Amyloid Fibrils Research Articles

Promotion and modulation of amyloid fibrillation of bovine beta-lactoglobulin by hydroxychalcones

Hydroxychalcones are naturally occurring compounds having biological relevance. This work demonstrates the synthesis of some chalcones having hydrophobic and polar groups and the investigation of their effect on the stability of a model protein beta-lactoglobulin (β-lg). An easy and simple method (Simon-Smith) was utilized to synthesize the four chalcone compounds. The binding of the chalcone molecules to the protein destabilized the protein structure and subsequently exposed the hydrophobic cores resulting in the aggregation of β-lg under thermal conditions. Using multiple spectroscopic methods (UV-Vis, fluorescence, FT-IR), imaging techniques like TEM, and theoretical tools were used to monitor the formation of protein aggregation and to understand the underlying mechanism. Exposures of hydrophobic amino acid residues in the aqueous medium by binding of the chalcones are responsible for protein aggregation through protein-protein interactions. It is sensitive to the polarity of the hydroxychalcones. In this process, polar group-containing hydroxychalcones are less effective, and unsubstituted hydroxychalcones are most efficient in promoting the amyloid fibrillation of β-lg.

Transthyretin monomers: a new plasma biomarker for pre-symptomatic transthyretin-related amyloidosis

Background Genotyping and amyloid fibril detection in tissues are generally considered the diagnostic gold standard in transthyretin-related amyloidosis. Patients carry less stable TTR homotetramers prone to dissociation into non-native monomers, which rapidly self-assemble into oligomers and, ultimately, amyloid fibrils. Thus, the initial event of the amyloid cascade produces the smallest transthyretin species: the monomers. This creates engineering opportunities for diagnosis that remain unexplored. Methods We hypothesise that molecular sieving represents a promising method for isolating and concentrating trace TTR monomers from the tetramers present in plasma samples. Subsequently, immunodetection can be utilised to distinguish monomeric TTR from other low molecular weight proteins within the adsorbed fraction. A two-step assay was devised (ImmunoSieve assay), combining molecular sieving and immunodetection for sensing monomeric transthyretin. This assay was employed to analyse plasma microsamples from 10 individuals, including 5 pre-symptomatic carriers of TTR-V30M, the most prevalent amyloidosis-associated TTR variant worldwide, and 5 healthy controls. Results The ImmunoSieve assay enable sensitive detection of monomeric transthyretin in plasma microsamples. Moreover, the circulating monomeric TTR levels were significantly higher in carriers of amyloidogenic TTR mutation. Conclusions Monomeric TTR can function as a biomarker for evaluating disease progression and assessing responses to therapies targeted at stabilising native TTR.

Designed De Novo α-Sheet Peptides Destabilize Bacterial Biofilms and Increase the Susceptibility of E. coli and S. aureus to Antibiotics.

Biofilm-associated microbes are 10-1000 times less susceptible to antibiotics. An emerging treatment strategy is to target the structural components of biofilm to weaken the extracellular matrix without introducing selective pressure. Biofilm-associated bacteria, including Escherichia coli and Staphylococcus aureus, generate amyloid fibrils to reinforce their extracellular matrix. Previously, de novo synthetic α-sheet peptides designed in silico were shown to inhibit amyloid formation in multiple bacterial species, leading to the destabilization of their biofilms. Here, we investigated the impact of inhibiting amyloid formation on antibiotic susceptibility. We hypothesized that combined administration of antibiotics and α-sheet peptides would destabilize biofilm formation and increase antibiotic susceptibility. Two α-sheet peptides, AP90 and AP401, with the same sequence but inverse chirality at every amino acid were tested: AP90 is L-amino acid dominant while AP401 is D-amino acid dominant. For E. coli, both peptides increased antibiotic susceptibility and decreased the biofilm colony forming units when administered with five different antibiotics, and AP401 caused a greater increase in all cases. For S. aureus, increased biofilm antibiotic susceptibility was also observed for both peptides, but AP90 outperformed AP401. A comparison of the peptide effects demonstrates how chirality influences biofilm targeting of gram-negative E. coli and gram-positive S. aureus. The observed increase in antibiotic susceptibility highlights the role amyloid fibrils play in the reduced susceptibility of bacterial biofilms to specific antibiotics. Thus, the co-administration of α-sheet peptides and existing antibiotics represents a promising strategy for the treatment of biofilm infections.

