Formation of toxic fibrils of Alzheimer's amyloid beta-protein-(1-40) by monosialoganglioside GM1, a neuronal membrane component

A pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid beta-protein (Abeta) in fibrillar form on neuronal cells. However, the role of Abeta fibrils in neuronal dysfunction is highly controversial. This study demonstrates that monosialoganglioside GM1 (GM1) released from damaged neurons catalyzes the formation of Abeta fibrils, the toxicity and the cell affinity of which are much stronger than those of Abeta fibrils formed in phosphate-buffered saline. Abeta-(1-40) was incubated with equimolar GM1 at 37 degrees C. After a lag period of 6-12 h, amyloid fibrils were formed, as confirmed by circular dichroism, thioflavin-T fluorescence, size-exclusion chromatography, and transmission electron microscopy. The fibrils showed significant cytotoxicity against PC12 cells differentiated with nerve growth factor. Trisialoganglioside GT1b also facilitated the fibrillization, although the effect was weaker than that of GM1. Our study suggests an exacerbation mechanism of AD and an importance of polymorphisms in Abeta fibrils during the pathogenesis of the disease.     

Design, synthesis, and biophysical properties of a helical Abeta1-42 analog: Inhibition of fibrillogenesis and cytotoxicity

The aggregation of amyloid β-peptide (Aβ) into β-sheet-rich aggregates is a crucial step in the etiology of Alzheimer’s disease. Helical forms of Aβ have been suggested to be intermediates in the aggregation process of the peptide in aqueous phase, micelles and membranes. A stable helical Aβ analog would be useful to investigate the role of helical intermediates in fibrillization by Aβ. Here we designed a helical analog by simply cross-linking the Cys residues of A30C, G37C-Aβ1-42 with 1,6-bismaleimidohexane. The analog assumed a weak α-helical conformation in model membranes mimicking lipid raft microdomains of neuronal membranes under conditions in which the wild-type Aβ1-42 formed a β-sheet, indicating the cross-linking locally induced a helical conformation. Furthermore, addition of equimolar helical Aβ analog significantly reduced the amyloid formation and cytotoxicity by Aβ1-42. Thus, our helical Aβ1-42 is not only a model peptide to investigate the role of helical intermediates in fibrillization by Aβ, but also an inhibitor of Aβ-induced cytotoxicity.     

Hsp104 targets multiple intermediates on the amyloid pathway and suppresses the seeding capacity of Abeta fibrils and protofibrils

The heat shock protein Hsp104 has been reported to possess the ability to modulate protein aggregation and toxicity and to “catalyze” the disaggregation and recovery of protein aggregates, including amyloid fibrils, in yeast, Escherichia coli, mammalian cell cultures, and animal models of Huntington's disease and Parkinson's disease. To provide mechanistic insight into the molecular mechanisms by which Hsp104 modulates aggregation and fibrillogenesis, the effect of Hsp104 on the fibrillogenesis of amyloid beta (Aβ) was investigated by characterizing its ability to interfere with oligomerization and fibrillogenesis of different species along the amyloid-formation pathway of Aβ. To probe the disaggregation activity of Hsp104, its ability to dissociate preformed protofibrillar and fibrillar aggregates of Aβ was assessed in the presence and in the absence of ATP. Our results show that Hsp104 inhibits the fibrillization of monomeric and protofibrillar forms of Aβ in a concentration-dependent but ATP-independent manner. Inhibition of Aβ fibrillization by Hsp104 is observable up to Hsp104/Aβ stoichiometric ratios of 1:1000, suggesting a preferential interaction of Hsp104 with aggregation intermediates (e.g., oligomers, protofibrils, small fibrils) on the pathway of Aβ amyloid formation. This hypothesis is consistent with our observations that Hsp104 (i) interacts with Aβ protofibrils, (ii) inhibits conversion of protofibrils into amyloid fibrils, (iii) arrests fibril elongation and reassembly, and (iv) abolishes the capacity of protofibrils and sonicated fibrils to seed the fibrillization of monomeric Aβ. Together, these findings suggest that the strong inhibition of Aβ fibrillization by Hsp104 is mediated by its ability to act at different stages and target multiple intermediates on the pathway to amyloid formation.     

