Amyloid-related Diseases Research Articles

Different Folding States from the Same Protein Sequence Determine Reversible vs Irreversible Amyloid Fate.

The propensity to self-assemble into amyloid fibrils with a shared cross-β architecture is a generic feature of proteins. Amyloid-related diseases affect millions of people worldwide, yet they are incurable and cannot be effectively prevented, largely due to the irreversible assembly and extraordinary stability of amyloid fibrils. Recent studies suggest that labile amyloids may be possible in certain proteins containing low-complexity domains often involved in the formation of subcellular membraneless organelles. Although the fundamental understanding of this reversible amyloid folding process is completely missing, the current view is that a given protein sequence will result in either irreversible, as in most of the cases, or reversible amyloid fibrils, as in few exceptions. Here we show that two common globular proteins, human lysozyme and its homologue from hen egg white, can self-assemble into both reversible and irreversible amyloid fibrils depending on the folding path followed by the protein. In both folding states, the amyloid nature of the fibrils is demonstrated at the molecular level by its cross-β structure, yet with substantial differences on the mesoscopic polymorphism and the labile nature of the amyloid state. Structural analysis shows that reversible and irreversible amyloid fibrils possess the same full-length protein sequence but different fibril core structures and β-sheet arrangements. These results illuminate a mechanistic link between the reversible and irreversible nature of amyloids and highlight the central role of protein folding states in regulating the lability and reversibility of amyloids.

Efficient Amyloid Fibrillation Inhibition and Remodeling of Preformed Fibrils of Bovine Insulin by Propolis Polyphenols-Based Nanosheets.

Among common strategies for amyloid fibrillation inhibition, the use of naturally occurring polyphenols as an efficient therapeutic approach has attracted a growing body of attention. However, the poor water solubility and low bioavailability of these compounds have greatly restricted their clinical application in amyloid-related diseases. Thus, different types of formulations have been developed to overcome these limitations; among them, nanonization appears to be one of the most notable approaches. Herein, we show that the polyphenolic fraction of propolis (PFP), in the nanosheet form (PFP nanosheet), exhibits an improved capacity for amyloid fibrillation inhibition as well as clearance of preformed fibrils of bovine insulin. This increased efficiency is suggested to be related to the aqueous solubility and surface area enhancement as well as surface modifications upon undergoing the nanonization process, which can lead to strong binding with and trapping of protein at the surface of the nanosheets. On the basis of thioflavin T results, it is suggested that although PFP may modulate the fibrillation process via shortening of the lag phase, prolongation of the nucleation phase through interaction with and stabilizing monomeric species is the mechanism of action of PFP nanosheets. We propose that nanonization of natural small molecules can be considered as a powerful approach to improve their anti-amyloidogenic properties and overcome obstacles originating from poor water solubility and low bioavailability of drug candidates relating to neurodegenerative diseases. Taken together, the obtained results may suggest PFP nanosheets as a potential candidate for use against neurological disorders.

Direct Observation of β-Barrel Intermediates in the Self-Assembly of Toxic SOD128-38 and Absence in Nontoxic Glycine Mutants.

Soluble low-molecular-weight oligomers formed during the early stage of amyloid aggregation are considered the major toxic species in amyloidosis. The structure-function relationship between oligomeric assemblies and the cytotoxicity in amyloid diseases are still elusive due to the heterogeneous and transient nature of these aggregation intermediates. To uncover the structural characteristics of toxic oligomeric intermediates, we compared the self-assembly dynamics and structures of SOD128-38, a cytotoxic fragment of the superoxide dismutase 1 (SOD1) associated with the amyotrophic lateral sclerosis, with its two nontoxic mutants G33V and G33W using molecular dynamics simulations. Single-point glycine substitutions in SOD128-38 have been reported to abolish the amyloid toxicity. Our simulation results showed that the toxic SOD128-38 and its nontoxic mutants followed different aggregation pathways featuring distinct aggregation intermediates. Specifically, wild-type SOD128-38 initially self-assembled into random-coil-rich oligomers, among which fibrillar aggregates composed of well-defined curved single-layer β-sheets were nucleated via coil-to-sheet conversions and the formation of β-barrels as intermediates. In contrast, the nontoxic G33V/G33W mutants readily assembled into small β-sheet-rich oligomers and then coagulated with each other into cross-β fibrils formed by two-layer β-sheets without forming β-barrels as the intermediates. The direct observation of β-barrel oligomers during the assembly of toxic SOD128-38 fragments but not the nontoxic glycine-substitution mutants strongly supports β-barrels as the toxic oligomers in amyloidosis, probably via interactions with the cell membrane and forming amyloid pores. With well-defined structures, the β-barrel might serve as a novel therapeutic target against amyloid-related diseases.

