An intersheet packing interaction in A beta fibrils mapped by disulfide cross-linking

Most models for the central cross-beta folding unit in amyloid fibrils of the Alzheimer's plaque protein Abeta align the peptides in register in H-bonded, parallel beta-sheet structure. Some models require the Abeta peptide to undergo a chain reversal when folding into the amyloid core, while other models feature very long extended chains, or zigzag chains, traversing the protofilament. In this paper we introduce the use of disulfide bond cross-linking to probe the fold within the core and the packing interactions between beta-sheets. In one approach, amyloid fibrils grown under reducing conditions from each of three double cysteine mutants (17/34, 17/35, and 17/36) of the Abeta(1-40) sequence were subjected to oxidizing conditions. Of these three mutants, only the Leu17Cys/Leu34Cys peptide could be cross-linked efficiently while resident in fibrils. In another approach, double Cys mutants were cross-linked as monomers before aggregation, and the resulting fibrils were assessed for stability, antibody binding, dye binding, and cross-seeding efficiency. Here too, fibrils from the 17/34 double Cys mutant most closely resemble wild-type Abeta(1-40) fibrils. These data support models of the Abeta fibril in which the Leu17 and Leu34 side chains of the same peptide pack against each other at the beta-sheet interface within the amyloid core. Related cross-linking strategies may reveal longer range spatial relationships. The ability of the cross-linked 17/35 double Cys mutant Abeta to also make amyloid fibrils illustrates a remarkable plasticity of the amyloid structure and suggests a structural mechanism for the generation of conformational variants of amyloid.     

Structure and orientation of peptide inhibitors bound to beta-amyloid fibrils

Polymerization of the soluble beta-amyloid peptide into highly ordered fibrils is hypothesized to be a causative event in the development of Alzheimer's disease. Understanding the interactions of Abeta with inhibitors on an atomic level is fundamental for the development of diagnostics and therapeutic approaches, and can provide, in addition, important indirect information of the amyloid fibril structure. We have shown recently that trRDCs can be measured in solution state NMR for peptide ligands binding weakly to amyloid fibrils. We present here the structures for two inhibitor peptides, LPFFD and DPFFL, and their structural models bound to fibrillar Abeta(14-23) and Abeta(1-40) based on transferred nuclear Overhauser effect (trNOE) and transferred residual dipolar coupling (trRDC) data. In a first step, the inhibitor peptide structure is calculated on the basis of trNOE data; the trRDC data are then validated on the basis of the trNOE-derived structure using the program PALES. The orientation of the peptide inhibitors with respect to Abeta fibrils is obtained from trRDC data, assuming that Abeta fibrils orient such that the fibril axis is aligned in parallel with the magnetic field. The trRDC-derived alignment tensor of the peptide ligand is then used as a restraint for molecular dynamics docking studies. We find that the structure with the lowest rmsd value is in agreement with a model in which the inhibitor peptide binds to the long side of an amyloid fibril. Especially, we detect interactions involving the hydrophobic core, residues K16 and E22/D23 of the Abeta sequence. Structural differences are observed for binding of the inhibitor peptide to Abeta14-23 and Abeta1-40 fibrils, respectively, indicating different fibril structure. We expect this approach to be useful in the rational design of amyloid ligands with improved binding characteristics.     

Probing the role of backbone hydrogen bonding in beta-amyloid fibrils with inhibitor peptides containing ester bonds at alternate positions

Protein-protein interactions are frequently mediated by stable, intermolecular beta-sheets. A number of cytokines and the HIV Protease, for example, dimerize through beta-sheet motifs. Evidence also suggests that the macromolecular assemblies of peptides and proteins in amyloid fibrils are stabilized by intermolecular beta-sheets. In this paper, we report that interfering with the backbone hydrogen bonding of an amyloidgenic peptide (Abeta16-20) by replacing amide bonds with ester bonds prevents the aggregation of the peptide. The ester bonds were incorporated in an alternating fashion so that the peptide presents two unique hydrogen bonding faces when arrayed in an extended, beta-strand conformation; one face of the peptide has normal hydrogen bonding capabilities, but the other face is missing amide protons and its ability to hydrogen bond is severely limited. Analytical ultracentrifugation experiments demonstrate that this ester peptide, Abeta16-20e, is predominantly monomeric under solution conditions, unlike the fibril-forming Abeta16-20 peptide. Abeta16-20e also inhibits the aggregation of the Abeta1-40 peptide and disassembles preformed Abeta1-40 fibrils. These results suggest that backbone hydrogen bonding is critical for the assembly of amyloid fibrils.     