Fibrillar seeds alleviate amyloid-β cytotoxicity by omitting formation of higher-molecular-weight oligomers

Amyloid-β (Aβ) peptides can exist in distinct forms including monomers, oligomers and fibrils, consisting of increased numbers of monomeric units. Among these, Aβ oligomers are implicated as the primary toxic species as pointed by multiple lines of evidence. It has been suggested that toxicity could be rendered by the soluble higher-molecular-weight (high-n) Aβ oligomers. Yet, the most culpable form in the pathogenesis of Alzheimer’s disease (AD) remains elusive. Moreover, the potential interaction among the insoluble fibrils that have been excluded from the responsible aggregates in AD development, Aβ monomers and high-n oligomers is undetermined. Here, we report that insoluble Aβ fibrillar seeds can interact with Aβ monomers at the stoichiometry of 1:2 (namely, each Aβ molecule of seed can bind to two Aβ monomers at a time) facilitating the fibrillization by omitting the otherwise mandatory formation of the toxic high-n oligomers during the fibril maturation. As a result, the addition of exogenous Aβ fibrillar seeds is seen to rescue neuronal cells from Aβ cytotoxicity presumably exerted by high-n oligomers, suggesting an unexpected protective role of Aβ fibrillar seeds.     

Upregulation of SIRT1 deacetylase in phenylephrine-treated cardiomyoblasts

Deposition of amyloid fibrils consisting of amyloid β (Aβ) protein as senile plaques in the brain is a pathological hallmark of Alzheimer’s disease. However, a growing body of evidence shows that soluble Aβ oligomers correlate better with dementia than fibrils, suggesting that Aβ oligomers may be the primary toxic species. The structure and oligomerization mechanism of these Aβ oligomers are crucial for developing effective therapeutics. Here we investigated the oligomerization of Aβ42 in the context of a fusion protein containing GroES and ubiquitin fused to the N-terminus of Aβ sequence. The presence of fusion protein partners, in combination with a denaturing buffer containing 8 M urea at pH 10, is unfavorable for Aβ42 aggregation, thus allowing only the most stable structures to be observed. Transmission electron microscopy showed that Aβ42 fusion protein formed globular oligomers, which bound weakly to thioflavin T and Congo red. SDS–PAGE shows that Aβ42 fusion protein formed SDS-resistant hexamers and tetramers. In contrast, Aβ40 fusion protein remained as monomers on SDS gel, suggesting that the oligomerization of Aβ42 fusion protein is not due to the fusion protein partners. Cysteine scanning mutagenesis at 22 residue positions further revealed that single cysteine substitutions of the C-terminal hydrophobic residues (I31, I32, L34, V39, V40, and I41) led to disruption of hexamer and tetramer formation, suggesting that hydrophobic interactions between these residues are most critical for Aβ42 oligomerization.     

Inhibitors of amyloid β-protein aggregation mediated by GM1-containing raft-like membranes

The aggregation (fibril formation) of amyloid β-protein (Aβ) is considered to be a crucial step in the etiology of Alzheimer's disease (AD). The inhibition of Aβ aggregation and/or decomposition of fibrils formed in aqueous solution by small compounds have been studied extensively for the prevention and treatment of AD. However, recent studies suggest that Aβ aggregation also occurs in lipid rafts mediated by a cluster of monosialoganglioside GM1. This study examined the effects of representative compounds on Aβ aggregation and fibril destabilization in the presence of GM1-containing raft-like liposomes. Among the compounds tested, nordihydroguaiaretic acid (NDGA), rifampicin (RIF), tannic acid (TA), and quercetin (QUE) showed strong fibrillization inhibitory activity. NDGA and RIF inhibited the binding of Aβ to GM1 liposomes by competitively binding to the membranes and/or direct interaction with Aβ in solution, thus at least partly preventing fibrils from forming. Coincubation of Aβ with NDGA, RIF, and QUE in the presence of GM1 liposomes resulted in elongate particles, whereas the presence of TA yielded protofibrillar structures. TA and RIF also destabilized fibrils. The most potent NDGA prevented Aβ-induced toxicity in PC12 cells by inhibiting Aβ accumulation. Furthermore, a comparison of the inhibitory effects of various compounds between aqueous-phase and GM1-mediated aggregation of Aβ suggested that the two aggregation processes are not identical.     