Polyphosphates induce amyloid fibril formation of α-synuclein in concentration-dependent distinct manners

Polyphosphates (polyPs), chains of phosphate residues found in species across nature from bacteria to mammals, were recently reported to accelerate the amyloid fibril formation of many proteins. How polyPs facilitate this process, however, remains unknown. To gain insight into their mechanisms, we used various physicochemical approaches to examine the effects of polyPs of varying chain lengths on ultrasonication-dependent α-synuclein (α-syn) amyloid formation. Although orthophosphate and diphosphate exhibited a single optimal concentration of amyloid formation, triphosphate and longer-chain phosphates exhibited two optima, with the second at a concentration lower than that of orthophosphate or diphosphate. The second optimum decreased markedly as the polyP length increased. This suggested that although the optima at lower polyP concentrations were caused by interactions between negatively charged phosphate groups and the positive charges of α-syn, the optima at higher polyP concentrations were caused by the Hofmeister salting-out effects of phosphate groups, where the effects do not depend on the net charge. NMR titration experiments of α-syn with tetraphosphate combined with principal component analysis revealed that, at low tetraphosphate concentrations, negatively charged tetraphosphates interacted with positively charged “KTK” segments in four KTKEGV repeats located at the N-terminal region. At high concentrations, hydrated tetraphosphates affected the surface-exposed hydrophilic groups of compact α-syn. Taken together, our results suggest that long-chain polyPs consisting of 60 to 70 phosphates induce amyloid formation at sub-μM concentrations, which are comparable with the concentrations of polyPs in the blood or tissues. Thus, these findings may identify a role for polyPs in the pathogenesis of amyloid-related diseases.

Effects of Dissolved Gases on the Amyloid Fibril Morphology.

The onset or progression of numerous neurodegenerative diseases occurs due to aggregation of proteins that ultimately form fibrils. The assembly and morphology of fibrils are susceptible to environmental factors. In this work, we used atomic force microscopy (AFM) to investigate the effects of dissolved nitrogen and oxygen molecules on the morphology of fibrils formed by a hydrophobic amyloid peptide implicated in amyotrophic lateral sclerosis, 15 repeats of glycine-alanine, on a highly oriented pyrolytic graphite substrate. We started with preformed fibril solutions that were then diluted with buffers of different gas conditions, resulting in the aggregation of the fibrils into different morphologies that were revealed by AFM after adsorption on the substrate. Straight fibrils were observed in both degassed and ambient buffers, but a stronger lateral association was seen in degassed buffers. Smaller and softer fibrils were observed in O2-supersaturated buffers, and plaque-like fibril aggregates of considerably large size were evident in N2-supersaturated buffers. In overnight incubation experiments, we observed changes in both the morphology and height of the fibril aggregates, and their evolution varied with different gas conditions. These findings indicate that the gas type and concentration affect the aggregation of amyloid fibrils and may facilitate the development of biomaterial applications and treatments for amyloid-related diseases.

The role of water in the primary nucleation of protein amyloid aggregation

The understanding of the complex conformational landscape of amyloid aggregation and its modulation by relevant physicochemical and cellular factors is a prerequisite for elucidating some of the molecular basis of pathology in amyloid related diseases, and for developing and evaluating effective disease-specific therapeutics to reduce or eliminate the underlying sources of toxicity in these diseases. Interactions of proteins with solvating water have been long considered to be fundamental in mediating their function and folding; however, the relevance of water in the process of protein amyloid aggregation has been largely overlooked. Here, we provide a perspective on the role water plays in triggering primary amyloid nucleation of intrinsically disordered proteins (IDPs) based on recent experimental evidences. The initiation of amyloid aggregation likely results from the synergistic effect between both protein intermolecular interactions and the properties of the water hydration layer of the protein surface. While the self-assembly of both hydrophobic and hydrophilic IDPs would be thermodynamically favoured due to large water entropy contributions, large desolvation energy barriers are expected, particularly for the nucleation of hydrophilic IDPs. Under highly hydrating conditions, primary nucleation is slow, being facilitated by the presence of nucleation-active surfaces (heterogeneous nucleation). Under conditions of poor water activity, such as those found in the interior of protein droplets generated by liquid-liquid phase separation, however, the desolvation energy barrier is significantly reduced, and nucleation can occur very rapidly in the bulk of the solution (homogeneous nucleation), giving rise to structurally distinct amyloid polymorphs. Water, therefore, plays a key role in modulating the transition free energy of amyloid nucleation, thus governing the initiation of the process, and dictating the type of preferred primary nucleation and the type of amyloid polymorph generated, which could vary depending on the particular microenvironment that the protein molecules encounter in the cell.

