Amyloid Fibril Formation Research Articles

The small molecule ZPD-2 inhibits the aggregation and seeded polymerisation of C-terminally truncated α-Synuclein.

Protein aggregation, particularly the formation of amyloid fibrils, is associated with numerous human disorders, including Parkinson's disease. This neurodegenerative condition is characterised by the accumulation of α-Synuclein amyloid fibrils within intraneuronal deposits known as Lewy bodies or neurites. C-terminally truncated forms of α-Synuclein are frequently observed in these inclusions in the brains of patients, and their increased aggregation propensity suggests a role in the disease's pathogenesis. This study demonstrates that the small molecule ZPD-2 acts as a potent inhibitor of both the spontaneous and seeded amyloid polimerisation of C-terminally truncated α-Synuclein by interfering with early aggregation intermediates. This dual activity positions this molecule as a promising candidate for therapeutic development in treating synucleinopathies.

Intravenous chaperone treatment of late-stage Alzheimer´s disease (AD) mouse model affects amyloid plaque load, reactive gliosis and AD-related genes

Treatment strategies that are efficient against established Alzheimer’s disease (AD) are needed. BRICHOS is a molecular chaperone domain that prevents amyloid fibril formation and associated cellular toxicity. In this study, we treated an AD mouse model seven months after pathology onset, using intravenous administration of recombinant human (rh) Bri2 BRICHOS R221E. Two injections of rh Bri2 BRICHOS R221E per week for three months in AD mice reduced amyloid β (Aβ) burden, and mitigated astro- and microgliosis, as determined by glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (Iba1) immunohistochemistry. Sequencing of RNA from cortical microglia cells showed that BRICHOS treatment normalized the expression of identified plaque-induced genes in mice and humans, including clusterin and GFAP. Rh Bri2 BRICHOS R221E passed the blood–brain barrier (BBB) in age-matched wild-type mice as efficiently as in the AD mice, but then had no effect on measures of AD-like pathology, and mainly affected the expression of genes that affect cellular shape and movement. These results indicate a potential of rh Bri2 BRICHOS against advanced AD and underscore the ability of BRICHOS to target amyloid-induced pathology.

Unveiling the Inhibitory Effect of Magnolol in the Aggregation of Human Calcitonin (hCT): A Comprehensive In-Silico Study.

Amyloid fibril formation by some peptides leads to several neurogenetic disorders. This limits their biological activity and increases cytotoxicity. Human calcitonin (hCT), 32 residue containing peptide, known for regulating calcium and phosphate concentration in the blood tends to form amyloids in aqueous medium. Polyphenols are very effective in inhibiting fibril formation. As part of our research, we have taken Magnolol (Mag), which is extracted from the Chinese herb Magnolia officinalis. To evaluate its effectiveness as an inhibitor in preventing hCT aggregation, we conducted an all-atom classical molecular dynamics simulation with varying concentrations of Mag. In presence of Mag, hCT maintains its helical conformation in higher order. Magnolol primarily interacts with hCT via van der Waals interaction. Asp15 residue of hCT, resides in the amyloid region (D15FNKF19) forms strong hydrogen bonding interaction with Mag. Moreover, aromatic residues of hCT interact with Mag through π-π stacking interactions. Our work gives insights into the molecular mechanism of Magnolol in the inhibition of hCT fibril formation to use it as a potential candidate for medicinal purpose.

Exogenous Amyloid Fibrils Can Cause Significant Upregulation of Neurodegenerative Disease Proteins.

Neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, are associated with the formation of amyloid fibrils. In familial cases, the mutant causative genes accentuate disease progression through overexpression or misfolding of amyloidogenic proteins. Besides, considerable amyloidosis cases arise from external factors, but their origin and mechanisms are not yet fully understood. Herein, we found that amyloid fibrils generated from egg and milk proteins, in addition to their nutritional effects to intestinal cells, can selectively reduce the viability of nervous cells as well as pancreatic islet cells. In contrast, soy protein amyloid fibrils lacked cytotoxicity to the aforementioned cells. This protein source and cell type-dependent cytotoxicity are demonstrated to be associated with the significant upregulation of amyloidogenic proteins. The finding was also confirmed by the vein injection of beta-lactoglobulin fibrils to mice, exhibiting the pronounced upregulations of amyloid beta1-42 (Aβ1-42) and islet amyloid polypeptide in vivo. The study therefore provides insight into the health implications of exogenous amyloid fibrils.

