Insulin Amyloid Fibril Formation Research Articles

Insulin amyloid fibril formation reduction by tripeptide stereoisomers.

Insulin fibrillation is a problem for diabetic patients that can occur during storage and transport, as well as at the subcutaneous injection site, with loss of bioactivity, inflammation, and various adverse effects. Tripeptides are ideal additives to stabilise insulin formulations, thanks to their low cost of production and inherent cytocompatibility. In this work, we analysed the ability of eight tripeptide stereoisomers to inhibit the fibrillation of human insulin in vitro. The sequences contain proline as β-breaker and Phe-Phe as binding motif for the amyloid-prone aromatic triplet found in insulin. Experimental data based on spectroscopy, fluorescence, microscopy, and calorimetric techniques reveal that one stereoisomer is a more effective inhibitor than the others, and cell live/dead assays confirmed its high cytocompatibility. Importantly, in silico data revealed the key regions of insulin engaged in the interaction with this tripeptide, rationalising the molecular mechanism behind insulin fibril formation reduction.

Biocompatible zeolite-dye composites with anti-amyloidogenic properties

One of the most attractive approaches in biomedicine and pharmacy is the application of multifunctional materials. The mesoporous structure of clinoptilolite (CZ) absorbs various types of substances and can be used as a model for studying the carriers for targeted drug delivery with controlled release. CZ-dye composites are fabricated by incorporation into clinoptilolite pores commonly used dyes, aluminum phthalocyanine, zinc porphine, and hypericin. We examined and compared the effect of pure dyes and CZ-dye composites on insulin amyloidogenesis. The formation of insulin amyloid fibrils and the disassembly of preformed fibrils is significantly affected by any of the three compounds, however, the strongest effect is observed for aluminum phthalocyanine indicating a structurally-dependent anti-amyloidogenic activity of the dyes. The incorporation of dyes into CZ particles resulted in enhanced anti-amyloidogenic activity in comparison to pure CZ particles. The cell metabolic activity, biocompatibility and fluorescence biodistribution of the dyes entrapped in the composites were tested in vitro (U87 MG cells) and in vivo in the quail chorioallantoic membrane model. Considering the photoactive properties of the dyes used, we assume their applicability in photodiagnostics and photodynamic therapy. It can also be expected that their anti-amyloidogenic potential can be enhanced by photodynamic effect.

Surface-modified cerium dioxide nanoparticles with improved anti-amyloid and preserved nanozymatic activity

Cerium dioxide nanoparticles (CeO2 NPs) are used increasingly in nanotechnology and particularly in biotechnology and bioresearch. Thus, CeO2 NPs have been successfully tested in vitro as a potential therapeutic agent for various pathologies associated with oxidative stress, including the formation of protein amyloid aggregates. In this study, to increase the anti-amyloidogenic efficiency and preserve the antioxidant potential, the surface of the synthesized CeO2 NPs is modified with a nonionic, sugar-based surfactant, dodecyl maltoside (DDM), which is known for its high anti-amyloidogenic activity and biocompatibility. Dynamic light scattering and Fourier transform infrared spectroscopy demonstrated successful modification by DDM. The apparent hydrodynamic diameters of CeO2 NPs and DDM-modified NPs (CeO2@DDM NPs) are found to be ⁓180 nm and ⁓260 nm, respectively. A positive zeta potential value of + 30.5 mV for CeO2 NPs and + 22.5 mV for CeO2 @DDM NPs suggest sufficient stability and good dispersion of NPs in an aqueous solution. A combination of Thioflavin T fluorescence analysis and atomic force microscopy is used to assess the effect of nanoparticles on the formation of insulin amyloid fibrils. Results show that the fibrillization of insulin is inhibited by both, naked and modified NPs in a dose-dependent manner. However, while the IC50 of naked NPs is found to be ∼270 ± 13 µg/mL, the surface-modified NPs are 50% more efficient with IC50 equaled to 135 ± 7 µg/mL. In addition, both, the naked CeO2 NPs and DDM-modified NPs displayed an antioxidant activity expressed as oxidase-, catalase- and SOD-like activity. Therefore, the resulting nanosized material is very well suited to prove or disprove the hypothesis that oxidative stress plays a role in the formation of amyloid fibrils.