Direct observation of Abeta amyloid fibril growth and inhibition

Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.     

Supramolecular structural constraints on Alzheimer's beta-amyloid fibrils from electron microscopy and solid-state nuclear magnetic resonance

We describe electron microscopy (EM), scanning transmission electron microscopy (STEM), and solid-state nuclear magnetic resonance (NMR) measurements on amyloid fibrils formed by the 42-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta(1)(-)(42)) and by residues 10-35 of the full-length peptide (Abeta(10)(-)(35)). These measurements place constraints on the supramolecular structure of the amyloid fibrils, especially the type of beta-sheets present in the characteristic amyloid cross-beta structural motif and the assembly of these beta-sheets into a fibril. EM images of negatively stained Abeta(10)(-)(35) fibrils and measurements of fibril mass per length (MPL) by STEM show a strong dependence of fibril morphology and MPL on pH. Abeta(10)(-)(35) fibrils formed at pH 3.7 are single "protofilaments" with MPL equal to twice the value expected for a single cross-beta layer. Abeta(10)(-)(35) fibrils formed at pH 7.4 are apparently pairs of protofilaments or higher order bundles. EM and STEM data for Abeta(1)(-)(42) fibrils indicate that protofilaments with MPL equal to twice the value expected for a single cross-beta layer are also formed by Abeta(1)(-)(42) and that these protofilaments exist singly and in pairs at pH 7.4. Solid-state NMR measurements of intermolecular distances in Abeta(10)(-)(35) fibrils, using multiple-quantum (13)C NMR, (13)C-(13)C dipolar recoupling, and (15)N-(13)C dipolar recoupling techniques, support the in-register parallel beta-sheet organization previously established by Lynn, Meredith, Botto, and co-workers [Benzinger et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13407-13412; Benzinger et al. (2000) Biochemistry 39, 3491-3499] and show that this beta-sheet organization is present at pH 3.7 as well as pH 7.4 despite the differences in fibril morphology and MPL. Solid-state NMR measurements of intermolecular distances in Abeta(1)(-)(42) fibrils, which represent the first NMR data on Abeta(1)(-)(42) fibrils, also indicate an in-register parallel beta-sheet organization. These results, along with previously reported data on Abeta(1)(-)(40) fibrils, suggest that the supramolecular structures of Abeta(10)(-)(35), Abeta(1)(-)(40), and Abeta(1)(-)(42) fibrils are quite similar. A schematic structural model of these fibrils, consistent with known experimental EM, STEM, and solid-state NMR data, is presented.     

Seeding specificity in amyloid growth induced by heterologous fibrils

Over residues 15-36, which comprise the H-bonded core of the amyloid fibrils it forms, the Alzheimer's disease plaque peptide amyloid beta (Abeta) possesses a very similar sequence to that of another short, amyloidogenic peptide, islet amyloid polypeptide (IAPP). Using elongation rates to quantify seeding efficiency, we inquired into the relationship between primary sequence similarity and seeding efficiency between Abeta-(1-40) and amyloid fibrils produced from IAPP as well as other proteins. In both a solution phase and a microtiter plate elongation assay, IAPP fibrils are poor seeds for Abeta-(1-40) elongation, exhibiting weight-normalized efficiencies of only 1-2% compared with Abeta-(1-40) fibrils. Amyloid fibrils of peptides with sequences completely unrelated to Abeta also exhibit poor to negligible seeding ability for Abeta elongation. Fibrils from a number of point mutants of Abeta-(1-40) exhibit intermediate seeding abilities for wild-type Abeta elongation, with differing efficiencies depending on whether or not the mutation is in the amyloid core region. The results suggest that amyloid fibrils from different proteins exhibit structural differences that control seeding efficiencies. Preliminary results also suggest that identical sequences can grow into different conformations of amyloid fibrils as detected by seeding efficiencies. The results have a number of implications for amyloid structure and biology.     

Structure and texture of fibrous crystals formed by Alzheimer's abeta(11-25) peptide fragment

Amyloid fibril deposition is central to the pathology of Alzheimer's disease. X-ray diffraction from amyloid fibrils formed from full-length Abeta(1-40) and from a shorter fragment, Abeta(11-25), have revealed cross-beta diffraction fingerprints. Magnetic alignment of Abeta(11-25) amyloid fibrils gave a distinctive X-ray diffraction texture, allowing interpretation of the diffraction data and a model of the arrangement of the peptides within the amyloid fiber specimen to be constructed. An intriguing feature of the structure of fibrillar Abeta(11-25) is that the beta sheets, of width 5.2 nm, stack by slipping relative to each other by the length of two amino acid units (0.70 nm) to form beta ribbons 4.42 nm in thickness. Abeta(1-40) amyloid fibrils likely consist of once-folded hairpins, consistent with the size of the fibers obtained using electron microscopy and X-ray diffraction.     