Zinc and pH modulate the ability of insulin to inhibit aggregation of islet amyloid polypeptide

Aggregation of the human islet amyloid polypeptide (hIAPP) contributes to the development and progression of Type 2 Diabetes (T2D). hIAPP aggregates within a few hours at few micromolar concentration in vitro but exists at millimolar concentrations in vivo. Natively occurring inhibitors of hIAPP aggregation might therefore provide a model for drug design against amyloid formation associated with T2D. Here, we describe the combined ability of low pH, zinc, and insulin to inhibit hIAPP fibrillation. Insulin dose-dependently slows hIAPP aggregation near neutral pH but had less effect on the aggregation kinetics at acidic pH. We determine that insulin alters hIAPP aggregation in two manners. First, insulin diverts the aggregation pathway to large nonfibrillar aggregates with ThT-positive molecular structure, rather than to amyloid fibrils. Second, soluble insulin suppresses hIAPP dimer formation, which is an important early aggregation event. Further, we observe that zinc significantly modulates the inhibition of hIAPP aggregation by insulin. We hypothesize that this effect arose from controlling the oligomeric state of insulin and show that hIAPP interacts more strongly with monomeric than oligomeric insulin.

Amyloid Neuropathy: From Pathophysiology to Treatment in Light-Chain Amyloidosis and Hereditary Transthyretin Amyloidosis.

Amyloid neuropathy is caused by deposition of insoluble β-pleated amyloid sheets in the peripheral nervous system. It is most common in: (1) light-chain amyloidosis, a clonal non-proliferative plasma cell disorder in which fragments of immunoglobulin, light or heavy chain, deposit in tissues, and (2) hereditary transthyretin (ATTRv) amyloidosis, a disorder caused by autosomal dominant mutations in the TTR gene resulting in mutated protein that has a higher tendency to misfold. Amyloid fibrils deposit in the endoneurium of peripheral nerves, often extensive in the dorsal root ganglia and sympathetic ganglia, leading to atrophy of Schwann cells in proximity to amyloid fibrils and blood-nerve barrier disruption. Clinically, amyloid neuropathy is manifested as a length-dependent sensory predominant neuropathy associated with generalized autonomic failure. Small unmyelinated nerves are involved early and prominently in early-onset Val30Met ATTRv, whereas other ATTRv and light-chain amyloidosis often present with large- and small-fiber involvement. Nerve conduction studies, quantitative sudomotor axon testing, and intraepidermal nerve fiber density are useful tools to evaluate denervation. Amyloid deposition can be demonstrated by tissue biopsy of the affected organ or surrogate site, as well as bone-avid radiotracer cardiac imaging. Treatment of light-chain amyloidosis has been revolutionized by monoclonal antibodies and stem cell transplantation with improved 5-year survival up to 77%. Novel gene therapy and transthyretin stabilizers have revolutionized treatment of ATTRv, improving the course of neuropathy (less change in the modified Neuropathy Impairment Score + 7 from baseline) and quality of life. With great progress in amyloidosis therapies, early diagnosis and presymptomatic testing for ATTRv family members has become paramount. ANN NEUROL 2024;96:423-440.

