Metal binding modes of Alzheimer's amyloid beta-peptide in insoluble aggregates and soluble complexes

Aggregation of the amyloid beta-peptide (Abeta) into insoluble fibrils is a key pathological event in Alzheimer's disease. Zn(II) induces the Abeta aggregation at acidic-to-neutral pH, while Cu(II) is an effective inducer only at mildly acidic pH. We have examined Zn(II) and Cu(II) binding modes of Abeta and their pH dependence by Raman spectroscopy. The Raman spectra clearly demonstrate that three histidine residues in the N-terminal hydrophilic region provide primary metal binding sites and the solubility of the metal-Abeta complex is correlated with the metal binding mode. Zn(II) binds to the N(tau) atom of the histidine imidazole ring and the peptide aggregates through intermolecular His(N(tau))-Zn(II)-His(N(tau)) bridges. The N(tau)-metal ligation also occurs in Cu(II)-induced Abeta aggregation at mildly acidic pH. At neutral pH, however, Cu(II) binds to N(pi), the other nitrogen of the histidine imidazole ring, and to deprotonated amide nitrogens of the peptide main chain. The chelation of Cu(II) by histidine and main-chain amide groups results in soluble Cu(II)-Abeta complexes. Under normal physiological conditions, Cu(II) is expected to protect Abeta against Zn(II)-induced aggregation by competing with Zn(II) for histidine residues of Abeta.     

Concentration dependent Cu2+ induced aggregation and dityrosine formation of the Alzheimer's disease amyloid-beta peptide

The Amyloid beta peptide (Abeta) of Alzheimer's diseases (AD) is closely linked to the progressive cognitive decline associated with the disease. Cu2+ ions can induce the de novo aggregation of the Abeta peptide into non-amyloidogenic aggregates and the production of a toxic species. The mechanism by which Cu2+ mediates the change from amyloid material toward Cu2+ induced aggregates is poorly defined. Here we demonstrate that the aggregation state of Abeta1-42 at neutral pH is governed by the Cu2+:peptide molar ratio. By probing amyloid content and total aggregation, we observed a distinct Cu2+ switching effect centered at equimolar Cu2+:peptide ratios. At sub-equimolar Cu2+:peptide molar ratios, Abeta1-42 forms thioflavin-T reactive amyloid; conversely, at supra-equimolar Cu2+:peptide molar ratios, Abeta1-42 forms both small spherical oligomers approximately 10-20 nm in size and large amorphous aggregates. We demonstrate that these insoluble aggregates form spontaneously via a soluble species without the presence of an observable lag phase. In seeding experiments, the Cu2+ induced aggregates were unable to influence fibril formation or convert into fibrillar material. Aged Cu2+ induced aggregates are toxic when compared to Abeta1-42 aged in the absence of Cu2+. Importantly, the formation of dityrosine crosslinked Abeta, by the oxidative modification of the peptide, only occurs at equimolar molar ratios and above. The formation of dityrosine adducts occurs following the initiation of aggregation and hence does not drive the formation of the Cu2+ induced aggregates. These results define the role Cu2+ plays in modulating the aggregation state and toxicity of Abeta1-42.     

Metal swap between Zn7-metallothionein-3 and amyloid-beta-Cu protects against amyloid-beta toxicity

Aberrant interactions of copper and zinc ions with the amyloid-beta peptide (Abeta) potentiate Alzheimer's disease (AD) by participating in the aggregation process of Abeta and in the generation of reactive oxygen species (ROS). The ROS production and the neurotoxicity of Abeta are associated with copper binding. Metallothionein-3 (Zn(7)MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain and downregulated in AD. This protein protects, by an unknown mechanism, cultured neurons from the toxicity of Abeta. Here, we show that a metal swap between Zn(7)MT-3 and soluble and aggregated Abeta(1-40)-Cu(II) abolishes the ROS production and the related cellular toxicity. In this process, copper is reduced by the protein thiolates forming Cu(I)(4)Zn(4)MT-3, in which an air-stable Cu(I)(4)-thiolate cluster and two disulfide bonds are present. The discovered protective effect of Zn(7)MT-3 from the copper-mediated Abeta(1-40) toxicity may lead to new therapeutic strategies for treating AD.     

