Aggregation and conformational studies on a pentapeptide derivative

Most of the disease causing proteins such as beta amyloid, amylin, and huntingtin protein, which are natively disordered, readily form fibrils consisting of beta-sheet polymers. Though all amyloid fibrils are made up of beta-sheet polymers, not all peptides with predominant beta-sheet content in the native state develop into amyloid fibrils. We hypothesize that stable amyloid like fibril formation may require mixture of different conformational states in the peptide. We have tested this hypothesis on amyloid forming peptide namely HCl(Ile)(5)NH(CH(2)CH(2)O)(3)CH(3) (I). We show peptide I, has propensity to form self-assembled structures of beta-sheets in aqueous solutions. When incubated over a period of time in aqueous buffer, I self assembled into beta sheet like structures with diameters ranging from 30 to 60 A that bind with amyloidophilic dyes like Congo red and Thioflavin T. Interestingly peptide I developed into unstable fibrils after prolonged aging at higher concentration in contrast with the general mature fibril-forming propensity of various amyloid petides known to date.     

Alzheimer beta-amyloid homodimers facilitate A beta fibrillization and the generation of conformational antibodies

We reported previously that stabilized beta-amyloid peptide dimers were derived from mutant amyloid precursor protein with a single cysteine in the ectodomain juxtamembrane position. In vivo studies revealed that two forms of SDS-stable A beta homodimers exist, species ending at A beta 40 and A beta 42. The phenomenon of the transformation of the initially deposited 42-residue beta-amyloid peptide into the amyloid fibrils of Alzheimer's disease plaques remains to be explained in physical terms, i.e. energetically and structurally. We therefore performed spectroscopic analyses revealing that engineered dimeric peptides ending at residue 42 displayed a much more pronounced beta-structural transition than corresponding monomers. Specifically, the single chemically induced dimerization of A beta peptides significantly increased the beta-sheet content by a factor of 2. The C-terminal residues Ile-41 and Ala-42 of dimeric forms further increased the beta-sheet content by roughly one-third. In contrast to A beta 42, the beta-sheet content of the alpha- and gamma-secretase-generated p3 fragments did not necessarily correlate with the tendency to form fibrils, although p3/17-42 had a pronounced thread forming character with fibril lengths of up to 2.5 microM. Electron microscopic images show that forms of p3/17-42 generated smaller granular particles than forms ending at residue 40. We discuss these findings in terms of A beta 1-42 dimers representing paranuclei, which self-aggregate into ribbon-like ordered fibrils by elongation. Based on A beta 42 dimer-specific titers of a polyclonal antiserum we propose that the A beta homodimer represents a nidus for plaque formation and a well defined novel therapeutic target.     

Core and heterogeneity of beta2-microglobulin amyloid fibrils as revealed by H/D exchange

Dialysis-related amyloidosis, which occurs in the patients receiving a long-term hemodialysis with high frequency, accompanies the deposition of amyloid fibrils composed of beta(2)-microglobulin (beta2-m). In vitro, beta2-m forms two kinds of fibrous structures at acidic pH. One is a rigid "mature fibril", and the other is a flexible thin filament often called an "immature fibril". In addition, a 22-residue peptide (K3 peptide) corresponding to Ser20 to Lys41 of intact beta2-m forms rigid amyloid-like fibrils similar to mature fibrils. We compared the core of these three fibrils at single-residue resolution using a recently developed hydrogen/deuterium (H/D) exchange method with the dissolution of fibrils by dimethylsulfoxide (DMSO). The exchange time-course of these fibrils showed large deviations from a single exponential curve showing that, because of the supramolecular structures, the same residue exists in different environments from molecule to molecule, even in a single fibril. The exchange profiles revealed that the core of the immature fibril is restricted to a narrow region compared to that of the mature fibril. In contrast, all residues were protected from exchange in the K3 fibril, indicating that a whole region of the peptide is engaged in the beta-sheet network. These results suggest the mechanism of amyloid fibril formation, in which the core beta-sheet formed by a minimal sequence propagates to form a rigid and extensive beta-sheet network.     

