Cross-β Sheet Research Articles

Amyloid-like Aggregation of Wheat Gluten and Its Components during Cooking: Mechanisms and Structural Characterization.

Amyloid-like aggregation widely occurs during the processing and production of natural proteins, with evidence indicating its presence following the thermal processing of wheat gluten. However, significant gaps remain in understanding the underlying fibrillation mechanisms and structural polymorphisms. In this study, the amyloid-like aggregation behavior of wheat gluten and its components (glutenin and gliadin) during cooking was systematically analyzed through physicochemical assessment and structural characterization. The presence of amyloid-like fibrils (AFs) was confirmed using X-ray diffraction and Congo red staining, while Thioflavin T fluorescence revealed different patterns and rates of AFs growth among wheat gluten, glutenin, and gliadin. AFs in gliadin exhibited linear growth curves, while those in gluten and glutenin showed S-shaped curves, with the shortest lag phase and fastest growth rate (t1/2 = 2.11 min) observed in glutenin. Molecular weight analyses revealed AFs primarily in the 10-15 kDa range, shifting to higher weights over time. Glutenin-derived AFs had the smallest ζ-potential value (-19.5 mV) and the most significant size increase post cooking (approximately 400 nm). AFs in gluten involve interchain reorganization, hydrophobic interactions, and conformational transitions, leading to additional cross β-sheets. Atomic force microscopy depicted varying fibril structures during cooking, notably longer, taller, and stiffer AFs from glutenin.

PH-Dependent Reversible Self-Assembly of β-Lactoglobulin-Derived Reducing Peptides.

Peptide-based self-assembled nanostructures are emerging vehicles for nutrient delivery and interface engineering. The present study screened eight β-lactoglobulin (β-Lg) derived peptides and found that two reducing peptides [EQSLVCQCLV (EV-10) and VCQCLVR (VR-7)] demonstrated pH-dependent reversible fibrilization. EV-10 formed fibrils at pH 2.0 but became unordered aggregates at pH 7.0. VR-7 showed the opposite trend. Both peptides could undergo repetitive transitions between fibrils and unordered aggregates during consecutive pH-cycling. Fibrilization of both peptides was dominated by charges carried by N- and C-terminals. Both fibrils were characterized by a cross-β sheet structure where the β-sheet was arranged in an antiparallel manner. Fe3+ was reduced by Cys and EV-10 (pH 5.0 and 7.0) simultaneously upon mixing. In contrast, EV-10 fibrils released Fe3+ reducing capacity progressively, which were beneficial to long-term protection Fe2+. The EV-10 fibrils remained intact after simulated gastric digestion and finally dissociated after intestinal digestion. The results shed light on the mechanisms of fibrilization of β-Lg derived peptides. This study was beneficial to the rational design of smart pH-responsive materials for drug delivery and antioxidants for nutrients susceptible to oxidation.

Maturation-dependent changes in the size, structure and seeding capacity of Aβ42 amyloid fibrils

Many proteins self-assemble to form amyloid fibrils, which are highly organized structures stabilized by a characteristic cross-β network of hydrogen bonds. This process underlies a variety of human diseases and can be exploited to develop versatile functional biomaterials. Thus, protein self-assembly has been widely studied to shed light on the properties of fibrils and their intermediates. A still open question in the field concerns the microscopic processes that underlie the long-time behaviour and properties of amyloid fibrillar assemblies. Here, we use atomic force microscopy with angstrom-sensitivity to observe that amyloid fibrils undergo a maturation process, associated with an increase in both fibril length and thickness, leading to a decrease of their density, and to a change in their cross-β sheet content. These changes affect the ability of the fibrils to catalyse the formation of new aggregates. The identification of these changes helps us understand the fibril maturation processes, facilitate the targeting of amyloid fibrils in drug discovery, and offer insight into the development of biocompatible and sustainable protein-based materials.

