Inhibitors of amyloid toxicity based on beta-sheet packing of Abeta40 and Abeta42

Amyloid fibrils associated with Alzheimer's disease and a wide range of other neurodegenerative diseases have a cross beta-sheet structure, where main chain hydrogen bonding occurs between beta-strands in the direction of the fibril axis. The surface of the beta-sheet has pronounced ridges and grooves when the individual beta-strands have a parallel orientation and the amino acids are in-register with one another. Here we show that in Abeta amyloid fibrils, Met35 packs against Gly33 in the C-terminus of Abeta40 and against Gly37 in the C-terminus of Abeta42. These packing interactions suggest that the protofilament subunits are displaced relative to one another in the Abeta40 and Abeta42 fibril structures. We take advantage of this corrugated structure to design a new class of inhibitors that prevent fibril formation by placing alternating glycine and aromatic residues on one face of a beta-strand. We show that peptide inhibitors based on a GxFxGxF framework disrupt sheet-to-sheet packing and inhibit the formation of mature Abeta fibrils as assayed by thioflavin T fluorescence, electron microscopy, and solid-state NMR spectroscopy. The alternating large and small amino acids in the GxFxGxF sequence are complementary to the corresponding amino acids in the IxGxMxG motif found in the C-terminal sequence of Abeta40 and Abeta42. Importantly, the designed peptide inhibitors significantly reduce the toxicity induced by Abeta42 on cultured rat cortical neurons.     

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.     

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.     

Spatial separation of beta-sheet domains of beta-amyloid: disruption of each beta-sheet by N-methyl amino acids

In a recent model of beta-amyloid (Abeta) fibrils, based mainly on solid-state NMR data, a molecular layer consists of two beta-sheets (residues 12-23 and 31-40 of Abeta1-40), folded onto one another by a connecting "bend" structure (residues 25-29) in the side-chain dimension. In this paper, we use two N-methyl amino acids to disrupt each of the two beta-sheets individually (2NMe(NTerm), residues 17 and 19; and 2NMe(CTerm), residues 37 and 39), or both of them at the same time (4NMe, with the above four N-methylated residues). Our data indicate that incorporation of two N-methyl amino acids into one beta-sheet is sufficient to disrupt that sheet while leaving the other, unmodified beta-sheet intact and able to form fibrils. We show, however, that disruption of each of the two beta-sheets has strikingly different effects on fibrillogenesis kinetics and fibril morphology. Both 2NMe(NTerm) and 2NMe(CTerm) form fibrils at similar rates, but more slowly than that of unmodified Abeta1-40. Electron microscopy shows that 2NMe(NTerm) forms straight fibrils with fuzzy amorphous material coating the edges, while 2NMe(CTerm) forms very regular, highly twisted fibrils-in both cases, distinct from the morphology of Abeta1-40 fibrils. Both 2NMe peptides show a "CMC" approximately four times greater than that of Abeta1-40. CD spectra of these peptides also evolve differently in time: whereas the CD spectra of 2NMe(NTerm) evolve little over 10 days, those of 2NMe(CTerm) show a transition to high beta-sheet content at about day 4-5. We also show that disruption of both beta-sheet domains, as in 4NMe, prevents fibril formation altogether, and renders Abeta1-40 highly water soluble and monomeric, and with solvent-exposed side chains. In summary, our data show (1) that the two beta-sheet domains fold in a semiautonomous manner, since disrupting each one still allows the other to fold; (2) that disruption of the N-terminal beta-sheet has a more profound effect on fibrillogenesis than disruption of the C-terminal beta-sheet, suggesting that the former is the more critical for the overall structure of the fibril; and (3) that disruption of both beta-sheet domains renders the peptide monomeric and unable to form fibrils.     