The influence of phospholipid membranes on bovine calcitonin peptide's secondary structure and induced neurotoxic effects

The peptide hormone, calcitonin, which is associated with medullary carcinoma of the thyroid, has a marked tendency to form amyloid fibrils and may be a useful model in probing the role of peptide-membrane interactions in beta-sheet and amyloid formation and amyloid neurotoxicity. Using bovine calcitonin, we found that, like other amyloids, the peptide was toxic only when in a beta-sheet-rich, amyloid form, but was non-toxic, when it lacked an amyloid structure. We found that the peptide bound with significant affinity to membranes that contained either cholesterol and gangliosides. In addition, incubation of calcitonin with cholesterol-rich and ganglioside-containing membranes resulted in significant changes in peptide structure yielding a peptide enriched in beta-sheet and amyloid content. Because the cholesterol- and ganglioside-rich phospholipid systems enhanced the calcitonin beta-sheet and amyloid contents, and peptide amyloid content was associated with neurotoxicity, we then investigated whether depleting cellular cholesterol and gangliosides affected calcitonin neurotoxicity. We found that cholesterol and ganglioside removal significantly reduced the calcitonin-induced PC12 cell neurotoxicity. Similar results have been observed with other amyloid-forming peptides such as beta-amyloid (A beta) of Alzheimer's disease and suggest that modulation of membrane composition and peptide-membrane interactions may prove useful in the control of amyloid formation and amyloid neurotoxicity.     

Stereospecific amyloid-like fibril formation by a peptide fragment of beta2-microglobulin

Understanding the role of the L/D-stereospecificity of amino acids is important in obtaining further insight into the mechanism of the formation of amyloid fibrils. Beta(2)-microglobulin is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. A 22-residue peptide of beta(2)-microglobulin, Ser20-Lys41 (L-K3 peptide), obtained by digestion with Acromobacter protease I, formed amyloid-like fibrils in 50% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl at 25 degrees C, as confirmed by thioflavin T fluorescence, circular dichroism spectra, and atomic force microscopy images. A synthetic K3 peptide composed of D-amino acids (D-K3 peptide) formed similar fibrils but with opposite chirality as indicated by circular dichroism spectra. A mixture of L-K3 and D-K3 peptides also formed fibrils, although the L- and D-amino acid composition of each fibril is unknown. To examine the possible cross-reactivity between L- and D-enantiomers, we carried out seeding experiments in which preformed seeds were extended by monomers. The results revealed that only the homologous extensions proceed smoothly, i.e., the growth of L-seeds by L-monomers or D-seeds by D-monomers. The results suggest that, while the fibrils derived from L- and D-peptides form in a similar manner but with opposite stereochemistry, a cross-reaction between them is prevented because the geometry of the mixed sheet cannot satisfy dominant factors for beta-sheet stabilization.     

Seeding-dependent propagation and maturation of amyloid fibril conformation

Recent studies of amyloid fibrils have focused on the presence of multiple amyloid forms even with one protein and their propagation by seeding, leading to conformational memory. To establish the structural basis of these critical features of amyloid fibrils, we used the amyloidogenic fragment Ser20-Lys41 (K3) of beta2-microglobulin, a protein responsible for dialysis-related amyloidosis. In 20% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl (pH approximately 2), K3 peptide formed two types of amyloid-like fibrils, f218 and f210, differing in the amount of beta-sheet as measured by circular dichroism spectroscopy and Fourier transform infrared spectroscopy. Atomic force microscopy showed that the fibril with a larger amount of beta-sheet (f210) is thinner and longer. Both fibrils were reproduced by seeding, showing the template-dependent propagation of a fibril's conformation. However, upon repeated self-seeding, f218 fibrils were gradually transformed into f210 fibrils, revealing the conformational maturation. The observed maturation can be explained fully by a competitive propagation of two fibrils. The maturation of amyloid fibrils might play a role during the development of amyloidosis.     