Zinc binding to Alzheimer's Abeta(1-16) peptide results in stable soluble complex

Aggregation of the human amyloid beta-peptide (Abeta) into insoluble plaques is a key event in Alzheimer's disease. Zinc sharply accelerates the Abeta aggregation in vitro, and the Abeta region 6-28 was suggested to be the obligatory zinc binding site. However, time-dependent aggregation of the zinc-bound Abeta species investigated so far prevented their structural analysis. By using CD spectroscopy, we have shown here for the first time that (i) the protected synthetic peptide spanning the fragment 1-16 of Abeta binds specifically zinc with 1:1 and 1:2 stoichiometry under physiologically relevant conditions; (ii) the peptide-zinc complex is soluble and stable for several months; (iii) zinc binding causes a conformational change of the peptide towards a more structured state. These findings suggest the region 1-16 to be the minimal autonomous zinc binding domain of Abeta.     

Gerstmann-Sträussler-Scheinker disease amyloid protein polymerizes according to the "dock-and-lock" model

Prion protein (PrP) amyloid formation is a central feature of genetic and acquired prion diseases such as Gerstmann-Str?ussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob disease. The major component of GSS amyloid is a PrP fragment spanning residues approximately 82-146, which when synthesized as a peptide, readily forms fibrils featuring GSS amyloid. The present study employed surface plasmon resonance (SPR) to characterize the binding events underlying PrP82-146 oligomerization at the first stages of fibrillization, according to evidence suggesting a pathogenic role of prefibrillar oligomers rather than mature amyloid fibrils. We followed in real time the binding reactions occurring during short term (seconds) addition of PrP82-146 small oligomers (1-5-mers, flowing species) onto soluble prefibrillar PrP82-146 aggregates immobilized on the sensor surface. SPR data confirmed very efficient aggregation/elongation, consistent with the hypothesis of nucleation-dependent polymerization process. Much lower binding was observed when PrP82-146 flowed onto the scrambled sequence of PrP82-146 or onto prefibrillar Abeta42 aggregates. As previously found with Abeta40, SPR data could be adequately fitted by equations modeling the "dock-and-lock" mechanism, in which the "locking" step is due to sequential conformational changes, each increasing the affinity of the monomer for the fibril until a condition of irreversible binding is reached. However, these conformational changes (i.e. the locking steps) appear to be faster and easier with PrP82-146 than with Abeta40. Such differences suggest that PrP82-146 has a greater propensity to polymerize and greater stability of the aggregates.     

Examining the zinc binding site of the amyloid-beta peptide.

The amyloid beta-peptide (Abeta) is a principal component of insoluble amyloid plaques which are characteristic neuropathological features of Alzheimer's disease. Abeta also exists as a normal soluble protein that undergoes a pathogenic transition to an aggregated, fibrous form. This transition can be affected by extraneous proteinaceous and nonproteinaceous elements, such as zinc ions, which may promote aggregation and/or stabilization of the fibrils. Protein chelation of zinc is typically mediated by histidines, cysteines and carboxylates. Previous studies have demonstrated that the Abeta-Zn2+ binding site is localized within residues 6-28 and that histidines may serve as the principal sites of interaction. To localize key residues within this region, a series of Abeta peptides (residues 1-28) were synthesized that contained systematic His/Ala substitutions. Circular dichroism and electron microscopy were used to monitor the effects of Zn2+ on the peptide beta-sheet conformation and fibril aggregation. Our results indicate that substitution of either His13 or His14 but not His6 eliminates the zinc-mediated effects. These observations indicate a specific zinc binding site within Abeta that involves these central histidine residues.     

Binding of zinc(II) and copper(II) to the full-length Alzheimer's amyloid-beta peptide

There is evidence that binding of metal ions like Zn2+ and Cu2+ to amyloid beta-peptides (Abeta) may contribute to the pathogenesis of Alzheimer's disease. Cu2+ and Zn2+ form complexes with Abeta peptides in vitro; however, the published metal-binding affinities of Abeta vary in an enormously large range. We studied the interactions of Cu2+ and Zn2+ with monomeric Abeta(40) under different conditions using intrinsic Abeta fluorescence and metal-selective fluorescent dyes. We showed that Cu(2+) forms a stable and soluble 1 : 1 complex with Abeta(40), however, buffer compounds act as competitive copper-binding ligands and affect the apparent K(D). Buffer-independent conditional K(D) for Cu(II)-Abeta(40) complex at pH 7.4 is equal to 0.035 micromol/L. Interaction of Abeta(40) with Zn2+ is more complicated as partial aggregation of the peptide occurs during zinc titration experiment and in the same time period (within 30 min) the initial Zn-Abeta(40) complex (K(D) = 60 micromol/L) undergoes a transition to a more tight complex with K(D) approximately 2 micromol/L. Competition of Abeta(40) with ion-selective fluorescent dyes Phen Green and Zincon showed that the K(D) values determined from intrinsic fluorescence of Abeta correspond to the binding of the first Cu2+ and Zn2+ ions to the peptide with the highest affinity. Interaction of both Zn2+ and Cu2+ ions with Abeta peptides may occur in brain areas affected by Alzheimer's disease and Zn2+-induced transition in the peptide structure might contribute to amyloid plaque formation.     