Transthyretin Cardiac Amyloidosis: Insights into Wild-Type Variants and Familial Amyloid Cardiomyopathy

Transthyretin (TTR) cardiac amyloidosis is a progressive condition characterized by amyloid fibril deposition in the heart, leading to heart failure. This review focuses on insights into wild-type (ATTRwt) and familial (ATTRm) variants of TTR amyloidosis. The pathophysiology involves TTR tetramer destabilization, monomer misfolding, and amyloid fibril formation. Therapeutic advances, including TTR stabilizers like tafamidis and Diflunisal, and gene-silencing agents such as patisiran and inotersen, show promise in managing the disease. Despite these advancements, early diagnosis remains challenging, and side effects of current therapies necessitate the development of safer, more effective treatments. Future research should focus on novel therapies, improved diagnostic methods, and personalized treatment strategies to enhance patient outcomes. This review underscores the importance of continued innovation and collaboration in tackling TTR cardiac amyloidosis.

Self-limiting multimerization of α-synuclein on membrane and its implication in Parkinson's diseases.

α-Synuclein (α-syn), a crucial molecule in Parkinson's disease (PD), is known for its interaction with lipid membranes, which facilitates vesicle trafficking and modulates its pathological aggregation. Deciphering the complexity of the membrane-binding behavior of α-syn is crucial to understand its functions and the pathology of PD. Here, we used single-molecule imaging to show that α-syn forms multimers on lipid membranes with huge intermultimer distances. The multimers are characterized by self-limiting growth, manifesting in concentration-dependent exchanges of monomers, which are fast at micromolar concentrations and almost stop at nanomolar concentrations. We further uncovered movement patterns of α-syn's occasional trapping on membranes, which may be attributed to sparse lipid packing defects. Mutations such as E46K and E35K may disrupt the limit on the growth, resulting in larger multimers and accelerated amyloid fibril formation. This work emphasizes sophisticated regulation of α-syn multimerization on membranes as a critical underlying factor in the PD pathology.

Flavones Suppress Aggregation and Amyloid Fibril Formation of Human Lysozyme under Macromolecular Crowding Conditions.

The crowded milieu of a biological cell significantly impacts protein aggregation and interactions. Understanding the effects of macromolecular crowding on the aggregation and fibrillation of amyloidogenic proteins is crucial for the treatment of many amyloid-related disorders. Most in vitro studies of protein amyloid formation and its inhibition by small molecules are conducted in dilute buffers, which do not mimic the complexity of the cellular environment. In this study, we used PEGs to simulate macromolecular crowding and examined the inhibitory effects of flavones DHF, baicalein, and luteolin on human lysozyme (HuL) aggregation at pH 2. Naturally occurring flavones have been effective inhibitors of amyloid formation in some proteins. Our findings indicate that while flavones inhibit HuL aggregation and fibrillation in dilute buffer solutions, complete inhibition is observed with a combination of flavones and PEGs, as shown by ThT fluorescence, light scattering, TEM, and AFM studies. The species formed in the presence of PEG 8000 and flavones were less hydrophobic, less toxic, and α-helix-rich compared to control samples, which were hydrophobic and β-sheet-rich, as demonstrated by ANS hydrophobicity, MTT assay, and CD spectroscopy. Fluorescence titration studies of flavones with HuL showed a significant increase in binding constant values under crowding conditions. These findings highlight the importance of macromolecular crowding in modulating protein aggregation and amyloid inhibition. Further studies using disease-causing mutants of HuL and other amyloidogenic proteins are needed to explore the role of macromolecular crowding in small-molecule-mediated modulation and inhibition of protein aggregation and amyloid formation.

TDP-43 Amyloid Fibril Formation via Phase Separation-Related and -Unrelated Pathways.