Mutational analysis of designed peptides that undergo structural transition from alpha helix to beta sheet and amyloid fibril formation

BACKGROUND: Conformational alteration and fibril formation of proteins have a key role in a variety of amyloid diseases. A simplified model peptide would lead to a better understanding of underlying mechanisms whereby protein misfolding and aggregation occur. Recently, we reported the design of peptides that undergo a self-initiated structural transition from an alpha helix to a beta sheet and form amyloid fibrils. In this study, we focus on two glutamine residues in the peptide, and report a mutational analysis of these residues. RESULTS: A coiled-coil alpha-helix structure bearing a hydrophobic adamantanecarbonyl (Ad) group at the N terminus was designed (parent peptide Ad-QQ). In neutral aqueous solution, the double Gln-->Ala mutant (Ad-AA) underwent the alpha-->beta structural transition within four hours, which was similar to the case of Ad-QQ. In contrast, two kinds of single Gln-->Ala mutant (Ad-QA and Ad-AQ) required three days for the transition. Furthermore, Ad-QQ and Ad-AA formed amyloid fibrils, whereas Ad-QA and Ad-AQ did not. Interestingly, however, Ad-QA and Ad-AQ complementarily assembled into the fibrils when they were mixed. CONCLUSIONS: The Gln-->Ala substitution in the peptide significantly alters the alpha-->beta transitional properties and the ability to form amyloid fibrils. A heterogeneous assembly of two peptide species into the fibrils is also presented. These results suggest that the secondary structural transition and self-assembly into the well-organized fibril may depend strictly on the primary structure, which determines the beta-sheet packing. The results might provide insights into misfolding and fibril formation of disease-associated mutant proteins.     

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.     

Amyloid fibril formation by peptide LYS (11-36) in aqueous trifluoroethanol

Peptide LYS (11-36), derived from the beta-sheet region of T4 lysozyme, forms an amyloid fibril in aqueous trifluoroethanol (TFE) at elevated temperature. The peptide has a moderate alpha-helix content in 20 and 50% (v/v) TFE solution; large quantities of fibrils were formed after incubation at 55 degrees C for 2 weeks as monitored by a thioflavin T fluorescence assay. No fibrils were observed when the peptide initially existed predominantly as a random coil or as a complete alpha helix. Our results suggest that a moderate amount of alpha helix and random coil present in the peptide initially facilitates the fibril-formation process, but a high alpha-helix content inhibits fibril formation. Transmission electron microscopy revealed several types of fibril morphologies at different TFE concentrations. The fibrils were highly twisted and consisted of interleaved protofilaments in 50% TFE, while smooth and flat ribbonlike fibrils were found in 20% TFE. In 50% TFE, the fibril growth rate of LYS (11-36) was found to depend strongly on peptide concentration and seeding but was insensitive to solution pH and ionic strength.     

New insights into the self-assembly of insulin amyloid fibrils: an H-D exchange FT-IR study

The solvent protection of the amide backbone in bovine insulin fibrils was studied by FT-IR spectroscopy. In the mature fibrils, approximately 85 +/- 2% of amide protons are protected. Of those "trapped" protons, a further 25 +/- 2 or 35 +/- 2% is H-D exchanged after incubation for 1 h at 1 GPa and 25 degrees C or 0.1 MPa and 100 degrees C, respectively. In contrast to the native or unfolded protein, fibrils do not H-D exchange upon incubation at 65 degrees C. A complete deuteration of H(2)O-grown fibrils occurs when the beta-sheet structure is reassembled in a 75 wt % DMSO/D(2)O solution. Our findings suggest a densely packed environment around the amide protons involved in the intermolecular beta-sheet motive. In disagreement with the concept of "amyloid fibers as water-filled nanotubes" [Perutz, M. F., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5591-5595], elution of D(2)O-grown fibrils with H(2)O is complete, which is reflected by the vanishing of D(2)O bending vibrations at 1214 cm(-)(1). This implies the absence of "trapped water" within insulin fibrils. The rigid conformations of the native and fibrillar insulin contrast with transient intermediate states docking at the fibrils' ends. Room-temperature seeding is accompanied by an accelerated H-D exchange in insulin molecules in the act of docking and integrating with the seeds, proving that the profound structural disruption is the sine qua non of forming an aggregation-competent conformation.     

