Ex situ atomic force microscopy analysis of beta-amyloid self-assembly and deposition on a synthetic template

The beta-amyloid (Abeta) deposition, which is the conversion of soluble Abeta peptides to insoluble plaques on a surface, is an essential pathological process in Alzheimer's disease (AD). The identification and characterization of possible environmental factors that may influence amyloid deposition in vivo are important to unveil the underlying etiology of AD. According to the amyloid cascade hypothesis, diffuse plaques are initial and visual deposits in the early event of AD, leading to amyloid plaques. To study amyloid deposition and growth in vitro, we prepared a synthetic template by immobilizing Abeta seeds on an N-hydroxysuccinimide ester-activated solid surface. According to our analysis with an ex situ atomic force microscope, the formation of amyloid plaque-like aggregates was mediated by the interaction between Abeta in a solution and on a synthetic template, suggesting that Abeta oligomers function well as seeds for amyloid deposition. It was observed that insoluble amyloid aggregates formed on the template surface serve as a sink of soluble Abeta in a solution as well as mediate the formation of intermediates in the pathway of amyloid fibrillization in a solution. Relative seeding efficiencies of fresh monomers, oligomers, and fully grown fibrils were analyzed by measuring the deposited plaque volume and its height distribution through atomic force microscopy. The result revealed that oligomeric forms of Abeta act more efficiently as seeds than monomers or fibrils do. Fluorescence spectroscopy with thioflavin T confirmed that amyloid aggregate formation proceeds in a concentration-dependent manner. Analysis with Fourier transform infrared spectroscopy indicated a progressive transition of soluble Abeta42 monomer to amyloid fibrils having antiparallel beta-sheet structure on the template. Furthermore, studies on the interaction between Abeta40 and 42, two major variants of Abeta derived from the amyloid precursor protein, showed that amyloid aggregate formation on the surface was accelerated further by the homogeneous association of soluble Abeta42 onto Abeta42 seeds than by other combinations. A slightly acidic condition was found to be unfavorable for amyloid formation. This study gives insight into understanding the effects of environmental factors on amyloid formation via the use of a synthetic template system.     

Pathway Dependence of the Formation and Development of Prefibrillar Aggregates in Insulin B Chain

Amyloid fibrils have been an important subject as they are involved in the development of many amyloidoses and neurodegenerative diseases. The formation of amyloid fibrils is typically initiated by nucleation, whereas its exact mechanisms are largely unknown. With this situation, we have previously identified prefibrillar aggregates in the formation of insulin B chain amyloid fibrils, which have provided an insight into the mechanisms of protein assembly involved in nucleation. Here, we have investigated the formation of insulin B chain amyloid fibrils under different pH conditions to better understand amyloid nucleation mediated by prefibrillar aggregates. The B chain showed strong propensity to form amyloid fibrils over a wide pH range, and prefibrillar aggregates were formed under all examined conditions. In particular, different structures of amyloid fibrils were found at pH 5.2 and pH 8.7, making it possible to compare different pathways. Detailed investigations at pH 5.2 in comparison with those at pH 8.7 have suggested that the evolution of protofibril-like aggregates is a common mechanism. In addition, different processes of evolution of the prefibrillar aggregates have also been identified, suggesting that the nucleation processes diversify depending on the polymorphism of amyloid fibrils.     

A designed protein interface that blocks fibril formation

Protein fibril formation is implicated in many diseases, and therefore much effort has been focused toward the development of inhibitors of this process. In a previous project, a monomeric protein was computationally engineered to bind itself and form a heterodimer complex following interfacial redesign. One of the protein monomers, termed monomer-B, was unintentionally destabilized and shown to form macroscopic fibrils. Interestingly, in the presence of the designed binding partner, fibril formation was blocked. Here we describe the complete characterization of the amyloid properties of monomer-B and the inhibition of fiber formation by the designed binding partner, monomer-A. Both proteins are mutants of the betal domain of streptococcal protein-G. The free monomer-B protein forms amyloid-type fibrils, as determined by transmission electron microscopy and the change in fluorescence of Thioflavin T, an amyloid-specific dye. Fibril formation kinetics are influenced by pH, protein concentration, and seeding with preformed fibrils. Under all conditions tested, monomer-A was able to inhibit the formation of monomer-B fibrils. This inhibition is specific to the engineered interaction, as incubation of monomer-B with wild-type protein-G (a structural homologue) did not result in inhibition under the same conditions. Thus, this de novo-designed heterodimeric complex is an excellent model system for the study of protein-based fibril formation and inhibition. This system provides additional insight into the development of pharmaceuticals for amyloid disorders, as well as the potential use of amyloid fibrils for self-assembling nanostructures.     