Intrinsically disordered regions (IDRs) in proteins can undergo liquid-liquid phase separation (LLPS) for functional assembly, but this increases the chance of forming disease-associated amyloid fibrils. Not all amyloid fibrils form through LLPS however, and the importance of LLPS relative to other pathways in fibril formation remains unclear. We investigated this question in TDP-43, a motor neuron disease and dementia-causing protein that undergoes LLPS, using thioflavin T (ThT) fluorescence, NMR, transmission electron microscopy (TEM), and wide-angle X-ray scattering (WAXS) experiments. Using a fluorescence probe modified from ThT strategically designed for targeting protein assembly rather than β-sheets and supported by TEM images, we propose that the biphasic ThT signals observed under LLPS-favoring conditions are due to the presence of amorphous aggregates. These aggregates represent an intermediate state that diverges from the direct pathway to β-sheet-dominant fibrils. Under non-LLPS conditions in contrast (at low pH or at physiological conditions in a construct with key LLPS residues removed), the protein forms a hydrogel. Real-time WAXS data, ThT signals, and TEM images collectively demonstrate that the gelation process circumvents LLPS and yet still results in the formation of fibril-like structural networks. We suggest that the IDR of TDP-43 forms disease-causing amyloid fibrils regardless of the formation pathway. Our findings shed light on why both LLPS-promoting and LLPS-inhibiting mutants are found in TDP-43-related diseases.

Different charged biopolymers induce α-synuclein to form fibrils with distinct structures

The aggregation of α-synuclein (α-syn) into amyloid fibrils, a key process in the development of Parkinson's disease (PD) and other synucleinopathies, is influenced by a range of factors such as charged biopolymers, chaperones, and metabolites. However, the specific impacts of different biopolymers on α-syn fibril structure are not well understood. In our work, we found that different polyanions and polycations, such as polyphosphate (polyP), polyuridine (polyU), and polyamines (including putrescine, spermidine, and spermine), markedly altered the fibrillation kinetics of α-syn in vitro. Furthermore, seeding assay revealed distinct cross-seeding capacities across different biopolymer-induced α-syn fibrils, suggesting the formation of structurally distinct strains under different conditions. Utilizing cryo-electron microscopy (cryo-EM), we further examined the detailed structural configuration of α-syn fibrils formed in the presence of these biopolymers. Notably, we found that while polyamines do not change the atomic structure of α-syn fibrils, polyU and polyP induce the formation of distinct amyloid fibrils, exhibiting a range of structural polymorphs. Our work offers valuable insights into how various charged biopolymers affect the aggregation process and the resultant structures of α-syn fibrils, thereby enhancing our understanding of the structural variations in α-syn fibrils across different pathological conditions.

Unraveling the impact of tungsten disulfide quantum dots on human serum albumin conformational dynamics and fibrillation pathways: An integrated multi-spectroscopic, biochemical, and molecular docking investigation

Herein, the intricate molecular interplay between human serum albumin (HSA) and tungsten disulfide quantum dots (WS2 QDs) was probed using spectroscopic techniques and sophisticated molecular simulation methods. Fluorescence spectroscopy demonstrated that under physiological conditions, WS2 QDs forge a non-fluorescent ground-state complex with HSA, facilitated by hydrogen bonding and van der Waals forces, ultimately resulting in the static quenching of the protein's intrinsic fluorescence. Complementary site competition experiments and molecular docking simulations reinforced a precise 1: 1 binding stoichiometry, predominantly targeting HSA's Site I. Three-dimensional fluorescence spectroscopy revealed that WS2 QDs perturb the HSA polypeptide backbone, subtly modifying the microenvironment surrounding aromatic amino acid residues. This alteration was further corroborated by circular dichroism spectroscopy, marked by a decrease in helical content and a transition towards irregular peptide conformations. Thermal stability assays illuminated the reduced thermal resilience of the HSA − WS2 QD complex. Laser confocal microscopy coupled with thioflavin T staining yielded compelling evidence that WS2 QDs effectively inhibit amyloid fibril formation in both HSA and lysozyme, underscoring their potential as potent anti-amyloidogenic agents. This comprehensive study offers pivotal insights into multifaceted impact of WS2 QDs on protein structure and function, thereby expanding their horizon of potential applications within the burgeoning field of nanomedicine.

An in vitro cell model for exploring inflammatory and amyloidogenic events in alkaptonuria.

