Multiscale Modeling of Amyloid Fibrils Formed by Aggregating Peptides Derived from the Amyloidogenic Fragment of the A-Chain of Insulin

Computational prediction of molecular structures of amyloid fibrils remains an exceedingly challenging task. In this work, we propose a multi-scale modeling procedure for the structure prediction of amyloid fibrils formed by the association of ACC_1-13 aggregation-prone peptides derived from the N-terminal region of insulin’s A-chain. First, a large number of protofilament models composed of five copies of interacting ACC_1-13 peptides were predicted by application of CABS-dock coarse-grained (CG) docking simulations. Next, the models were reconstructed to all-atom (AA) representations and refined during molecular dynamics (MD) simulations in explicit solvent. The top-scored protofilament models, selected using symmetry criteria, were used for the assembly of long fibril structures. Finally, the amyloid fibril models resulting from the AA MD simulations were compared with atomic force microscopy (AFM) imaging experimental data. The obtained results indicate that the proposed multi-scale modeling procedure is capable of predicting protofilaments with high accuracy and may be applied for structure prediction and analysis of other amyloid fibrils.     

Effect of 1-Ethyl-3-methylimidazolium Tetrafluoroborate and Acetate Ionic Liquids on Stability and Amyloid Aggregation of Lysozyme

Amyloid fibrils draw attention as potential novel biomaterials due to their high stability, strength, elasticity or resistance against degradation. Therefore, the controlled and fast fibrillization process is of great interest, which raises the demand for effective tools capable of regulating amyloid fibrillization. Ionic liquids (ILs) were identified as effective modulators of amyloid aggregation. The present work is focused on the study of the effect of 1-ethyl-3-methyl imidazolium-based ILs with kosmotropic anion acetate (EMIM-ac) and chaotropic cation tetrafluoroborate (EMIM-BF₄) on the kinetics of lysozyme amyloid aggregation and morphology of formed fibrils using fluorescence and CD spectroscopy, differential scanning calorimetry, AFM with statistical image analysis and docking calculations. We have found that both ILs decrease the thermal stability of lysozyme and significantly accelerate amyloid fibrillization in a dose-dependent manner at concentrations of 0.5%, 1% and 5% (v/v) in conditions and time-frames when no fibrils are formed in ILs-free solvent. The effect of EMIM-BF₄ is more prominent than EMIM-ac due to the different specific interactions of the anionic part with the protein surface. Although both ILs induced formation of amyloid fibrils with typical needle-like morphology, a higher variability of fibril morphology consisting of a different number of intertwining protofilaments was identified for EMIM-BF_4.     

Searching for the Best Transthyretin Aggregation Protocol to Study Amyloid Fibril Disruption

Several degenerative amyloid diseases, with no fully effective treatment, affect millions of people worldwide. These pathologies—amyloidoses—are known to be associated with the formation of ordered protein aggregates and highly stable and insoluble amyloid fibrils, which are deposited in multiple tissues and organs. The disruption of preformed amyloid aggregates and fibrils is one possible therapeutic strategy against amyloidosis; however, only a few compounds have been identified as possible fibril disruptors in vivo to date. To properly identify chemical compounds as potential fibril disruptors, a reliable, fast, and economic screening protocol must be developed. For this purpose, three amyloid fibril formation protocols using transthyretin (TTR), a plasma protein involved in several amyloidoses, were studied using thioflavin-T fluorescence assays, circular dichroism (CD), turbidity, dynamic light scattering (DLS), and transmission electron microscopy (TEM), in order to characterize and select the most appropriate fibril formation protocol. Saturation transfer difference nuclear magnetic resonance spectroscopy (STD NMR) was successfully used to study the interaction of doxycycline, a known amyloid fibril disruptor, with preformed wild-type TTR (TTRwt) aggregates and fibrils. DLS and TEM were also used to characterize the effect of doxycycline on TTRwt amyloid species disaggregation. A comparison of the TTR amyloid morphology formed in different experimental conditions is also presented.     