The crowded environment of a reverse micelle induces the formation of β-strand seed structures for nucleating amyloid fibril formation

A hallmark of Alzheimer's disease is the accumulation of insoluble fibrils in the brain composed of amyloid beta (Aβ) proteins with parallel in-register cross-β-sheet structure. It has been suggested that the aggregation of monomeric Aβ proteins into fibrils is promoted by "seeds" that form within compartments of the brain that have limited solvent due to macromolecular crowding. To characterize these seeds, a crowded macromolecular environment was mimicked by encapsulating Aβ40 monomers into reverse micelles. Fourier-transform infrared spectroscopy revealed that monomeric Aβ proteins form extended β-strands in reverse micelles, while an analogue with a scrambled sequence does not. This is a remarkable finding, because the formation of extended β-strands by monomeric Aβ proteins suggests a plausible mechanism whereby the formation of amyloid fibrils may be nucleated in the human brain.     

Silica Nanowires Templated by Amyloid‐like Fibrils

Many peptides self‐assemble to form amyloid fibrils. We previously explored the sequence propensity to form amyloid using variants of a designed peptide with sequence KFFEAAAKKFFE. These variant peptides form highly stable amyloid fibrils with varied lateral assembly and are ideal to template further assembly of non‐proteinaceous material. Herein, we show that the fibrils formed by peptide variants can be coated with a layer of silica to produce silica nanowires using tetraethyl‐orthosilicate. The resulting nanowires were characterized using electron microscopy (TEM), X‐ray fiber diffraction, FTIR and cross‐section EM to reveal a nanostructure with peptidic core. Lysine residues play a role in templating the formation of silica on the fibril surface and, using this library of peptides, we have explored the contributions of lysine as well as arginine to silica templating, and find that sequence plays an important role in determining the physical nature and structure of the resulting nanowires.     

Polymorphism of Amyloid Fibrils In Vivo

Polymorphism is a wide‐spread feature of amyloid‐like fibrils formed in vitro, but it has so far remained unclear whether the fibrils formed within a patient are also affected by this phenomenon. In this study we show that the amyloid fibrils within a diseased individual can vary considerably in their three‐dimensional architecture. We demonstrate this heterogeneity with amyloid fibrils deposited within different organs, formed from sequentially non‐homologous polypeptide chains and affecting human or animals. Irrespective of amyloid type or source, we found in vivo fibrils to be polymorphic. These data imply that the chemical principles of fibril assembly that lead to such polymorphism are fundamentally conserved in vivo and in vitro.     

Secondary nucleation and accessible surface in insulin amyloid fibril formation

At low pH insulin is highly prone to self-assembly into amyloid fibrils. The process has been proposed to be affected by the existence of secondary nucleation pathways, in which already formed fibrils are able to catalyze the formation of new fibrils. In this work, we studied the fibrillation process of human insulin in a wide range of protein concentrations. Thioflavin T fluorescence was used for its ability to selectively detect amyloid fibrils, by mechanisms that involve the interaction between the dye and the accessible surface of the fibrils. Our results show that the rate of fibrillation and the Thioflavin T fluorescence intensity saturate at high protein concentration and that, surprisingly, the two parameters are proportional to each other. Because Thioflavin T fluorescence is likely to depend on the accessible surface of the fibrils, we suggest that the overall fibrillation kinetics is mainly governed by the accessible surface, through secondary nucleation mechanisms. Moreover, a statistical study of the fibrillation kinetics suggests that the early stages of the process are affected by stochastic nucleation events.     