Alkaptonuria (AKU) is a progressive systemic inherited metabolic disorder primarily affecting the osteoarticular system, characterized by the degeneration of cartilage induced by ochronosis, ultimately leading to early osteoarthritis (OA). However, investigating AKU pathology in human chondrocytes, which is crucial for understanding the disease, encounters challenges due to limited availability and donor variability. To overcome this obstacle, an in vitro model has been established using homogentisic acid (HGA) to simulate AKU conditions. This model employed immortalized C20/A4 human chondrocytes and serves as a dependable platform for studying AKU pathogenesis. Significantly, the model demonstrates the accumulation of ochronotic pigment in HGA-treated cells, consistent with findings from previous studies. Furthermore, investigations into inflammatory processes during HGA exposure revealed notable oxidative stress, as indicated by elevated levels of reactive oxygen species and lipid peroxidation. Additionally, the model demonstrated HGA-induced inflammatory responses, evidenced by increased production of nitric oxide, overexpression of inducible nitric oxide synthase, and cyclooxygenase-2. These findings underscore the model's utility in studying inflammation associated with AKU. Moreover, analysis of serum amyloid A and serum amyloid P proteins revealed a potential interaction, corroborating evidence of amyloid fibril formation. This hypothesis was further supported by Congo red staining, which showed fibril formation exclusively in HGA-treated cells. Overall, the C20/A4 cell model provided valuable insights into AKU pathogenesis, emphasizing its potential for facilitating drug development and therapeutic interventions.

A structural rationale for reversible vs irreversible amyloid fibril formation from a single protein.

Reversible and irreversible amyloids are two diverging cases of protein (mis)folding associated with the cross-β motif in the protein folding and aggregation energy landscape. Yet, the molecular origins responsible for the formation of reversible vs irreversible amyloids have remained unknown. Here we provide evidence at the atomic level of distinct folding motifs for irreversible and reversible amyloids derived from a single protein sequence: human lysozyme. We compare the 2.8 Å structure of irreversible amyloid fibrils determined by cryo-electron microscopy helical reconstructions with molecular insights gained by solid-state NMR spectroscopy on reversible amyloids. We observe a canonical cross-β-sheet structure in irreversible amyloids, whereas in reversible amyloids, there is a less-ordered coexistence of β-sheet and helical secondary structures that originate from a partially unfolded lysozyme, thus carrying a "memory" of the original folded protein precursor. We also report the structure of hen egg-white lysozyme irreversible amyloids at 3.2 Å resolution, revealing another canonical amyloid fold, and reaffirming that irreversible amyloids undergo a complete conversion of the native protein into the cross-β structure. By combining atomic force microscopy, cryo-electron microscopy and solid-state NMR, we show that a full unfolding of the native protein precursor is a requirement for establishing irreversible amyloid fibrils.

Amyloid Fibril Formation on Neuronal Cells in the Coexistence of Aβ40 and Aβ42.

The abnormal aggregation and subsequent deposition of amyloid β-protein (Aβ) in the brain are considered central to the pathogenesis of Alzheimer's disease. The two major species of Aβ are Aβ40 and Aβ42, present at an approximate ratio of 9: 1. Accumulating evidence suggests that neuronal membranes are an important platform of amyloidogenesis by Aβ. However, information on the aggregational behaviors of coexistent Aβ40 and Aβ42 on membranes is lacking. In this study, the aggregation and resultant cytotoxicity of coexistent Aβ40 and Aβ42 at a physiologically relevant ratio were investigated by fluorescence techniques. We found that the degree of coexistence of both Aβs in aggregates increased as the assembly proceeded, and reached a maximum in fibrils. However, the cytotoxicity of the mixed fibrils was weaker than that of Aβ42 fibrils, indicating that Aβ40 attenuates the toxicity of Aβ42 by forming mixed fibrils. In contrast, the degree of coexistence was significantly lower in aqueous phase aggregation, highlighting different aggregation mechanisms between in membranes and in the aqueous phase.