Structural and dynamic features of Alzheimer's Abeta peptide in amyloid fibrils studied by site-directed spin labeling

Electron paramagnetic resonance spectroscopy analysis of 19 spin-labeled derivatives of the Alzheimer's amyloid beta (Abeta) peptide was used to reveal structural features of amyloid fibril formation. In the fibril, extensive regions of the peptide show an in-register, parallel arrangement. Based on the parallel arrangement and side chain mobility analysis we find the amyloid structure to be mostly ordered and specific, but we also identify more dynamic regions (N and C termini) and likely turn or bend regions (around residues 23-26). Despite their different aggregation properties and roles in disease, the two peptides, Abeta40 and Abeta42, homogeneously co-mix in amyloid fibrils suggesting that they possess the same structural architecture.     

Mapping abeta amyloid fibril secondary structure using scanning proline mutagenesis

Although the amyloid fibrils formed from the Alzheimer's disease amyloid peptide Abeta are rich in cross-beta sheet, the peptide likely also exhibits turn and unstructured regions when it becomes incorporated into amyloid. We generated a series of single-proline replacement mutants of Abeta(1-40) and determined the thermodynamic stabilities of amyloid fibrils formed from these mutants to characterize the susceptibility of different residue positions of the Abeta sequence to proline substitution. The results suggest that the Abeta peptide, when engaged in the amyloid fibril, folds into a conformation containing three highly structured segments, consisting of contiguous sequence elements 15-21, 24-28, and 31-36, that are sensitive to proline replacement and likely to include the beta-sheet portions of the fibrils. Residues relatively insensitive to proline replacement fall into two groups: (a) residues 1-14 and 37-40 are likely to exist in relatively unstructured, flexible elements extruded from the beta-sheet-rich amyloid core; (b) residues 22, 23, 29 and 30 are likely to occupy turn positions between these three structured elements. Although destabilized, fibrils formed from Abeta(1-40) proline mutants are very similar in structure to wild-type fibrils, as indicated by hydrogen-deuterium exchange and other analysis. Interestingly, however, some proline mutations destabilize fibrils while at the same time increasing the number of amide protons protected from hydrogen exchange. This suggests that the stability of amyloid fibrils, rather than being driven exclusively by the formation of H-bonded beta-sheet, is achieved, as in globular proteins, through a balance of stabilizing and destabilizing forces. The proline scanning data are most compatible with a model for amyloid protofilament structure loosely resembling the parallel beta-helix folding motif, such that each Abeta(15-36) core region occupies a single layer of a prismatic, H-bonded stack of peptides.     

Biophysical characterization of longer forms of amyloid beta peptides: possible contribution to flocculent plaque formation

Alzheimer's disease is characterized by amyloid deposits in the parenchyma and vasculature of the brain. The plaques are mainly composed of amyloid beta (Abeta) peptides ending in residues 40 and 42. Novel longer Abeta peptides were found in brain homogenates of mouse models of Alzheimer's disease and human brain tissue of patients carrying the familial amyloid precursor protein V717F mutation. The biophysical characteristics of these longer Abeta peptides and their role in plaque formation are not understood. We chose to focus our studies on Abeta peptides ending in residues Ile45, Val46 and Ile47 as these peptides were identified in human brain tissue. A combination of circular dichroism and electron microscopy was used to characterize the secondary and tertiary structures of these peptides. All three longer Abeta peptides consisted mainly of a beta-sheet secondary structure. Electron microscopy demonstrated that these beta-structured peptides formed predominantly amorphous aggregates, which convert to amyloid fibres over extended time periods. As these longer peptides may act as seeds for the nucleation of fibrils composed predominantly of shorter amyloid peptides, these interactions were studied. All peptides accelerated the random to beta-structural transitions and fibril formation of Abeta40 and 42.     

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.     