End Capping Alters the Structural Characteristics and Mechanical Properties of Transthyretin (105–115) Amyloid Protofibrils

Pathological amyloid proteins are associated with degenerative and neurodegenerative diseases. These amyloid proteins develop as oligomer, fibrillar, and plaque forms, due to the denatured and unstable status of the amyloid monomers. Specifically, the development of fibrillar amyloid proteins has been investigated through several experimental studies. To understand the generation of amyloid fibrils, environmental factors such as point mutations, pH, and polymorphic characteristics have been considered. Recently, amyloid fibril studies related to end‐capping effects have been conducted to understand amyloid fibril development. However, atomic‐level studies to determine the stability and mechanical properties of amyloid fibrils based on end capping have not been undertaken. In this study, we show that end capping alters the structural characteristics and conformations of transthyretin (TTR) amyloid fibrils by using molecular dynamics (MD) simulations. Variation in the structural conformations and characteristics of the TTR fibrils through end capping are observed, due to the resulting electrostatic energies and hydrophobicity characteristics. Moreover, the end capping changes the mechanical properties of TTR fibrils. Our results shed light on amyloid fibril formation under end‐capping conditions.     

Structural intermediates during α-synuclein fibrillogenesis on phospholipid vesicles

α-Synuclein (AS) fibrils are the main protein component of Lewy bodies, the pathological hallmark of Parkinson's disease and other related disorders. AS forms helices that bind phospholipid membranes with high affinity, but no atomic level data for AS aggregation in the presence of lipids is yet available. Here, we present direct evidence of a conversion from α-helical conformation to β-sheet fibrils in the presence of anionic phospholipid vesicles and direct conversion to β-sheet fibrils in their absence. We have trapped intermediate states throughout the fibril formation pathways to examine the structural changes using solid-state NMR spectroscopy and electron microscopy. The comparison between mature AS fibrils formed in aqueous buffer and those derived in the presence of anionic phospholipids demonstrates no major changes in the overall fibril fold. However, a site-specific comparison of these fibrillar states demonstrates major perturbations in the N-terminal domain with a partial disruption of the long β-strand located in the 40s and small perturbations in residues located in the "non-β amyloid component" (NAC) domain. Combining all these results, we propose a model for AS fibrillogenesis in the presence of phospholipid vesicles.     

Cold Denaturation of α‐Synuclein Amyloid Fibrils

Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α‐Synuclein fibrils cold‐denatured to monomers at 0–20 °C and heat‐denatured at 60–110 °C. Meanwhile, the fibrils of β2‐microglobulin, Alzheimer’s Aβ1‐40/Aβ1‐42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α‐synuclein fibrils. We propose that although cold‐denaturation is common to both native proteins and misfolded fibrillar states, the main‐chain dominated amyloid structures may explain amyloid‐specific cold denaturation arising from the unfavorable burial of charged side‐chains in fibril cores.     

Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer's disease amyloid-β protein

Background: Amyloid plaques composed of the fibrillar form of the amyloid-β protein (Aβ) are the defining neuropathological feature of Alzheimer's disease (AD). A detailed understanding of the time course of amyloid formation could define steps in disease progression and provide targets for therapeutic intervention. Amyloid fibrils, indistinguishable from those derived from an AD brain, can be produced in vitro using a seeded polymerization mechanism. In its simplest form, this mechanism involves a cooperative transition from monomeric Aβ to the amyloid fibril without the buildup of intermediates. Recently, however, a transient species, the Aβ amyloid protofibril, has been identified. Here, we report studies of Aβ amyloid protofibril and its seeded transition into amyloid fibrils using atomic force microscopy.Results: Seeding of the protofibril-to-fibril transition was observed. Preformed fibrils, but not protofibrils, effectively seeded this transition. The assembly state of Aβ influenced the rate of seeded growth, indicating that protofibrils are fibril assembly precursors. The handedness of the helical surface morphology of fibrils depended on the chirality of Aβ. Finally, branched and partially wound fibrils were observed.Conclusions: The temporal evolution of morphologies suggests that the protofibril-to-fibril transition is nucleation-dependent and that protofibril winding is involved in that transition. Fibril unwinding and branching may be essential for the post-nucleation growth process. The protofibrillar assembly intermediate is a potential target for AD therapeutics aimed at inhibiting amyloid formation and AD diagnostics aimed at detecting presymptomatic disease.     

