Flavonoids with Vicinal Hydroxyl Groups Inhibit Human Calcitonin Amyloid Formation

Human calcitonin (hCT) is a 32-residue peptide hormone that can aggregate into amyloid fibrils and cause cellular toxicity. In this study, we investigated the inhibition effects of a group of polyphenolic molecules on hCT amyloid formation. Our results suggest that the gallate moiety in epigallocatechin-3-gallate (EGCG), a well-recognized amyloid inhibitor, is not critical for its inhibition function in the hCT amyloid formation. Our results demonstrate that flavonoid compounds, such as myricetin, quercetin, and baicalein, that contain vicinal hydroxyl groups on the phenyl ring effectively prevent hCT fibrillization. This structural feature may also be applied to non-flavonoid polyphenolic inhibitors. Moreover, our results indicate a plausible mechanistic role of these vicinal hydroxyl groups which might include the oxidation to form a quinone and the subsequent covalent linkage with amino acid residues such as lysine or histidine in hCT. This may further disrupt the crucial electrostatic and aromatic interactions involved in the process of hCT amyloid fibril formation. The inhibition activity of the polyphenolic compounds against hCT fibril formation may likely be attributed to a combination of factors such as covalent linkage formation, aromatic stacking, and hydrogen bonding interactions.     

Microfluidic self-assembly of insulin monomers into amyloid fibrils on a solid surface

We report the self-assembly of insulin monomers into amyloid fibrils within microchannels. To demonstrate the microfluidic amyloid formation and fibril growth on a solid surface, we seeded the internal surfaces of the microchannels with insulin monomers via N-hydroxysuccinimide ester activation and continuously flushed a fresh insulin solution through the microchannels. According to our analysis using optical and fluorescence microscopy, insulin amyloid preferentially formed in the center of the microchannels and, after reaching a certain density, spread to the side walls of the microchannels. By using ex situ atomic force microscopy, we observed the growth of amyloid fibrils inside the microchannels, which occurred at a much higher rate than that in bulk systems. After 12 h of incubation, insulin formed amyloid spherulites having "Maltese cross" extinction patterns within the microchannels according to the polarized microscopic analysis. Microfluidic amyloid formation enabled low consumption of reagents, reduction of incubation time, and simultaneous observation of amyloid formation under different conditions. This work will contribute to the rapid analysis of amyloid formation associated with many protein misfolding diseases.     

Inhibition of α‐Synuclein Amyloid Fibril Elongation by Blocking Fibril Ends

Misfolding of the protein α‐synuclein (αSyn) into amyloid fibrils plays a central role in the development of Parkinson's disease. Most approaches for the inhibition of αSyn fibril formation are based on stabilizing the native monomeric form of the protein or destabilizing the fibrillized misfolded form. They require high concentrations of inhibitor and therefore cannot be easily used for therapies. In this work, we designed an inhibitor (Inh‐β) that selectively binds the growing ends of αSyn fibrils and creates steric hindrance for the binding of monomeric αSyn. This approach permits the inhibition of fibril formation at Inh‐β concentrations (IC₅₀=850 nm ) much lower than the concentration of monomeric αSyn. We studied its kinetic mechanism in vitro and identified the reactions that limit inhibition efficiency. It is shown that blocking of αSyn fibril ends is an effective approach to inhibiting fibril growth and provides insights for the development of effective inhibitors of αSyn aggregation.     

Structural regulation of a peptide-conjugated graft copolymer: a simple model for amyloid formation