Metabolic perturbations associated with hIAPP-induced insulin resistance in skeletal muscles: Implications to the development of type 2 diabetes

The human islet amyloid polypeptide (hIAPP) tends to misfold and self-assemble to form amyloid fibrils, which has been associated with the loss of function and viability of pancreatic β-cells in type 2 diabetes mellitus (T2DM). The role of hIAPP in the development of insulin resistance (a hallmark of T2DM) in skeletal muscles – the major sites for glucose utilization – needs further investigation. Even though, insulin-resistant conditions have been known to stimulate hIAPP aggregation, the events that lead to the development of insulin resistance due to hIAPP aggregation in skeletal muscles remain unidentified. Here, we have attempted to identify metabolic perturbations in L6 myotubes that were exposed to increasing concentrations of recombinant hIAPP for different time durations. It was observed that hIAPP exposure was associated with increased mitochondrial and cellular ROS levels, loss in mitochondrial membrane potential and viability of the myotubes. Metabolomic investigations of hIAPP-treated myotubes revealed significant perturbations in o-phosphocholine, sn-glycero-3-phosphocholine and dimethylamine levels (p < 0.05). Therefore, we anticipate that defects in glycerophospholipid metabolism and the associated oxidative stress and membrane damage may play key roles in the development of insulin resistance due to protein misfolding in skeletal muscles. In summary, the perturbed metabolites and their pathways have not only the potential to be used as early biomarkers to predict the onset of insulin resistance and T2DM but also as therapeutic targets for the effective management of the same.

Non-equilibrium physics of multi-species assembly applied to fibrils inhibition in biomolecular condensates and growth of online distrust

Self-assembly is a key process in living systems—from the microscopic biological level (e.g. assembly of proteins into fibrils within biomolecular condensates in a human cell) through to the macroscopic societal level (e.g. assembly of humans into common-interest communities across online social media platforms). The components in such systems (e.g. macromolecules, humans) are highly diverse, and so are the self-assembled structures that they form. However, there is no simple theory of how such structures assemble from a multi-species pool of components. Here we provide a very simple model which trades myriad chemical and human details for a transparent analysis, and yields results in good agreement with recent empirical data. It reveals a new inhibitory role for biomolecular condensates in the formation of dangerous amyloid fibrils, as well as a kinetic explanation of why so many diverse distrust movements are now emerging across social media. The nonlinear dependencies that we uncover suggest new real-world control strategies for such multi-species assembly.

Spatiotemporal formation of a single liquid-like condensate and amyloid fibrils of α-synuclein by optical trapping at solution surface

Liquid-like protein condensates have recently attracted much attention due to their critical roles in biological phenomena. They typically show high fluidity and reversibility for exhibiting biological functions, while occasionally serving as sites for the formation of amyloid fibrils. To comprehend the properties of protein condensates that underlie biological function and pathogenesis, it is crucial to study them at the single-condensate level; however, this is currently challenging due to a lack of applicable methods. Here, we demonstrate that optical trapping is capable of inducing the formation of a single liquid-like condensate of α-synuclein in a spatiotemporally controlled manner. The irradiation of tightly focused near-infrared laser at an air/solution interface formed a condensate under conditions coexisting with polyethylene glycol. The fluorescent dye-labeled imaging showed that the optically induced condensate has a gradient of protein concentration from the center to the edge, suggesting that it is fabricated through optical pumping-up of the α-synuclein clusters and the expansion along the interface. Furthermore, Raman spectroscopy and thioflavin T fluorescence analysis revealed that continuous laser irradiation induces structural transition of protein molecules inside the condensate to β-sheet rich structure, ultimately leading to the condensate deformation and furthermore, the formation of amyloid fibrils. These observations indicate that optical trapping is a powerful technique for examining the microscopic mechanisms of condensate appearance and growth, and furthermore, subsequent aging leading to amyloid fibril formation.

Salt-Induced Hydrophobic C-Terminal Region of α-Synuclein Triggers Its Fibrillation under the Mimic Physiologic Condition.

α-Synuclein (αS) causes Parkinson's disease due to the structural alteration into amyloid fibrils that form after the interaction with synaptic membranes in neurons. To understand the alternation mechanism, the effect of salt (NaCl) on the interaction of αS with synaptic mimic membrane was characterized at the molecular level because salt triggered the amyloid fibril formation. The membrane-bound conformation (or the initial conformation before fibrillation) showed that NaCl decreased the number of helical structures and Tyr residues interacting with the membrane surface compared to when NaCl was absent, implying an increase in solvent-exposed regions. The N-terminal region of αS interacted with the membrane, forming the helical structures regardless of NaCl, while the C-terminal region formed a random structure with weak membrane interaction, but NaCl inhibited the interaction of its hydrophobic area, suggesting that salt promoted amyloid fibril formations by exposing the hydrophobic C-terminal region, which can intermolecularly interact with free αS.