Lysozyme Amyloid Fibril Structural Variability Dependence on Initial Protein Folding State

Amyloid fibril formation is associated with several amyloidoses, including neurodegenerative Alzheimer’s or Parkinson’s diseases. The process of such fibrillar structure formation is still not fully understood, with new mechanistic insights appearing on a regular basis. This, in turn, has limited the development of potential anti-amyloid compounds, with only a handful of effective cures or treatment modalities available. One of the multiple amyloid aggregation factors that requires further examination is the ability of proteins to form multiple, structurally distinct aggregates, based on the environmental conditions. In this work, we examine how the initial folding state affects the fibrilization of lysozyme—an amyloidogenic protein, often used in protein aggregation studies. We show that there is a correlation between the initial state of the protein and the aggregate formation lag time, rate of elongation, resulting aggregate structural variability and dye-binding properties, as well as formation lag time and rate of elongation.     

Misfolding and Amyloid Aggregation of Apomyoglobin

Apomyoglobin is an excellent example of a monomeric all α-helical globular protein whose folding pathway has been extensively studied and well characterized. Structural perturbation induced by denaturants or high temperature as well as amino acid substitution have been described to induce misfolding and, in some cases, aggregation. In this article, we review the molecular mechanism of the aggregation process through which a misfolded form of a mutated apomyoglobin aggregates at physiological pH and room temperature forming an amyloid fibril. The results are compared with data showing that either amyloid or aggregate formation occurs under particular denaturing conditions or upon cleavage of the residues corresponding to the C-terminal helix of apomyoglobin. The results are discussed in terms of the sequence regions that are more important than others in determining the amyloid aggregation process.     

Protein Folding and Aggregation into Amyloid: The Interference by Natural Phenolic Compounds

Amyloid aggregation is a hallmark of several degenerative diseases affecting the brain or peripheral tissues, whose intermediates (oligomers, protofibrils) and final mature fibrils display different toxicity. Consequently, compounds counteracting amyloid aggregation have been investigated for their ability (i) to stabilize toxic amyloid precursors; (ii) to prevent the growth of toxic oligomers or speed that of fibrils; (iii) to inhibit fibril growth and deposition; (iv) to disassemble preformed fibrils; and (v) to favor amyloid clearance. Natural phenols, a wide panel of plant molecules, are one of the most actively investigated categories of potential amyloid inhibitors. They are considered responsible for the beneficial effects of several traditional diets being present in green tea, extra virgin olive oil, red wine, spices, berries and aromatic herbs. Accordingly, it has been proposed that some natural phenols could be exploited to prevent and to treat amyloid diseases, and recent studies have provided significant information on their ability to inhibit peptide/protein aggregation in various ways and to stimulate cell defenses, leading to identify shared or specific mechanisms. In the first part of this review, we will overview the significance and mechanisms of amyloid aggregation and aggregate toxicity; then, we will summarize the recent achievements on protection against amyloid diseases by many natural phenols.     

Aggregation Condition–Structure Relationship of Mouse Prion Protein Fibrils

Prion diseases are associated with conformational conversion of cellular prion protein into a misfolded pathogenic form, which resembles many properties of amyloid fibrils. The same prion protein sequence can misfold into different conformations, which are responsible for variations in prion disease phenotypes (prion strains). In this work, we use atomic force microscopy, FTIR spectroscopy and magic-angle spinning NMR to devise structural models of mouse prion protein fibrils prepared in three different denaturing conditions. We find that the fibril core region as well as the structure of its N- and C-terminal parts is almost identical between the three fibrils. In contrast, the central part differs in length of β-strands and the arrangement of charged residues. We propose that the denaturant ionic strength plays a major role in determining the structure of fibrils obtained in a particular condition by stabilizing fibril core interior-facing glutamic acid residues.     

Inhibition of Amyloid Protein Aggregation Using Selected Peptidomimetics

Aberrant protein aggregation leads to the formation of amyloid fibrils. This phenomenon is linked to the development of more than 40 irremediable diseases such as Alzheimer's disease, Parkinson's disease, type 2 diabetes, and cancer. Plenty of research efforts have been given to understanding the underlying mechanism of protein aggregation, associated toxicity, and the development of amyloid inhibitors. Recently, the peptidomimetic approach has emerged as a potential tool to modulate several protein-protein interactions (PPIs). In this review, we discussed selected peptidomimetic-based approaches for the modulation of important amyloid proteins (Islet Amyloid Polypeptide, Amyloid Beta, α-synuclein, mutant p53, and insulin) aggregation. This approach holds a powerful platform for creating an essential stepping stone for the vital development of anti-amyloid therapeutic agents.     

