Amyloid Protofibrils of Lysozyme Nucleate and Grow Via Oligomer Fusion

The mechanisms linking deposits of insoluble amyloid fibrils to the debilitating neuronal cell death characteristic of neurodegenerative diseases remain enigmatic. Recent findings implicate transiently formed intermediates of mature amyloid fibrils as the principal toxic agent. Hence, determining which intermediate aggregates represent on-pathway precursors or off-pathway side branches is critical for understanding amyloid self-assembly, and for devising therapeutic approaches targeting relevant toxic species. We examined amyloid fibril self-assembly in acidic solutions, using the model protein hen egg-white lysozyme. Combining in situ dynamic light scattering with calibrated atomic-force microscopy, we monitored the nucleation and growth kinetics of multiple transient aggregate species, and characterized both their morphologies and physical dimensions. Upon incubation at elevated temperatures, uniformly sized oligomers formed at a constant rate. After a lag period of several hours, protofibrils spontaneously nucleated. The nucleation kinetics of protofibrils and the tight match of their widths and heights with those of oligomers imply that protofibrils both nucleated and grew via oligomer fusion. After reaching several hundred nanometers in length, protofibrils assembled into mature fibrils. Overall, the amyloid fibril assembly of lysozyme followed a strict hierarchical aggregation pathway, with amyloid monomers, oligomers, and protofibrils forming on-pathway intermediates for assembly into successively more complex structures.     

Dependence on solution conditions of aggregation and amyloid formation by an SH3 domain

The formation of amyloid fibrils by the SH3 domain of the alpha-subunit of bovine phosphatidylinositol-3'-kinase (PI3-SH3) has been investigated under carefully controlled solution conditions. NMR and CD characterisation of the denatured states from which fibrils form at low pH show that their properties can be correlated with the nature of the resulting aggregates defined by EM and FTIR spectroscopy. Compact partially folded states, favoured by the addition of anions, are prone to precipitate rapidly into amorphous species, whilst well-defined fibrillar structures are formed slowly from more expanded denatured states. Kinetic data obtained by a variety of techniques show a clear lag phase in the formation of amyloid fibrils. NMR spectroscopy shows no evidence for a significant population of small oligomers in solution during or after this lag phase. EM and FTIR indicate the presence of amorphous aggregates (protofibrils) rich in beta-structure after the lag phase but prior to the development of well-defined amyloid fibrils. These observations strongly suggest a nucleation and growth mechanism for the formation of the ordered aggregates. The morphologies of the fibrillar structures were found to be highly sensitive to the pH at which the protein solutions are incubated. This can be attributed to the effect of small perturbations in the electrostatic interactions that stabilise the contacts between the protofilaments forming the amyloid fibrils. Moreover, different hydrogen bonding patterns related to the various aggregate morphologies can be distinguished by FTIR analysis.     

Beta(2)-microglobulin and its deamidated variant, N17D form amyloid fibrils with a range of morphologies in vitro.

Amyloid fibrils formed by incubation of recombinant wild-type human beta(2)-microglobulin (beta(2)M) ab initio in vitro at low pH and high ionic strength are short and highly curved. By contrast, fibrils extracted from patients suffering from haemodialysis-related amyloidosis and those formed by seeding growth of the wild-type protein in vitro with fibrils ex vivo are longer and straighter than those previously produced ab initio in vitro. Here we explore the effect of growth conditions on morphology of beta(2)M fibrils formed ab initio in vitro from the wild-type protein, as well as a variant form of beta(2)M in which Asn17 is deamidated to Asp (N17D). We show that deamidation results in significant destabilisation of beta(2)M at neutral pH. Despite this, acidification is still necessary to form amyloid from the mutant protein in vitro. Interestingly, at low pH and low ionic strength long, straight fibrils of recombinant beta(2)M are formed in vitro. The fibrils comprise three distinct morphological types when examined using electron microscopy (EM) and atomic force microscopy (AFM) that vary in periodicity and the number of constituent protofibrils. Using kinetic experiments we suggest that the immature fibrils observed previously do not represent intermediates in the assembly of fully mature amyloid, at least under the conditions studied here.     

