformation of Abeta(1-40) transfers to the beta-sheet structure, which promotes the formation of fibrils. At low surfactin micelle concentration, besides the electrostatic force and hydrophobic interaction, hydrogen bonds formed between surfactin micelles and adjacent Abeta(1-40) peptide chains may promote the ordered organization of these Abeta(1-40) peptide chains, thus leading to the formation of beta-sheets and fibrils to a great extent. At high surfactin micelle concentration, the separating of Abeta(1-40) chains by the excessive surfactin micelles and the aggregation of the complexes of Abeta(1-40) with surfactin micelles inhibit the formation of beta-sheets and fibrils.     

Quasihomogeneous nucleation of amyloid beta yields numerical bounds for the critical radius, the surface tension, and the free energy barrier for nucleus formation

Amyloid aggregates are believed to grow through a nucleation mediated pathway, but important aggregation parameters, such as the nucleation radius, the surface tension of the aggregate, and the free energy barrier toward aggregation, have remained difficult to measure. Homogeneous nucleation theory, if applicable, can directly relate these parameters to measurable quantities. We employ fluorescence correlation spectroscopy to measure the particle size distribution in an aggregating solution of Alzheimer's amyloid beta molecule (Abeta(1-40)) and analyze the data from a homogeneous nucleation theory perspective. We observe a reproducible saturation concentration and a critical dependence of various aspects of the aggregation process on this saturation concentration, which supports the applicability of the nucleation theory to Abeta aggregation. The measured size distributions show a valley between two peaks ranging from 5 to 50 nm, which defines a boundary for the value of the nucleation radius. By carefully controlling the conditions to inhibit heterogeneous nucleation, we can hold off nucleation in a 25 times supersaturated solution for at least up to 3 h at room temperature. This quasi-homogeneous kinetics implies that at room temperature, the surface energy of the Abeta/water interface is > or =4.8 mJ/m(2), the free energy barrier to nucleation (at 25 times supersaturation) is > or =1.93x10(-19) J, and the number of monomers in the nucleus is > or =29.     

Stoichiometric inhibition of amyloid beta-protein aggregation with peptides containing alternating alpha,alpha-disubstituted amino acids

We have prepared two peptides based on the hydrophobic core (Lys-Leu-Val-Phe-Phe) of amyloid beta-protein (Abeta) that contain alpha,alpha-disubstituted amino acids at alternating positions, but differ in the positioning of the oligolysine chain (AMY-1, C-terminus; AMY-2, N-terminus). We have studied the effects of AMY-1 and AMY-2 on the aggregation of Abeta and find that, at stoichiometric concentrations, both peptides completely stop Abeta fibril growth. Equimolar mixtures of AMY-1 and Abeta form only globular aggregates as imaged by scanning force microscopy and transmission electron microscopy. These samples show no signs of protofibrillar or fibrillar material even after prolonged periods of time (4.5 months). Also, 10 mol % of AMY-1 prevents Abeta self-assembly for long periods of time; aged samples (4.5 months) show only a few protofibrillar or fibrillar aggregates. Circular dichroism spectroscopy of equimolar mixtures of AMY-1 and Abeta show that the secondary structure of the mixture changes over time and progresses to a predominantly beta-sheet structure, which is consistent with the design of these inhibitors preferring a sheet-like conformation. Changing the position of the charged tail on the peptide, AMY-2 interacts with Abeta differently in that equimolar mixtures form large ( approximately 1 mum) globular aggregates which do not progress to fibrils, but precipitate out of solution. The differences in the aggregation mediated by the two peptides is discussed in terms of a model where the inhibitors act as cosurfactants that interfere with the native ability of Abeta to self-assemble by disrupting hydrophobic interactions either at the C-terminus or N-terminus of Abeta.     

Sequestration within biomolecular condensates inhibits Aβ-42 amyloid formation

Biomolecular condensates are emerging as an efficient strategy developed by cells to control biochemical reactions in space and time by locally modifying composition and environment. Yet, local increase in protein concentration within these compartments could promote aberrant aggregation events, including the nucleation and growth of amyloid fibrils. Understanding protein stability within the crowded and heterogeneous environment of biological condensates is therefore crucial, not only when the aggregation-prone protein is the scaffold element of the condensates but also when proteins are recruited as client molecules within the compartments. Here, we investigate the partitioning and aggregation kinetics of the amyloidogenic peptide Abeta42 (Aβ-42), the peptide strongly associated with Alzheimer''s disease, recruited into condensates based on low complexity domains (LCDs) derived from the DEAD-box proteins Laf1, Dbp1 and Ddx4, which are associated with biological membraneless organelles. We show that interactions between Aβ-42 and the scaffold proteins promote sequestration and local increase of the peptide concentration within the condensates. Yet, heterotypic interactions within the condensates inhibit the formation of amyloid fibrils. These results demonstrate that biomolecular condensates could sequester aggregation-prone proteins and prevent aberrant aggregation events, despite the local increase in their concentration. Biomolecular condensates could therefore work not only as hot-spots of protein aggregation but also as protective reservoirs, since the heterogenous composition of the condensates could prevent the formation of ordered fibrillar aggregates.