A conformation change in the carboxyl terminus of Alzheimer's Abeta (1-40) accompanies the transition from dimer to fibril as revealed by fluorescence quenching analysis

Alzheimer's disease is characterized by the presence of insoluble, fibrous deposits composed principally of amyloid beta (Abeta) peptide. A number of studies have provided information on the fibril structure and on the factors affecting fiber formation, but the details of the fibril structure are not known. We used fluorescence quenching to investigate the solvent accessibility and surface charge of the soluble Abeta(1-40) dimer and amyloid fibrils. Analogs of Abeta(1-40) containing a single tryptophan were synthesized by substituting residues at positions 4, 10, 34, and 40 with tryptophan. Quenching measurements in the dimeric state indicate that the amino-terminal analogs (AbetaF4W and AbetaY10W) are accessible to polar quenchers, and the more carboxyl-terminal analog AbetaV34W is less accessible. AbetaV40W, on the other hand, exhibits a low degree of quenching, indicating that this residue is highly shielded from the solvent in the dimeric state. Correcting for the effect of reduced translational and rotational diffusion, fibril formation was associated with a selective increase in solvent exposure of residues 34 and 40, suggesting that a conformation change may take place in the carboxyl-terminal region coincident with the dimer to fibril transition.     

Reversible mechanical unzipping of amyloid beta-fibrils

Amyloid fibrils are self-associating filamentous structures, the deposition of which is considered to be one of the most important factors in the pathogenesis of Alzheimer's disease and various other disorders. Here we used single molecule manipulation methods to explore the mechanics and structural dynamics of amyloid fibrils. In mechanically manipulated amyloid fibrils, formed from either amyloid beta (Abeta) peptides 1-40 or 25-35, beta-sheets behave as elastic structures that can be "unzipped" from the fibril with constant forces. The unzipping forces were different for Abeta1-40 and Abeta25-35. Unzipping was fully reversible across a wide range of stretch rates provided that coupling, via the beta-sheet, between bound and dissociated states was maintained. The rapid, cooperative zipping together of beta-sheets could be an important mechanism behind the self-assembly of amyloid fibrils. The repetitive force patterns contribute to a mechanical fingerprint that could be utilized in the characterization of different amyloid fibrils.     

Experimental constraints on quaternary structure in Alzheimer's beta-amyloid fibrils

We describe solid-state nuclear magnetic resonance (NMR) measurements on fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta(1-40)) that place constraints on the identity and symmetry of contacts between in-register, parallel beta-sheets in the fibrils. We refer to these contacts as internal and external quaternary contacts, depending on whether they are within a single molecular layer or between molecular layers. The data include (1) two-dimensional 13C-13C NMR spectra that indicate internal quaternary contacts between side chains of L17 and F19 and side chains of I32, L34, and V36, as well as external quaternary contacts between side chains of I31 and G37; (2) two-dimensional 15N-13C NMR spectra that indicate external quaternary contacts between the side chain of M35 and the peptide backbone at G33; (3) measurements of magnetic dipole-dipole couplings between the side chain carboxylate group of D23 and the side chain amine group of K28 that indicate salt bridge interactions. Isotopic dilution experiments allow us to make distinctions between intramolecular and intermolecular contacts. On the basis of these data and previously determined structural constraints from solid-state NMR and electron microscopy, we construct full molecular models using restrained molecular dynamics simulations and restrained energy minimization. These models apply to Abeta(1-40) fibrils grown with gentle agitation. We also present evidence for different internal quaternary contacts in Abeta(1-40) fibrils grown without agitation, which are morphologically distinct.     

Structural differences in Abeta amyloid protofibrils and fibrils mapped by hydrogen exchange--mass spectrometry with on-line proteolytic fragmentation

We report here structural differences between Abeta(1-40) protofibrils and mature amyloid fibrils associated with Alzheimer's disease as determined using hydrogen-deuterium exchange-mass spectrometry (HX-MS) coupled with on-line proteolysis. Specifically, we have identified regions of the Abeta(1-40) peptide containing backbone amide hydrogen atoms that are protected from HX or exposed when this peptide is incorporated into protofibrils or amyloid fibrils formed in phosphate-buffered saline without stirring at 37 degrees C. Study of protofibrils was facilitated by use of the protofibril-stabilizing agent calmidazolium chloride. Our data clearly show that both the C-terminal segment 35-40 and the N-terminal segment 1-19 are highly exposed to HX in both fibrils and protofibrils. In contrast, the internal fragment 20-34 is highly protected from exchange in fibrils but much less so in protofibrils. The data suggest that the beta-sheet elements comprising the amyloid fibril are already present in protofibrils, but that they are expanded into some adjacent residues upon the formation of mature amyloid. The N-terminal approximately ten residues appear to be unstructured in both protofibrils and fibrils. The 20-30 segment of Abeta(1-40) is more ordered in fibrils than in protofibrils, suggesting that, if protofibrils are a mechanistic precursor of fibrils, the transition from protofibril to fibril involves substantial ordering of this region of the Abeta peptide.     