Synthesis of New Tyrosol-Based Phosphodiester Derivatives: Effect on Amyloid β Aggregation and Metal Chelation Ability

Alzheimer's disease (AD) is a multifactorial pathology that requires multifaceted agents able to address its peculiar nature. Increasing evidence has shown that aggregation of amyloid β (Aβ) and oxidative stress are strictly interconnected, and their modulation might have a positive and synergic effect in contrasting AD-related impairments. Herein, a new and efficient fragment-based approach towards tyrosol phosphodiester derivatives (TPDs) has been developed starting from suitable tyrosol building blocks and exploiting the well-established phosphoramidite chemistry. The antioxidant activity of new TPDs has been tested as well as their ability to inhibit Aβ protein aggregation. In addition, their metal chelating ability has been evaluated as a possible strategy to develop new natural-based entities for the prevention or therapy of AD. Interestingly, TPDs containing a catechol moiety have demonstrated highly promising activity in inhibiting the aggregation of Aβ₄₀ and a strong ability to chelate biometals such as Cu^II and Zn^II.     

Liberation of GPI-Anchored Prion from Phospholipids Accelerates Amyloidogenic Conversion

Prion diseases or transmissible spongiform encephalopathies are a rare group of fatal neurodegenerative illnesses in humans and animals caused by misfolding of prion protein (PrP). Prion protein is a cell-surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed mostly in the central and peripheral nervous system, and this membrane-bound protein can be cleaved from the cell membranes by phosphoinositide phospholipase C. Numerous studies have investigated GPI-free recombinant PrP, but the role of GPI on misfolding of PrP is not well known. In this study, we synthesized a GPI analog that was covalently linking to a PrP S230C mutant, resulting in S230C-GPI. The structural changes in S230C-GPI upon binding to lipid vesicles composed of mixtures of the zwitterionic lipid (POPC) and the anionic lipid (POPG) were analyzed by circular dichroism spectroscopy, and the amyloid aggregation of S230C-GPI in the liberation from phospholipid vesicles was monitored by proteinase K-digestion assay. Our results indicate that S230C-GPI in the liberation of lipid vesicles has high tendency to misfold into amyloid fibrils, while the membrane-bound S230C-GPI proteins are highly stable and rarely convert into amyloid forms. In addition, the role of cholesterol in S230C-GPI was studied. The effect of GPI, cholesterol and phospholipid vesicles on misfolding of PrP is further discussed.     

Amyloid Aggregates of Smooth-Muscle Titin Impair Cell Adhesion

Various amyloid aggregates, in particular, aggregates of amyloid β-proteins, demonstrate in vitro and in vivo cytotoxic effects associated with impairment of cell adhesion. We investigated the effect of amyloid aggregates of smooth-muscle titin on smooth-muscle-cell cultures. The aggregates were shown to impair cell adhesion, which was accompanied by disorganization of the actin cytoskeleton, formation of filopodia, lamellipodia, and stress fibers. Cells died after a 72-h contact with the amyloid aggregates. To understand the causes of impairment, we studied the effect of the microtopology of a titin-amyloid-aggregate-coated surface on fibroblast adhesion by atomic force microscopy. The calculated surface roughness values varied from 2.7 to 4.9 nm, which can be a cause of highly antiadhesive properties of this surface. As all amyloids have the similar structure and properties, it is quite likely that the antiadhesive effect is also intrinsic to amyloid aggregates of other proteins. These results are important for understanding the mechanisms of the negative effect of amyloids on cell adhesion.     

Temperature-Dependent Structural Variability of Prion Protein Amyloid Fibrils

Prion protein aggregation into amyloid fibrils is associated with the onset and progression of prion diseases—a group of neurodegenerative amyloidoses. The process of such aggregate formation is still not fully understood, especially regarding their polymorphism, an event where the same type of protein forms multiple, conformationally and morphologically distinct structures. Considering that such structural variations can greatly complicate the search for potential antiamyloid compounds, either by having specific propagation properties or stability, it is important to better understand this aggregation event. We have recently reported the ability of prion protein fibrils to obtain at least two distinct conformations under identical conditions, which raised the question if this occurrence is tied to only certain environmental conditions. In this work, we examined a large sample size of prion protein aggregation reactions under a range of temperatures and analyzed the resulting fibril dye-binding, secondary structure and morphological properties. We show that all temperature conditions lead to the formation of more than one fibril type and that this variability may depend on the state of the initial prion protein molecules.     