Trace amounts of pyroglutaminated Aβ-(3–42) enhance aggregation of Aβ-(1–42) on neuronal membranes at physiological concentrations: FCS analysis of cell surface

Minor species of amyloid β-peptide (Aβ), such as Aβ-(1–43) and pyroglutaminated Aβ-(3–42) (Aβ-(3pE–42)), have been suggested to be involved in the initiation of the Aβ aggregation process, which is closely associated with the etiology of Alzheimer's disease. They can play important roles in aggregation not only in the aqueous phase but also on neuroral membranes; however, the latter behaviors remain mostly unexplored. Here, initial aggregation processes of Aβ on living cells were monitored at physiological nanomolar concentrations by fluorescence correlation spectroscopy. Membrane-bound Aβ-(1–42) and Aβ-(1–40) formed oligomers composed of ~4 Aβ molecules during 48-h incubation, whereas the peptides remained monomeric in the culture medium, indicating that the membranes facilitated Aβ aggregation. The presence of 5 mol% Aβ-(3pE–42), but not Aβ-(1–43), significantly enhanced the aggregation of Aβ-(1–42) up to ~10-mers. On the other hand, neither trace amounts of Aβ-(1–42) nor Aβ-(3pE–42) enhanced the aggregation of Aβ-(1–40). The observed small Aβ oligomers are expected to act as pathogenic seeds for amyloid fibrils responsible for neurotoxicity. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.     

High-molecular weight Aβ oligomers and protofibrils are the predominant Aβ species in the native soluble protein fraction of the AD brain

Alzheimer's disease (AD) is characterized by the aggregation and deposition of amyloid β protein (Aβ) in the brain. Soluble Aβ oligomers are thought to be toxic. To investigate the predominant species of Aβ protein that may play a role in AD pathogenesis, we performed biochemical analysis of AD and control brains. Sucrose buffer-soluble brain lysates were characterized in native form using blue native (BN)-PAGE and also in denatured form using SDS-PAGE followed by Western blot analysis. BN-PAGE analysis revealed a high-molecular weight smear (>1000 kD) of Aβ(42) -positive material in the AD brain, whereas low-molecular weight and monomeric Aβ species were not detected. SDS-PAGE analysis, on the other hand, allowed the detection of prominent Aβ monomer and dimer bands in AD cases but not in controls. Immunoelectron microscopy of immunoprecipitated oligomers and protofibrils/fibrils showed spherical and protofibrillar Aβ-positive material, thereby confirming the presence of high-molecular weight Aβ (hiMWAβ) aggregates in the AD brain. In vitro analysis of synthetic Aβ(40) - and Aβ(42) preparations revealed Aβ fibrils, protofibrils, and hiMWAβ oligomers that were detectable at the electron microscopic level and after BN-PAGE. Further, BN-PAGE analysis exhibited a monomer band and less prominent low-molecular weight Aβ (loMWAβ) oligomers. In contrast, SDS-PAGE showed large amounts of loMWAβ but no hiMWAβ(40) and strikingly reduced levels of hiMWAβ(42) . These results indicate that hiMWAβ aggregates, particularly Aβ(42) species, are most prevalent in the soluble fraction of the AD brain. Thus, soluble hiMWAβ aggregates may play an important role in the pathogenesis of AD either independently or as a reservoir for release of loMWAβ oligomers.     