pH-dependent amyloid and protofibril formation by the ABri peptide of familial British dementia

The ABri is a 34 residue peptide that is the major component of amyloid deposits in familial British dementia. In the amyloid deposits, the ABri peptide adopts aggregated beta-pleated sheet structures, similar to those formed by the Abeta peptide of Alzheimer's disease and other amyloid forming proteins. As a first step toward elucidating the molecular mechanisms of the beta-amyloidosis, we explored the ability of the environmental variables (pH and peptide concentration) to promote beta-sheet fibril structures for synthetic ABri peptides. The secondary structures and fibril morphology were characterized in parallel using circular dichroism, atomic force microscopy, negative stain electron microscopy, Congo red, and thioflavin-T fluorescence spectroscopic techniques. As seen with other amyloid proteins, the ABri fibrils had characteristic binding with Congo red and thioflavin-T, and the relative amounts of beta-sheet and amyloid fibril-like structures are influenced strongly by pH. In the acidic pH range 3.1-4.3, the ABri peptide adopts almost exclusively random structure and a predominantly monomeric aggregation state, on the basis of analytical ultracentrifugation measurements. At neutral pH, 7.1-7.3, the ABri peptide had limited solubility and produced spherical and amorphous aggregates with predominantly beta-sheet secondary structure, whereas at slightly acidic pH, 4.9, spherical aggregates, intermediate-sized protofibrils, and larger-sized mature amyloid fibrils were detected by atomic force microscopy. With aging at pH 4.9, the protofibrils underwent further association and eventually formed mature fibrils. The presence of small amounts of aggregated peptide material or seeds encourage fibril formation at neutral pH, suggesting that generation of such seeds in vivo could promote amyloid formation. At slightly basic pH, 9.0, scrambling of the Cys5-Cys22 disulfide bond occurred, which could lead to the formation of covalently linked aggregates. The presence of the protofibrils and the enhanced aggregation at slightly acidic pH is consistent with the behavior of other amyloid-forming proteins, which supports the premise that a common mechanism may be involved in protein misfolding and beta-amyloidosis.     

The binding of thioflavin-T to amyloid fibrils: localisation and implications

Amyloid fibrils are a polymeric form of protein, involving a continuous beta-sheet with the strands perpendicular to the long axis of the fibril. Although typically implicated in diseases such as Alzheimer's disease and the transmissible spongiform encephalopathies, non disease-associated protein can also be converted into amyloid fibrils. Traditionally, amyloid fibrils are identified via the use of specific dyes such as Congo red and thioflavin-T, although their specificity is ill understood. Recently, solutions of bovine insulin and bovine beta-lactoglobulin have been found to form spherulites, micron-sized spherical structures containing radially arranged amyloid fibrils. When studied by confocal microscopy using polarised laser light and thioflavin-T, a consistent pattern of emission, rather than a uniform disc, was observed. This suggests the dye binds in a specific, regular fashion to amyloid fibrils. Confocal microscopy studies of thioflavin-T aligned in stretched poly-vinyl alcohol films showed that the dye dipole excitation axis lies parallel to the long molecular axis. Therefore, thioflavin-T binds to amyloid fibrils such that their long axes are parallel. We propose binding occurs in 'channels' that run along the length of the beta-sheet. Steric interactions between dye molecules and side chains indicate why thioflavin-T fluoresces more intensely when bound to amyloid fibrils and can explain why this interaction with amyloid fibrils is specific, but with varying efficiency.     

Kinetics and energetics of assembly,nucleation, and growth of aggregates and fibrils for an amyloidogenic protein. Insights into transition states from pressure,temperature, and co-solute studies

The transition states for prenucleation assembly, nucleation, and growth of aggregates and amyloid fibrils were investigated for a dimeric immunoglobulin light chain variable domain, employing pressure, temperature, and solutes as variables. Pressure-induced aggregation was nucleation-dependent and first-order in protein concentration and could be seeded. The insoluble aggregates were mixtures of amyloid fibrils and amorphous aggregates. Activation volumes, activation surface areas, and activation waters of hydration were larger for aggregate growth than for prenucleation assembly or nucleation, although activation free energies were similar for the three processes. Activation free energies for each of the transition states were dominated by the unfavorable free energy of solvation of newly exposed surfaces. Equilibrium dissociation and unfolding of the dimer showed a much larger volume change than those required to form the transition states for the three processes. Thus, the transition states for these steps are similar to the native state, and their formation requires only small structural perturbations. Finally, the presence of Congo red during amyloid fibril formation shortened lag times and caused pressure insensitivity of nucleation, suggesting that this compound or its analogs may not be effective as inhibitors of amyloidosis.     