Ambiguous Cerebral Amyloidosis

Cerebral amyloid, also known as amyloid-related diseases of the CNS, is a heterogenous group of chronic, progressive disorders in which abnormal amyloid protein accumulates in the brain. Subtypes of cerebral amyloid include Alzheimer disease, cerebral amyloid angiopathy, inflammatory cerebral amyloid angiopathy, and amyloidoma. Abnormal amyloid accumulation often leads to adverse events within the CNS. Cerebral amyloid can present with multiple distinct imaging appearances that often overlap with several other CNS pathologies, which ultimately makes an accurate diagnosis a challenge. In this article, we discuss the various imaging patterns seen in cerebral amyloid. In addition, we review the differential diagnoses and highlight the radiologic features that aid in distinction.Learning Objective: Become familiar with the multiple subtypes of cerebral amyloidosis, also known as amyloid-related diseases of the CNS, and their imaging manifestations, and be able to differentiate these subtypes from common mimickers.

Regulation of Functional Protein Aggregation by Multiple Factors: Implications for the Amyloidogenic Behavior of the CAP Superfamily Proteins.

The idea that amyloid fibrils and other types of protein aggregates are toxic for cells has been challenged by the discovery of a variety of functional aggregates. However, an identification of crucial differences between pathological and functional aggregation remains to be explored. Functional protein aggregation is often reversible by nature in order to respond properly to changing physiological conditions of the cell. In addition, increasing evidence indicates that fast fibril growth is a feature of functional amyloids, providing protection against the long-term existence of potentially toxic oligomeric intermediates. It is becoming clear that functional protein aggregation is a complexly organized process that can be mediated by a multitude of biomolecular factors. In this overview, we discuss the roles of diverse biomolecules, such as lipids/membranes, glycosaminoglycans, nucleic acids and metal ions, in regulating functional protein aggregation. Our studies on the protein GAPR-1 revealed that several of these factors influence the amyloidogenic properties of this protein. These observations suggest that GAPR-1, as well as the cysteine-rich secretory proteins, antigen 5 and pathogenesis-related proteins group 1 (CAP) superfamily of proteins that it belongs to, require the assembly into an amyloid state to exert several of their functions. A better understanding of functional aggregate formation may also help in the prevention and treatment of amyloid-related diseases.

Date palm (Phoenix dactylifera L.) fruit's polyphenols as potential inhibitors for human amylin fibril formation and toxicity in type 2 diabetes

Backgroundβ-Cell death is the key feature of type 2 diabetes mellitus (T2DM). The misfolding of human Islet Amyloid Polypeptide (hIAPP) is regarded as one of the causative factors of T2DM. Recent studies suggested that a diet based on date fruits presents various health benefits, as these fruits are naturally enriched in plant polyphenols. MethodIn this study, we used a broad biophysical approach, using cell biology techniques and bioinformatic tools, to demonstrate that various polyphenols from date palm (Phoenix dactylifera L.) fruit significantly inhibited hIAPP aggregation and cytotoxicity. ResultOur results suggest that all of the polyphenols showed inhibitory effects, albeit varied, on the formation of toxic hIAPP amyloids. Correlation between cell viability assay, permeabilization of synthetic phospholipid vesicles tests, and ANS florescence measurements, revealed that both classes of polyphenols protected INS-1E cells from the toxicity of amylin aggregates. Docking results showed that the used polyphenols physically interacted with both hIAPP amyloidogenic region (residues Ser20-Ser29) and the non-amyloidogenic regions via hydrophobic and hydrogen interactions, thus reducing aggregation levels. ConclusionThese findings highlight the benefits of consuming dates and the great potential of its polyphenols as a potential therapy for the prevention and treatment of T2DM as well as for many other amyloid-related diseases.