Effect of the beta-sheet-breaker peptide LPFFD on oriented network of amyloid β25-35 fibrils

Amyloid fibrils are self-associating filamentous structures deposited in extracellular tissue in various neurodegenerative and protein misfolding disorders. It has been shown that beta-sheet-breaker (BSB) peptides may interfere with amyloid fibril assembly. Although BSB peptides are prospective therapeutic agents in amyloidosis, there is ambiguity about the mechanisms and generality of their action. In the present work we analyzed the effect of the BSB peptide LPFFD on the growth kinetics, morphologic, and mechanical properties of amyloid β25-35 (Aβ25-35) fibrils assembled in an oriented array on mica surface. Aβ25-35 is thought to represent the biologically active, toxic fragment of the full-length Aβ peptide. Growth kinetics and morphologic features were analyzed using in situ atomic force microscopy in the presence of various concentrations of LPFFD. We found that the addition of LPFFD only slightly altered the assembly kinetics of Aβ25-35 fibrils. Already formed fibrils did not disassemble in the presence of high concentrations of LPFFD. The mechanical stability of the fibrils was explored with force spectroscopy methods. The nanomechanical behavior of Aβ25-35 fibrils is characterized by the appearance of force staircases which correspond to the force-driven unzipping and dissociation of several protofilaments. In the presence of LPFFD single-plateau force traces dominated. The effects of LPFFD on Aβ25-35 fibril assembly and stability suggest that inter-protofilament interactions were slightly weakened. Complete disassembly of fibrils, however, was not observed. Thus, under the conditions explored here, LPFFD may not be considered as a BSB peptide with generalized beta-sheet breaking properties.     

Molecular modeling of the amyloid-beta-peptide using the homology to a fragment of triosephosphate isomerase that forms amyloid in vitro

The main component of the amyloid senile plaques found in Alzheimer's brain is the amyloid-beta-peptide (A beta), a proteolytic product of a membrane precursor protein. Previous structural studies have found different conformations for the A beta peptide depending on the solvent and pH used. In general, they have suggested an alpha-helix conformation at the N-terminal domain and a beta-sheet conformation for the C-terminal domain. The structure of the complete A beta peptide (residues 1-40) solved by NMR has revealed that only helical structure is present in A beta. However, this result cannot explain the large beta-sheet A beta aggregates known to form amyloid under physiological conditions. Therefore, we investigated the structure of A beta by molecular modeling based on extensive homology using the Smith and Waterman algorithm implemented in the MPsrch program (Blitz server). The results showed a mean value of 23% identity with selected sequences. Since these values do not allow a clear homology to be established with a reference structure in order to perform molecular modeling studies, we searched for detailed homology. A 28% identity with an alpha/beta segment of a triosephosphate isomerase (TIM) from Culex tarralis with an unsolved three-dimensional structure was obtained. Then, multiple sequence alignment was performed considering A beta, TIM from C.tarralis and another five TIM sequences with known three-dimensional structures. We found a TIM segment with secondary structure elements in agreement with previous experimental data for A beta. Moreover, when a synthetic peptide from this TIM segment was studied in vitro, it was able to aggregate and to form amyloid fibrils, as established by Congo red binding and electron microscopy. The A beta model obtained was optimized by molecular dynamics considering ionizable side chains in order to simulate A beta in a neutral pH environment. We report here the structural implications of this study.     

Dissolution of beta2-microglobulin amyloid fibrils by dimethylsulfoxide

Increasing numbers of proteins have been found to aggregate into insoluble fibers, collectively referred to as amyloid fibrils. To address the conformational stability of amyloid fibrils, we studied the effects of dimethylsulfoxide (DMSO), 2,2,2-trifluoroethanol (TFE), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) on beta(2)-microglobulin amyloid fibrils by circular dichroism, thioflavin T fluorescence, light scattering, and electron microscopy. When measured by circular dichroism and thioflavin T fluorescence, HFIP, and TFE dissolved the fibrils, producing predominantly helical conformations. However, these alcohols did not dissolve the amyloid fibrils completely as monitored by light scattering and electron microscopy. On the other hand, DMSO completely dissolved the amyloid fibrils although a high concentration [i.e., 80% (v/v)] was required. These results are consistent with the important role of hydrogen bonds in stabilizing amyloid fibrils.     