The self-assembly of peptides and proteins into beta-sheet-rich high-order structures has attracted much attention as a result of the characteristic nanostructure of these assemblies and because of their association with neurodegenerative diseases. Here we report the structural and conformational properties of a peptide-conjugated graft copolymer, poly(gamma-methyl-L-glutamate) grafted polyallylamine (1) in a water-2,2,2-trifluoroethanol solution as a simple model for amyloid formation. Atomic force microscopy revealed that the globular peptide 1 self-assembles into nonbranching fibrils that are about 4 nm in height under certain conditions. These fibrils are rich in beta-sheets and, similar to authentic amyloid fibrils, bind the amyloidophilic dye Congo red. The secondary and quaternary structures of the peptide 1 can be controlled by manipulating the pH, solution composition, and salt concentration; this indicates that the three-dimensional packing arrangement of peptide chains is the key factor for such fibril formation. Furthermore, the addition of carboxylic acid-terminated poly(ethylene glycol), which interacts with both of amino groups of 1 and hydrophobic PMLG chains, was found to obviously inhibit the alpha-to-beta structural transition for non-assembled peptide 1 and to partially cause a beta-to-alpha structural transition against the 1-assembly in the beta-sheet form. These findings demonstrate that the amyloid fibril formation is not restricted to specific protein sequences but rather is a generic property of peptides. The ability to control the assembled structure of the peptide should provide useful information not only for understanding the amyloid fibril formation, but also for developing novel peptide-based material with well-defined nanostructures.     

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.     

Monitoring and inhibition of insulin fibrillation by a small organic fluorogen with aggregation-induced emission characteristics

Amyloid fibrillation of proteins is associated with a great variety of pathologic conditions. Development of new molecules that can monitor amyloidosis kinetics and inhibit fibril formation is of great diagnostic and therapeutic value. In this work, we have developed a biocompatible molecule that functions as an ex situ monitor and an in situ inhibitor for protein fibrillation, using insulin as a model protein. 1,2-Bis[4-(3-sulfonatopropoxyl)phenyl]-1,2-diphenylethene salt (BSPOTPE) is nonemissive when it is dissolved with native insulin in an incubation buffer but starts to fluoresce when it is mixed with preformed insulin fibril, enabling ex situ monitoring of amyloidogenesis kinetics and high-contrast fluorescence imaging of protein fibrils. Premixing BSPOTPE with insulin, on the other hand, inhibits the nucleation process and impedes the protofibril formation. Increasing the dose of BSPOTPE boosts its inhibitory potency. Theoretical modeling using molecular dynamics simulations and docking reveals that BSPOTPE is prone to binding to partially unfolded insulin through hydrophobic interaction of the phenyl rings of BSPOTPE with the exposed hydrophobic residues of insulin. Such binding is assumed to have stabilized the partially unfolded insulin and obstructed the formation of the critical oligomeric species in the protein fibrillogenesis process.     

Investigation of mechanical properties of insulin crystals by atomic force microscopy

Mechanical properties of protein crystals and aggregates depend on the conformational and structural properties of individual protein molecules as well as on the packing density and structure within solid materials. An atomic force microscopy (AFM)-based approach is developed to measure the elastic modulus of small protein crystals by nanoindentation and is applied to measure the elasticity of insulin crystals. The top face of the crystals deposited on mica substrates is identified as the (001) face. Insulin crystals exhibit a nearly elastic response during the compression cycle. The elastic modulus measured on the top face has asymmetric distribution with a significant width. This width is related to the uncertainty in the deflection sensitivity. A model that takes into account the distribution of the sensitivity values is used to correct the elastic modulus. Measurements performed in aqueous buffer on several crystals at different locations with three different AFM probes give a mean elastic modulus of 164 +/- 10 MPa. This value is close to the static elastic moduli of other protein crystals measured by different techniques that are usually measured in the range from 100 MPa to 1 GPa. The measured modulus of insulin crystals falls between the elastic modulus values of insulin amyloid fibrils measured previously at two orthogonal directions (a modulus of 14 MPa was measured by compressing the fibril in the direction perpendicular to the fibril axis, and a modulus of 3.3 GPa was measured in the direction along the fibril axis). This comparison indicates the heterogeneous structure of fibrils in the direction perpendicular to the fibril axis, with a packing density of the amyloid fibril core that is higher than the average packing density in insulin crystals. The mechanical wear of insulin crystals is detected during AFM measurements. In nanoindentation experiments on insulin crystal, the compressive load by the AFM tip ( approximately 1 nN, corresponding to a pressure of around 5 MPa) occasionally removes protein molecules from the top or the second top layer of insulin crystal in a sequential manner. The molecular model of this surface damage is proposed. In addition, the removal of the multiple layers of molecules is observed during the AC-mode imaging in aqueous buffer. The number of removed layers depends on the scan size.     