Cryo-EM structure and polymorphic maturation of a viral transduction enhancing amyloid fibril

Amyloid fibrils have emerged as innovative tools to enhance the transduction efficiency of retroviral vectors in gene therapy strategies. In this study, we used cryo-electron microscopy to analyze the structure of a biotechnologically engineered peptide fibril that enhances retroviral infectivity. Our findings show that the peptide undergoes a time-dependent morphological maturation into polymorphic amyloid fibril structures. The fibrils consist of mated cross-β sheets that interact by the hydrophobic residues of the amphipathic fibril-forming peptide. The now available structural data help to explain the mechanism of retroviral infectivity enhancement, provide insights into the molecular plasticity of amyloid structures and illuminate the thermodynamic basis of their morphological maturation.

Anionic surfactant causes dual conformational changes in insulin

Amyloid fibrillation is a process by which proteins aggregate and form insoluble fibrils that are implicated in several neurodegenerative diseases. In n this study, we aimed to investigate the impact of the negatively charged detergent sodium dodecyl sulfate (SDS) on insulin amyloid fibrillation at pH 7.4 and 2.0, as SDS has been linked to the acceleration of amyloid fibrillation in vitro, but the underlying molecular mechanism is not fully understood. Our findings show that insulin forms amyloid-like aggregates in the presence of SDS at concentrations ranging from 0.05 to 1.8 mM at pH 2.0, while no aggregates were observed at SDS concentrations greater than 1.8 mM, and insulin remained soluble. However, at pH 7.4, insulin remained soluble regardless of the concentration of SDS. Interestingly, the aggregated insulin had a cross-β sheet secondary structure, and when incubated with higher SDS concentrations, it gained more alpha-helix. The electrostatics and hydrophobic interaction of SDS and insulin may contribute to amyloid induction. Moreover, the SDS-induced aggregation was not affected by the presence of salts. Furthermore, as the concentration of SDS increased, the preformed insulin amyloid induced by SDS began to disintegrate. Overall, our study sheds light on the mechanism of surfactant-induced amyloid fibrillation in insulin protein.

Cryo-EM structure of a catalytic amyloid fibril

Catalytic amyloid fibrils are novel types of bioinspired, functional materials that combine the chemical and mechanical robustness of amyloids with the ability to catalyze a certain chemical reaction. In this study we used cryo-electron microcopy to analyze the amyloid fibril structure and the catalytic center of amyloid fibrils that hydrolyze ester bonds. Our findings show that catalytic amyloid fibrils are polymorphic and consist of similarly structured, zipper-like building blocks that consist of mated cross-β sheets. These building blocks define the fibril core, which is decorated by a peripheral leaflet of peptide molecules. The observed structural arrangement differs from previously described catalytic amyloid fibrils and yielded a new model of the catalytic center.

Chiral LVFFARK enantioselectively inhibits amyloid-β protein fibrillogenesis

The modulation of protein aggregation is involved not only in biochemical engineering processes, but also in in vivo biological events such as Alzheimer’s disease (AD) that features amyloid-β protein (Aβ) deposits. Inspired by the different pharmacological efficacy of enantiomers, taking heptapeptide LVFFARK (LK7) as an example, herein the chiral influence of peptide inhibitors on Aβ fibrillogenesis and cytotoxicity was investigated by extensive biophysical and biological analyses. It was intriguing to find that although both L-LK7 and D-LK7 could inhibit Aβ aggregation in a concentration-dependent manner, it was the d-enantiomer that exhibited chirality preference and selectivity for modulation of Aβ self-assembly. As compared with L-LK7 at the same conditions, D-LK7 showed significantly enhanced potency on suppressing cross-β sheet formation, fibrillar Aβ aggregates deposition, Aβ conformational transition, and Aβ-triggered neurotoxicity on cultured cells. For instance, L-LK7 and D-LK7 rescued cells by increasing cell viability from 60% to 62% and 84% at 100 μmol·L−1, respectively. The chiral discrimination of L-LK7 and D-LK7 was further validated by the different elimination efficiency on amyloid accumulation in AD model nematodes. It is considered that the higher binding affinity of D-LK7 to Aβ monomers than that of L-LK7 resulted in the stronger inhibition effect. This work provided new insights into understanding chirality in the interaction with Aβ and the consequent inhibitory effect, and would contribute to the design of anti-amyloid agents.

Structure-specific amyloid precipitation in biofluids.