Oriented epitaxial growth of amyloid fibrils of the N27C mutant β25–35 peptide

Amyloid fibrils are present in the extracellular space of various tissues in neurodegenerative and protein misfolding diseases. Amyloid fibrils may be used in nanotechnology applications, because of their self-assembly properties and stability, if their growth and orientation can be controlled. Recently, we have shown that amyloid beta 25-35 (A beta 25-35) forms a highly oriented, K(+)-dependent network on mica. Here, we analyzed the properties of A beta 25-35_N27C, the cysteine residue of which may be used for subsequent chemical modifications. We find that A beta 25-35_N27C forms epitaxially growing fibrils on mica, which evolve into a trigonally oriented branched network. The binding is apparently more sensitive to cation concentration than that of the wild-type peptide. By nanomanipulating A beta 25-35_N27C fibrils with a gold-coated AFM tip, we show that the sulfhydryl of Cys27 is reactive and accessible from the solution. The oriented network of A beta 25-35_N27C fibrils can therefore be specifically labeled and may be used for constructing nanobiotechnological devices.     

Low concentrations of sodium dodecyl sulfate induce the extension of beta 2-microglobulin-related amyloid fibrils at a neutral pH

In beta(2)-microglobulin-related (Abeta2M) amyloidosis, partial unfolding of beta(2)-microglobulin (beta2-m) is believed to be prerequisite to its assembly into Abeta2M amyloid fibrils in vivo. Although low pH or 2,2,2-trifluoroethanol at a low concentration has been reported to induce partial unfolding of beta2-m and subsequent amyloid fibril formation in vitro, factors that induce them under near physiological conditions have not been determined. Using fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy, and electron microscopy, we here show that at low concentrations, sodium dodecyl sulfate (SDS) converts natively folded beta2-m monomers into partially folded, alpha-helix-containing conformers. Surprisingly, this results in the extension of Abeta2M amyloid fibrils at neutral pH, which could be explained basically by a first-order kinetic model. At low concentrations, SDS also stabilized the fibrils at neutral pH. These SDS effects were concentration-dependent and maximal at approximately 0.5 mM, around the critical micelle concentration of SDS (0.67 mM). As the concentration of SDS was increased above 1 mM, the alpha-helix content of beta2-m rose to approximately 10%, while the beta-sheet content decreased to approximately 20%, a change paralleled by a complete cessation of fibril extension and the destabilization of the fibrils. Detergents of other classes had no significant effect on the extension of fibrils. These findings are consistent with the hypothesis that in vivo, specific factors (e.g., phospholipids) that affect the conformation and stability of beta2-m and amyloid fibrils will have significant effects on the kinetics of Abeta2M fibril formation.     

Amyloid fibril formation by a synthetic peptide from a region of human acetylcholinesterase that is homologous to the Alzheimer's amyloid-beta peptide

A region near the C-terminus of human acetylcholinesterase (AChE) is weakly homologous with the N-terminus of the Alzheimer's disease amyloid-beta peptide. We report that a 14-amino acid synthetic polypeptide whose sequence corresponds to residues 586-599 of the human synaptic or T form of AChE assembles into amyloid fibrils under physiological conditions. The fibrils have all the classical characteristics of amyloid: they have a diameter of 6-7 nm and bind both Congo red and thioflavin-T. Furthermore, the kinetics of assembly indicate that fibril formation proceeds via a two-step nucleation-dependent polymerization pathway, and a transition in the peptide conformation from random coil to beta-sheet is observed during fibril formation using far-UV circular dichroism spectroscopy. We also show that the peptide in aggregated fibrillar form has a toxic effect upon PC-12 cells in vitro. AChE normally resides mainly on cholinergic neuronal membranes, but is abnormally localized to senile plaques in Alzheimer's disease. Recently, an in vitro interaction between AChE and A beta, the principal constituent of the amyloid fibrils in senile plaques, has been documented. The presence of a fibrillogenic region within AChE may be relevant to the interaction of AChE with amyloid fibrils formed by Abeta.     