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.     

Differential pattern of glycogen accumulation after protein phosphatase 1 glycogen-targeting subunit PPP1R6 overexpression, compared to PPP1R3C and PPP1R3A, in skeletal muscle cells

Background

Insulin is a hormone that regulates blood glucose homeostasis and is a central protein in a medical condition termed insulin injection amyloidosis. It is intimately associated with glycaemia and is vulnerable to glycation by glucose and other highly reactive carbonyls like methylglyoxal, especially in diabetic conditions. Protein glycation is involved in structure and stability changes that impair protein functionality, and is associated with several human diseases, such as diabetes and neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Familiar Amyloidotic Polyneuropathy. In the present work, methylglyoxal was investigated for their effects on the structure, stability and fibril formation of insulin.

Results

Methylglyoxal was found to induce the formation of insulin native-like aggregates and reduce protein fibrillation by blocking the formation of the seeding nuclei. Equilibrium-unfolding experiments using chaotropic agents showed that glycated insulin has a small conformational stability and a weaker dependence on denaturant concentration (smaller m-value). Our observations suggest that methylglyoxal modification of insulin leads to a less compact and less stable structure that may be associated to an increased protein dynamics.

Conclusions

We propose that higher dynamics in glycated insulin could prevent the formation of the rigid cross-β core structure found in amyloid fibrils, thereby contributing to the reduction in the ability to form fibrils and to the population of different aggregation pathways like the formation of native-like aggregates.     

Inhibiting and Reversing Amyloid‐β Peptide (1–40) Fibril Formation with Gramicidin S and Engineered Analogues

In Alzheimer’s disease, amyloid‐β (Aβ) peptides aggregate into extracellular fibrillar deposits. Although these deposits may not be the prime cause of the neurodegeneration that characterizes this disease, inhibition or dissolution of amyloid fibril formation by Aβ peptides is likely to affect its development. ThT fluorescence measurements and AFM images showed that the natural antibiotic gramicidin S significantly inhibited Aβ amyloid formation in vitro and could dissolve amyloids that had formed in the absence of the antibiotic. In silico docking suggested that gramicidin S, a cyclic decapeptide that adopts a β‐sheet conformation, binds to the Aβ peptide hairpin‐stacked fibril through β‐sheet interactions. This may explain why gramicidin S reduces fibril formation. Analogues of gramicidin S were also tested. An analogue with a potency that was four‐times higher than that of the natural product was identified.     

Cytochrome display on amyloid fibrils

Protein amyloid fibrils can be functionalized by coating the core protofilament with high concentrations of proteins and enzymes. This can be done elegantly by appending a functional domain to an amyloidogenic protein monomer, then assembling the monomers into a fibril. To display an array of biologically functional porphyrins on the surface of protein fibrils, we have fused the sequence of the small, soluble cytochrome b562 to an SH3 dimer sequence that can form classical amyloid fibrils rapidly under well-defined conditions. The resulting fusion protein also forms amyloid fibrils and, in addition, binds metalloporphyrins, at half of the porphyrin binding sites as shown by UV-vis and NMR spectroscopies. Once metalloporphyrins are bound to the fibrils, the resulting holo-cytochrome domains are spectroscopically identical to the wild type cytochrome. The concentration of metalloporphyrins on a saturated fibril is estimated to be of the order of approximately 20 mM, suggesting that they could be interesting systems for applications in nanotechnology.     

The organization of aromatic side groups in an amyloid fibril probed by solid-state 2H and 19F NMR spectroscopy

Some 25 diseases are associated with proteins and peptides that assemble into amyloid fibrils composed of beta-strands connected by hydrogen bonds oriented parallel to the fiber long axis. There is mounting evidence that amyloid formation involves specific interactions between amino acid side groups, which bring together beta-sheets to form layers with buried and exposed faces. This work demonstrates how a combination of solid-state 2H and 19F NMR experiments can provide constraints on fibril architecture by probing the environment and spatial organisation of aromatic side groups. It is shown that phenylalanine rings within fibrils formed by a decapeptide fragment of the islet amyloid polypeptide, amylin, are highly motionally restrained and are situated within 6.5 A of one another. Taken together with existing structural constraints for this peptide, these results are consistent with a fibril architecture that comprises layers of two or more beta-sheets, with the aromatic residues facing into the inter-sheet space and possibly engaged in pi-pi interactions. The methods presented will be of general utility in exploring the architecture of fibrils of larger, full-length peptides and proteins, including amylin itself.     