Chickpea Peptide: A Nutraceutical Molecule Corroborating Neurodegenerative and ACE-I Inhibition.

Chickpea seeds are the source of proteins in human nutrition and attribute some nutraceutical properties. Herein, we report the effects of chickpea seed bioactive peptide on albumin, insulin, lactoglobulin and lysozyme amyloid fibril formation. Employing thioflavin T (ThT) assays and circular dichroism (CD), amyloid structural binding transition was experimented to analyze the inhibition of amyloid fibril formation. The purified active peptide with a molecular mass of 934.53 Da was evaluated in vitro for its ACE-I inhibitory, antibacterial, antifungal and antidiabetic activities. Further, in vivo animal studies were carried out in wistar rats for blood pressure lowering action. In hypertensive rats, chickpea peptide decreased 131 ± 3.57 mm of Hg for systolic blood pressure and 86 ± 1.5 mm of Hg for diastolic blood pressure after 8 h intraperitoneal administration. Additionally, the peptide suppressed the fibrillation of amyloid and destabilized the preformed mature fibrils. Data emphasize efficacy of chickpea peptide vis-a-vis ACE-Inhibitory, antibacterial, antifungal, antidiabetic and anti-amyloidogenic activities, allowing us to propose this novel peptide as a suitable candidate for nutraceutical-based drugs and seems the first kind of its nature.

Tuning the surface charge of phospholipid bilayers inhibits insulin fibrilization

The interactions between proteins and materials, in particular lipid bilayers, have been studied extensively for their relevance in diseases and for the formulation of protein-based therapeutics and vaccines. However, the precise rules by which material properties induce favorable or unfavorable structural states in biomolecules are incompletely understood, and as a result, the rational design of materials remains challenging. Here, we investigated the influence of lipid bilayers (in the form of small unilamellar vesicles) on the formation of insulin amyloid fibrils using a fibril-specific assay (thioflavin T), polyacrylamide gel electrophoresis, and circular dichroism spectroscopy. Lipid bilayers composed of equal mixtures of cationic and anionic lipids effectively inhibited fibril formation and stabilized insulin in its native conformation. However, other lipid bilayer compositions failed to inhibit fibril formation or even destabilized insulin, exacerbating fibrilization and/or non-amyloid aggregation. Our findings suggest that electrostatic interactions with lipid bilayers can play a critical role in stabilizing or destabilizing insulin, and preventing the conversion of insulin to its amyloidogenic, disease-associated state.

Sulfated polysaccharide ascophyllan prevents amyloid fibril formation of human insulin and inhibits amyloid-induced hemolysis and cytotoxicity in PC12 cells.

We found that ascophyllan significantly inhibited the fibrillation of human insulin and was the most effective among the sulfated polysaccharides tested. Gel-filtration analysis suggested that ascophyllan was capable of forming a complex with insulin through a weak interaction. Secondary structure transition from native α-helix to β-sheet predominant structure of insulin under the fibrillation conditions was suppressed in the presence of ascophyllan. Interestingly, ascophyllan attenuated insulin fibril-induced hemolysis of human erythrocytes. Moreover, ascophyllan attenuated insulin amyloid-induced cytotoxicity on rat pheochromocytoma PC12 cells and reduced the level of intracellular reactive oxygen species. This is the first report indicating that a sulfated polysaccharide, ascophyllan, can suppress the insulin amyloid fibril formation and inhibit the fibril-induced detrimental bioactivities.

Inhibition of Insulin Amyloid Fibrillation by Salvianolic Acids and Calix[n]arenes: Molecular Docking Insight

In this study, the ability of salvianolic acids A, B, C, F, G and calix[[Formula: see text]]arenes ([Formula: see text], 5, 6 and 8) with different upper rims in the inhibition of insulin amyloid fibril formation was studied using molecular docking. The results were analyzed from a molecular point of view. All of the considering ligands interacted with significant residues of insulin, which had a crucial role in the process of insulin fibrillation. The interactions among the ligands and insulin residues could be done through hydrogen bonding and hydrophobic interactions with good binding affinity. So, these ligands could prevent the formation of the insulin fibril. The good consistency of the docking results of [Formula: see text]-sulfonatocalix[4]arene and [Formula: see text]-sulfonatocalix[6]arene with the experimental results in the previous literature represented the capacity of the current theoretical method to supplement and interpret experimental findings. Also, in this study, salvianolic acids A, C, F and G were suggested as new inhibitors of the insulin amyloid fibril.