Machine learning-assisted fluorescence/fluorescence colorimetric sensor array for discriminating amyloid fibrils

Misfolding and aggregation of proteins often lead to the development of diseases, and amyloid has gained widespread attention as a biomarker for a variety of diseases. In this study, we developed a fluorescence/fluorescence colorimetric dual-mode sensor array for the detection of amyloid fibrils using several commercially available organic small molecular dyes and alkaloids, including Thioflavin T, Congo Red, 8-anilino-1-naphthalenesulfonic acid, Safranine T, berberine and coptisine, as the elements. Herein, the array could not only use the fluorescence intensities change before and after protein interaction as a pattern recognition signal, but also read the ΔR/ΔG/ΔB values of the photos taken in the UV dark box on a smartphone-based platform, which converted the chromaticity information into intuitive data. Five studied amyloid fibrils, i.e. insulin, lysozyme, bovine serum albumin, amyloid-β 42 and α-synuclein fibrils, were properly distinguished with data processing assisted by machine learning algorithms, i.e. linear discriminant analysis, principal component analysis and hierarchical cluster analysis. After reducing the number of elements by principal component analysis, a simplified array quantified individual amyloid fibrils at 0.05–5 μM and 0.5–10 μM with fluorescence and fluorescence colorimetric signals, respectively, and successfully identified 25 unknown samples with high accuracy in diluted human plasma matrix and artificial cerebrospinal fluid. The array had good selectivity and sensitivity, providing a simple and inexpensive method for amyloid discrimination.

Conformational Dynamics of an Amyloidogenic Intermediate of Transthyretin: Implications for Structural Remodeling and Amyloid Formation

The aggregation pathway of transthyretin (TTR) proceeds through rate-limiting dissociation of the tetramer (a dimer of dimers) and partial misfolding of the resulting monomer, which assembles into amyloid structures through a downhill polymerization mechanism. The structural features of the aggregation-prone monomeric intermediate are poorly understood. NMR relaxation dispersion offers a unique opportunity to characterize amyloidogenic intermediates when they exchange on favorable timescales with NMR-visible ground states. Here we use NMR to characterize the structure and conformational dynamics of the monomeric F87E mutant of human TTR. Chemical shifts derived from analysis of multinuclear relaxation dispersion data provide insights into the structure of a low-lying excited state that exchanges with the ground state of the F87E monomer at a rate of 3800 s−1. Disruption of the subunit interfaces of the TTR tetramer leads to destabilization of edge strands in both β-sheets of the F87E monomer. Conformational fluctuations are propagated through the entire hydrogen bonding network of the DAGH β-sheet, from the inner β-strand H, which forms the strong dimer-dimer interface in the TTR tetramer, to outer strand D which is unfolded in TTR fibrils. Fluctuations are also propagated from the AB loop in the weak dimer-dimer interface to the EF helix, which undergoes structural remodeling in fibrils. The conformational fluctuations in both regions are enhanced at acidic pH where amyloid formation is most favorable. The relaxation dispersion data provide insights into the conformational dynamics of the amyloidogenic state of monomeric TTR that predispose it for structural remodeling and progression to amyloid fibrils.

Hierarchical Protofilament Intertwining Rules the Formation of Mixed-Curvature Amyloid Polymorphs.

Amyloid polymorphism is a hallmark of almost all amyloid species, yet the mechanisms underlying the formation of amyloid polymorphs and their complex architectures remain elusive. Commonly, two main mesoscopic topologies are found in amyloid polymorphs characterized by non-zero Gaussian and mean curvatures: twisted ribbons and helical fibrils, respectively. Here, a rich heterogeneity of configurations is demonstrated on insulin amyloid fibrils, where protofilament packing can occur, besides the common polymorphs, also in a combined mode forming mixed-curvature polymorphs. Through AFM statistical analysis, an extended array of heterogeneous architectures that are rationalized by mesoscopic theoretical arguments are identified. Notably, an unusual fibrillization pathway is also unraveled toward mixed-curvature polymorphs via the widespread recruitment and intertwining of protofilaments and protofibrils. The results present an original view of amyloid polymorphism and advance the fundamental understanding of the fibrillization mechanism from single protofilaments into mature amyloid fibrils.