Simultaneous monitoring of peptide aggregate distributions, structure, and kinetics using amide hydrogen exchange: application to Abeta(1-40) fibrillogenesis

Increasing evidence indicates that soluble aggregates of amyloid beta protein (Abeta) are neurotoxic. However, difficulty in isolating these unstable, dynamic species impedes studies of Abeta and other aggregating peptides and proteins. In this study, hydrogen-deuterium exchange (HX) detected by mass spectrometry (MS) was used to measure Abeta(1-40) aggregate distributions without purification or modification that might alter the aggregate structure or distribution. Different peaks in the mass spectra were assigned to monomer, low molecular weight oligomer, intermediate, and fibril based on HX labeling behavior and complementary assays. After 1 h labeling, the intermediates incorporated approximately ten more deuterons relative to fibrils, indicating a more solvent exposed structure of such intermediates. HX-MS also showed that the intermediate species dissociated much more slowly to monomer than did the very low molecular weight oligomers that were formed at very early times in Abeta aggregation. Atomic force microscopy (AFM) measurements revealed the intermediates were roughly spherical with relatively homogenous diameters of 30-50 nm. Quantitative analysis of the HX mass spectra showed that the amount of intermediate species was correlated with Abeta toxicity patterns reported in a previous study under the same conditions. This study also demonstrates the potential of the HX-MS approach to characterizing complex, multi-component oligomer distributions of aggregating peptides and proteins.     

Alzheimer Aβ peptide interactions with lipid membranes

Fibrillar aggregates of misfolded amyloid proteins are involved in a variety of diseases such as Alzheimer disease (AD), type 2 diabetes, Parkinson, Huntington and prion-related diseases. In the case of AD amyloid β (Aβ) peptides, the toxicity of amyloid oligomers and larger fibrillar aggregates is related to perturbing the biological function of the adjacent cellular membrane. We used atomistic molecular dynamics (MD) simulations of Aβ_9–40 fibrillar oligomers modeled as protofilament segments, including lipid bilayers and explicit water molecules, to probe the first steps in the mechanism of Aβ-membrane interactions. Our study identified the electrostatic interaction between charged peptide residues and the lipid headgroups as the principal driving force that can modulate the further penetration of the C-termini of amyloid fibrils or fibrillar oligomers into the hydrophobic region of lipid membranes. These findings advance our understanding of the detailed molecular mechanisms and the effects related to Aβ-membrane interactions, and suggest a polymorphic structural character of amyloid ion channels embedded in lipid bilayers. While inter-peptide hydrogen bonds leading to the formation of β-strands may still play a stabilizing role in amyloid channel structures, these may also present a significant helical content in peptide regions (e.g., termini) that are subject to direct interactions with lipids rather than with neighboring Aβ peptides.     

The Osaka FAD mutation E22Δ leads to the formation of a previously unknown type of amyloid β fibrils and modulates Aβ neurotoxicity

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cerebral deposition of amyloid fibrils formed by the amyloid β (Aβ) peptide. Aβ has a length of 39-43 amino acid residues; the predominant Aβ isoforms are Aβ1-40 and Aβ1-42. While the majority of AD cases occur spontaneously, a subset of early-onset familial AD cases is caused by mutations in the genes encoding the Aβ precursor protein or presenilin 1/presenilin 2. Recently, a deletion of glutamic acid at position 22 within the Aβ sequence (E22Δ) was identified in Japanese patients with familial dementia, but the aggregation properties of the deletion variant of Aβ are not well understood. We investigated the aggregation characteristics and neurotoxicity of recombinantly expressed Aβ isoforms 1-40 and 1-42 with and without the E22Δ mutation. We show that the E22Δ mutation strongly accelerates the fibril formation of Aβ1-42 E22Δ compared to Aβ1-42 wild type (wt). In addition, we demonstrate that fibrils of Aβ1-40 E22Δ form a unique quaternary structure characterized by a strong tendency to form fibrillar bundles and a strongly increased thioflavin T binding capacity. Aβ1-40 E22Δ was neurotoxic in rat primary neuron cultures as compared to nontoxic Aβ1-40 wt. Aβ1-42 E22Δ was less toxic than Aβ1-42 wt, but it significantly decreased neurite outgrowth per cell in neuronal primary cultures. Because Aβ1-40 is the major Aβ form in vivo, the gain of toxic function caused by the E22 deletion may explain the development of familial AD in mutation carriers.     