Disaggregation of Islet Amyloid Polypeptide Fibrils as a Potential Anti-Fibrillation Mechanism of Tetrapeptide TNGQ

Islet amyloid polypeptide (IAPP) fibrillation has been commonly associated with the exacerbation of type 2 diabetes prognosis. Consequently, inhibition of IAPP fibrillation to minimize β-cell cytotoxicity is an important approach towards β-cell preservation and type 2 diabetes management. In this study, we identified three tetrapeptides, TNGQ, MANT, and YMSV, that inhibited IAPP fibrillation. Using thioflavin T (ThT) fluorescence assay, circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and molecular docking, we evaluated the potential anti-fibrillation mechanism of the tetrapeptides. ThT fluorescence kinetics and microscopy as well as transmission electron microscopy showed that TNGQ was the most effective inhibitor based on the absence of normal IAPP fibrillar morphology. CD spectroscopy showed that TNGQ maintained the α-helical conformation of monomeric IAPP, while DLS confirmed the presence of varying fibrillation species. Molecular docking showed that TNGQ and MANT interact with monomeric IAPP mainly by hydrogen bonding and electrostatic interaction, with TNGQ binding at IAPP surface compared to YMSV, which had the highest docking score, but interact mainly through hydrophobic interaction in IAPP core. The highly polar TNGQ was the most active and appeared to inhibit IAPP fibrillation by disaggregation of preformed IAPP fibrils. These findings indicate the potential of TNGQ in the development of peptide-based anti-fibrillation and antidiabetic nutraceuticals.     

Molecular Simulations of Amyloid Structures,Toxicity, and Inhibition

The misfolding and aggregation of proteins and peptides into amyloid fibrils are believed to be responsible for the dysfunction and death of neuron cells in many neurodegenerative diseases. Resolving the atomic structures of amyloid peptides at different aggregation stages by molecular simulations has opened new ways to probe the molecular mechanisms of amyloid aggregation, toxicity, and inhibition, as well as to validate computational data with available experimental ones. In this review article, we summarize some recent and important findings on: 1) a number of atomic structures of amyloid oligomers with typical β-sheet-rich conformations, related to amyloid aggregation; 2) different amyloid peptide-induced membrane-disruption mechanisms, related to amyloid toxicity; and 3) rational design of different amyloid inhibitors capable of preventing amyloid aggregation and toxicity, related to amyloid inhibition. All these findings will provide some mechanistic implications for molecular mechanisms of amyloid aggregation, toxicity, and inhibition, which are fundamentally and practically important for the treatment of amyloid diseases.     

New Perspectives in Reducing Amyloid Aggregation and Toxicity

One of the fundamental biochemical characteristics of Alzheimer′s Disease (AD) is the intercellular accumulation of amyloid beta (Aβ) peptide plaques in the brain. Although there is not yet a cure for AD, major efforts have been made to identify antiamyloidogenic agents and therapeutic strategies to treat this disease and related conditions. Our group focuses on inhibiting the aggregation and toxicity of Aβ using two distinct approaches. The first involves covalently attaching the sequence-derived core amyloidogenic amino acid fragment KLVFF to the surfaces of serum albumin microspheres. These particles bind soluble Aβ with high affinity, inhibit its aggregation, reduce its toxicity, and decrease the inflammation induced by its aggregates. Microsphere-bound Aβ stimulates microglia and is phagocytosed via a different mechanism from that used for Aβ removal, so enabling its clearance even by defective cells. Our second approach utilizes self-assembled cyclic D,L-α-peptides as conformational mimics of the amyloid fibril architecture that inhibit the aggregation and cytotoxicity of Aβ. Here, we review these strategies and their mechanisms of action.     

Polymorphism of Alpha-Synuclein Amyloid Fibrils Depends on Ionic Strength and Protein Concentration

Protein aggregate formation is linked with multiple amyloidoses, including Alzheimer‘s and Parkinson‘s diseases. Currently, the understanding of such fibrillar structure formation and propagation is still not sufficient, the outcome of which is a lack of potent, anti-amyloid drugs. The environmental conditions used during in vitro protein aggregation assays play an important role in determining both the aggregation kinetic parameters, as well as resulting fibril structure. In the case of alpha-synuclein, ionic strength has been shown as a crucial factor in its amyloid aggregation. In this work, we examine a large sample size of alpha-synuclein aggregation reactions under thirty different ionic strength and protein concentration combinations and determine the resulting fibril structural variations using their dye-binding properties, secondary structure and morphology. We show that both ionic strength and protein concentration determine the structural variability of alpha-synuclein amyloid fibrils and that sometimes even identical conditions can result in up to four distinct types of aggregates.     

