The deposition of amyloid ? (A?) peptides is a pathological hallmark of Alzheimer's disease. A? peptides were previously considered to interact specifically with ganglioside-containing membranes. Several studies have suggested that A? peptides also bind to phosphatidylcholine (PC) membranes, which lead to deformation of membranes and fibrillation of A?. Moreover, the role of membrane curvature, one type of deformation produced by binding of proteins to a membrane, in the binding and fibrillation of A? remains unclear. To clearly understand the relationship between the binding, consequent membrane deformation and fibrillation of A?, we examined the amyloid fibrillation of A? (1-40) in the presence of liposomes of various sizes. Membrane curvature increased with a decrease in the size of the liposomes. We used liposomes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine to eliminate electrostatic effects. The results obtained showed that liposomes of smaller sizes (? 50 nm) significantly accelerated the nucleation step, thereby shortening the lag time of fibrillation. On the other hand, liposomes of larger sizes decreased the amount of fibrils, but did not notably affect the lag time. The morphologies of fibrils, which were monitored by total internal reflection fluorescence microscopy, atomic force microscopy, and transmission electron microscopy, revealed that the length of A? (1-40) fibrils became shorter and the amount of amorphous aggregates became larger as liposomes increased in size. These results suggest that the curvature of membranes coupled with an increase in water-accessible hydrophobic regions is important for binding and concentrating A? monomers, leading to amyloid nucleation. Furthermore, amyloid fibrillation on membranes may compete with non-productive binding to produce amorphous aggregates.
Amyloid fibrils are fibrillar aggregates of denatured proteins associated with a large number of amyloidoses. The formation of amyloid fibrils has been considered to occur by nucleation and elongation. Real-time imaging of the elongation as well as linear morphology of amyloid fibrils suggests that all elongation events occur at the growing ends of fibrils. On the other hand, we suggested that monomers also bind to the lateral sides of preformed fibrils during the seed-dependent elongation, diffuse to the growing ends, and finally make further conformation changes to the mature amyloid fibrils. To examine lateral binding during the elongation of fibrils, we used islet amyloid polypeptide (IAPP), which has been associated with type II diabetes, and prepared IAPP modified with the fluorescence dye, Alexa532. By monitoring the elongation process with amyloid specific thioflavin T and Alexa532 fluorescence, we obtained overlapping images of the two fluorescence probes, which indicated lateral binding. These results are similar to the surface diffusion-dependent growth of crystals, further supporting the similarities between amyloid fibrillation and the crystallization of substances.
Amyloid fibrils form in supersaturated solutions of precursor proteins by a nucleation and growth mechanism characterized by a lag time. Although the lag time provides a clue to understanding the complexity of nucleation events, its long period and low reproducibility have been obstacles for exact analysis. Ultrasonication is known to effectively break supersaturation and force fibrillation. By constructing a Handai amyloid burst inducer, which combines a water bath-type ultrasonicator and a microplate reader, we examined the ultrasonication-forced fibrillation of several proteins, with a focus on the fluctuation in the lag time. Amyloid fibrillation of hen egg white lysozyme was examined at pH 2.0 in the presence of 1.0-5.0 M guanidine hydrochloride (GdnHCl), in which the dominant species varied from the native to denatured conformations. Although fibrillation occurred at various concentrations of GdnHCl, the lag time varied largely, with a minimum being observed at ?3.0 M, the concentration at which GdnHCl-dependent denaturation ended. The coefficient of variation of the lag time did not depend significantly on the GdnHCl concentration and was 2-fold larger than that of the ultrasonication-dependent oxidation of iodide, a simple model reaction. These results suggest that the large fluctuation observed in the lag time for amyloid fibrillation originated from a process associated with a common amyloidogenic intermediate, which may have been a relatively compact denatured conformation. We also suggest that the Handai amyloid burst inducer system will be useful for studying the mechanism of crystallization of proteins because proteins form crystals by the same mechanism as amyloid fibrils under supersaturation.