Parallel beta-sheets and polar zippers in amyloid fibrils formed by residues 10-39 of the yeast prion protein Ure2p

We report the results of solid-state nuclear magnetic resonance (NMR) and atomic force microscopy measurements on amyloid fibrils formed by residues 10-39 of the yeast prion protein Ure2p (Ure2p(10)(-)(39)). Measurements of intermolecular (13)C-(13)C nuclear magnetic dipole-dipole couplings indicate that Ure2p(10)(-)(39) fibrils contain in-register parallel beta-sheets. Measurements of intermolecular (15)N-(13)C dipole-dipole couplings, using a new solid-state NMR technique called DSQ-REDOR, are consistent with hydrogen bonds between side chain amide groups of Gln18 residues. Such side chain hydrogen bonding interactions have been called "polar zippers" by M. F. Perutz and have been proposed to stabilize amyloid fibrils formed by peptides with glutamine- and asparagine-rich sequences, such as Ure2p(10)(-)(39). We propose that polar zipper interactions account for the in-register parallel beta-sheet structure in Ure2p(10)(-)(39) fibrils and that similar peptides will also exhibit parallel beta-sheet structures in amyloid fibrils. We present molecular models for Ure2p(10)(-)(39) fibrils that are consistent with available experimental data. Finally, we show that solid-state (13)C NMR chemical shifts for (13)C-labeled Ure2p(10)(-)(39) fibrils are insensitive to hydration level, indicating that the fibril structure is not affected by the presence or absence of bulk water.     

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.     

Site-specific identification of non-beta-strand conformations in Alzheimer's beta-amyloid fibrils by solid-state NMR

The most well-established structural feature of amyloid fibrils is the cross-beta motif, an extended beta-sheet structure formed by beta-strands oriented perpendicular to the long fibril axis. Direct experimental identification of non-beta-strand conformations in amyloid fibrils has not been reported previously. Here we report the results of solid-state NMR measurements on amyloid fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta(1-40)), prepared synthetically with pairs of (13)C labels at consecutive backbone carbonyl sites. The measurements probe the peptide backbone conformation in residues 24-30, a segment where a non-beta-strand conformation has been suggested by earlier sequence analysis, cross-linking experiments, and molecular modeling. Data obtained with the fpRFDR-CT, DQCSA, and 2D MAS exchange solid-state NMR techniques, which provide independent constraints on the phi and psi backbone torsion angles between the labeled carbonyl sites, indicate non-beta-strand conformations at G25, S26, and G29. These results represent the first site-specific identification and characterization of non-beta-strand peptide conformations in an amyloid fibril.     

Review: model peptides and the physicochemical approach to beta-amyloids

beta-Amyloid peptides are the main protein components of neuritic plaques and may be important in the pathogenesis of Alzheimer's Disease. The determination of the structure of beta-amyloid fibrils poses a challenge because of the limited solubility of beta-amyloid peptides and the noncrystalline nature of fibrils formed from these peptides. In this paper, we describe several physicochemical approaches which have been used to examine fibrils and the fibrillogenesis of peptide models of beta-amyloid. Recent advances in solid state NMR, such as the DRAWS pulse sequence, have made this approach a particularly attractive one for peptides such as beta-amyloid, which are not yet amenable to high-resolution solution phase NMR and crystallography. The application of solid state NMR techniques has yielded information on a model peptide comprising residues 10-35 of human beta-amyloid and indicates that in fibrils, this peptide assumes a parallel beta-strand conformation, with all residues in exact register. In addition, we discuss the use of block copolymers of Abeta peptides and polyethylene glycol as probes for the pathways of fibrillogenesis. These methods can be combined with other new methods, such as high-resolution synchrotron X-ray diffraction and small angle neutron and X-ray scattering, to yield structural data of relevance not only to disease, but to the broader question of protein folding and self-assembly.     

Investigation of alpha-synuclein fibril structure by site-directed spin labeling

The misfolding and fibril formation of alpha-synuclein plays an important role in neurodegenerative diseases such as Parkinson disease. Here we used electron paramagnetic resonance spectroscopy, together with site-directed spin labeling, to investigate the structural features of alpha-synuclein fibrils. We generated fibrils from a total of 83 different spin-labeled derivatives and observed single-line, exchange-narrowed EPR spectra for the majority of all sites located within the core region of alpha-synuclein fibrils. Such exchange narrowing requires the orbital overlap between multiple spin labels in close contact. The core region of alpha-synuclein fibrils must therefore be arranged in a parallel, in-register structure wherein same residues from different molecules are stacked on top of each other. This parallel, in-register core region extends from residue 36 to residue 98 and is tightly packed. Only a few sites within the core region, such as residues 62-67 located at the beginning of the NAC region, as well as the N- and C-terminal regions outside the core region, are significantly less ordered. Together with the accessibility measurements that suggest the location of potential beta-sheet regions within the fibril, the data provide significant structural constraints for generating three-dimensional models. Furthermore, the data support the emerging view that parallel, in-register structure is a common feature shared by a number of naturally occurring amyloid fibrils.     