Non-toxic conformer of amyloid β may suppress amyloid β-induced toxicity in rat primary neurons: Implications for a novel therapeutic strategy for Alzheimer’s disease

The 42-mer amyloid β-protein (Aβ42) oligomers cause neurotoxicity and cognitive impairment in Alzheimer’s disease (AD). We previously identified the toxic conformer of Aβ42 with a turn at positions 22–23 (“toxic” turn) to form oligomers and to induce toxicity in rat primary neurons, along with the non-toxic conformer with a turn at positions 25–26. G25P-Aβ42 and E22V-Aβ42 are non-toxic mutants that disfavor the “toxic” turn. Here we hypothesize that these non-toxic mutants of Aβ42 could suppress Aβ42-induced neurotoxicity, and examined their effects on the neurotoxicity, aggregation, and levels of the toxic conformer, which was evaluated by dot blotting using a monoclonal antibody (11A1) against the toxic conformer. G25P-Aβ42 and E22V-Aβ42 suppressed the neurotoxicity and aggregation of Aβ42 as well as the formation of the toxic conformer. The neurotoxicity induced by Aβ42 was also significantly reduced by the treatment of 11A1, but not of Aβ-sequence specific antibodies (6E10 and 4G8). Since recent studies indicate that Aβ oligomers contain parallel β-sheet, the present results suggest that the non-toxic mutants of Aβ42 without the “toxic” turn could prevent the propagation process of the toxic conformer of Aβ42 resulting in suppression of the formation of the toxic oligomers. This could be a promising strategy for AD therapeutics.     

Amyloid-β Membrane Binding and Permeabilization are Distinct Processes Influenced Separately by Membrane Charge and Fluidity

The 40 and 42 residue amyloid-β (Aβ) peptides are major components of the proteinaceous plaques prevalent in the Alzheimer's disease-afflicted brain and have been shown to have an important role in instigating neuronal degeneration. Whereas it was previously thought that Aβ becomes cytotoxic upon forming large fibrillar aggregates, recent studies suggest that soluble intermediate-sized oligomeric species cause cell death through membrane permeabilization. The present study examines the interactions between Aβ40 and lipid membranes using liposomes as a model system to determine how changes in membrane composition influence the conversion of Aβ into these toxic species. Aβ40 membrane binding was monitored using fluorescence-based assays with a tryptophan-substituted peptide (Aβ40 [Y10W]). We extend previous observations that Aβ40 interacts preferentially with negatively charged membranes, and show that binding of nonfibrillar, low molecular mass oligomers of Aβ40 to anionic, but not neutral, membranes involves insertion of the peptide into the bilayer, as well as sequential conformational changes corresponding to the degree of oligomerization induced. Significantly, while anionic membranes in the gel, liquid crystalline, and liquid ordered phases induce these conformational changes equally, membrane permeabilization is reduced dramatically as the fluidity of the membrane is decreased. These findings demonstrate that binding alone is not sufficient for membrane permeabilization, and that the latter is also highly dependent on the fluidity and phase of the membrane. We conclude that binding and pore formation are two distinct steps. The differences in Aβ behavior induced by membrane composition may have significant implications on the development and progression of AD as neuronal membrane composition is altered with age.     

Abeta40 protects non-toxic Abeta42 monomer from aggregation

Abeta40 and Abeta42 are the predominant Abeta species in the human body. Toxic Abeta42 oligomers and fibrils are believed to play a key role in causing Alzheimer's disease (AD). However, the role of Abeta40 in AD pathogenesis is not well established. Emerging evidence indicates a protective role for Abeta40 in AD pathogenesis. Although Abeta40 is known to inhibit Abeta42 fibril formation, it is not clear whether the inhibition acts on the non-toxic monomer or acts on the toxic Abeta42 oligomers. In contrast to conventional methods that detect the appearance of fibrils, in our study Abeta42 aggregation was monitored by the decreasing NMR signals from Abeta42 monomers. In addition, differential NMR isotope labelling enabled the selective observation of Abeta42 aggregation in a mixture of Abeta42 and Abeta40. We found Abeta40 monomers inhibit the aggregation of non-toxic Abeta42 monomers, in an Abeta42/Abeta40 ratio-dependent manner. NMR titration revealed that Abeta40 monomers bind to Abeta42 aggregates with higher affinity than Abeta42 monomers. Abeta40 can also release Abeta42 monomers from Abeta42 aggregates. Thus, Abeta40 likely protects Abeta42 monomers by competing for the binding sites on pre-existing Abeta42 aggregates. Combining our data with growing evidence from transgenic mice and human genetics, we propose that Abeta40 plays a critical, protective role in Alzheimer's by inhibiting the aggregation of Abeta42 monomer. Abeta40 itself, a peptide already present in the human body, may therefore be useful for AD prevention and therapy.     