Investigation of a peptide responsible for amyloid fibril formation of beta 2-microglobulin by achromobacter protease I.

To obtain insight into the mechanism of amyloid fibril formation from beta(2)-microglobulin (beta2-m), we prepared a series of peptide fragments using a lysine-specific protease from Achromobacter lyticus and examined their ability to form amyloid fibrils at pH 2.5. Among the nine peptides prepared by the digestion, the peptide Ser(20)-Lys(41) (K3) spontaneously formed amyloid fibrils, confirmed by thioflavin T binding and electron microscopy. The fibrils composed of K3 peptide induced fibril formation of intact beta2-m with a lag phase, distinct from the extension reaction without a lag phase observed for intact beta2-m seeds. Fibril formation of K3 peptide with intact beta2-m seeds also exhibited a lag phase. On the other hand, the extension reaction of K3 peptide with the K3 seeds occurred without a lag phase. At neutral pH, the fibrils composed of either intact beta2-m or K3 peptide spontaneously depolymerized. Intriguingly, the depolymerization of K3 fibrils was faster than that of intact beta2-m fibrils. These results indicated that, although K3 peptide can form fibrils by itself more readily than intact beta2-m, the K3 fibrils are less stable than the intact beta2-m fibrils, suggesting a close relation between the free energy barrier of amyloid fibril formation and its stability.     

Optimum amyloid fibril formation of a peptide fragment suggests the amyloidogenic preference of beta2-microglobulin under physiological conditions

Beta(2)-microglobulin (beta(2)m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Although full-length beta(2)m readily forms amyloid fibrils in vitro by seed-dependent extension with a maximum at pH 2.5, fibril formation under physiological conditions as detected in patients has been difficult to reproduce. A 22-residue K3 peptide of beta(2)m, Ser(20)-Lys(41), obtained by digestion with Acromobacter protease I, forms amyloid fibrils without seeding. To obtain further insight into the mechanism of fibril formation, we studied the pH dependence of fibril formation of the K3 peptide and its morphology using a ThT fluorescence assay and electron microscopy, respectively. K3 peptide formed amyloid fibrils over a wide range of pH values with an optimum around pH 7 and contrasted with the pH profile of the seed-dependent extension reaction of full-length beta(2)m. This suggests that once the rigid native-fold of beta(2)m is unfolded and additional factors triggering the nucleation process are provided, full-length beta(2)m discloses an intrinsic potential to form amyloid fibrils at neutral pH. The fibril formation was strongly promoted by dimerization of K3 through Cys(25). The morphology of the fibrils varied depending on the fibril formation conditions and the presence or absence of a disulfide bond. Various fibrils had the potential to seed fibril formation of full-length beta(2)m accompanied with a characteristic lag phase, suggesting that the internal structures are similar.     

Self-assembly of Mutant Huntingtin Exon-1 Fragments into Large Complex Fibrillar Structures Involves Nucleated Branching

Huntingtin (HTT) fragments with extended polyglutamine tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington's disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high-resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modeling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends.     

