Amyloid Fibril Assembly Research Articles

AFM-based force spectroscopy measurements of mature amyloid fibrils of the peptideglucagon

We report on the mechanical characterization of individual mature amyloid fibrils byatomic force microscopy (AFM) and AFM-based single-molecule force spectroscopy(SMFS). These self-assembling materials, formed from the 29-residue amphiphatic peptidehormone glucagon, were found to display a reversible elastic behaviour. Based onAFM morphology and SMFS studies, we suggest that the observed elasticity isdue to a force-induced conformational transition which is reversible due to theβ-helical conformation of protofibrils, allowing a high degree of extension. The elasticproperties of such mature fibrils contribute to their high stability, suggesting that theinternal hydrophobic interactions of amyloid fibrils are likely to be of fundamentalimportance in the assembly of amyloid fibrils and therefore for the understanding of theprogression of their associated pathogenic disorders. In addition, such biological amyloidfibril structures with highly stable mechanical properties can potentially be used toproduce nanofibres (nanowires) that may be suitable for nanotechnological applications.

Heparin Strongly Enhances the Formation of β2-Microglobulin Amyloid Fibrils in the Presence of Type I Collagen

The tissue specificity of fibrillar deposition in dialysis-related amyloidosis is most likely associated with the peculiar interaction of beta2-microglobulin (beta2-m) with collagen fibers. However, other co-factors such as glycosaminoglycans might facilitate amyloid formation. In this study we have investigated the role of heparin in the process of collagen-driven amyloidogenesis. In fact, heparin is a well known positive effector of fibrillogenesis, and the elucidation of its potential effect in this type of amyloidosis is particularly relevant because heparin is regularly given to patients subject to hemodialysis to prevent blood clotting. We have monitored by atomic force microscopy the formation of beta2-m amyloid fibrils in the presence of collagen fibers, and we have discovered that heparin strongly accelerates amyloid deposition. The mechanism of this effect is still largely unexplained. Using dynamic light scattering, we have found that heparin promotes beta2-m aggregation in solution at pH 6.4. Morphology and structure of fibrils obtained in the presence of collagen and heparin are highly similar to those of natural fibrils. The fibril surface topology, investigated by limited proteolysis, suggests that the general assembly of amyloid fibrils grown under these conditions and in vitro at low pH is similar. The exposure of these fibrils to trypsin generates a cleavage at the C-terminal of lysine 6 and creates the 7-99 truncated form of beta2-m (DeltaN6beta2-m) that is a ubiquitous constituent of the natural beta2-m fibrils. The formation of this beta2-m species, which has a strong propensity to aggregate, might play an important role in the acceleration of local amyloid deposition.

Apolipoprotein C-II Amyloid Fibrils Assemble via a Reversible Pathway that Includes Fibril Breaking and Rejoining

Alzheimer's and several other diseases are characterized by the misfolding and assembly of protein subunits into amyloid fibrils. Current models propose that amyloid fibril formation proceeds via the self-association of several monomers to form a nucleus, which then elongates by the addition of monomer to form mature fibrils. We have examined the concentration-dependent kinetics of apolipoprotein C-II amyloid fibril formation and correlated this with the final size distribution of the fibrils determined by sedimentation velocity experiments. In contrast to predictions of the nucleation-elongation model, the final size distribution of the fibrils was found to be relatively independent of the starting monomer concentration. To explain these results, we extended the nucleation-elongation model to include fibril breaking and rejoining as integral parts of the amyloid fibril assembly mechanism. The system was examined under conditions that affected the stability of the mature fibrils including the effect of dilution on the free pool of monomeric apolipoprotein C-II and the time-dependent recovery of fibril size following sonication. Antibody-labelling transmission electron microscopy studies provided direct evidence for spontaneous fibril breaking and rejoining. These studies establish the importance of breaking and rejoining in amyloid fibril formation and identify prospective new therapeutic targets in the assembly pathway.

Role of Different Regions of α-Synuclein in the Assembly of Fibrils

Elucidating the details of the assembly of amyloid fibrils is a key step to understanding the mechanism of amyloid deposition diseases including Parkinson's disease. Although several models have been proposed, based on analyses of polypeptides and short peptides, a detailed understanding of the structure and mechanism of alpha-synuclein fibrillation remains elusive. In this study, we used trypsin and endoproteinase GluC to digest intact alpha-synuclein fibrils and to analyze the detailed morphology of the resultant fibrils/remnants. We also created three mutants of alpha-synuclein, in which the N-terminal and C-terminal regions were removed, both individually and in combination, and investigated the detailed morphology of the fibrils from these mutants. Our results indicate that the assembly of mature alpha-synuclein fibrils is hierarchical: protofilaments --> protofibrils --> mature fibrils. There is a core region of approximately 70 amino acids, from residues approximately 32 to 102, which comprises the beta-rich core of the protofilaments and fibrils. In contrast, the two terminal regions show no evidence of participating in the assembly of the protofilament core but play a key role in the interactions between the protofilaments, which is necessary for the fibril maturation.