Protofilaments, filaments, ribbons, and fibrils from peptidomimetic self-assembly: implications for amyloid fibril formation and materials science

Deciphering the mechanism(s) of β-sheet mediated self-assembly is essential for understanding amyloid fibril formation and for the fabrication of polypeptide materials. Herein, we report a simple peptidomimetic that self-assembles into polymorphic β-sheet quaternary structures including protofilaments, filaments, fibrils, and ribbons that are reminiscent of the highly ordered structures displayed by the amyloidogenic peptides Aβ, calcitonin, and amylin. The distribution of quaternary structures can be controlled by and in some cases specified by manipulating the pH, buffer composition, and the ionic strength. The ability to control β-sheet-mediated assembly takes advantage of quaternary structure dependent pK(a) perturbations. Biophysical methods including analytical ultracentrifugation studies as well as far-UV circular dichroism and FT-IR spectroscopy demonstrate that linked secondary and quaternary structural changes mediate peptidomimetic self-assembly. Electron and atomic force microscopy reveal that peptidomimetic assembly involves numerous quaternary structural intermediates that appear to self-assemble in a convergent fashion affording quaternary structures of increasing complexity. The ability to control the assembly pathway(s) and the final quaternary structure(s) afforded should prove to be particularly useful in deciphering the quaternary structural requirements for amyloid fibril formation and for the construction of noncovalent macromolecular structures.     

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.     

硫酸钠对胰岛素淀粉样纤维化及其细胞毒性的作用

超过20种人类疾病与蛋白质或者多肽淀粉样纤维化密切相关,探究影响蛋白质的结构稳定性及其淀粉样纤维化的环境条件具有重要意义.本文采用牛胰岛素作为模型蛋白质,研究了Na2SO4对蛋白质淀粉样纤维化的作用.实验结果表明,不同浓度的Na2SO4对胰岛素淀粉样纤维化过程具有不同的影响,低浓度条件下可促进纤维化,较高浓度可明显抑制淀粉样纤维的形成,更高的浓度则使胰岛素形成非纤维状聚集体.ANS荧光分析结果表明,所有浓度的Na2SO4均可减小胰岛素聚集体的表面疏水性,并导致聚集体对细胞膜的损害作用降低.Na2SO4的上述作用可能与其改变蛋白质分子间的静电作用力及溶剂效应有关.     

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.     

The Prenucleation Equilibrium of the Parathyroid Hormone Determines the Critical Aggregation Concentration and Amyloid Fibril Nucleation

Nucleation and growth of amyloid fibrils were found to only occur in supersaturated solutions above a critical concentration (c_crit). The biophysical meaning of c_crit remained mostly obscure, since typical low values of c_crit in the sub-μM range hamper investigations of potential oligomeric states and their structure. Here, we investigate the parathyroid hormone PTH₈₄ as an example of a functional amyloid fibril forming peptide with a comparably high c_crit of 67±21 μM. We describe a complex concentration dependent prenucleation ensemble of oligomers of different sizes and secondary structure compositions and highlight the occurrence of a trimer and tetramer at c_crit as possible precursors for primary fibril nucleation. Furthermore, the soluble state found in equilibrium with fibrils adopts to the prenucleation state present at c_crit. Our study sheds light onto early events of amyloid formation directly related to the critical concentration and underlines oligomer formation as a key feature of fibril nucleation. Our results contribute to a deeper understanding of the determinants of supersaturated peptide solutions. In the current study we present a biophysical approach to investigate c_crit of amyloid fibril formation of PTH₈₄ in terms of secondary structure, cluster size and residue resolved intermolecular interactions during oligomer formation. Throughout the investigated range of concentrations (1 μM to 500 μM) we found different states of oligomerization with varying ability to contribute to primary fibril nucleation and with a concentration dependent equilibrium. In this context, we identified the previously described c_crit of PTH₈₄ to mark a minimum concentration for the formation of homo-trimers/tetramers. These investigations allowed us to characterize molecular interactions of various oligomeric states that are further converted into elongation competent fibril nuclei during the lag phase of a functional amyloid forming peptide.     