Variable (V) domains of antibodies are essential for antigen recognition by our adaptive immune system. However, some variants of the light chain V domains (VL) form pathogenic amyloid fibrils in patients. It is so far unclear which residues play a key role in governing these processes. Here, we show that the conserved residue 2 of VL domains is crucial for controlling its thermodynamic stability and fibril formation. Hydrophobic side chains at position 2 stabilize the domain, whereas charged residues destabilize and lead to amyloid fibril formation. NMR experiments identified several segments within the core of the VL domain to be affected by changes in residue 2. Furthermore, molecular dynamic simulations showed that hydrophobic side chains at position 2 remain buried in a hydrophobic pocket, and charged side chains show a high flexibility. This results in a predicted difference in the dissociation free energy of ?10 kJ mol(-1), which is in excellent agreement with our experimental values. Interestingly, this switch point is found only in VL domains of the ? family and not in VL? or in VH domains, despite a highly similar domain architecture. Our results reveal novel insight into the architecture of variable domains and the prerequisites for formation of amyloid fibrils. This might also contribute to the rational design of stable variable antibody domains.
Interaction between monomer peptides and seeds is essential for clarifying the fibrillation mechanism of amyloid ? (A?) peptides. We monitored the deposition reaction of A?1-40 peptides on immobilized seeds grown from A?1-42, which caused formation of oligomers in the early stage. The deposition reaction and fibrillation procedure were monitored throughout by novel total-internal-reflection-fluorescence microscopy with a quartz-crystal microbalance (TIRFM-QCM) system. This system allows simultaneous evaluation of the amount of deposited peptides on the surface seeds by QCM and fibril nucleation and elongation by TIRFM. Most fibrils reached other nuclei, forming the fibril network across the nucleus hubs in a short time. We found a fibril-elongation rate two-orders-of-magnitude higher in an oligomeric cloud than reported values, indicating ultrafast transition of oligomers into fibrils.
Protein nanoassemblies possess unique advantage in biomedical applications such as drug delivery, biocatalysis and vaccine development. Despite recent accomplishment in atomic structure data, the underlying molecular mechanism of protein self-assembly remains elusive, where considerable heterogeneity is often involved. Here we use E. coli chaperonin GroEL, a tetradecameric protein with a molecular weight of 805 kDa, to probe its transformation from cage-like oligomers to protein nanofibers. We show that sodium dodecyl sulfate (SDS), a widely-used protein denaturant, at submicellar concentration binds to and causes partial distortion of GroEL apical domain. Subsequently, the GroEL apical domain with altered secondary structural content converts the GroEL oligomers into modular structural units which are observed to self-assemble into cylindrical nanofibers under an agitated incubation in a physiological buffer. Interestingly, through targeted mutagenesis where two cysteine residues are introduced at the entry site of GroEL cage, we found that the formation of GroEL nanoassembly could be modulated depending on the redox condition of incubation. Without the need of chemical engineering, tunable GroEL nanofibers built by controlled-assembly are among the largest nanoscale bioassembly with broad applications.
Amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. We proposed that ultrasonication may be an effective agitation to trigger nucleation that would otherwise not occur under the persistent metastability of supersaturation. However, the roles of supersaturation and effects of ultrasonication have not been elucidated in detail except for limited cases. Insulin is an amyloidogenic protein that is useful for investigating the mechanisms underlying amyloid fibrillation with biological relevance. We studied the alcohol-induced amyloid fibrillation of insulin using various concentrations of 2,2,2-trifluoroethanol and 1,1,1,3,3,3-hexafluoro-2-propanol at pH 2.0 and 4.8. Ultrasonic irradiation effectively triggered fibrillation under conditions in which insulin retained persistent supersaturation. Structural analyses by circular dichroism, Fourier transform infrared spectroscopy, transmission electron microscopy, and atomic force microscopy revealed that the dominant structures of fibrils varied between parallel and antiparallel ?-sheets depending on the solvent conditions. pH and alcohol concentration-dependent phase diagrams showed a marked difference before and after the ultrasonic treatment, which indicated that the persistent metastability of supersaturation determined the conformations of insulin. These results indicate the importance of an alternative view of amyloid fibrils as supersaturation-limited crystal-like aggregates formed above the solubility limit.
Amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. Although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. Furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. Here, with ?2-microglobulin, a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetry (ITC). The spontaneous fibrillation after a lag phase was accompanied by exothermic heat. The thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. We also characterized the thermodynamics of amorphous aggregation, enabling the comparison of protein folding, amyloid fibrillation, and amorphous aggregation. These results indicate that ITC will become a promising approach for clarifying comprehensively the thermodynamics of protein folding and misfolding.
Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. ?-Synuclein fibrils cold-denatured to monomers at 0-20?°C and heat-denatured at 60-110?°C. Meanwhile, the fibrils of ?2-microglobulin, Alzheimer's A?1-40/A?1-42 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 ?-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.