Morphology and persistence length of amyloid fibrils are correlated to peptide molecular structure

The formation of amyloid fibrils is a self-assembly process of peptides or proteins. The superior mechanical properties of these fibrils make them interesting for materials science but constitute a problem in amyloid-related diseases. Amyloid structures tend to be polymorphic, and their structure depends on growth conditions. To understand and control the assembly process, insights into the relation between the mechanical properties and molecular structure are essential. We prepared long, straight as well as short, worm-like β-lactoglobulin amyloid fibrils and determined their morphology and persistence length by atomic force microscopy (AFM) and the molecular conformation using vibrational sum-frequency generation (VSFG) spectroscopy. We show that long fibrils with near-100% β-sheet content have a 40-times higher persistence length than short, worm-like fibrils with β-sheet contents below 80%.     

酚化合物对胰岛素淀粉样纤维化的抑制作用

采用牛胰岛素作为模型多肽分子, 对几种结构相近的简单多酚的抗多肽淀粉样纤维化作用进行了研究. 结果表明, 邻苯二酚和对苯二酚对胰岛素纤维化具有抑制作用, 并通过形成醌中间体对多肽链进行修饰, 与对苯醌作用类似; 而苯酚和间二苯酚在相同条件下, 既不能修饰多肽也无抑制纤维化作用. 在无氧条件下, 邻苯二酚和对苯二酚对胰岛素纤维化的抑制作用明显降低, 说明酚化合物经氧化形成的醌中间体是其抗胰岛素纤维化的主要活性结构.     

Co‐existence of Two Different α‐Synuclein Oligomers with Different Core Structures Determined by Hydrogen/Deuterium Exchange Mass Spectrometry

Neurodegenerative disorders are characterized by the formation of protein oligomers and amyloid fibrils, which in the case of Parkinson’s disease involves the protein α‐synuclein (αSN). Cytotoxicity is mainly associated with the oligomeric species, but we still know little about their assembly and structure. Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is used to analyze oligomers formed by wild‐type (wt) αSN and also three familial αSN mutants (A30P, E46K, and A53T). All four variants show co‐existence of two different oligomers. The backbone amides of oligomer type I are protected from exchange with D₂O until they dissociate into monomeric αSN by EX1 exchange kinetics. Fewer residues are protected against exchange in oligomer type II, but this type does not revert to αSN monomers. Both oligomers are protected in the core sequence Y39–A89. Based on incubation studies, oligomer type I appears to form straight fibrils, while oligomer type II forms amorphous clusters that do not directly contribute to the fibrillation process.     

Pyrroloquinoxaline hydrazones as fluorescent probes for amyloid fibrils

Here we describe the identification and preliminary characterization of a new class of pyrrolo(imidazo)quinoxaline hydrazones as florescent probes for Aβ(1-42) fibrils. All the newly developed compounds were able to bind amyloid fibrils formed in vitro and some of them displayed an increase of their fluorescence upon binding. When tested on brain tissue preparations presenting Aβ deposits, the described hydrazones selectively stained amyloid structures and did not display aspecific binding. The hydrazones did not show antifibrillogenic activity and electron microscopy analysis revealed that they do not interfere with fibrils structure. The described pyrrolo(imidazo)quinoxalines could be useful for studying amyloid structures in vitro. Moreover, their experimentally proven ability to cross the blood-brain barrier in mouse opens the possibility of developing these compounds as potential amyloid imaging agents for in vivo applications.     

Gelation of Misfolded Proteins

Insulin protein was exposed to mildly denaturing conditions (heat and low pH) to encourage the formation of beta-sheet rich amyloid fibrils. This resulted in an increase in viscosity of our protein samples and the morphology and thermodynamics of the resulting hydrogel were monitored using environmental scanning electron microscopy and micro differential scanning calorimetry respectively. It was found that the beta-sheet fibrils aggregated further to form macrofibrils, 2 μm in diameter and several microns in length. These long, flexible macrofibrils became entangled to form hydrogels with controllable mesh size: the higher the incubation temperature the higher the number of entanglements, and consequently the smaller the mesh size.     