Biomolecular condensates sequester an aggregation-prone peptide and prevent its aggregation, showing that heterotypic interactions within the condensates can prevent the formation of amyloid fibrils, despite the local increase in concentration.     

Kinetic pathways to peptide aggregation on surfaces: the effects of β-sheet propensity and surface attraction

Mechanisms of peptide aggregation on hydrophobic surfaces are explored using molecular dynamics simulations with a coarse-grained peptide representation. Systems of peptides are studied with varying degrees of backbone rigidity (a measure of β-sheet propensity) and degrees of attraction between their hydrophobic residues and the surface. Multiple pathways for aggregation are observed, depending on the surface attraction and peptide β-sheet propensity. For the case of a single-layered β-sheet fibril forming on the surface (a dominant structure seen in all simulations), three mechanisms are observed: (a) a condensation-ordering transition where a bulk-formed amorphous aggregate binds to the surface and subsequently rearranges to form a fibril; (b) the initial formation of a single-layered fibril in the bulk depositing flat on the surface; and (c) peptides binding individually to the surface and nucleating fibril formation by individual peptide deposition. Peptides with a stiffer chiral backbone prefer mechanism (b) over (a), and stronger surface attractions prefer mechanism (c) over (a) and (b). Our model is compared to various similar experimental systems, and an agreement was found in terms of the surface increasing the degree of fibrillar aggregation, with the directions of fibrillar growth matching the crystallographic symmetry of the surface. Our simulations provide details of aggregate growth mechanisms on scales inaccessible to either experiment or atomistic simulations.     

Effect of lysine-28 side-chain acetylation on the nanomechanical behavior of alzheimer amyloid beta25-35 fibrils

Amyloid fibrils are self-associating filamentous structures formed from the 39- to 42-residue-long amyloid beta peptide (Abeta peptide). The deposition of Abeta fibrils is one of the most important factors in the pathogenesis of Alzheimer's disease. Abeta25-35 is a fibril-forming peptide that is thought to represent the biologically active, toxic form of the full-length Abeta peptide. We have recently shown that beta sheets can be mechanically unzipped from the fibril surface with constant forces in a reversible transition, and the unzipping forces differ in fibrils composed of different peptides. In the present work, we explored the effect of epsilon-amino acetylation of the Lys28 residue on the magnitude of the unzipping force of Abeta25-35 fibrils. Although the gross structure of the Lys28-acetylated (Abeta25-35_K28Ac) and wild-type Abeta25-35 (Abeta25-35wt) fibrils were similar, as revealed by atomic force microscopy, the fundamental unzipping forces were significantly lower for Abeta25-35_K28Ac (20 +/- 4 pN SD) than for Abeta25-35wt (42 +/- 9 pN SD). Simulations based on a simple two-state model suggest that the decreased unzipping forces, caused most likely by steric constraints, are likely due to a destabilized zippered state of the fibril.     

Unraveling the Pressure Effect on Nucleation Processes of Amyloidogenic Proteins

The influence of pressure on the nucleation rate of insulin under fibril‐forming conditions was studied and subsequently analysed using classical nucleation theory. The aim was a better understanding and quantification of the influence of pressure on protein aggregation/fibrillation reactions. The application of pressure has a drastic accelerating effect on the nucleation and growth process of insulin fibrils. We show that this effect arises from a volume decrease upon nucleus formation, due to formation of a less hydrated and more compact transition state that can be quantified extending nucleation theory by a pressure–volume term. Conversely, the absolute values of the lag time and the critical size of the nucleus cannot be satisfactorily described by the classical nucleation theory, which might be due to the presence of secondary effects, such as parallel aggregation pathways or fragmentation processes.     

Energy landscapes of the monomer and dimer of the Alzheimer's peptide Abeta(1-28)

The cytotoxicity of Alzheimer's disease has been linked to the self-assembly of the 4042 amino acid of the amyloid-beta (Abeta) peptide into oligomers. To understand the assembly process, it is important to characterize the very first steps of aggregation at an atomic level of detail. Here, we focus on the N-terminal fragment 1-28, known to form fibrils in vitro. Circular dichroism and NMR experiments indicate that the monomer of Abeta(1-28) is alpha-helical in a membranelike environment and random coil in aqueous solution. Using the activation-relaxation technique coupled with the OPEP coarse grained force field, we determine the structures of the monomer and of the dimer of Abeta(1-28). In agreement with experiments, we find that the monomer is predominantly random coil in character, but displays a non-negligible beta-strand probability in the N-terminal region. Dimerization impacts the structure of each chain and leads to an ensemble of intertwined conformations with little beta-strand content in the region Leu17-Ala21. All these structural characteristics are inconsistent with the amyloid fibril structure and indicate that the dimer has to undergo significant rearrangement en route to fibril formation.     

beta-Sheet ligands in action: KLVFF recognition by aminopyrazole hybrid receptors in water