Amyloid-induced aggregation and precipitation of soluble proteins: an electrostatic contribution of the Alzheimer's beta(25-35) amyloid fibril

Amyloid-induced aggregation and precipitation of soluble proteins were investigated in vitro using the amyloid fibrils of the beta(25--35) peptide, a cytotoxic fragment of the Alzheimer's beta-peptide at positions 25--35. The aggregation rate of firefly luciferase was found to be modulated by both a chaperone molecule DnaK and the beta(25--35) amyloid, but their effects were opposite in direction. The amyloid fibril drastically facilitated the luciferase aggregation, which may define a kind of anti-chaperone activity. The effect of the beta(25--35) amyloid to promote protein aggregation and precipitation was further demonstrated for a wide variety of target proteins. The amount of coprecipitation was well correlated with the predicted isoelectric point of the target proteins, indicating that the interaction between the beta(25--35) amyloid and the target was driven by an electrostatic force between them. This view was confirmed by the experiments using an electrically neutral mutant peptide, beta(25--35)KA. It was also found that clustering of the beta(25--35) peptide to form amyloid and the conformation of the target protein are additional factors that determine the strength of the amyloid-protein interaction. Spectroscopic and electron microscopic methods have revealed that the proteins coprecipitated with the beta(25--35) amyloid formed amorphous aggregates deposited together with the amyloid fibrils. The conformation of protein molecules left in the residual soluble fraction was also damaged in the amyloid-containing solution. As a summary, this study has proposed a scheme for events related to the nonspecific amyloid-protein interaction, which may play substantial roles in in vivo conditions.     

4,4(')-Dianilino-1,1(')-binaphthyl-5,5(')-disulfonate: report on non-beta-sheet conformers of Alzheimer's peptide beta(1-40)

The venerable fluorescent probe of protein hydrophobic regions, 4,4(')-dianilino-1,1(')-binaphthyl-5,5(')-disulfonate (bis-ANS), unexpectedly increases in fluorescence with soluble beta(1-40) in acidic buffer solutions but reacts weakly with amyloid fibrils while other hydrophobic probes react with the fibrils. CD analysis correlates reaction with the probe with random coil/mixed conformations and alpha-helical forms of beta(1-40) in buffer solutions but less so with soluble beta-sheet forms or amyloid fibrils. The kinetics of the fluoroalcohol-induced interconversion of conformers can be followed by changes in bis-ANS fluorescence. Formation of the beta-sheet form in aqueous buffer is limited by a slow component (minutes) while fluoroalcohol-promoted changes between beta-sheet and alpha-helix occur over seconds. Variants of beta(1-40) such as beta(1-42) or the Dutch E22Q mutation of beta(1-40) and fragments beta(1-28), beta(12-28), beta(10-20 amide), and beta(10-35 amide) react with bis-ANS under conditions that do not support fibril formation. Primary amino acid sequence is important as beta(1-11) does not cause bis-ANS fluorescence while beta(1-16) does, but hydrophobicity is not as beta(25-35) and beta(15-20 amide) are unreactive. bis-ANS is a useful biophysical tool for characterizing particular, but not all, soluble Abeta conformations distinct from the fibrillar form of amyloid peptides detected by Thioflavin T.     

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.     

Inhibition of Alzheimer amyloid aggregation with sulfated glycopolymers

Glycopolymers carrying sulfated saccharides with modest sugar contents (11% and 28%) were found to suppress the formation of amyloid fibrils by amyloid beta peptides (Abeta(1-42), Abeta(1-40), and Abeta(25-35)), as evaluated by thioflavin T assays and atomic force microscopy observation. Circular dichroism spectra showed that the conformation of amyloid beta peptides depended on the glycopolymer additives, and that the glycopolymer additives reduced the beta-sheet contents. Neutralization activity was confirmed by in vitro assay with HeLa cells. The sulfate group and the appropriate sugar contents were essential for the inhibitory effect.