Biopolymer-coated gold nanoparticles inhibit human insulin amyloid fibrillation

Deposits of protein misfolding and/or aggregates are a pathological hallmark of amyloid-related diseases. For instance, insulin amyloid fibril deposits have been observed in patients with insulin-dependent diabetes mellitus after insulin administration. Here, we report on the use of AuNPs functionalized with linear- (i.e. dextrin and chitosan) and branched- (i.e. dextran-40 and dextran-10) biopolymers as potential agents to inhibit insulin fibril formation. Our dynamic light scattering analyses showed a size decrease of the amyloid fibrils in the presence of functionalized AuNPs. Circular dichroism spectroscopy as well as enzyme-linked immunosorbent assay data demonstrated that the secondary structural transition from α-helix to β-sheet (which is characteristic for insulin amyloid fibril formation) was significantly suppressed by all biopolymer-coated AuNPs, and in particular, by those functionalized with linear biopolymers. Both transmission electron microscopy and atomic force microscopy analyses showed that the long thick amyloid fibrils formed by insulin alone become shorter, thinner or cluster when incubated with biopolymer-coated AuNPs. Dextrin- and chitosan-coated AuNPs were found to be the best inhibitors of the fibril formation. Based on these results, we propose a mechanism for the inhibition of insulin amyloid fibrils: biopolymer-coated AuNPsstrongly interact with the insulin monomers and inhibit the oligomer formation as well as elongation of the protofibrils.Moreover, cytotoxicity experiments showed that AuNP-insulin amyloid fibrils are less toxic compared to insulin amyloid fibrils alone. Our results suggest that both dextrin- and chitosan-AuNPs could be used as therapeutic agents for the treatment of amyloid-related disorders.

Development of Gold Nanorods-Insulin Amyloid Fibril Assemblies: A Hybrid Nanoscaffold with Enhanced Conductivity

Gold nanoparticles are metallic nanostructures with wide range of applications in biomedicine and biosensing. Among diverse morphologies of gold nanostructures, gold nanorods have attracted much more attention for their outstanding structure, optical property and function. The unique characteristic of these nanostructures encourages their integration with biomolecules of interest for versatile biomedical proposes. In this effort, amyloid fibrils with well-known mechanical strength and chemical stability have been used as biotemplates for assembly of gold nanorods to exploit dual property of both nano and bio counterparts in a hybrid nanoscaffold. To maximize the interaction of the nanostructures with the biotemplate, surface of the gold nanorods was modified with biocompatible polystyrene sulfonate (PSS). Spectroscopic and microscopic techniques including UV–Vis, CD, ThT and TEM were used to characterize formation of insulin amyloid fibrils, gold nanorods and their assemblies on the biotemplate. Morphology of gold nanorods was also monitored upon interaction with amyloid fibrils. Electrochemical impedance spectroscopy of the hybrid nanoscaffold showed that increase in the concentration of the assembled nanostructures on the biotemplate has clearly led to enhancement of conductivity. Results of this investigation highlight gold nanorods as promising candidates in design and fabrication of novel hybrid nanoscaffolds of enhanced conductivity, especially for biosensing and tissue engineering purposes.

Dual effect of non-ionic detergent Triton X-100 on insulin amyloid formation.

Atomic force microscopy, Thioflavin T (ThT) fluorescence assay, circular dichroism spectroscopy, differential scanning calorimetry, and molecular modeling techniques have been employed to investigate the amyloid aggregation of insulin in the presence of non-ionic detergent, Triton X-100 (TX-100). In contrast to recently described inhibition of lysozyme amyloid formation by non-ionic detergents (Siposova, 2017), the amyloid aggregation of insulin in the presence of sub-micellar TX-100 concentration exhibits two dissimilar phases. The first, inhibition phase, is observed at the protein to detergent molar ratio of 1:0.1 to 1:1. During this phase, the insulin amyloid fibril formation is inhibited by TX-100 up to ∼60%. The second, "morphological" phase, is observed at increasing detergent concentration, corresponding to protein:detergent molar ratio of ∼1:1 - 1:10. Under these conditions a significant increase of the steady-state ThT fluorescence intensities and a dramatically changed morphology of the insulin fibrils were observed. Increasing of the detergent concentration above the CMC led to complete inhibition of amyloidogenesis. Analysis of the experimental and molecular modeling results suggests an existence of up to six TX-100 binding sites within dimer of insulin with different binding energy. The physiological relevance of the results is discussed.