Hetero-oligomerization of TDP-43 carboxy-terminal fragments with cellular proteins contributes to proteotoxicity

Carboxy terminal fragments (CTFs) of TDP-43 contain an intrinsically disordered region (IDR) and form cytoplasmic condensates containing amyloid fibrils. Such condensates are toxic and associated with pathogenicity in amyotrophic lateral sclerosis. However, the molecular details of how the domain of TDP-43 CTFs leads to condensation and cytotoxicity remain elusive. Here, we show that truncated RNA/DNA-recognition motif (RRM) at the N-terminus of TDP-43 CTFs leads to the structural transition of the IDR, whereas the IDR itself of TDP-43 CTFs is difficult to assemble even if they are proximate intermolecularly. Hetero-oligomers of TDP-43 CTFs that have recruited other proteins are more toxic than homo-oligomers, implicating loss-of-function of the endogenous proteins by such oligomers is associated with cytotoxicity. Furthermore, such toxicity of TDP-43 CTFs was cell-nonautonomously affected in the nematodes. Therefore, misfolding and oligomeric characteristics of the truncated RRM at the N-terminus of TDP-43 CTFs define their condensation properties and toxicity.

TSPO in pancreatic beta cells and its possible involvement in type 2 diabetes

Amyloidosis forms a large family of pathologies associated with amyloid deposit generated by the formation of amyloid fibrils or plaques. The amyloidogenic proteins and peptides involved in these processes are targeted against almost all organs. In brain they are associated with neurodegenerative disease, and the Translocator Protein (TSPO), overexpressed in these inflammatory conditions, is one of the target for the diagnostic. Moreover, TSPO ligands have been described as promising therapeutic drugs for neurodegenerative diseases. Type 2 diabetes, another amyloidosis, is due to a beta cell mass decrease that has been linked to hIAPP (human islet amyloid polypeptide) fibril formation, leading to the reduction of insulin production. In the present study, in a first approach, we link overexpression of TSPO and inflammation in potentially prediabetic patients. In a second approach, we observed that TSPO deficient rats have higher level of insulin secretion in basal conditions and more IAPP fibrils formation compared with wild type animals. In a third approach, we show that diabetogenic conditions also increase TSPO overexpression and IAPP fibril formation in rat beta pancreatic cell line (INS-1E). These data open the way for further studies in the field of type 2 diabetes treatment or prevention.

WITHDRAWAL: Quantitative modeling of approved and emerging therapeutics for modifying TTR levels in patients with Transthyretin Amyloid Cardiomyopathy.

Withdrawal: McGirr, K., Sarkar, S., Subramanian, K. (2023) "Quantitative modeling of approved and emerging therapeutics for modifying TTR levels in patients with Transthyretin Amyloid Cardiomyopathy" CPT: Pharmacometrics & Systems Pharmacology. https://doi.org/10.1002/psp4.13078. The above article, published online on November 6, 2023, in Wiley Online Library (http://wileyonlinelibrary.com), has been withdrawn by agreement between the authors, journal Editor-in-Chief, France Mentré, the American Society for Clinical Pharmacology and Therapeutics (ASCPT), and Wiley Periodicals LLC. The withdrawal has been agreed to at the authors' request due to a disagreement over the ownership of some data contained therein.

Aggregation Mechanisms and Molecular Structures of Amyloid-β in Alzheimer's Disease.

Amyloid plaques are a major pathological hallmark involved in Alzheimer's disease and consist of deposits of the amyloid-β peptide (Aβ). The aggregation process of Aβ is highly complex, which leads to polymorphous aggregates with different structures. In addition to aberrant aggregation, Aβ oligomers can undergo liquid-liquid phase separation (LLPS) and form dynamic condensates. It has been hypothesized that these amyloid liquid droplets affect and modulate amyloid fibril formation. In this review, we briefly introduce the relationship between stress granules and amyloid protein aggregation that is associated with neurodegenerative diseases. Then we highlight the regulatory role of LLPS in Aβ aggregation and discuss the potential relationship between Aβ phase transition and aggregation. Furthermore, we summarize the current structures of Aβ oligomers and amyloid fibrils, which have been determined using nuclear magnetic resonance (NMR) and cryo-electron microscopy (cryo-EM). The structural variations of Aβ aggregates provide an explanation for the different levels of toxicity, shed light on the aggregation mechanism and may pave the way towards structure-based drug design for both clinical diagnosis and treatment.