The composition of soluble toxic protein aggregates formed in vivo is currently unknown in neurodegenerative diseases, due to their ultra-low concentration in human biofluids and their high degree of heterogeneity. Here we report a method to capture amyloid-containing aggregates in human biofluids in an unbiased way, a process we name amyloid precipitation. We use a structure-specific chemical dimer, a Y-shaped, bio-inspired small molecule with two capture groups, for amyloid precipitation to increase affinity. Our capture molecule for amyloid precipitation (CAP-1) consists of a derivative of Pittsburgh Compound B (dimer) to target the cross β-sheets of amyloids and a biotin moiety for surface immobilization. By coupling CAP-1 to magnetic beads, we demonstrate that we can target the amyloid structure of all protein aggregates present in human cerebrospinal fluid, isolate them for analysis and then characterize them using single-molecule fluorescence imaging and mass spectrometry. Amyloid precipitation enables unbiased determination of the molecular composition and structural features of the in vivo aggregates formed in neurodegenerative diseases.

Deciphering the Structure and Formation of Amyloids in Neurodegenerative Diseases With Chemical Biology Tools.

Protein aggregation into highly ordered, regularly repeated cross-β sheet structures called amyloid fibrils is closely associated to human disorders such as neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases, or systemic diseases like type II diabetes. Yet, in some cases, such as the HET-s prion, amyloids have biological functions. High-resolution structures of amyloids fibrils from cryo-electron microscopy have very recently highlighted their ultrastructural organization and polymorphisms. However, the molecular mechanisms and the role of co-factors (posttranslational modifications, non-proteinaceous components and other proteins) acting on the fibril formation are still poorly understood. Whether amyloid fibrils play a toxic or protective role in the pathogenesis of neurodegenerative diseases remains to be elucidated. Furthermore, such aberrant protein-protein interactions challenge the search of small-molecule drugs or immunotherapy approaches targeting amyloid formation. In this review, we describe how chemical biology tools contribute to new insights on the mode of action of amyloidogenic proteins and peptides, defining their structural signature and aggregation pathways by capturing their molecular details and conformational heterogeneity. Challenging the imagination of scientists, this constantly expanding field provides crucial tools to unravel mechanistic detail of amyloid formation such as semisynthetic proteins and small-molecule sensors of conformational changes and/or aggregation. Protein engineering methods and bioorthogonal chemistry for the introduction of protein chemical modifications are additional fruitful strategies to tackle the challenge of understanding amyloid formation.

Visualizing the nanoscale architecture of amyloid aggregates using amyloidophilic probes and a polarized vortex microscope

Protein misfolding and amyloid aggregation underlie various aging-related neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. Although amyloid aggregates share a common cross-β sheet structural motif formed by similar amino acid sequences, aggregation intermediates are highly dynamic, transitory, and heterogeneous in terms of their structures and sizes. Transient amyloid binding (TAB) superresolution (SR) microscopy is a versatile method for studying a variety of amyloid structures without the need for covalent labeling or immunostaining.

Brazilin Removes Toxic Alpha-Synuclein and Seeding Competent Assemblies from Parkinson Brain by Altering Conformational Equilibrium

Alpha-synuclein (α-syn) fibrils, a major constituent of the neurotoxic Lewy Bodies in Parkinson’s disease, form via nucleation dependent polymerization and can replicate by a seeding mechanism. Brazilin, a small molecule derived from red cedarwood trees in Brazil, has been shown to inhibit the fibrillogenesis of amyloid-beta (Aβ) and α-syn as well as remodel mature fibrils and reduce cytotoxicity. Here we test the effects of Brazilin on both seeded and unseeded α-syn fibril formation and show that the natural polyphenol inhibits fibrillogenesis of α-syn by a unique mechanism that alters conformational equilibria in two separate points of the assembly mechanism: Brazilin preserves the natively unfolded state of α-syn by specifically binding to the compact conformation of the α-syn monomer. Brazilin also eliminates seeding competence of α-syn assemblies from Parkinson’s disease patient brain tissue, and reduces toxicity of pre-formed assemblies in primary neurons by inducing the formation of large fibril clusters. Molecular docking of Brazilin shows the molecule to interact both with unfolded α-syn monomers and with the cross-β sheet structure of α-syn fibrils. Our findings suggest that Brazilin has substantial potential as a neuroprotective and therapeutic agent for Parkinson’s disease.