Gelatin as a Potential Inhibitor of Insulin Amyloid Fibril Formation

AbstractDirect inhibition of amyloid fibrillation, the primary cause of many neurodegenerative diseases, using natural and synthetic molecules is one of the therapeutic strategies to prohibit the progression of amyloid diseases. Herein, we have demonstrated that, for the first time, gelatin, a commonly used gelling agent in food, was a potent inhibitor of amyloid fibril formation using insulin, a model protein for amyloid fibrillation. Thioflavin T and turbidity data indicated concentration‐dependent inhibition of insulin amyloid fibrillation by gelatin. Atomic force microscopy envisaged the inhibition of insulin fibrillation by gelatin. The spectroscopic analysis suggested that gelatin inhibits the insulin amyloid fibrillation by stabilising the native structure of insulin. 8‐anilinonaphthalene‐1‐sulfonic acid fluorescence data indicated that gelatin prevented the accessible exposed hydrophobic regions of insulin, which is required for insulin aggregation. Studies also suggested that the anti‐amyloidogenic efficiency of a mixture containing gelatin and a known amyloid inhibitor (Ferulic acid) was higher than their individual efficiency. Since gelatin is commonly used in drug formulation, its inherent anti‐amyloidogenic character may be beneficial in the formulation of anti‐amyloidogenic compounds. Hence, the current findings may have significant implications for the development of therapeutic agents for the prevention of disease‐related amyloid proteins.

A-type dimeric epigallocatechin-3-gallate (EGCG) is a more potent inhibitor against the formation of insulin amyloid fibril than EGCG monomer.

Because fibrillary protein aggregates is regarded to be closely associated with many diseases such as Alzheimer's disease, diabetes, and Parkinson's disease, growing interest and researches have been focused on finding potential fibrillation inhibitors. In the present study, the inhibitory effects of epigallocatechin-3-gallate (EGCG) and A-type dimeric epigallocatechin-3-gallate (A-type EGCG dimer) on the formation of insulin fibrillation were compared by multi-dimensional approaches including thioflavin-T (ThT) fluorescence assay, 1-anilinonaphthalene-8-sulfonic (ANS) fluorescence assay, dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD) spectroscopy. Our results confirmed that A-type EGCG dimer is a more potent inhibitor against the formation of bovine insulin amyloid fibril than EGCG. In addition, A-type EGCG dimer could not only inhibit insulin amyloid fibril formation, but also change the aggregation pathway and induce bovine insulin into amorphous aggregates. The results of the present study may provide a new guide on finding novel anti-amyloidogenic agents.

Phospholipids as inhibitors of amyloid fibril formation

Amyloid fibrils are the protein aggregates, whose formation is involved in the pathogenesis of Alzheimer’s disease, systemic amyloidosis, etc. Since there is no effective ways to treat these diseases, developing the new anti-amyloid drugs is of great importance. In this study a series of phospholipids have been tested for their ability to inhibit lysozyme and insulin amyloid fibril formation at acidic or neutral pH and elevated temperature. The lag time, elongation rate and fibrillization extent were estimated using Thioflavin T fluorescence assay. It is found that the oxidized and charged phospholipids, included into the liposomes, were the most effective inhibitors of the protein fibrillization. By comparing the magnitude and direction of the lipid effect in different lipid-protein systems it was concluded that the reduction of the amyloid fibril formation is governed by hydrophobic and specific liposome-protein interactions. It is hypothesized that the presence of the surface formed by the lipid polar heads is critical for reducing the protein fibrillization extent.