Amyloid-beta(1-42) rapidly forms protofibrils and oligomers by distinct pathways in low concentrations of sodium dodecylsulfate

Alzheimer's disease (AD) is characterized by large numbers of senile plaques in the brain that consist of fibrillar aggregates of 40- and 42-residue amyloid-beta (Abeta) peptides. However, the degree of dementia in AD correlates better with the concentration of soluble Abeta species assayed biochemically than with histologically determined plaque counts, and several investigators now propose that soluble aggregates of Abeta are the neurotoxic agents that cause memory deficits and neuronal loss. These endogenous aggregates are minor components in brain extracts from AD patients and transgenic mice that express human Abeta, but several species have been detected by gel electrophoresis in sodium dodecylsulfate (SDS) and isolated by size exclusion chromatography (SEC). Endogenous Abeta aggregation is stimulated at cellular interfaces rich in lipid rafts, and anionic micelles that promote Abeta aggregation in vitro may be good models of these interfaces. We previously found that micelles formed in dilute SDS (2 mM) promote Abeta(1-40) fiber formation by supporting peptide interaction on the surface of a single micelle complex. In contrast, here we report that monomeric Abeta(1-42) undergoes an immediate conversion to a predominant beta-structured conformation in 2 mM SDS which does not proceed to amyloid fibrils. The conformational change is instead rapidly followed by the near quantitative conversion of the 4 kDa monomer SDS gel band to 8-14 kDa bands consistent with dimers through tetramers. Removal of SDS by dialysis gave a shift in the predominant SDS gel bands to 30-60 kDa. While these oligomers resemble the endogenous aggregates, they are less stable. In particular, they do not elute as discrete species on SEC, and they are completed disaggregated by boiling in 1% SDS. It appears that endogenous oligomeric Abeta aggregates are stabilized by undefined processes that have not yet been incorporated into in vitro Abeta aggregation procedures.     

Interaction between amyloid beta-protein aggregates and membranes.

The conversion of soluble, nontoxic amyloid beta-protein (Abeta) to aggregated, toxic Abeta rich in beta-sheet structures is considered to be the key step in the development of Alzheimer's disease. Therefore, extensive studies have been carried out on the mechanisms involved in Abeta aggregation and the characterization of Abeta aggregates formed in aqueous solutions mimicking biological fluids. On the other hand, several investigators pointed out that membranes play an important role in Abeta aggregation. However, it remains unclear whether Abeta aggregates formed in solution and membranes are identical and whether the former can bind to membranes. In this study, using a dye-labeled Abeta-(1-40) as well as native Abeta-(1-40), the properties of Abeta aggregates formed in buffer and raft-like membranes composed of monosialoganglioside GM1/cholesterol/sphingomyelin were compared. Fourier transform infrared spectroscopic measurements suggested that Abeta aggregates formed in buffer and in membranes have different beta-sheet structures. Fluorescence experiments revealed that Abeta aggregated in buffer did not show any affinity for membranes.     

Secondary structure and interfacial aggregation of amyloid-beta(1-40) on sodium dodecyl sulfate micelles