A Detailed Analysis of the Morphology of Fibrils of Selectively Mutated Amyloid β (1–40)

A small library of rationally designed amyloid β [Aβ(1–40)] peptide variants is generated, and the morphology of their fibrils is studied. In these molecules, the structurally important hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) is systematically mutated to introduce defined physical forces to act as specific internal constraints on amyloid formation. This Aβ(1–40) peptide library is used to study the fibril morphology of these variants by employing a comprehensive set of biophysical techniques including solution and solid‐state NMR spectroscopy, AFM, fluorescence correlation spectroscopy, and XRD. Overall, the findings demonstrate that the introduction of significant local physical perturbations of a crucial early folding contact of Aβ(1–40) only results in minor alterations of the fibrillar morphology. The thermodynamically stable structure of mature Aβ fibrils proves to be relatively robust against the introduction of significantly altered molecular interaction patterns due to point mutations. This underlines that amyloid fibril formation is a highly generic process in protein misfolding that results in the formation of the thermodynamically most stable cross‐β structure.     

Supersaturation-Dependent Formation of Amyloid Fibrils

The supersaturation of a solution refers to a non-equilibrium phase in which the solution is trapped in a soluble state, even though the solute’s concentration is greater than its thermodynamic solubility. Upon breaking supersaturation, crystals form and the concentration of the solute decreases to its thermodynamic solubility. Soon after the discovery of the prion phenomena, it was recognized that prion disease transmission and propagation share some similarities with the process of crystallization. Subsequent studies exploring the structural and functional association between amyloid fibrils and amyloidoses solidified this paradigm. However, recent studies have not necessarily focused on supersaturation, possibly because of marked advancements in structural studies clarifying the atomic structures of amyloid fibrils. On the other hand, there is increasing evidence that supersaturation plays a critical role in the formation of amyloid fibrils and the onset of amyloidosis. Here, we review the recent evidence that supersaturation plays a role in linking unfolding/folding and amyloid fibril formation. We also introduce the HANABI (HANdai Amyloid Burst Inducer) system, which enables high-throughput analysis of amyloid fibril formation by the ultrasonication-triggered breakdown of supersaturation. In addition to structural studies, studies based on solubility and supersaturation are essential both to developing a comprehensive understanding of amyloid fibrils and their roles in amyloidosis, and to developing therapeutic strategies.     

Molecular Dynamics Simulation Studies on the Aggregation of Amyloid-β Peptides and Their Disaggregation by Ultrasonic Wave and Infrared Laser Irradiation

Alzheimer’s disease is understood to be caused by amyloid fibrils and oligomers formed by aggregated amyloid-β (Aβ) peptides. This review article presents molecular dynamics (MD) simulation studies of Aβ peptides and Aβ fragments on their aggregation, aggregation inhibition, amyloid fibril conformations in equilibrium, and disruption of the amyloid fibril by ultrasonic wave and infrared laser irradiation. In the aggregation of Aβ, a β-hairpin structure promotes the formation of intermolecular β-sheet structures. Aβ peptides tend to exist at hydrophilic/hydrophobic interfaces and form more β-hairpin structures than in bulk water. These facts are the reasons why the aggregation is accelerated at the interface. We also explain how polyphenols, which are attracting attention as aggregation inhibitors of Aβ peptides, interact with Aβ. An MD simulation study of the Aβ amyloid fibrils in equilibrium is also presented: the Aβ amyloid fibril has a different structure at one end from that at the other end. The amyloid fibrils can be destroyed by ultrasonic wave and infrared laser irradiation. The molecular mechanisms of these amyloid fibril disruptions are also explained, particularly focusing on the function of water molecules. Finally, we discuss the prospects for developing treatments for Alzheimer’s disease using MD simulations.