Although alcohols are useful cosolvents for producing amyloid fibrils, the underlying mechanism of alcohol-dependent fibrillation is unclear. We studied the alcohol-induced fibrillation of hen egg-white lysozyme at various concentrations of ethanol, 2,2,2-trifluoroethanol (TFE), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Under the conditions where the alcohol-denatured lysozyme retained metastability, ultrasonication effectively triggered fibrillation. The optimal alcohol concentration depended on the alcohol species. HFIP showed a sharp maximum at 12-16%. For TFE, a broad maximum at 40-80% was observed. Ethanol exhibited only an increase in fibrillation above 60%. These profiles were opposite to the equilibrium solubility of lysozyme in water/alcohol mixtures. The results indicate that although fibrillation is determined by solubility, supersaturation prevents conformational transitions and ultrasonication is highly effective in minimizing an effect of supersaturation. We propose an alcohol-dependent protein misfolding funnel useful for examining amyloidogenicity. This misfolding funnel will apply to fibrillation under physiological conditions where biological environments play important roles in decreasing the solubility.
Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins, which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. The formation of amyloid fibrils in vivo is poorly understood. We recently identified the first naturally occurring structural variant, D76N, of human ?2-microglobulin (?2m), the ubiquitous light chain of class I major histocompatibility antigens, as the amyloid fibril protein in a family with a new phenotype of late onset fatal hereditary systemic amyloidosis. Here we show that, uniquely, D76N ?2m readily forms amyloid fibrils in vitro under physiological extracellular conditions. The globular native fold transition to the fibrillar state is primed by exposure to a hydrophobic-hydrophilic interface under physiological intensity shear flow. Wild type ?2m is recruited by the variant into amyloid fibrils in vitro but is absent from amyloid deposited in vivo. This may be because, as we show here, such recruitment is inhibited by chaperone activity. Our results suggest general mechanistic principles of in vivo amyloid fibrillogenesis by globular proteins, a previously obscure process. Elucidation of this crucial causative event in clinical amyloidosis should also help to explain the hitherto mysterious timing and location of amyloid deposition.
Protein crystals form in supersaturated solutions via a nucleation and growth mechanism. The amyloid fibrils of denatured proteins also form via a nucleation and growth mechanism. This similarity suggests that, although protein crystals and amyloid fibrils are distinct in their morphologies, both processes can be controlled in a similar manner. It has been established that ultrasonication markedly accelerates the formation of amyloid fibrils and simultaneously breaks them down into fragmented fibrils. In this study, we investigated the effects of ultrasonication on the crystallization of hen egg white lysozyme and glucose isomerase from Streptomyces rubiginosus. Protein crystallization was monitored by light scattering, tryptophan fluorescence, and light transmittance. Repeated ultrasonic irradiations caused the crystallization of lysozyme and glucose isomerase after cycles of irradiations. The size of the ultrasonication-induced crystals was small and homogeneous, and their numbers were larger than those obtained under quiescent conditions. Switching off ultrasonic irradiation when light scattering or tryptophan fluorescence began to change resulted in the formation of larger crystals due to the suppression of the further nucleation and fractures in preformed crystals. The results indicate that protein crystallization and amyloid fibrillation are explained on the basis of a common phase diagram in which ultrasonication accelerates the formation of crystals or crystal-like amyloid fibrils as well as fragmentation of preformed crystals or fibrils.
Corneal dystrophies are genetic disorders resulting in progressive corneal clouding due to the deposition of amyloid fibrils derived from keratoepithelin, also called transforming growth factor ?-induced protein (TGFBI). The formation of amyloid fibrils is often accelerated by surfactants such as sodium dodecyl sulfate (SDS). Most eye drops contain benzalkonium chloride (BAC), a cationic surfactant, as a preservative substance. In the present study, we aimed to reveal the role of BAC in the amyloid fibrillation of keratoepithelin-derived peptides in vitro. We used three types of 22-residue synthetic peptides covering Leu110-Glu131 of the keratoepithelin sequence: an R-type peptide with wild-type R124, a C-type peptide with C124 associated with lattice corneal dystrophy type I, and a H-type peptide with H124 associated with granular corneal dystrophy type II. The time courses of spontaneous amyloid fibrillation and seed-dependent fibril elongation were monitored in the presence of various concentrations of BAC or SDS using thioflavin T fluorescence. BAC and SDS accelerated the fibrillation of all synthetic peptides in the absence and presence of seeds. Optimal acceleration occurred near the CMC, which suggests that the unstable and dynamic interactions of keratoepithelin peptides with amphipathic surfactants led to the formation of fibrils. These results suggest that eye drops containing BAC may deteriorate corneal dystrophies and that those without BAC are preferred especially for patients with corneal dystrophies.