Formation of amyloids by Abeta-(1-42) on NGF-differentiated PC12 cells: roles of gangliosides and cholesterol

The conversion of soluble, non-toxic amyloid beta-protein (Abeta) to aggregated, toxic Abeta could be the key step in the development of Alzheimer's disease. Liposomal studies have proposed that Abeta-(1-40) preferentially recognizes a cholesterol-dependent cluster of gangliosides and a conformationally altered form of Abeta promotes the aggregation of the protein. Cell experiments using fluorescein-labeled Abeta-(1-40) supported this model. Here, the interaction of native Abeta-(1-42) with unfixed rat pheochromocytoma PC12 cells was visualized using the amyloid-specific dye Congo red. Abeta-(1-42) preferentially bound to ganglioside and cholesterol-rich domains of cell membranes and formed amyloids in a time-dependent manner. These observations corroborate the model involving ganglioside-mediated accumulation of Abeta. The NGF-induced differentiation of PC12 cells into neuron-like cells caused a marked increase in both gangliosides and cholesterol, and thereby greatly potentiated the accumulation and cytotoxicity of Abeta-(1-42). NGF-differentiated cells exposed to Abeta-(1-42) had degenerated neurites, in which ganglioside and cholesterol-rich domains were localized, preceding cell death. A reduction in the amount of cholesterol by the cholesterol synthesis inhibitor compactin almost nullified the formation of amyloids by Abeta-(1-42). Our system using NGF-differentiated PC12 cells and Congo red is useful for screening inhibitors of the formation of amyloids by and cytotoxicity of Abeta.     

Pattern recognition with a fibril-specific antibody fragment reveals the surface variability of natural amyloid fibrils

Amyloid immunotherapy has led to the rise of antibodies, which target amyloid fibrils or structural precursors of fibrils, based on their specific conformational properties. Recently, we reported the biotechnological generation of the B10 antibody fragment, which provides conformation-specific binding to amyloid fibrils. B10 strongly interacts with fibrils from Alzheimer's β amyloid (Aβ) peptide, while disaggregated Aβ peptide or Aβ oligomers are not explicitly recognized. B10 also enables poly-amyloid-specific binding and recognizes amyloid fibrils derived from different types of amyloidosis or different polypeptide chains. Based on our current data, however, we find that B10 does not recognize all tested amyloid fibrils and amyloid tissue deposits. It also does not specifically interact with intrinsically unfolded polypeptide chains or globular proteins even if the latter encompass high β-sheet content or β-solenoid domains. By contrast, B10 binds amyloid fibrils from d-amino acid or l-amino acid peptides and non-proteinaceous biopolymers with highly regular and anionic surface properties, such as heparin and DNA. These data establish that B10 binding does not depend on an amyloid-specific or protein-specific backbone structure. Instead, it involves the recognition of a highly regular and anionic surface pattern. This specificity mechanism is conserved in nature and occurs also within a group of natural amyloid receptors from the innate immune system, the pattern recognition receptors. Our data illuminate the structural diversity of naturally occurring amyloid scaffolds and enable the discrimination of distinct fibril populations in vitro and within diseased tissues.     