Endoscopic submucosal dissection for localized amyloidosis of the sigmoid colon.

Amyloidosis is a rare disease caused by the extracellular deposition of amyloid, which results from the self-assembly of various precursor proteins with a highly ordered and abnormal cross-β sheet structure [1] [2].

The Effects of Resveratrol, Caffeine, β-Carotene, and Epigallocatechin Gallate (EGCG) on Amyloid- Aggregation in Synthetic Brain Membranes.

Alzheimer's disease is a neurodegenerative condition marked by the formation and aggregation of amyloid-β (Aβ) peptides. There exists, to this day, no cure or effective prevention for the disease; however, there is evidence that a healthy diet and certain food products can slow down first occurrence and progression. To investigate if food ingredients can interact with peptide aggregates, synthetic membranes that contained aggregates consisting of cross-β sheets of the membrane active fragment A areprepared. The impact of resveratrol, found in grapes, caffeine, the main active ingredient in coffee, β-carotene, found in orange fruits and vegetables, and epigallocatechin gallate (EGCG), a component of green tea, on the size and volume fraction of Aβ aggregates is studied using optical and fluorescence microscopy, X-ray diffraction, UV-vis spectroscopy, and molecular dynamics simulations. All compounds are membrane active and spontaneously partitioned in the synthetic brain membranes. While resveratrol and caffeine lead to membrane thickening and reduced membrane fluidity, β-carotene and EGCG preserve or increasefluidity. Resveratrol and caffeine do not reduce the volume fraction of peptide aggregates while β-carotene significantly reduces plaque size. Interestingly, EGCG dissolves peptide aggregates and significantly decreases the corresponding cross-β and β-sheet signals.

Influence of hydrophobic interfaces and shear on ovalbumin amyloid-like fibril formation in oil-in-water emulsions

Animal proteins are widely used because of their good techno-functional (e.g. emulsification) properties. However, the large ecological impact of animal-derived proteins is often debated and therefore alternatives are evaluated in which animal proteins with increased functionality are employed. Amyloid fibrils are a distinct type of protein fibrils characterized by an intermolecular cross β-sheet structure, mostly studied because of concerns regarding their role in certain human diseases. However, they have also been detected in processed food proteins such as heated ovalbumin (OVA) and other egg proteins. Previous work also mentioned superior emulsification properties of amyloid-like fibrils. Here, the effect of different shear conditions during the emulsification of oil in water emulsions stabilized by heated OVA was investigated. Emulsification with intensive shear treatment (using both an ultra-turrax and a microfluidizer) resulted in submicron sized droplets, whilst ultra-turrax only gave rise to rapidly creaming emulsions (>10 μm). Thioflavin T (ThT) fluorescence, used as an indicator of cross β-sheets, did significantly increase for submicron emulsions, whereas supermicron emulsions showed a similar fluorescence as the aqueous phase used for emulsification. The increase in ThT fluorescence in submicron emulsions was in line with a higher amount of larger fibrillar structures, which showed high ThT fluorescence, measured using size exclusion-HPLC and transmission electron microscopy images. Similar observations were also made for unheated OVA, suggesting that high-shear emulsification induced amyloid-like fibril formation. In contrast, shearing of aqueous solutions without the creation of interfaces did not show increased fibril formation. Therefore, it is claimed that the large specific surface area induced amyloid-like fibril formation or maturation as a result of a better interaction between hydrophobic amyloid-like fibril fragments. In this work, we were able to characterize the protein fibrillar structures present in emulsions by considering commonly used techniques for the study of fibril formation. This creates opportunities towards the implementation of these protein mixtures in food applications.