Inhibition of insulin amyloid fibril formation by cyclodextrins

Localized insulin-derived amyloid masses occasionally form at the site of repeated insulin injections in patients with insulin-dependent diabetes and cause subcutaneous insulin resistance. Various kinds of insulin including porcine insulin, human insulin, and insulin analogues reportedly formed amyloid fibrils in vitro and in vivo, but the impact of the amino acid replacement in insulin molecules on amyloidogenicity is largely unknown. In the present study, we demonstrated the difference in amyloid fibril formation kinetics of human insulin and insulin analogues, which suggests an important role of the C-terminal domain of the insulin B chain in nuclear formation of amyloid fibrils. Furthermore, we determined that cyclodextrins, which are widely used as drug carriers in the pharmaceutical field, had an inhibitory effect on the nuclear formation of insulin amyloid fibrils. These findings have significant implications for the mechanism underlying insulin amyloid fibril formation and for developing optimal additives to prevent this subcutaneous adverse effect.

Acceleration of Insulin Amyloid Fibrillation at Liquid–Liquid Interfaces

Abstract The formation of insulin amyloid fibrils under acidic conditions was accelerated in the liquid–liquid systems, especially in the toluene/water system. High interfacial adsorptivity of insulin monomer was confirmed by interfacial tension measurement. Fluorescence microscope observation using a thioflavine T (ThT) probe proved directly the interfacial formation and adsorption of the amyloid fibril at the dodecane/water interface. Far-UV-circular dichroism (CD) spectra of insulin in the dodecane/water system suggested the conversion of α-helix form to the β-sheet in the aqueous phase. Furthermore, the total internal reflection-induced CD measurement of the amyloid fibril–ThT complex at the dodecane/water interface led to the conclusion that the amyloid formation at the interface is faster than the conversion of α-helix form in the aqueous phase.

Intermediates caught in the act: tracing insulin amyloid fibril formation in time by combined optical spectroscopy, light scattering, mass spectrometry and microscopy.

Interest in the topic of amyloid formation by peptides and proteins has increased dramatically in recent years, transforming it from a puzzling phenomenon associated with a small number of diseases into a major subject of study in disciplines ranging from material science to biology and medicine. The tendency of numerous (also non-pathogenic) proteins such as insulin to self-assemble into amyloid-like fibrils is well known. While fibrils are usually easily detected, the observation of transient intermediates is a big challenge in general. They are the key and the 'holy grail' for a molecular understanding of mechanisms in this context. Here we show that intermediates, i.e. oligomers, can be detected and their hydrodynamic radius RH as well as their overall conformation and structure can be monitored and the aggregation dynamics as well as structure formation can be detected in time with a suitable combination of experimental techniques. We have observed transient intermediates that resemble large oligomers held together in solution by non-covalent forces. The oligomers appear to convert into building blocks for mature fibrils with largely beta-sheet conformation resembling key players in a mechanism, which is termed 'nucleated conformation conversion' in the literature. Structural transformations of oligomers in time towards dominant beta-sheet conformations have been observed for the first time. The structures can even be observed in liquid phase AFM experiments. With this approach we have successfully shed new light into the aggregation and fibrilization process of insulin being possibly a model system for other amyloid systems.

Inhibition of insulin amyloid fibril formation by ferulic acid, a natural compound found in many vegetables and fruits

The highly ordered structures from the self-assembly of proteins/peptides, referred to as amyloid fibrils, are the major cause for many neurodegenerative diseases. Herein, we have analyzed the effect of ferulic acid (FA), one of the most abundant phenolic acids in plants, fruits and vegetables, on the inhibition of insulin (associated with type II diabetes) fibrillation. It was found that the amount of fibril formation was greatly reduced with increasing concentration of FA as evidenced from turbidity measurements. ThT binding experiments confirm the inhibition in which enhanced fluorescence, characteristic of amyloid fibrillation, was not observed in the presence of FA. Atomic force microscopy further confirms inhibition by FA where no typical insulin amyloid fibril has been detected. The characteristic conformational transition from α-helix to β-sheet was arrested in the presence of FA demonstrating that FA protects the native structure of insulin and thereby prevents the conformational change required for its amyloid fibril formation in vitro. The percentage of insulin fibril inhibition was found to be dependent on the concentration of FA. The current results demonstrate that FA can inhibit the insulin amyloid fibril formation and thus FA, in addition to other phenolic compounds, provides a possible therapeutic strategy to prevent or treat insulin amyloidosis.