An evolutionarily conserved function of C-reactive protein is to prevent the formation of amyloid fibrils.

C-reactive protein (CRP) binds to phosphocholine (PCh)-containing substances and subsequently activates the complement system to eliminate the ligand. The PCh-binding function of CRP has been conserved throughout evolution from arthropods to humans. Human CRP, in its structurally altered conformation at acidic pH, also binds to amyloid-β (Aβ) and prevents the formation of Aβ fibrils. It is unknown whether the Aβ-binding function of CRP has also been evolutionarily conserved. The aim of this study was to determine whether CRP isolated from American horseshoe crab Limulus polyphemus was also anti-amyloidogenic and whether this function required structural alteration of Limulus CRP (Li-CRP). Two CRP species Li-CRP-I and Li-CRP-II were purified from hemolymph by employing PCh-affinity chromatography and phosphoethanolamine-affinity chromatography, respectively. Both Li-CRP-I and Li-CRP-II bound to immobilized Aβ at physiological pH. Unlike human CRP, Li-CRP did not require any changes in its overall structure to bind to Aβ. Both Li-CRP-I and Li-CRP-II bound to Aβ in the fluid phase also and prevented the fibrillation of Aβ. Additionally, ion-exchange chromatography of purified Li-CRP indicated that a variety of Li-CRP molecules of different subunit compositions were present in Limulus hemolymph, raising the possibility that the presence of various Li-CRP species in hemolymph facilitates the recognition of a range of proteins with differing amyloidogenicity. We conclude that the binding of CRP to Aβ is an ancient function of CRP. In invertebrates, the Aβ-binding function of CRP can protect the host from toxicity caused by amyloidogenic and pathogenic proteins. In humans, the Aβ-binding function of CRP can protect against inflammatory diseases in which the host proteins are ectopically deposited on either host cells or foreign cells in an inflammatory milieu since immobilized proteins may expose Aβ-like structures after deposition at places where they are not supposed to be.

Cavity Lasing of Thioflavin T in the Condensed Phase for Discrimination between Surface Interaction and β-Sheet Groove Binding in Alzheimer-Linked Peptides.

This study investigates the lasing effects in a Fabry-Perot cavity to discern the binding interactions of thioflavin T (ThT) with various peptides associated with Alzheimer's disease, including Aβ(1-42), KLVFFA, and diphenylalanine (FF) in the condensed phase. Utilizing kinetic lasing measurements, the research explores ThT emission enhancements due to specific groove binding in β-sheet structures and highlights additional contributions from weak surface interactions and solvent-solute interactions. Lasing spectroscopy reveals a lack of transition of the FF system from its native state to an amyloid-like structure, challenging traditional ThT assay interpretations. These findings show the potential of lasing spectroscopy in elucidating the molecular basis of amyloid fibril formation and the development of diagnostic tools for amyloidogenic diseases.

Comparison of formation and structural characteristics of amyloid fibrils of peanut protein isolate after hydration treatment at different pH

In this paper, the process of fibrillation and the structure of fibrils formed from peanut protein after hydration treatment at pH 5.0 (group Q) and pH 2.0 (group S) were studied. The results showed that compared with the peanut protein obtained after hydration at pH 5.0, the particle size of the peanut protein obtained after hydration at pH 2.0 increased, and the surface hydrophobicity and intrinsic fluorescence intensity decreased, indicating that the protein aggregated. During fibrillation, the maximum Thioflavin T fluorescence intensity of protein in group Q was higher than that in group S, and the time for group S protein to reach the maximum fluorescence intensity was slower than that in group Q protein. Besides, the group Q protein first formed short worm-like fibrils and then long flexible fibrils, and the group S protein first formed short rigid fibrils and then worm-like fibrils. In conclusion, hydration treatment at pH 2.0 led to the aggregation of peanut protein, reduced the fibrils forming ability of peanut protein and affected subsequent self-assembly processes. This study could provide new insights of the fibril formation mechanism and structural properties of peanut protein after hydration treatment at different pH.