Inhibition of toxicity and protofibril formation in the amyloid-beta peptide beta(25-35) using N-methylated derivatives

Beta (25-35) is a fragment of beta-amyloid that retains its wild-type properties. N-methylated derivatives of beta(25-35) can block hydrogen bonding on the outer edge of the assembling amyloid, so preventing the aggregation and inhibiting the toxicity of the wild-type peptide. The effects are assayed by Congo Red and thioflavin T binding, electron microscopy and an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] toxicity assay. N-methyl-Gly-25 has similar properties to the wild- type, while five other methylation sites have varying effects on prefolded fibrils and fibril assembly. In particular, N-methyl-Gly-33 is able to completely prevent fibril assembly and reduces the toxicity of prefolded amyloid. With N-methyl-Leu-34 the fibril morphology is altered and toxicity reduced. A preliminary study of beta(25-35) structure in aqueous solution was made by small-angle neutron scattering (SANS). The protofibrillar aggregates are best described as a disc of radius 140 A and height 53 A (1 A = 0.1 nm), though the possibility of polydisperse aggregates cannot be ruled out. No aggregates form in the presence of N-methyl-Gly-33. We suggest that the use of N-methylated derivatives of amyloidogenic peptides and proteins could provide a general solution to the problem of amyloid deposition and toxicity and that SANS is an important technique for the direct observation of protofibril formation and destruction in solution.     

Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates

Propagation and infectivity of prions in human prionopathies are likely associated with conversion of the mainly α-helical human prion protein, HuPrP, into an aggregated form with amyloid-like properties. Previous reports on efficient conversion of recombinant HuPrP have used mild to harsh denaturing conditions to generate amyloid fibrils in vitro. Herein we report on the in vitro conversion of four forms of truncated HuPrP (sequences 90-231 and 121-231 with and without an N-terminal hexa histidine tag) into amyloid-like fibrils within a few hours by using a protocol (phosphate buffered saline solutions at neutral pH with intense agitation) close to physiological conditions. The conversion process monitored by thioflavin T, ThT, revealed a three stage process with lag, growth and equilibrium phases. Seeding with preformed fibrils shortened the lag phase demonstrating the classic nucleated polymerization mechanism for the reaction. Interestingly, comparing thioflavin T kinetics with solubility and turbidity kinetics it was found that the protein initially formed non-thioflavionophilic, morphologically disordered aggregates that over time matured into amyloid fibrils. By transmission electron microscopy and by fluorescence microscopy of aggregates stained with luminescent conjugated polythiophenes (LCPs); we demonstrated that HuPrP undergoes a conformational conversion where spun and woven fibrils protruded from morphologically disordered aggregates. The initial aggregation functioned as a kinetic trap that decelerated nucleation into a fibrillation competent nucleus, but at the same time without aggregation there was no onset of amyloid fibril formation. The agitation, which was necessary for fibril formation to be induced, transiently exposes the protein to the air-water interface suggests a hitherto largely unexplored denaturing environment for prion conversion.     

Quantitative morphological analysis reveals ultrastructural diversity of amyloid fibrils from alpha-synuclein mutants

High resolution atomic force microscopy is a powerful tool to characterize nanoscale morphological features of protein amyloid fibrils. Comparison of fibril morphological properties between studies has been hampered by differences in analysis procedures and measurement error determination used by various authors. We describe a fibril morphology analysis method that allows for quantitative comparison of features of amyloid fibrils of any amyloidogenic protein measured by atomic force microscopy. We have used tapping mode atomic force microscopy in liquid to measure the morphology of fibrillar aggregates of human wild-type alpha-synuclein and the disease-related mutants A30P, E46K, and A53T. Analysis of the images shows that fibrillar aggregates formed by E46K alpha-synuclein have a smaller diameter (9.0 +/- 0.8 nm) and periodicity (mode at 55 nm) than fibrils of wild-type alpha-synuclein (height 10.0 +/- 1.1 nm; periodicity has a mode at 65 nm). Fibrils of A30P have smaller diameter still (8.1 +/- 1.2 nm) and show a variety of periodicities. This quantitative analysis procedure enables comparison of the results with existing models for assembly of amyloid fibrils.     