Characterizing the Oligomers Distribution along the Aggregation Pathway of Amyloid Aβ1-40 by NMR.

This study delves into the early aggregation process of the Aβ1-40 amyloid peptide, elucidating the associated oligomers distribution. Motivated by the acknowledged role of small oligomers in the neurotoxic damage linked to Alzheimer's disease, we present an experimental protocol for preparing 26-O-acyl isoAβ1-40, a modified Aβ1-40 peptide facilitating rapid isomerization to the native amide form at neutral pH. This ensures seed-free solutions, minimizing experimental variability. Additionally, we demonstrate the efficacy of coupling NMR diffusion ordered spectroscopy (DOSY) with the Inverse Laplace Transform (ILT) reconstruction method, for effective characterization of early aggregation processes. This innovative approach efficiently maps oligomers distributions across a wide spectrum of initial peptide concentrations offering unique insights into the evolution of oligomers relative populations. As a proof of concept, we demonstrate the efficacy of our approach assessing the impact of Epigallocathechin gallate, a known remodeling agent of amyloid fibrils, on the oligomeric distributions of aggregated Aβ1-40. The DOSY-ILT proposed approach stands as a robust and discriminating asset, providing a powerful strategy for rapidly gaining insight into potential inhibitors' impact on the aggregation process.

Formation of tannic acid-binding ovalbumin amyloid fibril hydrogels: Enhanced antibacterial and antioxidant properties

The protein-induced amyloid fibrils (AFs) are limited in food industry due to their poor antioxidant and antibacterial activities. However, polyphenol shows the potential functional modifications of AFs, and extend their application in food processing. In present study, tannic acid-binding ovalbumin-induced AFs (TA-OAFs) hydrogels were fabricated via different concentrations of TA and ovalbumin amyloid fibrils, and the rheological properties, thermal stability, and morphology of the hydrogels were investigated. Then, the physiochemical, antioxidant, and antibacterial of TA-OAFs hydrogels were further analyzed. The obtained results indicated that the addition of TA could increase the average diameter of nanofibrils with increased β-sheet contents, and showed a TA concentration-dependent tendency. Moreover, TA-OAFs hydrogels also possessed better thermal stability, antioxidant activity, and antibacterial capacity for both Gram-negative and Gram-positive bacteria. The present study might contribute to a better understanding of antimicrobial materials designing of protein-induced amyloid fibrils in the food field with beneficial properties.

The role of shear forces in primary and secondary nucleation of amyloid fibrils

Shear forces affect self-assembly processes ranging from crystallization to fiber formation. Here, the effect of mild agitation on amyloid fibril formation was explored for four peptides and investigated in detail for A[Formula: see text]42, which is associated with Alzheimer's disease. To gain mechanistic insights into the effect of mild agitation, nonseeded and seeded aggregation reactions were set up at various peptide concentrations with and without an inhibitor. First, an effect on fibril fragmentation was excluded by comparing the monomer-concentration dependence of aggregation kinetics under idle and agitated conditions. Second, using a secondary nucleation inhibitor, Brichos, the agitation effect on primary nucleation was decoupled from secondary nucleation. Third, an effect on secondary nucleation was established in the absence of inhibitor. Fourth, an effect on elongation was excluded by comparing the seeding potency of fibrils formed under idle or agitated conditions. We find that both primary and secondary nucleation steps are accelerated by gentle agitation. The increased shear forces facilitate both the detachment of newly formed aggregates from catalytic surfaces and the rate at which molecules are transported in the bulk solution to encounter nucleation sites on the fibril and other surfaces. Ultrastructural evidence obtained with cryogenic transmission electron microscopy and free-flow electrophoresis in microfluidics devices imply that agitation speeds up the detachment of nucleated species from the fibril surface. Our findings shed light on the aggregation mechanism and the role of detachment for efficient secondary nucleation. The results inform on how to modulate the relative importance of different microscopic steps in drug discovery and investigations.