Site-Directed Mutations in the C-Terminal Extension of Human αB-Crystallin Affect Chaperone Function and Block Amyloid Fibril Formation

BackgroundAlzheimer's, Parkinson's and Creutzfeldt-Jakob disease are associated with inappropriate protein deposition and ordered amyloid fibril assembly. Molecular chaperones, including αB-crystallin, play a role in the prevention of protein deposition.Methodology/Principal FindingsA series of site-directed mutants of the human molecular chaperone, αB-crystallin, were constructed which focused on the flexible C-terminal extension of the protein. We investigated the structural role of this region as well as its role in the chaperone function of αB-crystallin under different types of protein aggregation, i.e. disordered amorphous aggregation and ordered amyloid fibril assembly. It was found that mutation of lysine and glutamic acid residues in the C-terminal extension of αB-crystallin resulted in proteins that had improved chaperone activity against amyloid fibril forming target proteins compared to the wild-type protein.Conclusions/SignificanceTogether, our results highlight the important role of the C-terminal region of αB-crystallin in regulating its secondary, tertiary and quaternary structure and conferring thermostability to the protein. The capacity to genetically modify αB-crystallin for improved ability to block amyloid fibril formation provides a platform for the future use of such engineered molecules in treatment of diseases caused by amyloid fibril formation.

Structural Reorganisation and Potential Toxicity of Oligomeric Species Formed during the Assembly of Amyloid Fibrils

Increasing evidence indicates that oligomeric protein assemblies may represent the molecular species responsible for cytotoxicity in a range of neurological disorders including Alzheimer and Parkinson diseases. We use all-atom computer simulations to reveal that the process of oligomerization can be divided into two steps. The first is characterised by a hydrophobic coalescence resulting in the formation of molten oligomers in which hydrophobic residues are sequestered away from the solvent. In the second step, the oligomers undergo a process of reorganisation driven by interchain hydrogen bonding interactions that induce the formation of β sheet rich assemblies in which hydrophobic groups can become exposed. Our results show that the process of aggregation into either ordered or amorphous species is largely determined by a competition between the hydrophobicity of the amino acid sequence and the tendency of polypeptide chains to form arrays of hydrogen bonds. We discuss how the increase in solvent-exposed hydrophobic surface resulting from such a competition offers an explanation for recent observations concerning the cytotoxicity of oligomeric species formed prior to mature amyloid fibrils.

Engineering metal ion coordination to regulate amyloid fibril assembly and toxicity

Protein and peptide assembly into amyloid has been implicated in functions that range from beneficial epigenetic controls to pathological etiologies. However, the exact structures of the assemblies that regulate biological activity remain poorly defined. We have previously used Zn(2+) to modulate the assembly kinetics and morphology of congeners of the amyloid beta peptide (Abeta) associated with Alzheimer's disease. We now reveal a correlation among Abeta-Cu(2+) coordination, peptide self-assembly, and neuronal viability. By using the central segment of Abeta, HHQKLVFFA or Abeta(13-21), which contains residues H13 and H14 implicated in Abeta-metal ion binding, we show that Cu(2+) forms complexes with Abeta(13-21) and its K16A mutant and that the complexes, which do not self-assemble into fibrils, have structures similar to those found for the human prion protein, PrP. N-terminal acetylation and H14A substitution, Ac-Abeta(13-21)H14A, alters metal coordination, allowing Cu(2+) to accelerate assembly into neurotoxic fibrils. These results establish that the N-terminal region of Abeta can access different metal-ion-coordination environments and that different complexes can lead to profound changes in Abeta self-assembly kinetics, morphology, and toxicity. Related metal-ion coordination may be critical to the etiology of other neurodegenerative diseases.