Polymorphic fibril formation by residues 10-40 of the Alzheimer's beta-amyloid peptide

We report investigations of the morphology and molecular structure of amyloid fibrils comprised of residues 10-40 of the Alzheimer's beta-amyloid peptide (Abeta(10-40)), prepared under various solution conditions and degrees of agitation. Omission of residues 1-9 from the full-length Alzheimer's beta-amyloid peptide (Abeta(1-40)) did not prevent the peptide from forming amyloid fibrils or eliminate fibril polymorphism. These results are consistent with residues 1-9 being disordered in Abeta(1-40) fibrils, and show that fibril polymorphism is not a consequence of disorder in residues 1-9. Fibril morphology was analyzed by atomic force and electron microscopy, and secondary structure and inter-side-chain proximity were probed using solid-state NMR. Abeta(1-40) fibrils were found to be structurally compatible with Abeta(10-40): Abeta(1-40) fibril fragments were used to seed the growth of Abeta(10-40) fibrils, with propagation of fibril morphology and molecular structure. In addition, comparison of lyophilized and hydrated fibril samples revealed no effect of hydration on molecular structure, indicating that Abeta(10-40) fibrils are unlikely to contain bulk water.     

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.     

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.     

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.     

Solid-state NMR as a method to reveal structure and membrane-interaction of amyloidogenic proteins and peptides

It is important to understand the Amyloid fibril formation in view of numerous medical and biochemical aspects. Structural determination of amyloid fibril has been extensively studied using electron microscopy. Subsequently, solid state NMR spectroscopy has been realized to be the most important means to determine not only microscopic molecular structure but also macroscopic molecular packing. Molecular structure of amyloid fibril was first predicted to be parallel beta-sheet structure, and subsequently, was further refined for Abeta(1-40) to be cross beta-sheet with double layered in register parallel beta-sheet structure by using solid state NMR spectroscopy. On the other hand, anti-parallel beta-sheet structure has been reported to short fragments of Abeta-amyloid and other amyloid forming peptides. Kinetic study of amyloid fibril formation has been studied using a variety of methods, and two-step autocatalytic reaction mechanism used to explain fibril formation. Recently, stable intermediates or proto-fibrils have been observed by electron microscope (EM) images. Some of the intermediates have the same microscopic structure as the matured fibril and subsequently change to matured fibrils. Another important study on amyloid fibril formation is determination of the interaction with lipid membranes, since amyloid peptide are cleaved from amyloid precursor proteins in the membrane interface, and it is reported that amyloid lipid interaction is related to the cytotoxicity. Finally it is discussed how amyloid fibril formation can be inhibited. Firstly, properly designed compounds are reported to have inhibition ability of amyloid fibril formation by interacting with amyloid peptide. Secondly, it is revealed that site directed mutation can inhibit amyloid fibril formation. These inhibitors were developed by knowing the fibril structure determined by solid state NMR.     

Abeta40-Lactam(D23/K28) models a conformation highly favorable for nucleation of amyloid

Recent solid-state NMR data (1) demonstrate that Abeta(1)(-)(40) adopts a conformation in amyloid fibrils with two in-register, parallel beta-sheets, connected by a bend structure encompassing residues D(23)VGSNKG(29), with a close contact between the side chains of Asp23 and Lys28. We hypothesized that forming this bend structure might be rate-limiting in fibril formation, as indicated by the lag period typically observed in the kinetics of Abeta(1)(-)(40) fibrillogenesis. We synthesized Abeta(1)(-)(40)-Lactam(D23/K28), a congener Abeta(1)(-)(40) peptide that contains a lactam bridge between the side chains of Asp23 and Lys28. Abeta(1)(-)(40)-Lactam(D23/K28) forms fibrils similar to those formed by Abeta(1)(-)(40). The kinetics of fibrillogenesis, however, occur without the typical lag period, and at a rate approximately 1000-fold greater than is seen with Abeta(1)(-)(40) fibrillogenesis. The strong tendency toward self-association is also shown by size exclusion chromatography in which Abeta(1)(-)(40)-Lactam(D23/K28) forms oligomers even at concentrations of approximately 1-5 microM. Under the same conditions, Abeta(1)(-)(40) shows no detectable oligomers by size exclusion chromatography. Our data suggest that Abeta(1)(-)(40)-Lactam(D23/K28) could bypass an unfavorable folding step in fibrillogenesis, because the lactam linkage "preforms" a bendlike structure in the peptide. Consistent with this view Abeta(1)(-)(40) growth is efficiently nucleated by Abeta(1)(-)(40)-Lactam(D23/K28) fibril seeds.     