Small-molecule conversion of toxic oligomers to nontoxic β-sheet-rich amyloid fibrils

Several lines of evidence indicate that prefibrillar assemblies of amyloid-β (Aβ) polypeptides, such as soluble oligomers or protofibrils, rather than mature, end-stage amyloid fibrils cause neuronal dysfunction and memory impairment in Alzheimer's disease. These findings suggest that reducing the prevalence of transient intermediates by small molecule-mediated stimulation of amyloid polymerization might decrease toxicity. Here we demonstrate the acceleration of Aβ fibrillogenesis through the action of the orcein-related small molecule O4, which directly binds to hydrophobic amino acid residues in Aβ peptides and stabilizes the self-assembly of seeding-competent, β-sheet-rich protofibrils and fibrils. Notably, the O4-mediated acceleration of amyloid fibril formation efficiently decreases the concentration of small, toxic Aβ oligomers in complex, heterogeneous aggregation reactions. In addition, O4 treatment suppresses inhibition of long-term potentiation by Aβ oligomers in hippocampal brain slices. These results support the hypothesis that small, diffusible prefibrillar amyloid species rather than mature fibrillar aggregates are toxic for mammalian cells.     

Characterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme

The aggregation process of wild-type human lysozyme at pH 3.0 and 60 °C has been analyzed by characterizing a series of distinct species formed on the aggregation pathway, specifically the amyloidogenic monomeric precursor protein, the oligomeric soluble prefibrillar aggregates, and the mature fibrils. Particular attention has been focused on the analysis of the structural properties of the oligomeric species, since recent studies have shown that the oligomers formed by lysozyme prior to the appearance of mature amyloid fibrils are toxic to cells. Here, soluble oligomers of human lysozyme have been analyzed by a range of techniques including binding to fluorescent probes such as thioflavin T and 1-anilino-naphthalene-8-sulfonate, Fourier transform infrared spectroscopy, and controlled proteolysis. Oligomers were isolated after 5 days of incubation of the protein and appear as spherical particles with a diameter of 8-17 nm when observed by transmission electron microscopy. Unlike the monomeric protein, oligomers have solvent-exposed hydrophobic patches able to bind the fluorescent probe 1-anilino-naphthalene-8-sulfonate. Fourier transform infrared spectroscopy spectra of oligomers are indicative of misfolded species when compared to monomeric lysozyme, with a prevalence of random structure but with significant elements of the β-sheet structure that is characteristic of the mature fibrils. Moreover, the oligomeric lysozyme aggregates were found to be more susceptible to proteolysis with pepsin than both the monomeric protein and the mature fibrils, indicating further their less organized structure. In summary, this study shows that the soluble lysozyme oligomers are locally unfolded species that are present at low concentration during the initial phases of aggregation. The nonnative conformational features of the lysozyme molecules of which they are composed are likely to be the factors that confer on them the ability to interact inappropriately with a variety of cellular components including membranes.     

Dimerization of Aβ40 inside dipalmitoylphosphatidylcholine bilayer and its effect on bilayer integrity: Atomistic simulation at three temperatures

Amyloid-beta (Aβ) protein is related to Alzheimer disease (AD), and various experiments have shown that oligomers as small as dimers are cytotoxic. Recent studies have concluded that interactions of Aβ with neuronal cell membranes lead to disruption of membrane integrity and toxicity and they play a key role in the development of AD. Molecular dynamics (MD) simulations have been used to investigate Aβ in aqueous solution and membranes. We have previously studied monomeric Aβ40 embedded in dipalmitoylphosphatidylcholine (DPPC) membrane using MD simulations. Here, we explore interactions of two Aβ40 peptides in DPPC bilayer and its consequences on dimer distribution in a lipid bilayer and on the secondary structure of the peptides. We explored that N-terminals played an important role in dimeric Aβ peptide aggregations and Aβ-bilayer interactions, while C-terminals bound peptides to bilayer like anchors. We did not observe exiting of peptides in our simulations although we observed insertion of peptides into the core of bilayer in some of our simulations. So it seems that the presence of Aβ on membrane surface increases its aggregation rate, and as diffusion occurs in two dimensions, it can increase the probability of interpeptide interactions. We found that dimeric Aβ, like monomeric one, had the ability to cause structural destabilization of DPPC membrane, which in turn might ultimately lead to cell death in an in vivo system. This information could have important implications for understanding the affinity of Aβ oligomers (here dimer) for membranes and the mechanism of Aβ oligomer toxicity in AD.