Gallic acid, one of the components in many plant tissues, is a potential inhibitor for insulin amyloid fibril formation

Proteins under stressful conditions can lead to the formation of an ordered self-assembled structure, referred to as amyloid fibrils, to which many neurodegenerative diseases such as Type II diabetes, Alzheimer's, Parkinson's, Huntington's, etc., are attributed. Inhibition of amyloid fibril formation using natural products is one of the main therapeutic strategies to prevent the progression of these diseases. Polyphenols are the mostly consumed as antioxidants in a human nutrition. Herein, we have studied the effect of a simple polyphenol, gallic acid (GA), one of the main components in plant tissues, especially in tea leaves, on the insulin amyloid fibril formation. Different biophysical characterizations such as turbidity, atomic force microscopy (AFM), Thioflavin T (ThT) assays, circular dichroism, and Fourier transform-infrared spectroscopy have been used to analyze the inhibition of amyloid fibril formation. The occurrence of fibrils in an AFM image and ThT fluorescence enhancement confirms the formation of insulin amyloid fibrils when incubated under acidic pH 2 at 65 °C. In the presence of GA, absence of fibrils in AFM image and no change in the intensity of ThT fluorescence confirms the inhibition of insulin amyloid fibrils by GA. Spectroscopic results reveal that GA inhibits the conformational transition of α-helix → β-sheet, which is generally induced during the insulin fibril formation. It was found that the inhibitory effect of GA is concentration dependent and non-linear. Based on the observed results, we propose that GA interacts with native insulin, preventing nuclei formation, which is essential for fibril growth, thereby inhibiting the amyloid fibril formation. The present results thus demonstrate that GA can effectively inhibit insulin amyloid fibril formation in vitro.

Stepwise Organization of the β-Structure Identifies Key Regions Essential for the Propagation and Cytotoxicity of Insulin Amyloid Fibrils

Amyloid fibrils are supramolecular assemblies, the deposition of which is associated with many serious diseases including Alzheimer, prion, and Huntington diseases. Several smaller aggregates such as oligomers and protofibrils have been proposed to play a role in early stages of the fibrillation process; however, little is known about how these species contribute to the formation of mature amyloid fibrils with a rigid cross-β structure. Here, we identified a new pathway for the formation of insulin amyloid fibrils at a high concentration of salt in which mature fibrils were formed in a stepwise manner via a prefibrillar intermediate: minute prefibrillar species initially accumulated, followed by the subsequent formation of thicker amyloid fibrils. Fourier transform infrared spectra suggested the sequential formation of two types of β-sheets with different strength hydrogen bonds, one of which was developed concomitantly with the mutual assembly of the prefibrillar intermediate to form mature fibrils. Interestingly, fibril propagation and cellular toxicity appeared only after the later step of structural organization, and a comparison of β-sheet regions between the prefibrillar intermediate and mature fibrils using proteolysis led to the proposal of specific regions essential for manifestation of these properties.

Ion-specific Effects on Prion Nucleation and Strain Formation

Ordered, fibrous, self-seeding aggregates of misfolded proteins known as amyloids are associated with important diseases in mammals and control phenotypic traits in fungi. A given protein may adopt multiple amyloid conformations, known as variants or strains, each of which leads to a distinct disease pattern or phenotype. Here, we study the effect of Hofmeister ions on amyloid nucleation and strain generation by the prion domain-containing fragment (Sup35NM) of a yeast protein Sup35p. Strongly hydrated anions (kosmotropes) initiate nucleation quickly and cause rapid fiber elongation, whereas poorly hydrated anions (chaotropes) delay nucleation and mildly affect the elongation rate. For the first time, we demonstrate that kosmotropes favor formation of amyloid strains that are characterized by lower thermostability and higher frangibility in vitro and stronger phenotypic and proliferation patterns effectively in vivo as compared with amyloids formed in chaotropes. These phenomena point to inherent differences in the biochemistry of Hofmeister ions. Our work shows that the ionic composition of a solution not only influences the kinetics of amyloid nucleation but also determines the amyloid strain that is preferentially formed.