Cupric-amyloid beta peptide complex stimulates oxidation of ascorbate and generation of hydroxyl radical

A growing body of evidence supports an important role for oxidative stress in the pathogenesis of Alzheimer's disease. Recently, a number of papers have shown a synergistic neurotoxicity of amyloid beta peptide and cupric ions. We hypothesized that complexes of cupric ions with neurotoxic amyloid beta peptides (Abeta) can stimulate copper-mediated free radical formation. We found that neurotoxic Abeta (1-42), Abeta (1-40), and Abeta (25-35) stimulated copper-mediated oxidation of ascorbate, whereas nontoxic Abeta (40-1) did not. Formation of ascorbate free radical was significantly increased by Abeta (1-42) in the presence of ceruloplasmin. Once cupric ion is reduced to cuprous ion, it can be oxidized by oxygen to generate superoxide radical or it can react with hydrogen peroxide to form hydroxyl radical. Hydrogen peroxide greatly increased the oxidation of cyclic hydroxylamines and ascorbate by cupric-amyloid beta peptide complexes, implying redox cycling of copper ions. Using the spin-trapping technique, we have shown that toxic amyloid beta peptides led to a 4-fold increase in copper-mediated hydroxyl radical formation. We conclude that toxic Abeta peptides do indeed stimulate copper-mediated oxidation of ascorbate and generation of hydroxyl radicals. Therefore, cupric-amyloid beta peptide-stimulated free radical generation may be involved in the pathogenesis of Alzheimer's disease.     

Interactions of Alzheimer amyloid-beta peptides with glycosaminoglycans effects on fibril nucleation and growth.

Proteoglycans and their constituent glycosaminoglycans are associated with all amyloid deposits and may be involved in the amyloidogenic pathway. In Alzheimer's disease, plaques are composed of the amyloid-beta peptide and are associated with at least four different proteoglycans. Using CD spectroscopy, fluorescence spectroscopy and electron microscopy, we examined glycosaminoglycan interaction with the amyloid-beta peptides 1-40 (Abeta40) and 1-42 (Abeta42) to determine the effects on peptide conformation and fibril formation. Monomeric amyloid-beta peptides in trifluoroethanol, when diluted in aqueous buffer, undergo a slow random to amyloidogenic beta sheet transition. In the presence of heparin, heparan sulfate, keratan sulfate or chondroitin sulfates, this transition was accelerated with Abeta42 rapidly adopting a beta-sheet conformation. This was accompanied by the appearance of well-defined amyloid fibrils indicating an enhanced nucleation of Abeta42. Incubation of preformed Abeta42 fibrils with glycosaminoglycans resulted in extensive lateral aggregation and precipitation of the fibrils. The glycosaminoglycans differed in their relative activities with the chondroitin sulfates producing the most pronounced effects. The less amyloidogenic Abeta40 isoform did not show an immediate structural transition that was dependent upon the shielding effect by the phosphate counter ion. Removal or substitution of phosphate resulted in similar glycosaminoglycan-induced conformational and aggregation changes. These findings clearly demonstrate that glycosaminoglycans act at the earliest stage of fibril formation, namely amyloid-beta nucleation, and are not simply involved in the lateral aggregation of preformed fibrils or nonspecific adhesion to plaques. The identification of a structure-activity relationship between amyloid-beta and the different glycosaminoglycans, as well as the condition dependence for glycosaminoglycan binding, are important for the successful development and evaluation of glycosaminoglycan-specific therapeutic interventions.     

Stabilization of discordant helices in amyloid fibril-forming proteins

Several proteins and peptides that can convert from alpha-helical to beta-sheet conformation and form amyloid fibrils, including the amyloid beta-peptide (Abeta) and the prion protein, contain a discordant alpha-helix that is composed of residues that strongly favor beta-strand formation. In their native states, 37 of 38 discordant helices are now found to interact with other protein segments or with lipid membranes, but Abeta apparently lacks such interactions. The helical propensity of the Abeta discordant region (K16LVFFAED23) is increased by introducing V18A/F19A/F20A replacements, and this is associated with reduced fibril formation. Addition of the tripeptide KAD or phospho-L-serine likewise increases the alpha-helical content of Abeta(12-28) and reduces aggregation and fibril formation of Abeta(1-40), Abeta(12-28), Abeta(12-24), and Abeta(14-23). In contrast, tripeptides with all-neutral, all-acidic or all-basic side chains, as well as phosphoethanolamine, phosphocholine, and phosphoglycerol have no significant effects on Abeta secondary structure or fibril formation. These data suggest that in free Abeta, the discordant alpha-helix lacks stabilizing interactions (likely as a consequence of proteolytic removal from a membrane-associated precursor protein) and that stabilization of this helix can reduce fibril formation.     

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 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.