Little is known about the precise mechanism of action of beta-sheet ligands, hampered by the notorious solubility problems involved with protein misfolding and amyloid formation. Recently the nucleation site for the pathogenic aggregation of the Alzheimer's peptide was identified as the KLVFF sequence in the central region of Abeta. A combination of two aminopyrazole ligands with di- or tripeptides taken from this key fragment now furnished water-soluble Abeta-specific ligands which allow model investigations in water. A detailed conformational analysis provides experimental evidence for an increased beta-sheet content induced in the peptide. Strong indications were also found for the peptide backbone recognition via hydrogen bonds plus hydrophobic contributions between aminopyrazole nuclei and Phe residues. The affinity of these new ligands toward the KKLVFF fragment is highly dependent on their sequence and composition from natural and artificial amino acids. Thus, for the first time, detailed insight is gained into the complexation of beta-sheet ligands with model peptides taken directly from Abeta.     

The effect of nanoparticles on amyloid aggregation depends on the protein stability and intrinsic aggregation rate

Nanoparticles interfere with protein amyloid formation. Catalysis of the process may occur due to increased local protein concentration and nucleation on the nanoparticle surface, whereas tight binding or a large particle/protein surface area may lead to inhibition of protein aggregation. Here we show a clear correlation between the intrinsic protein stability and the nanoparticle effect on the aggregation rate. The results were reached for a series of five mutants of single-chain monellin differing in intrinsic stability toward denaturation, for which a correlation between protein stability and aggregation propensity has been previously documented by Szczepankiewicz et al. [Mol. Biosyst.20107 (2), 521-532]. The aggregation process was monitored by thioflavin T fluorescence in the absence and presence of copolymeric nanoparticles with different hydrophobic characters. For mutants with a high intrinsic stability and low intrinsic aggregation rate, we find that amyloid fibril formation is accelerated by nanoparticles. For mutants with a low intrinsic stability and high intrinsic aggregation rate, we find the opposite--a retardation of amyloid fibril formation by nanoparticles. Moreover, both catalytic and inhibitory effects are most pronounced with the least hydrophobic nanoparticles, which have a larger surface accessibility of hydrogen-bonding groups in the polymer backbone.     

Noncovalent interaction between amyloid-β-peptide (1–40) and oleuropein studied by electrospray ionization mass spectrometry

Beta amyloid peptide (Abeta) is the major proteinaceous component of senile plaques formed in Alzheimer's disease (AD) brain. The aggregation of Abeta is associated with neurodegeneration, loss of cognitive ability, and premature death. It has been suggested that oxidative stress and generation of free radical species have implications in the fibrillation of Abeta and its subsequent neurotoxicity. For this reason, it is proposed that antioxidants may offer a protective or therapeutic alternative against amyloidosis. This study is the first report of the formation of the noncovalent complex between Abeta or its oxidized form and the natural derived antioxidant oleuropein (OE) by electrospray ionization mass spectrometry (ESI MS). ESI MS allowed the real time monitoring of the complex formation between Abeta, OE, and variants thereof. Several experimental conditions, such as elevated orifice potential, low pH values, presence of organic modifier, and ligand concentration were examined, to assess the specificity and the stability of the formed noncovalent complexes.     

Annular structures as intermediates in fibril formation of Alzheimer Abeta17-42

We report all-atom molecular dynamics simulations of annular beta-amyloid (17-42) structures, single- and double-layered, in solution. We assess the structural stability and association force of Abeta annular oligomers associated through different interfaces, with a mutated sequence (M35A), and with the oxidation state (M35O). Simulation results show that single-layered annular models display inherent structural instability: one is broken down into linear-like oligomers, and the other collapses. On the other hand, a double-layered annular structure where the two layers interact through their C-termini to form an NC-CN interface (where N and C are the N and C termini, respectively) exhibits high structural stability over the simulation time due to strong hydrophobic interactions and geometrical constraints induced by the closed circular shape. The observed dimensions and molecular weight of the oligomers from atomic force microscopy (AFM) experiments are found to correspond well to our stable double-layered model with the NC-CN interface. Comparison with K3 annular structures derived from the beta 2-microglobulin suggests that the driving force for amyloid formation is sequence specific, strongly dependent on side-chain packing arrangements, structural morphologies, sequence composition, and residue positions. Combined with our previous simulations of linear-like Abeta, K3 peptide, and sup35-derived GNNQQNY peptide, the annular structures provide useful insight into oligomeric structures and driving forces that are critical in amyloid fibril formation.     

Amyloid beta-peptide oligomerization in silico: dimer and trimer

Soluble oligomers of Alzheimer's amyloid beta protein (Abeta) may act as effectors of neurotoxicity in early stages of Alzheimer's disease. Detailed information about the structure of Abeta in atomistic level and the dynamics of assembly of monomeric Abeta into oligomeric structures is rather elusive. We have performed replica exchange molecular dynamics (REMD) simulations on the formation of the dimer and trimer of Abeta10-35 peptide. We have observed spontaneous formation of several basic structural units that may act as a template or an intermediate for further aggregation of Alzheimer's Abeta protein. Various conformers, including interlocking structures of experimentally known bend double beta strands, are identified.