Dissociation of Aβ 16–22 Amyloid Fibrils Probed by Molecular Dynamics

The mechanisms of deposition and dissociation are implicated in the assembly of amyloid fibrils. To investigate the kinetics of unbinding of Abeta(16-22) monomers from preformed fibrils, we use molecular dynamics (MD) simulations and the structures for Abeta(16-22) amyloid fibrils. Consistent with experimental studies, the dissociation of Abeta(16-22) peptides involves two main stages, locked and docked, after which peptides unbind. The lifetime of the locked state, in which a peptide retains fibril-like structure and interactions, extends up to 0.5 micros under normal physiological conditions. Upon cooperative rupture of all fibril-like hydrogen bonds (HBs) with the fibril, a peptide enters a docked state. This state is populated by disordered random coil conformations and its lifetime ranges from approximately 10 to 200 ns. The docked state is stabilized by hydrophobic side chain interactions, while the contribution from HBs is small. Our simulations also suggest that the peptides located on fibril edges may form stable beta-strand conformations distinct from the fibril "bulk". We propose that such edge peptides can act as fibril caps, which impede fibril elongation. Our results indicate that the interactions between unbinding peptides constitute the molecular basis for cooperativity of peptide dissociation. The kinetics of fibril growth is reconstructed from unbinding assuming the reversibility of deposition/dissociation pathways. The relation of in silica dissociation kinetics to experimental observations is discussed.

Mimicking phosphorylation of αB-crystallin affects its chaperone activity

AlphaB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to alphaB-crystallin. In the present study, we produced recombinant proteins designed to mimic phosphorylation of alphaB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural investigation of the effect of phosphorylation on the chaperone activity of alphaB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of alphaB-crystallin results in more efficient chaperone activity against both heat-induced and reduction-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of alphaB-crystallin against one amyloid-forming target protein (kappa-casein), but decreased it against another (ccbeta-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated alphaB-crystallins. The present study provides evidence for the regulation of the chaperone activity of alphaB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases.

Quantitative Morphological Analysis Reveals Ultrastructural Diversity of Amyloid Fibrils from α-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.

Kinetics and Thermodynamics of Amyloid Fibril Assembly

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Real-time and Single Fibril Observation of the Formation of Amyloid β Spherulitic Structures

In Alzheimer disease, amyloid beta, a 39-43-residue peptide produced by cleavage from a large amyloid precursor protein, undergoes conformational change to form amyloid fibrils and deposits as senile amyloid plaques in the extracellular cerebral cortices of the brain. However, the mechanism of how the intrinsically linear amyloid fibrils form spherical senile plaques is unknown. With total internal reflection fluorescence microscopy combined with the use of thioflavin T, an amyloid-specific fluorescence dye, we succeeded in observing the formation of the senile plaque-like spherulitic structures with diameters of around 15 microm on the chemically modified quartz surface. Real-time observation at a single fibrillar level revealed that, in the absence of tight contact with the surface, the cooperative and radial growth of amyloid fibrils from the core leads to a huge spherulitic structure. The results suggest the underlying physicochemical mechanism of senile plaque formation, essential for obtaining insight into prevention of Alzheimer disease.

Kinetics and Thermodynamics of Amyloid Fibril Assembly

With some exceptions, amyloids appear to be accidental aggregated structures whose formation was not selected for in molecular evolution. Despite this, amyloid fibrils are in many respects surprisingly well-behaved molecules. For example, Huntington's disease-related polyglutamine sequences aggregate via a relatively simple nucleated growth polymerization mechanism. In addition, the Alzheimer's plaque protein Abeta has been shown to undergo reversible amyloid fibril formation to a position of dynamic equilibrium such that reaction thermodynamics can be quantified. Studies of these well-behaved amyloid systems are allowing us to peer more deeply into the process and products of off-pathway misfolding and aggregation.

Dichotomous versus palm‐type mechanisms of lateral assembly of amyloid fibrils

Despite possessing a common cross-beta core, amyloid fibrils are known to exhibit great variations in their morphologies. To date, the mechanism responsible for the polymorphism in amyloid fibrils is poorly understood. Here we report that two variants of mammalian full-length prion protein (PrP), hamster (Ha) and mouse (Mo) PrPs, produced morphologically distinguishable subsets of mature fibrils under identical solvent conditions. To gain insight into the origin of this morphological diversity we analyzed the early stages of polymerization. Unexpectedly, we found that despite a highly conserved amyloidogenic region (94% identity within the residues 90-230), Ha and Mo PrPs followed two distinct pathways for lateral assembly of protofibrils into mature, higher order fibrils. The protofibrils of Ha PrP first formed irregular bundles characterized by a peculiar palm-type shape, which ultimately condensed into mature fibrils. The protofibrils of Mo PrP, on the other hand, associated in pairs in a pattern resembling dichotomous coalescence. These pathways are referred to here as the palm-type and dichotomous mechanisms. Two distinct mechanisms for lateral assembly explain striking differences in morphology of mature fibrils produced from closely related Mo and Ha PrPs. Remarkable similarities between subtypes of amyloid fibrils generated from different proteins and peptides suggest that the two mechanisms of lateral assembly may not be limited to prion proteins but may be a common characteristic of polymerization of amyloidogenic proteins and peptides in general.