Investigating the inhibitory effects of entacapone on amyloid fibril formation of human lysozyme

The misfolding of soluble protein to amyloid fibers or oligomers leads to cell membrane rupture, cell death, and a variety of amyloid-related diseases. Hence, inhibition of protein fibrillation is an important and promising method to prevent and treat these diseases. In this study, we have investigated the inhibitory effect of entacapone (Ent) on human lysozyme (HL) amyloid fibrillation using a combination of biophysical techniques; Rayleigh scattering (RLS) data indicated that Ent can reduce the aggregation of HL amyloid fibrillation with the inhibition constant (Λ) of (3.0 ± 0.5) × 103 M−1. This finding was further confirmed by thioflavin-T (ThT), 8-Anilino-1-naphthalenesulfonic acid (ANS) fluorescence assays and congo red (CR) binding absorption assays with an IC50 value of 125.89 ± 1.25 μM. Meanwhile, dynamic light scattering (DLS) showed that the size of HL amyloids decreases sharply after Ent treatment. This effect was positively correlated with Ent concentration. Atomic force microscopy (AFM) techniques confirmed that the formation of the fibril decreased significantly when HL was co-incubated with Ent. In addition, steady-state fluorescence spectra and synchronous fluorescence analysis suggested that the formation of stable complexes between Ent and HL contributes to maintain the alpha-helical structure of HL. The molecular docking study revealed that the Ent binds at the active pocket of HL with Glu35, Asp53, Gln58, Trp 64, Ala108 and Trp109 residues via hydrogen bonds, van-der-Waals forces and hydrophobic interactions. The epitope mapping of HL for its interaction with Ent was further elucidated using two-dimensional solution-state nuclear magnetic resonance (NMR) experiments. NMR results showed that the Trp64 and Trp109 of HL plays an important role for binding to Ent, correlating well with our docking result. Thus our study showed the potential of Ent to serve as an effective therapeutic agent for the therapy of amyloid-related diseases.

Crosstalk Between Alpha-Synuclein and Other Human and Non-Human Amyloidogenic Proteins: Consequences for Amyloid Formation in Parkinson's Disease.

It was recently shown (Sampson et al., Elife 9, 2020) that an amyloidogenic protein, CsgA, present in E. coli biofilms in the gut can trigger Parkinson’s disease in mice. This study emphasizes the possible role of the gut microbiome in modulation (and even initiation) of human neurodegenerative disorders, such as Parkinson’s disease. As the CsgA protein was found to accelerate alpha-synuclein (the key amyloidogenic protein in Parkinson’s disease) amyloid formation in vitro, this result suggests that also other amyloidogenic proteins from gut bacteria, and even from the diet (such as stable allergenic proteins), may be able to affect human protein conformations and thereby modulate amyloid-related diseases. In this review, we summarize what has been reported in terms of in vitro cross-reactivity studies between alpha-synuclein and other amyloidogenic human and non-human proteins. It becomes clear from the limited data that exist that there is a fine line between acceleration and inhibition, but that cross-reactivity is widespread, and it is more common for other proteins (among the studied cases) to accelerate alpha-synuclein amyloid formation than to block it. It is of high importance to expand investigations of cross-reactivity between amyloidogenic proteins to both reveal underlying mechanisms and links between human diseases, as well as to develop new treatments that may be based on an altered gut microbiome.

Stochastic master equation for early protein aggregation in the transthyretin amyloid disease

It is significant to understand the earliest molecular events occurring in the nucleation of the amyloid aggregation cascade for the prevention of amyloid related diseases such as transthyretin amyloid disease. We develop chemical master equation for the aggregation of monomers into oligomers using reaction rate law in chemical kinetics. For this stochastic model, lognormal moment closure method is applied to track the evolution of relevant statistical moments and its high accuracy is confirmed by the results obtained from Gillespie’s stochastic simulation algorithm. Our results show that the formation of oligomers is highly dependent on the number of monomers. Furthermore, the misfolding rate also has an important impact on the process of oligomers formation. The quantitative investigation should be helpful for shedding more light on the mechanism of amyloid fibril nucleation.

Alzheimer's Therapeutic Strategy: Photoactive Platforms for Suppressing the Aggregation of Amyloid β Protein.