Amyloid fibrils, rigid and filamentous aggregates associated with various diseases, are often difficult to depolymerize into monomers. Ultrasonication is a strong agitation that accelerates nucleation above the critical concentration of amyloid fibrillation. We examined the effects of ultrasonication on the fibrils of amyloid ?(1-40) as well as on monomers. Ultrasonic pulses accelerated spontaneous fibrillation when the peptide concentration was above 1?M. On the other hand, ultrasonic pulses accelerated the depolymerization of fibrils into monomers at 1?M. These results indicate that, although amyloid fibrillation is a reversible process determined by thermodynamic stability, kinetically trapped supersaturation and physical difficulty of dissolving rigid fibrils prevent the smooth phase transitions. We propose that, in addition to accelerating the nucleation of fibrillation and fragmentation of fibrils above the critical concentration, ultrasonication is useful for dissolving fibrils below the critical concentration.
Real-time monitoring of the deposition processes of A?1-40 and A?1-42 peptides on various seeds has been performed using a 55 MHz wireless quartz-crystal microbalance (QCM) over long-time periods (~40 h). Dissolved peptide solutions were stirred for nucleation and growth of seeds at pH = 7.4 and 4.6, which were immobilized on the sensor chips. The isolated A? peptides were then flowed at the neutral pH, focusing on the interaction between the seeds and the monomers (or small multimers), excluding other interactions among seeds and other aggregates. The thioflavin-T fluorescence assay and atomic-force microscopy were used for evaluating structures of the seeds and deposited aggregates. The deposition rate, determined by the frequency decrease, is about 100 monomers/nm(2)/year in the case of fibril formation. The notable deposition behavior was observed in the deposition of A?1-40 peptide on A?1-42 seeds grown at the lower pH, which can be an important model for Alzheimers disease.
Although amyloid fibrils deposit with various proteins, the comprehensive mechanism by which they form remains unclear. We studied the formation of fibrils of human islet amyloid polypeptide associated with type II diabetes in the presence of various concentrations of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) under acidic and neutral pH conditions using CD, amyloid-specific thioflavin T fluorescence, fluorescence imaging with thioflavin T, and atomic force microscopy. At low pH, the formation of fibrils was promoted by HFIP with an optimum at 5% (v/v). At neutral pH in the absence of HFIP, significant amounts of amorphous aggregates formed in addition to the fibrils. The addition of HFIP suppressed the formation of amorphous aggregates, leading to a predominance of fibrils with an optimum effect at 25% (v/v). Under both conditions, higher concentrations of HFIP dissolved the fibrils and stabilized the ?-helical structure. The results indicate that fibrils and amorphous aggregates are different types of precipitates formed by exclusion from water-HFIP mixtures. The exclusion occurs through the combined effects of hydrophobic interactions and electrostatic interactions, both of which are strengthened by low concentrations of HFIP, and a subtle balance between the two types of interactions determines whether the fibrils or amorphous aggregates dominate. We suggest a general view of how the structure of precipitates varies dramatically from single crystals to amyloid fibrils and amorphous aggregates.
Hydrodynamic forces are capable of inducing structural order in dispersed solid phases, and of causing symmetry-breaking when chiral crystals precipitate from an achiral liquid phase. Until it was observed upon vortex-assisted fibrillation of insulin, such behavior had been thought to be confined to few unbiological systems. In this paper we are discussing chiroptical properties of two chiral variants of insulin amyloid, termed +ICD and -ICD, which form during the process of chiral bifurcation in vortexed solutions of aggregating insulin. As conventional measurements of circular dichroism of solid, anisotropic substances are particularly vulnerable to overlapping influences of linear birefringence and linear dichroism, we have employed complementary tools including dedicated universal chiroptical spectrophotometer to rule out such artifacts. We propose that the strong chiroptical properties of +ICD and -ICD insulin fibrils are an aspect of genuine superstructural chirality of amyloid fibrils and of powerful excitonic couplings taking place within them. A comparison of thioflavin T complexes with fibrils formed by insulin and polyglutamic acid suggests that the extrinsic Cotton effect stemming from the level of single twisted dye molecules is weaker, although diagnostically useful, and cannot account for the overall magnitude of ICD of the dye bound to ±ICD insulin amyloid.