A molecular model of Alzheimer amyloid beta-peptide fibril formation

Polymerization of the amyloid beta (Abeta) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer's disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals. In this work, we present experimental results leading to a molecular level model for fibril formation. Systematically selected Abeta-fragments containing the Abeta16-20 sequence, previously shown essential for Abeta-Abeta binding, were incubated in a physiological buffer. Electron microscopy revealed that the shortest fibril-forming sequence was Abeta14-23. Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from Abeta1-42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers. Molecular modeling of Abeta14-23 oligomers in an antiparallel beta-sheet conformation displayed favorable hydrophobic interactions stabilized by salt bridges between all charged residues. We propose that this decapeptide sequence forms the core of Abeta-fibrils, with the hydrophobic C terminus folding over this core. The identification of this fundamental sequence and the implied molecular model could facilitate the design of potential inhibitors of amyloidogenesis.     

Self-assembly in aqueous solution of a modified amyloid beta peptide fragment

The self-assembly in films dried from aqueous solutions of a modified amyloid beta peptide fragment is studied. We focus on sequence Abeta(16-20), KLVFF, extended by two alanines at the N-terminus to give AAKLVFF. Self-assembly into twisted ribbon fibrils is observed, as confirmed by transmission electron microscopy (TEM). Dynamic light scattering reveals the semi-flexible nature of the AAKLVFF fibrils, while polarized optical microscopy shows that the peptide fibrils crystallize after an aqueous solution of AAKLVFF is matured over 5 days. The secondary structure of the fibrils is studied by FT-IR, circular dichroism and X-ray diffraction (XRD), which provide evidence for beta-sheet structure in the fibril. From high resolution TEM it is concluded that the average width of an AAKLVFF fibril is (63+/-18) nm, indicating that these fibrils comprise beta-sheets with multiple repeats of the unit cell, determined by XRD to have b and c dimensions 1.9 and 4.4 nm with an a axis 0.96 nm, corresponding to twice the peptide backbone spacing in the antiparallel beta-sheet.     

An atomic model for the pleated beta-sheet structure of Abeta amyloid protofilaments.

Synchrotron x-ray studies on amyloid fibrils have suggested that the stacked pleated beta-sheets are twisted so that a repeating unit of 24 beta-strands forms a helical turn around the fibril axis (. J. Mol. Biol. 273:729-739). Based on this morphological study, we have constructed an atomic model for the twisted pleated beta-sheet of human Abeta amyloid protofilament. In the model, 48 monomers of Abeta 12-42 stack (four per layer) to form a helical turn of beta-sheet. Each monomer is in an antiparallel beta-sheet conformation with a turn located at residues 25-28. Residues 17-21 and 31-36 form a hydrophobic core along the fibril axis. The hydrophobic core should play a critical role in initializing Abeta aggregation and in stabilizing the aggregates. The model was tested using molecular dynamics simulations in explicit aqueous solution, with the particle mesh Ewald (PME) method employed to accommodate long-range electrostatic forces. Based on the molecular dynamics simulations, we hypothesize that an isolated protofilament, if it exists, may not be twisted, as it appears to be when in the fibril environment. The twisted nature of the protofilaments in amyloid fibrils is likely the result of stabilizing packing interactions of the protofilaments. The model also provides a binding mode for Congo red on Abeta amyloid fibrils. The model may be useful for the design of Abeta aggregation inhibitors.     