Neurodegenerative diseases such as Alzheimer's disease (AD) have become a public health problem. Progressive cerebral accumulation of amyloid protein (Aβ) was widely considered as the cause of AD. One promising strategy for AD preclinical study is to degrade and clear the deposited amyloid aggregates with β-sheet-rich secondary structure in the brain. Based on the requirement, photo-active materials with the specific excitation and the standardization of the photosensitizer preparation and application in clinics, have attracted increased attention in the study and treatment of neurodegenerative disease as a novel method termed as photodynamic therapy (PDT). This review will focus on the new photosensitizing materials and discuss the trend of PDT techniques for the possible application in the treatment strategy of amyloid-related diseases.

Fullerenes as an Effective Amyloid Fibrils Disaggregating Nanomaterial.

Nowadays, determining the disassembly mechanism of amyloids under nanomaterials action is a crucial issue for their successful future use in therapy of neurodegenerative and overall amyloid-related diseases. In this study, the antiamyloid disassembly activity of fullerenes C60 and C70 dispersed in 1-methyl-2-pyrrolidinone (NMP) toward amyloid fibrils preformed from lysozyme and insulin was investigated using a combination of different experimental techniques. Thioflavin T fluorescence assay and atomic force microscopy were applied for monitoring of disaggregation activity of fullerenes. It was demonstrated that both types of fullerene-based complexes are very effective in disassembling preformed fibrils, and characterized by the low apparent half-maximal disaggregation concentration (DC50) in the range of ∼22-30 μg mL-1. Small-angle neutron scattering was employed to monitor the different stages of the disassembly process with respect to the size and morphology of the aggregates. Based on the obtained results, a possible disassembly mechanism for amyloid fibrils interacting with fullerene/NMP complexes was proposed. The study is a principal step in understanding of the fullerenes destruction mechanism of the protein amyloids, as well as providing valuable information on how macromolecules can be engineered to disassemble unwanted amyloid aggregates by different mechanisms.

Inhibition of human islet amyloid polypeptide aggregation and cellular toxicity by oleuropein and derivatives from olive oil

Loss of β-cell function and β-cell death is the key feature of type 2 diabetes mellitus (T2DM). One hypothesis for the mechanism of this feature is amyloid formation by the human islet amyloid polypeptide (hIAPP). Despite the global prevalence of T2DM, there are no therapeutic strategies for the treatment of or prevention of amylin amyloidosis. Clinical trials and population studies indicate the healthy virtues of the Mediterranean diet, especially the extra virgin olive oil (EVOO) found in this diet. This oil is enriched in phenolic compounds shown to be effective against several aging and lifestyle diseases. Oleuropein (Ole), one of the most abundant polyphenols in EVOO, has been reported to be anti-diabetic. Some of Ole's main derivative have attracted our interest due to their multi-targetted effects, including interference with amyloid aggregation path. However, the structure-function relationship of Ole and its metabolites in T2DM are not yet clear. We report here a broad biophysical approach and cell biology techniques that enabled us to characterize the different molecular mechanisms by which tyrosol (TYR), hydroxytyrosol (HT), oleuropein (Ole) and oleuropein aglycone (OleA) modulate the hIAPP fibrillation in vitro and their effects on cell cytotoxicity. The OleA formed by enolic acid and hydroxytyrosol moiety was found to be more active than the Ole and HT at low micromolar concentrations. We further demonstrated that OleA inhibit the cytotoxicity induced by hIAPP aggregates by protecting more the cell membrane from permeabilization and then from death. These findings highlight the benefits of consuming EVOO and the great potential of its polyphenols, mainly OleA. Moreover, they support the possibility to validate and optimize the possible pharmacological use of EVOO polyphenols for T2DM prevention and therapy and also for many other amyloid related diseases.

The Positive Side of the Alzheimer's Disease Amyloid Cross-Interactions: The Case of the Aβ 1-42 Peptide with Tau, TTR, CysC, and ApoA1.

Alzheimer’s disease (AD) represents a progressive amyloidogenic disorder whose advancement is widely recognized to be connected to amyloid-β peptides and Tau aggregation. However, several other processes likely contribute to the development of AD and some of them might be related to protein-protein interactions. Amyloid aggregates usually contain not only single type of amyloid protein, but also other type of proteins and this phenomenon can be rationally explained by the process of protein cross-seeding and co-assembly. Amyloid cross-interaction is ubiquitous in amyloid fibril formation and so a better knowledge of the amyloid interactome could help to further understand the mechanisms of amyloid related diseases. In this review, we discuss about the cross-interactions of amyloid-β peptides, and in particular Aβ1-42, with other amyloids, which have been presented either as integrated part of Aβ neurotoxicity process (such as Tau) or conversely with a preventive role in AD pathogenesis by directly binding to Aβ (such as transthyretin, cystatin C and apolipoprotein A1). Particularly, we will focus on all the possible therapeutic strategies aiming to rescue the Aβ toxicity by taking inspiration from these protein-protein interactions.