Lysozyme amyloidogenesis is accelerated by specific nicking and fragmentation but decelerated by intact protein binding and conversion

We have revisited the well-studied heat and acidic amyloid fibril formation pathway (pH 1.6, 65 degrees C) of hen egg-white lysozyme (HEWL) to map the barriers of the misfolding and amyloidogenesis pathways. A comprehensive kinetic mechanism is presented where all steps involving protein hydrolysis, fragmentation, assembly and conversion into amyloid fibrils are accounted for. Amyloid fibril formation of lysozyme has multiple kinetic barriers. First, HEWL unfolds within minutes, followed by irreversible steps of partial acid hydrolysis affording a large amount of nicked HEWL, the 49-101 amyloidogenic fragment and a variety of other species over 5-40 h. Fragmentation forming the 49-101 fragment is a requirement for efficient amyloid fibril formation, indicating that it forms the rate-determining nucleus. Nicked full-length HEWL is recruited efficiently into amyloid fibrils in the fibril growth phase or using mature fibrils as seeds, which abolished the lag phase completely. Mature amyloid fibrils of HEWL are composed mainly of nicked HEWL in the early equilibrium phase but go through a "fibril shaving" process, affording fibrils composed of the 49-101 fragment and 53-101 fragment during more extensive maturation (incubation for longer than ten days). Seeding of the amyloid fibril formation process using sonicated mature amyloid fibrils accelerates the fibril formation process efficiently; however, addition of intact full-length lysozyme at the end of the lag phase slows the rate of amyloidogenesis. The intact full-length protein, in contrast to nicked lysozyme, slows fibril formation due to its slow conversion into the amyloid fold, probably due to inclusion of the non-amyloidogenic 1-48/102-129 portion of HEWL in the fibrils, which can function as a "molecular bumper" stalling further growth.     

Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates

Propagation and infectivity of prions in human prionopathies are likely associated with conversion of the mainly a-helical human prion protein, HuPrP, into an aggregated form with amyloid-like properties. Previous reports on efficient conversion of recombinant HuPrP have used mild to harsh denaturing conditions to generate amyloid fibrils in vitro. Herein we report on the in vitro conversion of four forms of truncated HuPrP (sequences 90–231 and 121–231 with and without an N-terminal hexa histidine tag) into amyloid-like fibrils within a few hours by using a protocol (phosphate buffered saline solutions at neutral pH with intense agitation) close to physiological conditions. The conversion process monitored by thioflavin T, ThT, revealed a three stage process with lag, growth and equilibrium phases. Seeding with preformed fibrils shortened the lag phase demonstrating the classic nucleated polymerization mechanism for the reaction. Interestingly, comparing thioflavin T kinetics with solubility and turbidity kinetics it was found that the protein initially formed non- thioflavionophilic, morphologically disordered aggregates that over time matured into amyloid fibrils. By transmission electron microscopy and by fluorescence microscopy of aggregates stained with luminescent conjugated polythiophenes (LCPs); we demonstrated that HuPrP undergoes a conformational conversion where spun and woven fibrils protruded from morphologically disordered aggregates. The initial aggregation functioned as a kinetic trap that decelerated nucleation into a fibrillation competent nucleus, but at the same time without aggregation there was no onset of amyloid fibril formation. The agitation, which was necessary for fibril formation to be induced, transiently exposes the protein to the air-water interface suggests a hitherto largely unexplored denaturing environment for prion conversion.Key words: misfolding, aggregation, amyloid, prion, conformational conversion, fluorescence     

Phospholipid interaction induces molecular-level polymorphism in apolipoprotein C-II amyloid fibrils via alternative assembly pathways

A common feature of many of the most important and prominent amyloid-forming proteins is their ability to bind lipids and lipid complexes. Lipids are ubiquitous components of disease-associated amyloid plaques and deposits in humans, yet the specific roles of lipid in the process of amyloid fibril formation are poorly understood. This study investigated the effect of phospholipids on amyloid fibril formation by human apolipoprotein (apo) C-II using phosphatidylcholine derivatives comprising acyl chains of up to 14 carbon atoms. Submicellar concentrations of short-chain phospholipids increase the rate of apoC-II fibril formation in an acyl-chain-length- and concentration-dependent fashion, while high micellar concentrations of phospholipids completely inhibited amyloid formation. At lower concentrations of soluble phospholipid complexes, fibril formation by apoC-II was only partially inhibited, and under these conditions, aggregation followed a two-phase process. Electron microscopy showed that the fibrils resulting from the second phase of aggregation were straight, cablelike, and about 13 nm wide, in contrast to the homogeneous twisted-ribbon morphology of apoC-II fibrils formed under lipid-free conditions. Seeding experiments showed that this alternative fibril structure could be templated both in the presence and in the absence of lipid complex, suggesting that the two morphologies result from distinct assembly pathways. Circular dichroism spectroscopy studies indicated that the secondary structural conformation within the straight-type and ribbon-type fibrils were distinct, further suggesting divergent assembly pathways. These studies show that phospholipid complexes can change the structural architecture of mature fibrils and generate new fibril morphologies with the potential to alter the in vivo behaviour of amyloid. Such lipid interactions may play a role in defining the structural features of fibrils formed by diverse amyloidogenic proteins.     