Acetylcholinesterase (AChE) - Amyloid-β-Peptide Complexes in Alzheimers Disease. The Wnt Signaling Pathway

Alzheimer's disease (AD) is characterized by selective neuronal cell death, which is probably caused by amyloid beta-peptide (Abeta) oligomers and fibrils. We have found that acetylcholinesterase (AChE), a senile plaque component, increases amyloid fibril assembly with the formation of highly toxic complexes (Abeta-AChE). The neurotoxic effect induced by Abeta-AChE complexes was higher than that induced by the Abeta peptide alone as shown both in vitro (hippocampal neurons) and in vivo (rats injected with Abeta peptide in the dorsal hippocampus). Interestingly, treatment with Abeta-AChE complexes decreases the cytoplasmic beta-catenin level, a key component of Wnt signaling. Conversely, the activation of this signaling pathway by Wnt-3a promotes neuronal survival and rescues changes in Wnt components (activation or subcellular localization). Moreover Frzb-1, a Wnt antagonist reverses the Wnt-3a neuroprotection effect against Abeta neurotoxicity. Compounds that mimic the Wnt signaling or modulate the cross-talking with this pathway could be used as neuroprotective agents for therapeutic strategies in AD patients.

A General Model for Amyloid Fibril Assembly Based on Morphological Studies Using Atomic Force Microscopy

Based on atomic force microscopy analysis of the morphology of fibrillar species formed during fibrillation of alpha-synuclein, insulin, and the B1 domain of protein G, a previously described model for the assembly of amyloid fibrils of immunoglobulin light-chain variable domains is proposed as a general model for the assembly of protein fibrils. For all of the proteins studied, we observed two or three fibrillar species that vary in diameter. The smallest, protofilaments, have a uniform height, whereas the larger species, protofibrils and fibrils, have morphologies that are indicative of multiple protofilaments intertwining. In all cases, protofilaments intertwine to form protofibrils, and protofibrils intertwine to form fibrils. We propose that the hierarchical assembly model describes a general mechanism of assembly for all amyloid fibrils.

Hierarchical Assembly of β 2-Microglobulin Amyloid In Vitro Revealed by Atomic Force Microscopy

The kinetics of spontaneous assembly of amyloid fibrils of wild-type beta(2)-microglobulin (beta(2)M) in vitro, under acid conditions (pH 2.5) and low ionic strength, has been followed using thioflavin-T (ThT) binding. In parallel experiments, the morphology of the different fibrillar species present at different time-points during the growth process were characterised using tapping-mode atomic force microscopy (TM-AFM) in air and negative stain electron microscopy (EM). The thioflavin-T assay shows a characteristic lag phase during which the nucleation of fibrils occurs before a rapid growth in fibril density. The volume of fibrils deposited on mica measured from TM-AFM images at each time-point correlates well with the fluorescence data. TM-AFM and negative-stain EM revealed the presence of various kinds of protein aggregates in the lag phase that disappear concomitantly with a rise in the density of amyloid fibrils, suggesting that these aggregates precede fibril growth and may act as nucleation sites. Three distinct morphologies of mature amyloid fibrils were observed within a single growth experiment, as observed previously for the wild-type protein and the variant N17D. Additional supercoiled morphologies of the lower-order fibrils were observed. Comparative height analysis from the TM-AFM data allows each of the mature fibril types and single protofilaments to be identified unambiguously, and reveals that the assembly occurs via a hierarchy of morphological states.

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

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

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.

The protofilament structure of insulin amyloid fibrils.

Under solution conditions where the native state is destabilized, the largely helical polypeptide hormone insulin readily aggregates to form amyloid fibrils with a characteristic cross-beta structure. However, there is a lack of information relating the 4.8 A beta-strand repeat to the higher order assembly of amyloid fibrils. We have used cryo-electron microscopy (EM), combining single particle analysis and helical reconstruction, to characterize these fibrils and to study the three-dimensional (3D) arrangement of their component protofilaments. Low-resolution 3D structures of fibrils containing 2, 4, and 6 protofilaments reveal a characteristic, compact shape of the insulin protofilament. Considerations of protofilament packing indicate that the cross-beta ribbon is composed of relatively flat beta-sheets rather than being the highly twisted, beta-coil structure previously suggested by analysis of globular protein folds. Comparison of the various fibril structures suggests that very small, local changes in beta-sheet twist are important in establishing the long-range coiling of the protofilaments into fibrils of diverse morphology.