Alzheimer's disease (AD) is characterized by the presence of large numbers of fibrillar amyloid deposits in the form of senile plaques in the brain. The fibrils in senile plaques are composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that fibrillar Abeta and especially soluble Abeta aggregates are important in the pathogenesis of AD, and many laboratories have investigated soluble Abeta aggregates generated from monomeric Abeta in vitro. Of these in vitro aggregates, the best characterized are called protofibrils. They are composed of globules and short rods, show primarily beta-structure by circular dichroism (CD), enhance the fluorescence of bound thioflavin T, and readily seed the growth of long fibrils. However, one difficulty in correlating soluble Abeta aggregates formed in vitro with those in vivo is the high probability that cellular interfaces affect the aggregation rates and even the aggregate structures. Reports that focus on the features of interfaces that are important in Abeta aggregation have found that amphiphilic interactions and micellar-like Abeta structures may play a role. We previously described the formation of Abeta(1-40) aggregates at polar-nonpolar interfaces, including those generated at microdroplets formed in dilute hexafluoro-2-propanol (HFIP). Here we compared the Abeta(1-40) aggregates produced on sodium dodecyl sulfate (SDS) micelles, which may be a better model of biological membranes with phospholipids that have anionic headgroups. At both HFIP and SDS interfaces, changes in peptide secondary structure were observed by CD immediately when Abeta(1-40) was introduced. With HFIP, the change involved an increase in predominant beta-structure content and in fluorescence with thioflavin T, while with SDS, a partial alpha-helical conformation was adopted that gave no fluorescence. However, in both systems, initial amorphous clustered aggregates progressed to soluble fibers rich in beta-structure over a roughly 2 day period. Fiber formation was much faster than in the absence of an interface, presumably because of the close intermolecular proximity of peptides at the interfaces. While these fibers resembled protofibrils, they failed to seed the aggregation of Abeta(1-40) monomers effectively.     

Oxidative metabolites accelerate Alzheimer's amyloidogenesis by a two-step mechanism, eliminating the requirement for nucleation

The process of amyloid formation by the amyloid beta peptide (Abeta), i.e., the misassembly of Abetapeptides into soluble quaternary structures and, ultimately, amyloid fibrils, appears to be at the center of Alzheimer's disease (AD) pathology. We have shown that abnormal oxidative metabolites, including cholesterol-derived aldehydes, modify Abeta and accelerate the early stages of amyloidogenesis (the formation of spherical aggregates). This process, which we have termed metabolite-initiated protein misfolding, could explain why hypercholesterolemia and inflammation are risk factors for sporadic AD. Herein, the mechanism by which cholesterol metabolites hasten Abeta 1-40 amyloidogenesis is explored, revealing a process that has at least two steps. In the first step, metabolites modify Abeta peptides by Schiff base formation. The Abeta-metabolite adducts form spherical aggregates by a downhill polymerization that does not require a nucleation step, dramatically accelerating Abeta aggregation. In agitated samples, a second step occurs in which fibrillar aggregates form, a step also accelerated by cholesterol metabolites. However, the metabolites do not affect the rate of fibril growth in seeded aggregation assays; their role appears to be in initiating amyloidogenesis by lowering the critical concentration for aggregation into the nanomolar range. Small molecules that block Schiff base formation inhibit the metabolite effect, demonstrating the importance of the covalent adduct. Metabolite-initiated amyloidogenesis offers an explanation for how Abeta aggregation could occur at physiological nanomolar concentrations.     

Copper selectively triggers beta-sheet assembly of an N-terminally truncated amyloid beta-peptide beginning with Glu3

Metal ions have been suggested to induce aggregation of amyloid beta-peptide (Abeta), which is a key event in Alzheimer's disease. However, direct evidence that specific metal-peptide interactions are responsible for the amyloid formation has not previously been provided. Here we present the first example of the metal-induced amyloid formation by an Abeta fragment, which exhibits a clear-cut dependence on the amino acid sequence. A heptapeptide, EFRHDSG, corresponding to the amino acid residues 3-9 of Abeta (Abeta(3-9)) undergoes a conformational transition from irregular to beta-sheet and self-associates into insoluble aggregates upon Cu(II) binding. A Raman spectrum analysis of the Cu(II)-Abeta(3-9) complex and aggregation assays of mutated Abeta(3-9) peptides demonstrated that a concerted Cu(II) coordination of the imidazole side chain of His6, the carboxyl groups of Glu3 and Asp7, and the amino group at the N-terminus is essential for the amyloid formation. Although Abeta(1-9) and Abeta(2-9) also contain the metal binding sites, neither of these peptides forms amyloid depositions in the presence of Cu(II). The results of this study may not only provide new insight into the mechanism of amyloid formation, but also be important as a step toward the construction of proteinaceous materials with a specific function under the control of Cu(II).     

Neurotoxicity of acetylcholinesterase amyloid beta-peptide aggregates is dependent on the type of Abeta peptide and the AChE concentration present in the complexes.