Mutations in keratoepithelin are associated with blinding ocular diseases, including lattice corneal dystrophy type 1 and granular corneal dystrophy type 2. These diseases are characterized by deposits of amyloid fibrils and/or granular non-amyloid aggregates in the cornea. Removing the deposits in the cornea is important for treatment. Previously, we reported the destruction of amyloid fibrils of ?(2)-microglobulin K3 fragments and amyloid ? by laser irradiation coupled with the binding of an amyloid-specific thioflavin T. Here, we studied the effects of this combination on the amyloid fibrils of two 22-residue fragments of keratoepithelin. The direct observation of individual amyloid fibrils was performed in real time using total internal reflection fluorescence microscopy. Both types of amyloid fibrils were broken up by the laser irradiation, dependent on the laser power. The results suggest the laser-induced destruction of amyloid fibrils to be a useful strategy for the treatment of these corneal dystrophies.
Amyloid fibrils, similar to crystals, form through nucleation and growth. Because of the high free-energy barrier of nucleation, the spontaneous formation of amyloid fibrils occurs only after a long lag phase. Ultrasonication is useful for inducing amyloid nucleation and thus for forming fibrils, while the use of a microplate reader with thioflavin T fluorescence is suitable for detecting fibrils in many samples simultaneously. Combining the use of ultrasonication and microplate reader, we propose an efficient approach to studying the potential of proteins to form amyloid fibrils. With ?(2)-microglobulin, an amyloidogenic protein responsible for dialysis-related amyloidosis, fibrils formed within a few minutes at pH 2.5. Even under neutral pH conditions, fibrils formed after a lag time of 1.5 h. The results propose that fibril formation is a physical reaction that is largely limited by the high free-energy barrier, which can be effectively reduced by ultrasonication. This approach will be useful for developing a high-throughput assay of the amyloidogenicity of proteins.
The misfolding and self-assembly of proteins into amyloid fibrils, which occur in several debilitating and age-related diseases, are affected by common components of amyloid deposits, notably lipids and lipid complexes. Previously, the effects of phospholipids on amyloid fibril formation by apolipoprotein (apo) C-II have been examined, where low concentrations of micellar phospholipids and lipid bilayers induce a new, straight rod-like morphology for apoC-II fibrils. This fibril appearance is distinct from the twisted-ribbon morphology observed when apoC-II fibrils are formed in the absence of lipids. We used total internal reflection fluorescence microscopy (TIRFM) to visualize the described polymorphism of apoC-II amyloid fibrils. The spontaneous assembly of apoC-II into either twisted-ribbon fibrils in the absence of lipids or into fibrils of straight rod-like morphology when lipids are present was captured by TIRFM. The latter was found to be better suited for visualization using TIRFM. The difference between seeding of apoC-II straight fibrils on microscopic quartz slide and in test tube suggested a role for the effects of incubation surface on fibril formation. Seed-dependent growth of apoC-II straight fibrils was probed further by using a dual-labelling construct, giving insights into the straight fibril growth pattern.
The conversion of the soluble, nontoxic amyloid-beta (Abeta) peptide into an aggregated, toxic form rich in beta-sheets is considered a key step in the development of Alzheimers disease. Whereas growing evidence indicates that the Abeta amyloid fibrils consist of in-register parallel beta-sheets, little is known about the structure of soluble oligomeric intermediates because of their transient nature. To understand the mechanism by which amyloid fibrils form, especially the initial development of the "nucleus" oligomeric intermediates, we prepared covalently linked dimeric Abeta peptides and analyzed the kinetics of the fibril-forming process. A covalent bond introduced between two Abeta molecules dramatically facilitated the spontaneous formation of aggregates with a beta-sheet structure and affinity for thioflavin T. Transmission electron microscopy revealed, however, that these aggregates differed in morphology from amyloid fibrils, more closely resembling protofibrils. The protofibril-like aggregates were not the most thermodynamically stable state but were a kinetically trapped state. The results emphasize the importance of the conformational flexibility of the Abeta molecule and a balance in the association and dissociation rate for the formation of rigid amyloid fibrils.