Cooperative hydrogen bonding in amyloid formation

Amyloid diseases, including Alzheimer's and prion diseases, are each associated with unbranched protein fibrils. Each fibril is made of a particular protein, yet they share common properties. One such property is nucleation-dependent fibril growth. Monomers of amyloid-forming proteins can remain in dissolved form for long periods, before rapidly assembly into fibrils. The lag before growth has been attributed to slow kinetics of formation of a nucleus, on which other molecules can deposit to form the fibril. We have explored the energetics of fibril formation, based on the known molecular structure of a fibril-forming peptide from the yeast prion, Sup35, using both classical and quantum (density functional theory) methods. We find that the energetics of fibril formation for the first three layers are cooperative using both methods. This cooperativity is consistent with the observation that formation of amyloid fibrils involves slow nucleation and faster growth.     

Visualization and classification of amyloid beta supramolecular assemblies

Deposition of amyloid beta (Abeta) fibrils has been suggested to play a central role in Alzheimer's disease. In clarifying the mechanism by which fibrils form and moreover in developing new treatments for amyloidosis, direct observation is important. Focusing on the interactions with surfaces at the early stages, we studied the spontaneous formation of Abeta(1-40) fibrils on quartz slides, monitored by total internal reflection fluorescence microscopy combined with thioflavin T, an amyloid-specific fluorescence dye. Self-assembly of Abeta(1-40), accelerated by a low concentration of sodium dodecyl sulfate, produced various remarkable amyloid assemblies. Densely packed spherulitic structures with radial fibril growth were typically observed. When the packing of fibrils was coarse, extremely long fibrils often protruded from the spherulitic cores. In other cases, a large number of wormlike fibrils were formed. Transmission electron microscopy and atomic force microscopy revealed relatively short and straight fibrillar blocks associated laterally without tight interaction, leading to random-walk-like fibril growth. These results suggest that, during spontaneous fibrillation, the nucleation occurring in contact with surfaces is easily affected by environmental factors, creating various types of nuclei, and hence variations in amyloid morphology. A taxonomy of amyloid supramolecular assemblies will be useful in clarifying the structure-function relationship of amyloid fibrils.     

Ultrasonication-induced amyloid fibril formation of beta2-microglobulin

To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded beta2-m solution at pH 2.5, thioflavin T fluorescence increased markedly after a lag time of 1-2 h with a simultaneous increase of light scattering. Atomic force microscopy images showed the formation of a large number of short fibrils 3 nm in diameter. When the sonication-induced fibrils were used as seeds in the next seeding experiment at pH 2.5, a rapid and intense formation of long fibrils 3 nm in diameter was observed demonstrating seed-dependent fibril growth. We then examined the effects of sonication on the native beta2-m at neutral pH, conditions under which amyloid deposits occur in patients. In the presence of 0.5 mm sodium dodecyl sulfate, a model compound of potential trigger and stabilizer of amyloid fibrils in patients, a marked increase of thioflavin T fluorescence was observed after 1 day of sonication at pH 7.0. The products of sonication caused the accelerated fibril formation at pH 7.0. Atomic force microscopy images showed that the fibrils formed at pH 7.0 have a diameter of more than 7 nm, thicker than those prepared at pH 2.5. These results indicate that ultrasonication is one form of agitation triggering the formation of amyloid fibrils of beta2-m, producing fibrils adapted to the respective pH.     

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.