Alzheimer's disease (AD) is a neurodegenerative disorder whose hallmark is the presence of senile plaques and neurofibrillary tangles. Senile plaques are mainly composed of amyloid beta-peptide (Abeta) fibrils and several proteins including acetylcholinesterase (AChE). AChE has been previously shown to stimulate the aggregation of Abeta1-40 into amyloid fibrils. In the present work, the neurotoxicity of different amyloid aggregates formed in the absence or presence of AChE was evaluated in rat pheochromocytoma PC12 cells. Stable AChE-Abeta complexes were found to be more toxic than those formed without the enzyme, for Abeta1-40 and Abeta1-42, but not for amyloid fibrils formed with AbetaVal18-Ala, a synthetic variant of the Abeta1-40 peptide. Of all the AChE-Abeta complexes tested the one containing the Abeta1-40 peptide was the most toxic. When increasing concentrations of AChE were used to aggregate the Abeta1-40 peptide, the neurotoxicity of the complexes increased as a function of the amount of enzyme bound to each complex. Our results show that AChE-Abeta1-40 aggregates are more toxic than those of AChE-Abeta1-42 and that the neurotoxicity depends on the amount of AChE bound to the complexes, suggesting that AChE may play a key role in the neurodegeneration observed in Alzheimer brain.     

Metal ion-dependent effects of clioquinol on the fibril growth of an amyloid {beta} peptide

Although metal ions such as Cu(2+), Zn(2+), and Fe(3+) are implicated to play a key role in Alzheimer disease, their role is rather complex, and comprehensive understanding is not yet obtained. We show that Cu(2+) and Zn(2+) but not Fe(3+) renders the amyloid beta peptide, Abeta(1-40), nonfibrillogenic in nature. However, preformed fibrils of Abeta(1-40) were stable when treated with these metal ions. Consequently, fibril growth of Abeta(1-40) could be switched on/off by switching the molecule between its apo- and holo-forms. Clioquinol, a potential drug for Alzheimer disease, induced resumption of the Cu(2+)-suppressed but not the Zn(2+)-suppressed fibril growth of Abeta(1-40). The observed synergistic effect of clioquinol and Zn(2+) suggests that Zn(2+)-clioquinol complex effectively retards fibril growth. Thus, clioquinol has dual effects; although it disaggregates the metal ion-induced aggregates of Abeta(1-40) through metal chelation, it further retards the fibril growth along with Zn(2+). These results indicate the mechanism of metal ions in suppressing Abeta amyloid formation, as well as providing information toward the use of metal ion chelators, particularly clioquinol, as potential drugs for Alzheimer disease.     

Amyloid-beta protofibrils differ from amyloid-beta aggregates induced in dilute hexafluoroisopropanol in stability and morphology

The brains of Alzheimer's disease (AD) patients contain large numbers of amyloid plaques that are rich in fibrils composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that fibrillar Abeta and especially soluble Abeta aggregates are important in the etiology of AD. Recent reports also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we demonstrate that Abeta-(1-40) can form soluble aggregates with predominant beta-structures that differ in stability and morphology. One class of aggregates involved soluble Abeta protofibrils, prepared by vigorous overnight agitation of monomeric Abeta-(1-40) at low ionic strength. Dilution of these aggregation reactions induced disaggregation to monomers as measured by size exclusion chromatography. Protofibril concentrations monitored by thioflavin T fluorescence decreased in at least two kinetic phases, with initial disaggregation (rate constant approximately 1 h(-1)) followed by a much slower secondary phase. Incubation of the reactions without agitation resulted in less disaggregation at slower rates, indicating that the protofibrils became progressively more stable over time. In fact, protofibrils isolated by size exclusion chromatography were completely stable and gave no disaggregation. A second class of soluble Abeta aggregates was generated rapidly (<10 min) in buffered 2% hexafluoroisopropanol (HFIP). These aggregates showed increased thioflavin T fluorescence and were rich in beta-structure by circular dichroism. Electron microscopy and atomic force microscopy revealed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP, these aggregates initially were very unstable and disaggregated completely within 2 min. However, their stability increased as they progressed to fibers. Relative to Abeta protofibrils, the HFIP-induced aggregates seeded elongation by Abeta monomer deposition very poorly. The techniques used to distinguish these two classes of soluble Abeta aggregates may be useful in characterizing Abeta aggregates formed in vivo.     