Drying mode and hydrothermal treatment conditions govern the formation of amyloid-like protein fibrils in solutions of dried hen egg white

The recipe of some food products contains dried hen egg white (EW). It is convenient in use and available with a range of functionalities as a result of being stored under different time, temperature and moisture conditions following drying. Amyloid fibrils are fibrillary protein structures composed of highly ordered stacking of cross-β sheets. They can contribute to the foaming and gelling capacity of EW proteins. We here for the first time report that dried EW contains amyloid-like fibrils and that such fibrils in spray-dried EW (EWSD) are longer (ca. 100–200 nm) than those in freeze-dried EW [EWFD] (ca. 20–100 nm). In addition, conditions for optimal fibrillation were determined for both EWFD and freeze-dried ovalbumin (OVAFD) using a response surface design. Dilute solutions of OVAFD and EWFD were incubated at different pH values, times and temperatures. After storage at optimal conditions OVAFD [2.0% (wprotein/v), pH 7.0, 23 h, 76 °C] and EWFD [0.5% (wprotein/v), pH 7.0, 24 h, 85 °C], a higher level of cross-β sheet structures and larger worm-like protein fibrils were observed for OVAFD than for EWFD. Lastly, when EWSD stored for one week at either 50 °C and 50% relative humidity (RH) [EWSD 50°C/50%] or at 60 °C and 80% RH (EWSD 60°C/80%) was submitted to heating [0.5% (wprotein/v), pH 7.0, 24 h, 85 °C], a higher extent of fibrillation was found for EWSD 60°C/80% than for EWSD 50°C/50%. Thus, drying induces EW amyloid-like protein fibrillation. Also dry heating EWSD at 60 °C and 80% RH further enhances such fibrillation during heating in excess of water.

SDS induces cross beta-sheet amyloid as well as alpha-helical structure in conconavalin A

Surfactants stimulate amyloid fibril formation, but the mechanism of action is still not very clear. In the present study, we examined the effects of negatively charged surfactants, i.e., sodium dodecyl sulfate (SDS) on the concanavalin A (ConA) lectin fibrillation process, at pH 3.5. To determine the effect of SDS treatment on amyloid fibrillation of ConA protein in vitro, we used a variety of techniques for example turbidity, intrinsic fluorescence, ThT fluorescence, far-UV CD and transmission electron microscope (TEM) to assess the amyloid fibrillation process of ConA. Assessing turbidity, it was found that ConA aggregation was dependent on the concentration of SDS. ConA aggregated with incubation with 0.1 to 2.5 mM SDS concentration i.e., (SDS/ConA molar ratio in between 13.3 and 333). However, ConA aggregation was not detected below 0.1 and above 2.5 mM SDS. Thioflavin-T (ThT) strongly binds with aggregated ConA and led to increased fluorescence intensity. The secondary structure of ConA changed from β-sheeted into a cross-β sheet structure with ConA aggregation. Upon interaction with a concentration of SDS higher than 2.5 mM at the same pH, the ConA becomes a total α-helical structure. TEM suggests that in the presence of SDS, the aggregate form of ConA develops into a curly-shaped amyloid structure. This study provides insight into the molecular mechanisms of ConA amyloid fibrillation, which is essential for understanding and preventing surfactant or lipid-induced fibrillation for therapeutic purposes.

A DFT study of structure and stability of pleated and rippled cross-β sheets with hydrophobic sidechains.

The rippled cross-β sheet, a topography, in which mirror-image peptides are arranged with alternating chirality into a periodic two-dimensional network, is burgeoning as a new design principle for materials and biomedical applications. Experiments by the Schneider, Nilsson, and Raskatov labs have independently shown diverse racemic mixtures of aggregation-prone peptide of different sizes to favor the rippled over the pleated topography. Yet, systematic ab initio studies are lacking, and the field is yet to develop rules that would enable the design of new rippled cross-β frameworks from first principles. Here, DFT calculations were performed on a set of model systems, designed to begin understanding the impact that bulky, hydrophobic sidechains have upon the formation of pleated and rippled cross-β frameworks. It is hoped that this study will help stimulate the development of a predictive, general framework to enable rational design of rippled cross-β sheets in the future.

Self- and Cross-Seeding on α-Synuclein Fibril Growth Kinetics and Structure Observed by High-Speed Atomic Force Microscopy.