Identification of apolipoprotein E-derived amyloid within cholesterol granulomas of leopard geckos (Eublepharis macularius)

Apolipoprotein E (ApoE) is involved in cholesterol transport among cells and also plays an important role in amyloid formation, co-depositing with amyloid fibrils in various types of amyloidosis. Although the in vivo amyloidogenicity of ApoE has not been previously demonstrated, this study provides evidence of ApoE amyloidogenicity in leopard geckos (Eublepharis macularius), belonging to the class Reptilia. Histologically, amyloid deposits were localized within cholesterol granulomas and exhibited positive Congo red staining, with yellow to green birefringence under polarized light. On mass spectrometry-based proteomic analysis, ApoE was detected as a dominant component of amyloid; of the full length of the 274 amino acid residues, peptides derived from Leu185-Arg230 were frequently detected with non-tryptic truncations. Immunohistochemistry with anti-leopard gecko ApoE antibody showed positive reactions of amyloid deposits. These results show that ApoE is an amyloid precursor protein within the cholesterol granulomas of leopard geckos. Although further investigations are needed, the C-terminal region of ApoE involved in amyloid formation is a lipid-binding region, and there should be a relationship between amyloidogenesis and the development of cholesterol granulomas in leopard geckos. This study provides novel insights into the pathogenesis of ApoE-related diseases.

Neochlorogenic Acid, Quercetin 3-β-O-Glucoside and Ruby by Flavon Dietary Supplement Inhibit Amyloid Fibril Formation

Neochlorogenic Acid, Quercetin 3-β-O-Glucoside and Ruby by Flavon Dietary Supplement Inhibit Amyloid Fibril Formation

Role of Amyloidogenic and Non-Amyloidogenic Protein Spaces in Neurodegenerative Diseases and their Mitigation Using Theranostic Agents.

Neurodegenerative diseases (NDDs) refer to a complex heterogeneous group of diseases which are associated with the accumulation of amyloid fibrils or plaques in the brain leading to progressive loss of neuronal functions. Alzheimer's disease is one of the major NDD responsible for 60-80 % of all dementia cases. Currently, there are no curative or disease-reversing/modifying molecules for many of the NDDs except a few such as donepezil, rivastigmine, galantamine, carbidopa and levodopa which treat the disease-associated symptoms. Similarly, there are very few FDA-approved tracers such as flortaucipir (Tauvid) for tau fibril imaging and florbetaben (Neuraceq), flutemetamol (Vizamyl), and florbetapir (Amyvid) for amyloid imaging available for diagnosis. Recent advances in the cryogenic electron microscopy reported distinctly different microstructures for tau fibrils associated with different tauopathies highlighting the possibility to develop tauopathy-specific imaging agents and therapeutics. In addition, it is important to identify the proteins that are associated with disease development and progression to know about their 3D structure to develop various diagnostics, therapeutics and theranostic agents. The current article discusses in detail the disease-associated amyloid and non-amyloid proteins along with their structural insights. We comprehensively discussed various novel proteins associated with NDDs and their implications in disease pathology. In addition, we document various emerging chemical compounds developed for diagnosis and therapy of different NDDs with special emphasis on theranostic agents for better management of NDDs.

Inhibiting insulin aggregation by chaperone-like green cholinium-based DESs as additives

Protein microenvironment is critical to study protein function and stability. In the current study, the ability of synthesized choline-based deep eutectic solvents (DESs) as additives to inhibit and disaggregate amyloid fibrils of human insulin was investigated. The DESs synthesized in this study were nontoxic and can be classified as green chemistry. Utilizing various biophysical methods indicated that choline-chloride glycine (Ch/Gly) and choline-chloride ascorbic acid (Ch/Asb) exhibited chaperone-like properties. This was attributed to their inhibitory effects on the lag phase by stabilizing insulin monomers and on the saturation phase by disaggregating mature insulin fibrils. In contrast, choline-chloride histidine (Ch/His) accelerated the aggregation of insulin. Our results suggest that antioxidant and hydrophobic properties, combined with lower surface activity and higher hydrogen bond formation ability, are the main features describing the inhibitory effect of DESs on protein aggregation.

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.