Self-Assembled Peptide Nanofibrils Designed to Release Membrane-Lysing Antimicrobial Peptides.

Membrane-disrupting antimicrobial peptides continue to attract increasing attention due to their potential to combat multidrug-resistant bacteria. However, some limitations are found in the success of clinical setting-based antimicrobial peptide agents, for instance, the poor stability of antimicrobial peptides in vivo and their short-term activity. Self-assembled peptide materials can improve the stability of antimicrobial peptides, but the biosafety of peptide-based materials is the main concern, although they are considered to be biocompatible, because some peptide aggregates would possibly induce protein misfolding, which could be related to amyloid-related diseases. Therefore, in this work, we designed two peptides and constructed peptide-based nanofibrils by self-assembly before its utilization. It is found that the fibrils could release the antimicrobial peptide by disassembly for microbial membrane lysis in the presence of bacteria. The designed peptide-based fibrils presented a good and long-term antimicrobial activity with bacterial membrane disruption and the efflux of calcium from bacteria. Furthermore, it could be used to construct hybrid macrofilms displaying low cytotoxicity, low hemolytic activity, and good biocompatibility. The innovative design strategy could be beneficial for the development of smart antimicrobial nanomaterials.

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.

Contradictory effect of gold nanoparticle-decorated molybdenum sulfide nanocomposites on amyloid-β-40 aggregation

The effect of gold nanoparticle-decorated molybdenum sulfide (AuNP-MoS2) nanocomposites on amyloid-β-40 (Aβ40) aggregation was investigated. The interesting discovery was that the effect of AuNP-MoS2 nanocomposites on Aβ40 aggregation was contradictory. Low concentration of AuNP-MoS2 nanocomposites could enhance the nucleus formation of Aβ40 peptides and accelerate Aβ40 fibrils aggregation. However, although high concentration of AuNP-MoS2 nanocomposites could enhance the nucleus formation of Aβ40 peptides, it eventually inhibited Aβ40 aggregation process. It might be attributed to the interaction between AuNP-MoS2 nanocomposites and Aβ40 peptides. For low concentration of AuNP-MoS2 nanocomposites, it was acted as nuclei, resulting in the acceleration of the nucleation process. However, the structural flexibility of Aβ40 peptides was limited as the concentration of AuNP-MoS2 nanocomposites was increased, resulting in the inhibition of Aβ40 aggregation. These findings suggested that AuNP-MoS2 nanocomposites might have a great potential to design new multifunctional material for future treatment of amyloid-related diseases.

Exploration of Insulin Amyloid Polymorphism Using Raman Spectroscopy and Imaging

We aimed to investigate insulin amyloid fibril polymorphism caused by salt effects and heating temperature and to visualize the structural differences of the polymorphisms in situ using Raman imaging without labeling. The time course monitoring for amyloid formation was carried out in an acidic condition without any salts and with two species of salts (NaCl and Na2SO4) by heating at 60, 70, 80, and 90°C. The intensity ratio of two Raman bands at 1672 and 1657 cm−1 due to antiparallel β-sheet and α-helix structures, respectively, was revealed to be an indicator of amyloid fibril formation, and the relative proportion of the β-sheet structure was higher in the case with salts, especially at a higher temperature with Na2SO4. In conjunction with the secondary structural changes of proteins, the S-S stretching vibrational mode of a disulfide bond (∼514 cm−1) and the ratio of the tyrosine doublet I850/I826 were also found to be markers distinguishing polymorphisms of insulin amyloid fibrils by principal component analysis. Especially, amyloid fibrils with Na2SO4 media formed the gauche-gauche-gauche conformation of disulfide bond at a higher rate, but without any salts, the gauche-gauche-gauche conformation was partially transformed into the gauche-gauche-trans conformation at higher temperatures. The different environments of the hydroxyl groups of the tyrosine residue were assumed to be caused by fibril polymorphism. Raman imaging using these marker bands also successfully visualized the two- and three- dimensional structural differences of amyloid polymorphisms. These results demonstrate the potential of Raman imaging as a diagnostic tool for polymorphisms in tissues of amyloid-related diseases.