Experimental evidence for the reorganization of beta-strands within aggregates of the Abeta(16-22) peptide

Amyloidogenic deposits that accumulate in brain tissue with the progression of Alzheimer's disease contain large amounts of the amyloid beta-peptide. A small fragment of this peptide, comprising residues 16-22 (Abeta(16-22)), forms beta-sheets in isolation, which then aggregate into amyloid fibrils. Here, using isotope edited infrared spectroscopy to probe the secondary structure of the peptide with residue level specificity, we are able to show conclusively that the beta-sheets formed are antiparallel and, following an anneal cycle or prolonged incubation, are in register with the central residue (Phe19) in alignment across all strands. The alignment of strands proceeds via a rapid interchange from one sheet to another. This realignment of the peptide strands into a more favorable registry may have important implications for therapeutics since previous work has shown that well aligned beta-sheets form more stable amyloid fibrils.     

Investigating the structural properties of amyloid-like fibrils formed in vitro from beta2-microglobulin using limited proteolysis and electrospray ionisation mass spectrometry

The protein beta(2)-microglobulin (beta(2)m) aggregates to form classical amyloid fibrils in patients undergoing long-term haemodialysis. Amyloid-like fibrils with a cross-beta fold can also be formed from wild-type beta(2)m under acidic conditions in vitro. The morphology of such fibrils depends critically on the conditions used: incubation of beta(2)m in low ionic strength buffers at pH 2.5 results in the formation of long (microm), straight fibrils while, at pH 3.6, short (<500 nm) fibrils form. At higher ionic strengths (0.2-0.4 M) at pH 1.5-3.6, the fibrils have a distinct curved and nodular morphology. To determine the conformational properties of beta(2)m within in vitro fibrils of different morphologies, limited proteolysis of each fibril type using pepsin was performed and the resulting peptide fragments identified by tandem mass spectrometry. For comparison, the proteolytic degradation patterns of monomeric beta(2)m and seven synthetic peptides spanning the entire sequence of the intact protein were similarly analysed. The results show that fibrils with different morphologies result in distinct digestion patterns. While the curved, worm-like fibrils are relatively weakly protected from proteolysis, the long, straight fibrils formed at pH 2.5 at low ionic strength show only a single cut-site at Val9, demonstrating that substantial refolding of the initially acid-denatured and unprotected state of beta(2)m occurs during assembly. The data demonstrate that the organisation of the polypeptide chain in fibrils with different morphological features differs considerably, despite the fact that the fibrils possess a common cross-beta architecture.     

Inhibitory effect of safranal and crocin,two principle compounds of <Emphasis Type="Italic">Crocus sativus</Emphasis>, on fibrillation of lysozyme

A key feature in more than twenty amyloid-related diseases is the aggregation of intra-and/or extracellular misfolded proteins as amyloid fibrils. Therefore, preventing or reversing amyloid aggregation by using of small molecules is considered as useful approaches to the treatment of these diseases. We have evaluated the ability of safranal and crocin, to inhibit amyloid self-assembly of hen egg white lysozyme (HEWL), as an in vitro model system. Structural properties of HEWL in the presence of these compounds were investigated individually using thioflavin T, anilinonaphthalene-8-sulfonic acid fluorescence assays, far-UV circular dichroism and scanning electron microscopy as well as docking method. Our results showed that incubation of HEWL with either crocin or safranal at various concentrations leads a significant inhibition in the rate of amyloid formation. Docking analysis revealed crocin and safranal interact with the central hydrophobic region of lysozyme through van der Waals interaction. Hydroxyl group in crocin through hydrogen bonds connected to the several hydrophilic amino acids of lysozyme, while in safranal there are just one aldehyde group that through hydrogen bonds connected to aspartic acid in lysozyme. It can be concluded that both hydrophobic and hydrophilic groups contribute to lower lysozyme fibril accumulation.     

Amyloid fibril formation and other aggregate species formed by human serum albumin association

Under in vitro solution conditions where the native state is destabilized, many proteins present an abnormal structure and metabolism associated with a strong tendency to self-aggregation into a polymeric amyloid fibril structure, suggesting that this ability is a generic feature of the polypeptide chains. Such structures play a key role in different pathogenesis of neurodegenerative diseases such as Alzheimer, Parkinson, or Creutzfeldt-Jakob. Here, we report the formation of amyloid fibrils in the plasma protein human serum albumin under different in vitro conditions monitored using a combination of spectrophotometric and microscopic techniques. Amyloid fibril formation, therefore, is also allowed in a protein with a high degree of structural complexity. We also infer from experimental data the existence of other protein aggregated species than fibrils, some of which seem to be formed by a structural rearrangement of the proper fibrils.