alpha-Synuclein is one of the causative proteins of the neurodegenerative disorder, Parkinsons disease. Deposits of alpha-synuclein called Lewy bodies are a hallmark of this disorder, which is implicated in its progression. In order to understand the mechanism of amyloid fibril formation of alpha-synuclein in more detail, in this study we have isolated a specific, ~20 residue peptide region of the alpha-synuclein fibril core, using a combination of Edman degradation and mass-spectroscopy analyses of protease-resistant samples. Starting from this core peptide sequence, we then synthesized a series of peptides that undergo aggregation and fibril formation under similar conditions. Interestingly, in a derivative peptide where a crucial phenylalanine residue was changed to a glycine, the ability to initiate spontaneous fibril formation was abolished, while the ability to extend from preexisting fibril seeds was conserved. This fibril extension occurred irrespective of the source of the initial fibril seed, as demonstrated in experiments using fibril seeds of insulin, lysozyme, and GroES. This interesting ability suggests that this peptide might form the basis for a possible diagnostic tool useful in detecting the presence of various fibrillogenic factors.
Many human diseases are associated with protein aggregation and fibrillation. We present experiments on in vitro glucagon fibrillation using total internal reflection fluorescence microscopy, providing real-time measurements of single-fibril growth. We find that amyloid fibrils grow in an intermittent fashion, with periods of growth followed by long pauses. The observed exponential distributions of stop and growth times support a Markovian model, in which fibrils shift between the two states with specific rates. Even if the individual rates vary considerably, we observe that the probability of being in the growing (stopping) state is very close to 1/4 (3/4) in all experiments.
Light chain-associated (AL) amyloidosis is characterized by dominant fibril deposition of the variable domain (VL) of an immunoglobulin light chain, and thus its constant domain (CL) has been considered not to be amyloidogenic. We examined the in vitro fibril formation of the isolated CL in comparison with beta2-microglobulin (beta2-m), an immunoglobulin domain-like amyloidogenic protein responsible for dialysis-related amyloidosis. Two methods useful for beta2-m at neutral pH also induced amyloid fibrils of CL, which were monitored by thioflavin-T binding and electron microscopy (EM). These results suggest that CL plays an important role, more than previously assumed, in the development of AL-amyloidosis.
The amyloid deposition of amyloid beta (Abeta) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of beta(2)-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Abeta fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Abeta fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.
The inhibition of fibril formation of amyloid beta proteins (A beta) would be attractive therapeutic targets for the treatment of Alzheimers disease (AD). Dopamine (DA) and other catechol derivatives were used as inhibitory factors for A beta fibril formation. The fibril formation of A beta was monitored by Thioflavin T fluorescence, a transmission electron microscopy (TEM) and a total internal reflection fluorescence microscopy (TIRFM). Catechol and its derivatives showed the dose-dependent inhibitory effects on the spontaneous A beta fibril formation. The inhibitory activity depended on the chemical structure of catechol derivatives both in the presence and absence of the liposome a model of biomembrane. Formation of catechol quinone-conjugated-A beta adduct by a Schiff-base is a key step for the inhibition effect of A beta fibril formation.
Because of the insolubility and polymeric properties of amyloid fibrils, techniques used conventionally to analyze protein structure and dynamics have often been hampered. Ultrasonication can induce the monomeric solution of amyloidogenic proteins to form amyloid fibrils. However, ultrasonication can break down preformed fibrils into shorter fibrils. Here, combining these 2 opposing effects on beta(2)-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis, we present that ultrasonication pulses are useful for preparing monodispersed amyloid fibrils of minimal size with an average molecular weight of approximately 1,660,000 (140-mer). The production of minimal and monodispersed fibrils is achieved by the free energy minimum under competition between fibril production and breakdown. The small homogeneous fibrils will be of use for characterizing the structure and dynamics of amyloid fibrils, advancing molecular understanding of amyloidosis.