Copper and zinc binding modulates the aggregation and neurotoxic properties of the prion peptide PrP106-126.

The abnormal form of the prion protein (PrP) is believed to be responsible for the transmissible spongiform encephalopathies. A peptide encompassing residues 106-126 of human PrP (PrP106-126) is neurotoxic in vitro due its adoption of an amyloidogenic fibril structure. The Alzheimer's disease amyloid beta peptide (Abeta) also undergoes fibrillogenesis to become neurotoxic. Abeta aggregation and toxicity is highly sensitive to copper, zinc, or iron ions. We show that PrP106-126 aggregation, as assessed by turbidometry, is abolished in Chelex-100-treated buffer. ICP-MS analysis showed that the Chelex-100 treatment had reduced Cu(2+) and Zn(2+) levels approximately 3-fold. Restoring Cu(2+) and Zn(2+) to their original levels restored aggregation. Circular dichroism showed that the Chelex-100 treatment reduced the aggregated beta-sheet content of the peptide. Electron paramagnetic resonance spectroscopy identified a 2N1S1O coordination to the Cu(2+) atom, suggesting histidine 111 and methionine 109 or 112 are involved. Nuclear magnetic resonance confirmed Cu(2+) and Zn(2+) binding to His-111 and weaker binding to Met-112. An N-terminally acetylated PrP106-126 peptide did not bind Cu(2+), implicating the free amino group in metal binding. Mutagenesis of either His-111, Met-109, or Met-112 abolished PrP106-126 neurotoxicity and its ability to form fibrils. Therefore, Cu(2+) and/or Zn(2+) binding is critical for PrP106-126 aggregation and neurotoxicity.     

Rapid assembly of amyloid-beta peptide at a liquid/liquid interface produces unstable beta-sheet fibers

Accumulation of aggregated amyloid-beta peptide (Abeta) in the brain is a pathological hallmark of Alzheimer's disease (AD). In vitro studies indicate that the 40- to 42-residue Abeta peptide in solution will undergo self-assembly leading to the transient appearance of soluble protofibrils and ultimately to insoluble fibrils. The Abeta peptide is amphiphilic and accumulates preferentially at a hydrophilic/hydrophobic interface. Solid surfaces and air-water interfaces have been shown previously to promote Abeta aggregation, but detailed characterization of these aggregates has not been presented. In this study Abeta(1-40) introduced to aqueous buffer in a two-phase system with chloroform aggregated 1-2 orders of magnitude more rapidly than Abeta in the buffer alone. The interface-induced aggregates were released into the aqueous phase and persisted for 24-72 h before settling as a visible precipitate at the interface. Thioflavin T fluorescence and circular dichroism analyses confirmed that the Abeta aggregates had a beta-sheet secondary structure. However, these aggregates were far less stable than Abeta(1-40) protofibrils prepared in buffer alone and disaggregated completely within 3 min on dilution. Atomic force microscopy revealed that the aggregates consisted of small globules 4-5 nm in height and long flexible fibers composed of these globules aligned roughly along a longitudinal axis, a morphology distinct from that of Abeta protofibrils prepared in buffer alone. The relative instability of the fibers was supported by fiber interruptions apparently introduced by brief washing of the AFM grids. To our knowledge, unstable aggregates of Abeta with beta-sheet structure and fibrous morphology have not been reported previously. Our results provide the clearest evidence yet that the intrinsic beta-sheet structure of an in vitro Abeta aggregate depends on the aggregation conditions and is reflected in the stability of the aggregate and the morphology observed by atomic force microscopy. Resolution of these structural differences at the molecular level may provide important clues to the further understanding of amyloid formation in vivo.     

Characterization of copper interactions with alzheimer amyloid beta peptides: identification of an attomolar-affinity copper binding site on amyloid beta1-42