Fibril formation is an obligatory process in amyloid diseases and is characterized by nucleation and elongation phases that result in the formation of long filaments with cross-β sheet structure. The kinetics of this process, as well as that of secondary nucleation, is controlled by a variety of factors, including nucleus (seed) structure, monomer conformation, and biochemical milieu. Some fibrillar amyloid assemblies act as prions, replicating themselves from protein monomers templated by existing prion seeds. Prion strains, which are characterized by distinct physicochemical and pathologic properties, may also form due to perturbation of the templating process within the susceptible organism. Understanding the types and effects of perturbations occurring during the development and progression of Parkinson's disease is an area requiring more study. Here, we used high-speed atomic force microscopy to determine the kinetics and structural dynamics of α-synuclein fibril elongation initiated by self-seeding or cross-seeding of wild-type (WT) or mutant α-synuclein with WT or mutant α-synuclein seeds. We found that cross-seeding modulated not only elongation rates but also the structures of the growing fibrils. Some fibrils produced in this manner had structures distinct from their "parent" seeds. In other cases, cross-seeding was not observed at all. These findings suggest that α-synuclein sequence variants can produce different types of strains by self- or cross-seeding. Perpetuation of specific strains then would depend on the relative rates of fibril growth and the relative stabilities of the fibrils formed by each strain.

Conformational Selection as the Driving Force of Amyloid β Chiral Inactivation.

We recently introduced amyloid β chiral inactivation (Aβ-CI) as a molecular approach that uses mirror-image peptides to chaperone the natural Aβ stereoisomer into a less toxic state. The oligomer-to-fibril conversion mechanism remains the subject of active research. Perhaps the most striking feature of Aβ-CI is the virtual obliteration of the incubation/induction phase that is so characteristic of Aβ fibril formation kinetics. This qualitative change is indicative of the distinct mechanistic pathway Aβ-CI operates through. The current working model of Aβ-CI invokes the formation of "rippled" cross-β sheets, in which alternating l- and d-peptide strands form periodic networks. However, the assumption of rippled cross-β sheets does not per se explain the dramatic changes in reaction kinetics upon mixing of Aβ enantiomers. Herein, it is shown by DFT computational methods that the individual peptide strands in rippled cross-β networks are less conformationally strained than their pleated counterparts. This means that the adoption of fibril-seeding conformations is more probable for rippled cross-β. Conformational selection is thus suggested as the mechanistic rationale for the acceleration of fibril formation upon Aβ-CI.

Phagocytosis of full-length Tau oligomers by Actin-remodeling of activated microglia

BackgroundAlzheimer’s disease is associated with the accumulation of intracellular Tau tangles within neurons and extracellular amyloid-β plaques in the brain parenchyma, which altogether results in synaptic loss and neurodegeneration. Extracellular concentrations of oligomers and aggregated proteins initiate microglial activation and convert their state of synaptic surveillance into a destructive inflammatory state. Although Tau oligomers have fleeting nature, they were shown to mediate neurotoxicity and microglial pro-inflammation. Due to the instability of oligomers, in vitro experiments become challenging, and hence, the stability of the full-length Tau oligomers is a major concern.MethodsIn this study, we have prepared and stabilized hTau40WT oligomers, which were purified by size-exclusion chromatography. The formation of the oligomers was confirmed by western blot, thioflavin-S, 8-anilinonaphthaalene-1-sulfonic acid fluorescence, and circular dichroism spectroscopy, which determine the intermolecular cross-β sheet structure and hydrophobicity. The efficiency of N9 microglial cells to phagocytose hTau40WT oligomer and subsequent microglial activation was studied by immunofluorescence microscopy with apotome. The one-way ANOVA was performed for the statistical analysis of fluorometric assay and microscopic analysis.ResultsFull-length Tau oligomers were detected in heterogeneous globular structures ranging from 5 to 50 nm as observed by high-resolution transmission electron microscopy, which was further characterized by oligomer-specific A11 antibody. Immunocytochemistry studies for oligomer treatment were evidenced with A11+ Iba1high microglia, suggesting that the phagocytosis of extracellular Tau oligomers leads to microglial activation. Also, the microglia were observed with remodeled filopodia-like actin structures upon the exposure of oligomers and aggregated Tau.ConclusionThe peri-membrane polymerization of actin filament and co-localization of Iba1 relate to the microglial movements for phagocytosis. Here, these findings suggest that microglia modified actin cytoskeleton for phagocytosis and rapid clearance of Tau oligomers in Alzheimer’s disease condition.