Calorimetric measurements were carried out using a differential scanning calorimeter to characterize the thermal response of beta(2)-microglobulin amyloid fibrils, the deposition of which results in dialysis-related amyloidosis. The fibril solution showed a large decrease in heat capacity (exothermic effect) before the temperature-induced depolymerization of the fibrils, which was characterized by a definite dependence on heating rate. To understand the factors that determine the heating-rate-dependent thermal response, the concentration dependence of polyethylene glycol, which inhibits the association of amyloid fibrils with heating, on exothermic effect was examined in detail and showed a causal link between the exothermic effect and fibril association. The results suggest that the transient association driven by a spatial approach and the concomitant dehydration of hydrophobic areas of amyloid fibrils may be significant factors determining the thermal response with exothermic effect, which has not been observed in calorimetric studies of monomolecular globular proteins. The heating-rate-dependent thermal response with the exothermic effect was observed not only for other amyloid fibrils formed from amyloid beta-peptides but also during the processes of the temperature-induced conversion of beta(2)-microglobulin protofibrils and hen egg-white lysozyme into amyloid fibrils. These results highlight the physics related to the heating-rate-dependent behaviors of heat capacity in terms of interactions between the specific structures of amyloid fibrils and water molecules.
To understand the mechanism by which amyloid fibrils form, we have been making real-time observations of the growth of individual fibrils, using total internal fluorescence microscopy combined with an amyloid-specific fluorescence dye, thioflavin T (ThT). At neutral pH, irradiation at 442 nm with a laser beam to excite ThT inhibited the fibril growth of beta(2)-microglobulin (beta2-m), a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Examination with a 22-residue K3 fragment of beta2-m showed that the inhibition of fibril growth and moreover the destruction of preformed fibrils were coupled with the excitation of ThT. Several pieces of evidence suggest that the excited ThT transfers energy to ground state molecular oxygen, producing active oxygen, which causes various types of chemical modifications. The results imply a novel strategy for preventing the deposition of amyloid fibrils and for destroying preformed amyloid deposits.
Using the peptide hormone glucagon and Abeta(1-40) as model systems, we have sought to elucidate the mechanisms by which fibrils grow and multiply. We here present real-time observations of growing fibrils at a single-fibril level. Growing from preformed seeds, glucagon fibrils were able to generate new fibril ends by continuously branching into new fibrils. To our knowledge, this is the first time amyloid fibril branching has been observed in real-time. Glucagon fibrils formed by branching always grew in the forward direction of the parent fibril with a preferred angle of 35-40 degrees . Furthermore, branching never occurred at the tip of the parent fibril. In contrast, in a previous study by some of us, Abeta(1-40) fibrils grew exclusively by elongation of preformed seeds. Fibrillation kinetics in bulk solution were characterized by light scattering. A growth process with branching, or other processes that generate new ends from existing fibrils, should theoretically give rise to different fibrillation kinetics than growth without such a process. We show that the effect of adding seeds should be particularly different in the two cases. Our light-scattering data on glucagon and Abeta(1-40) confirm this theoretical prediction, demonstrating the central role of fibril-dependent nucleation in amyloid fibril growth.
?-Synuclein (140 amino acids), one of the causative proteins of Parkinsons disease, forms amyloid fibrils in brain neuronal cells. In order to further explore the contributions of the C-terminal region of ?-synuclein in fibril formation and also to understand the overall mechanism of fibril formation, we reduced the number of negatively charged residues in the C-terminal region using mutagenesis. Mutants with negative charges deleted displayed accelerated fibril formation compared with wild-type ?-synuclein, demonstrating that negative charges located in the C-terminal region of ?-synuclein modulate fibril formation. Additionally, when tyrosine residues located at position 125, 133, and 136 in the C-terminal region were changed to alanine residue(s), we found that all mutants containing the Tyr136Ala mutation showed delays in fibril formation compared with wild type. Mutation of Tyr136 to various amino acids revealed that aromatic residues located at this position act favorably toward fibril formation. In mutants where charge neutralization and tyrosine substitution were combined, we found that these two factors influence fibril formation in complex fashion. These findings highlight the importance of negative charges and aromatic side chains in the C-terminal region of ?-synuclein in fibril formation.
Amyloid fibrils and amorphous aggregates are two types of aberrant aggregates associated with protein misfolding diseases. Although they differ in morphology, the two forms are often treated indiscriminately. ?(2)-microglobulin (?2m), a protein responsible for dialysis-related amyloidosis, forms amyloid fibrils or amorphous aggregates depending on the NaCl concentration at pH 2.5. We compared the kinetics of their formation, which was monitored by measuring thioflavin T fluorescence, light scattering, and 8-anilino-1-naphthalenesulfonate fluorescence. Thioflavin T fluorescence specifically monitors amyloid fibrillation, whereas light scattering and 8-anilino-1-naphthalenesulfonate fluorescence monitor both amyloid fibrillation and amorphous aggregation. The amyloid fibrils formed via a nucleation-dependent mechanism in a supersaturated solution, analogous to crystallization. The lag phase of fibrillation was reduced upon agitation with stirring or ultrasonic irradiation, and disappeared by seeding with preformed fibrils. In contrast, the glass-like amorphous aggregates formed rapidly without a lag phase. Neither agitation nor seeding accelerated the amorphous aggregation. Thus, by monitoring the kinetics, we can distinguish between crystal-like amyloid fibrils and glass-like amorphous aggregates. Solubility and supersaturation will be key factors for further understanding the aberrant aggregation of proteins.