Cu and Zn have been shown to accumulate in the brains of Alzheimer's disease patients. We have previously reported that Cu(2+) and Zn(2+) bind amyloid beta (Abeta), explaining their enrichment in plaque pathology. Here we detail the stoichiometries and binding affinities of multiple cooperative Cu(2+)-binding sites on synthetic Abeta1-40 and Abeta1-42. We have developed a ligand displacement technique (competitive metal capture analysis) that uses metal-chelator complexes to evaluate metal ion binding to Abeta, a notoriously self-aggregating peptide. This analysis indicated that there is a very-high-affinity Cu(2+)-binding site on Abeta1-42 (log K(app) = 17.2) that mediates peptide precipitation and that the tendency of this peptide to self-aggregate in aqueous solutions is due to the presence of trace Cu(2+) contamination (customarily approximately 0.1 microM). In contrast, Abeta1-40 has much lower affinity for Cu(2+) at this site (estimated log K(app) = 10.3), explaining why this peptide is less self-aggregating. The greater Cu(2+)-binding affinity of Abeta1-42 compared with Abeta1-40 is associated with significantly diminished negative cooperativity. The role of trace metal contamination in inducing Abeta precipitation was confirmed by the demonstration that Abeta peptide (10 microM) remained soluble for 5 days only in the presence of high-affinity Cu(2+)-selective chelators.     

The ratio of monomeric to aggregated forms of Abeta40 and Abeta42 is an important determinant of amyloid-beta aggregation, fibrillogenesis, and toxicity

Aggregation and fibril formation of amyloid-beta (Abeta) peptides Abeta40 and Abeta42 are central events in the pathogenesis of Alzheimer disease. Previous studies have established the ratio of Abeta40 to Abeta42 as an important factor in determining the fibrillogenesis, toxicity, and pathological distribution of Abeta. To better understand the molecular basis underlying the pathologic consequences associated with alterations in the ratio of Abeta40 to Abeta42, we probed the concentration- and ratio-dependent interactions between well defined states of the two peptides at different stages of aggregation along the amyloid formation pathway. We report that monomeric Abeta40 alters the kinetic stability, solubility, and morphological properties of Abeta42 aggregates and prevents their conversion into mature fibrils. Abeta40, at approximately equimolar ratios (Abeta40/Abeta42 approximately 0.5-1), inhibits (> 50%) fibril formation by monomeric Abeta42, whereas inhibition of protofibrillar Abeta42 fibrillogenesis is achieved at lower, substoichiometric ratios (Abeta40/Abeta42 approximately 0.1). The inhibitory effect of Abeta40 on Abeta42 fibrillogenesis is reversed by the introduction of excess Abeta42 monomer. Additionally, monomeric Abeta42 and Abeta40 are constantly recycled and compete for binding to the ends of protofibrillar and fibrillar Abeta aggregates. Whereas the fibrillogenesis of both monomeric species can be seeded by fibrils composed of either peptide, Abeta42 protofibrils selectively seed the fibrillogenesis of monomeric Abeta42 but not monomeric Abeta40. Finally, we also show that the amyloidogenic propensities of different individual and mixed Abeta species correlates with their relative neuronal toxicities. These findings, which highlight specific points in the amyloid peptide equilibrium that are highly sensitive to the ratio of Abeta40 to Abeta42, carry important implications for the pathogenesis and current therapeutic strategies of Alzheimer disease.     

Secondary structure conversions of Alzheimer's Abeta(1-40) peptide induced by membrane-mimicking detergents

The amyloid beta peptide (Abeta) with 39-42 residues is the major component of amyloid plaques found in brains of Alzheimer's disease patients, and soluble oligomeric peptide aggregates mediate toxic effects on neurons. The Abeta aggregation involves a conformational change of the peptide structure to beta-sheet. In the present study, we report on the effect of detergents on the structure transitions of Abeta, to mimic the effects that biomembranes may have. In vitro, monomeric Abeta(1-40) in a dilute aqueous solution is weakly structured. By gradually adding small amounts of sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate to a dilute aqueous solution, Abeta(1-40) is converted to beta-sheet, as observed by CD at 3 degrees C and 20 degrees C. The transition is mainly a two-state process, as revealed by approximately isodichroic points in the titrations. Abeta(1-40) loses almost all NMR signals at dodecyl sulfate concentrations giving rise to the optimal beta-sheet content (approximate detergent/peptide ratio = 20). Under these conditions, thioflavin T fluorescence measurements indicate a maximum of aggregated amyloid-like structures. The loss of NMR signals suggests that these are also involved in intermediate chemical exchange. Transverse relaxation optimized spectroscopy NMR spectra indicate that the C-terminal residues are more dynamic than the others. By further addition of SDS or lithium dodecyl sulfate reaching concentrations close to the critical micellar concentration, CD, NMR and FTIR spectra show that the peptide rearranges to form a micelle-bound structure with alpha-helical segments, similar to the secondary structures formed when a high concentration of detergent is added directly to the peptide solution.