The Role of Initial Oligomers in Amyloid Fibril Formation by Human Stefin B

Oligomers are commonly observed intermediates at the initial stages of amyloid fibril formation. They are toxic to neurons and cause decrease in neural transmission and long-term potentiation. We describe an in vitro study of the initial steps in amyloid fibril formation by human stefin B, which proved to be a good model system. Due to relative stability of the initial oligomers of stefin B, electrospray ionization mass spectrometry (ESI MS) could be applied in addition to size exclusion chromatography (SEC). These two techniques enabled us to separate and detect distinguished oligomers from the monomers: dimers, trimers, tetramers, up to decamers. The amyloid fibril formation process was followed at different pH and temperatures, including such conditions where the process was slow enough to detect the initial oligomeric species at the very beginning of the lag phase and those at the end of the lag phase. Taking into account the results of the lower-order oligomers transformations early in the process, we were able to propose an improved model for the stefin B fibril formation.     

Effect of Ionic Strength on Thioflavin-T Affinity to Amyloid Fibrils and Its Fluorescence Intensity

The formation of amyloid fibrils is linked to multiple neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. Despite years of research and countless studies on the topic of such aggregate formation, as well as their resulting structure, the current knowledge is still fairly limited. One of the main aspects prohibiting effective aggregation tracking is the environment’s effect on amyloid-specific dyes, namely thioflavin-T (ThT). Currently, there are only a few studies hinting at ionic strength being one of the factors that modulate the dye’s binding affinity and fluorescence intensity. In this work we explore this effect under a range of ionic strength conditions, using insulin, lysozyme, mouse prion protein, and α-synuclein fibrils. We show that ionic strength is an extremely important factor affecting both the binding affinity, as well as the fluorescence intensity of ThT.     

Rational Design of a Peptidomimetic Inhibitor of Gelsolin Amyloid Aggregation

Gelsolin amyloidosis (AGel) is characterized by multiple systemic and ophthalmic features resulting from pathological tissue deposition of the gelsolin (GSN) protein. To date, no cure is available for the treatment of any form of AGel. More than ten single-point substitutions in the GSN gene are responsible for the occurrence of the disease and, among them, D187N/Y is the most widespread variant. These substitutions undergo an aberrant proteolytic cascade, producing aggregation-prone peptides of 5 and 8 kDa, containing the Gelsolin Amyloidogenic Core, spanning residues 182–192 (GAC_182–192). Following a structure-based approach, we designed and synthesized three novel sequence-specific peptidomimetics (LB-5, LB-6, and LB-7) built on a piperidine-pyrrolidine unnatural amino acid. LB-5 and LB-6, but not LB-7, efficiently inhibit the aggregation of the GAC_182–192 amyloidogenic peptides at sub-stoichiometric concentrations. These peptidomimetics resulted also effective in vivo, in a C. elegans-based assay, in counteracting the proteotoxicity of aggregated GAC_182–192. These data pave the way to a novel pharmacological strategy against AGel and also validate a toolbox exploitable in other amyloidogenic diseases.     

PMEL Amyloid Fibril Formation: The Bright Steps of Pigmentation

In pigment cells, melanin synthesis takes place in specialized organelles, called melanosomes. The biogenesis and maturation of melanosomes is initiated by an unpigmented step that takes place prior to the initiation of melanin synthesis and leads to the formation of luminal fibrils deriving from the pigment cell-specific pre-melanosomal protein (PMEL). In the lumen of melanosomes, PMEL fibrils optimize sequestration and condensation of the pigment melanin. Interestingly, PMEL fibrils have been described to adopt a typical amyloid-like structure. In contrast to pathological amyloids often associated with neurodegenerative diseases, PMEL fibrils represent an emergent category of physiological amyloids due to their beneficial cellular functions. The formation of PMEL fibrils within melanosomes is tightly regulated by diverse mechanisms, such as PMEL traffic, cleavage and sorting. These mechanisms revealed increasing analogies between the formation of physiological PMEL fibrils and pathological amyloid fibrils. In this review we summarize the known mechanisms of PMEL fibrillation and discuss how the recent understanding of physiological PMEL amyloid formation may help to shed light on processes involved in pathological amyloid formation.