High-frequency (~ 55 MHz) wireless quartz-crystal microbalance biosensor was used for studying heterogeneous deposition behavior of A?(1-40) peptide on A?(1-42) nuclei, which were grown under the stirring agitation and 200-kHz ultrasonication at pH 2.2, 4.6, and 7.4. The deposition reaction was monitored over 40 h, and the deposition rate was deduced. Among the agitation nuclei, the maximum deposition rate was observed on the nucleus grown at pH 4.6. However, ultrasonication nucleus grown at pH 7.4 produced much larger deposition rate, despite the same ?-sheet concentration. This result indicates that local structural modulation is caused in the nucleus by ultrasonication, which adsorbs the A? peptide more actively than other nuclei. The resultant deposits clearly show oligomeric structure.
Alzheimers disease is the most common form of senile dementia. This neurodegenerative disorder is characterized by an amyloid deposition in senile plaques, composed primarily of fibrils of an aggregated peptide, amyloid ? (A?). The modeling of a senile plaque formation on a model neuronal membrane under the physiological condition is an attractive issue. In this study, we used anionic liposomes to model the senile plaque formation by A?. The growth behavior of amyloid A? fibrils was directly observed, revealing that the induction of the spherulitic A? aggregates could result from the growth of seeds in the presence of anionic liposomes. The seeds of A? fibrils strongly interacted with negatively charged liposome and the subsequent association of the seeds were induced to form the seed cluster with many growth ends, which is advantageous for the formation of spherulitic A? aggregates. Therefore, anionic liposomes mediated not only fibril growth but also the aggregation process. These results imply that anionic liposome membranes would affect the aggregate form of A? fibrils. The modeling of senile plaque reported here is considered to have great potential for study on the amyloidosis.
The chaperonin GroEL plays an essential role in promoting protein folding and in protecting against misfolding and aggregation in the cellular environment. In this study, we report that both GroEL and its isolated apical domain form amyloid-like fibrils under physiological conditions, and that the fibrillation of the apical domain is accelerated under acidic conditions. We also found, however, that despite its fibrillation propensity, the apical domain exhibits a pronounced inhibitory effect on the fibril growth of ?(2)-microglobulin. Thus, the analysis of the behaviour of the apical domain reveals how aggregation and chaperone-mediated anti-aggregation processes can be closely related.
The polymorphic property of amyloid structures has been focused on as a molecular basis of the presence and propagation of different phenotypes of amyloid diseases, although little is known about the molecular mechanism for expressing diverse structures from only one protein sequence. Here, we have found that, in combination with an enhancing effect of ultrasonication on nucleation, ?(2)-microglobulin, a protein responsible for dialysis-related amyloidosis, generates distinct fibril conformations in a concentration-dependent manner in the presence of 2,2,2-trifluoroethanol (TFE). Although the newly formed fibrils all exhibited a similar needle-like morphology with an extensive cross-? core, as suggested by Fourier transform infrared absorption spectra, they differed in thioflavin T intensity, extension kinetics, and tryptophan fluorescence spectra even in the same solvents, representing polymorphic structures. The hydrophobic residues seemed to be more exposed in the fibrils originating at higher concentrations of TFE, as indicated by the increased binding of 1-anilinonaphthalene-8-sulfonic acid, suggesting that the modulation of hydrophobic interactions is critical to the production of polymorphic amyloid structures. Interestingly, the fibrils formed at higher TFE concentrations showed significantly higher stability against guanidium hydrochloride, the perturbation of ionic strength, and, furthermore, pressurization. The cross-? structure inside the fibrils seems to have been more idealized, resulting in increased stability when nucleation occurred in the presence of the alcohol, indicating that a weaker contribution of hydrophobic interactions is intrinsically more amenable to the formation of a non-defective amyloid structure.
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