Inhibition of beta-amyloid(40) fibrillogenesis and disassembly of beta-amyloid(40) fibrils by short beta-amyloid congeners containing N-methyl amino acids at alternate residues.

A potential goal in the prevention or therapy of Alzheimer's disease is to decrease or eliminate neuritic plaques composed of fibrillar beta-amyloid (Abeta). In this paper we describe N-methyl amino acid containing congeners of the hydrophobic "core domain" of Abeta that inhibit the fibrillogenesis of full-length Abeta. These peptides also disassemble preformed fibrils of full-length Abeta. A key feature of the inhibitor peptides is that they contain N-methyl amino acids in alternating positions of the sequence. The most potent of these inhibitors, termed Abeta16-22m, has the sequence NH(2)-K(Me-L)V(Me-F)F(Me-A)E-CONH(2). In contrast, a peptide, NH(2)-KL(Me-V)(Me-F)(Me-F)(Me-A)-E-CONH(2), with N-methyl amino acids in consecutive order, is not a fibrillogenesis inhibitor. Another peptide containing alternating N-methyl amino acids but based on the sequence of a different fibril-forming protein, the human prion protein, is also not an inhibitor of Abeta40 fibrillogenesis. The nonmethylated version of the inhibitor peptide, NH(2)-KLVFFAE-CONH(2) (Abeta16-22), is a weak fibrillogenesis inhibitor. Perhaps contrary to expectations, the Abeta16-22m peptide is highly soluble in aqueous media, and concentrations in excess of 40 mg/mL can be obtained in buffers of physiological pH and ionic strength, compared to only 2 mg/mL for Abeta16-22. Analytical ultracentrifugation demonstrates that Abeta16-22m is monomeric in buffer solution. Whereas Abeta16-22 is susceptible to cleavage by chymotrypsin, the methylated inhibitor peptide Abeta16-22m is completely resistant to this protease. Circular dichroic spectroscopy of Abeta16-22m indicates that this peptide is a beta-strand, albeit with an unusual minimum at 226 nm. In summary, the inhibitor motif is that of alternating N-methyl and nonmethylated amino acids in a sequence critical for Abeta40 fibrillogenesis. These inhibitors appear to act by binding to growth sites of Abeta nuclei and/or fibrils and preventing the propagation of the network of hydrogen bonds that is essential for the formation of an extended beta-sheet fibril.     

Analysis of the secondary structure of beta-amyloid (Abeta42) fibrils by systematic proline replacement

Amyloid fibrils in Alzheimer's disease mainly consist of 40- and 42-mer beta-amyloid peptides (Abeta40 and Abeta42) that exhibit aggregative ability and neurotoxicity. Although the aggregates of Abeta peptides are rich in intermolecular beta-sheet, the precise secondary structure of Abeta in the aggregates remains unclear. To identify the amino acid residues involved in the beta-sheet formation, 34 proline-substituted mutants of Abeta42 were synthesized and their aggregative ability and neurotoxicity on PC12 cells were examined. Prolines are rarely present in beta-sheet, whereas they are easily accommodated in beta-turn as a Pro-X corner. Among the mutants at positions 15-32, only E22P-Abeta42 extensively aggregated with stronger neurotoxicity than wild-type Abeta42, suggesting that the residues at positions 15-21 and 24-32 are involved in the beta-sheet and that the turn at positions 22 and 23 plays a crucial role in the aggregation and neurotoxicity of Abeta42. The C-terminal proline mutants (A42P-, I41P-, and V40P-Abeta42) hardly aggregated with extremely weak cytotoxicity, whereas the C-terminal threonine mutants (A42T- and I41T-Abeta42) aggregated potently with significant cytotoxicity. These results indicate that the hydrophobicity of the C-terminal two residues of Abeta42 is not related to its aggregative ability and neurotoxicity, rather the C-terminal three residues adopt the beta-sheet. These results demonstrate well the large difference in aggregative ability and neurotoxicity between Abeta42 and Abeta40. In contrast, the proline mutants at the N-terminal 13 residues showed potent aggregative ability and neurotoxicity similar to those of wild-type Abeta42. The identification of the beta-sheet region of Abeta42 is a basis for designing new aggregation inhibitors of Abeta peptides.