Amyloid-like Assemblies Research Articles

Uncovering the membrane and pore formation mechanisms of the lysozyme membrane made via the amyloid-like assembly

Biomimetic protein membranes have attracted increasing interests because of their distinctive separation properties in the membrane field. Low-cost and stable protein lysozyme has been intensively invetigated to fabricate high-performance sepration membranes via a facile and scalable method of amyloid-like assembly. However, the pore formation mechnism of the lysozyme membrane remains unclear. Herein, the lysozyme membranes were fabricated and the membrane formation mechnism and separation properties were investigated experimentally. Molecular dynamics simulation (MDS) was also employed to reveal the pore formation mechanism of the lysozyme membrane. The fabricated lysozyme membrane achieved a stable high water permeance of 10.2 L m−2 h−1 bar−1, as well as a relatively high separation factor (9.5) towards antibiotic and sodium chloride. MDS results found that the membrane pore size is governed by the amino acid compositions and is negatively dependent on the intermolecular forces among amino acids around the pores. Selective pores with 0.54 nm in diameter are primarily formed by these amino acids including alanine (35 %), aspartic acid (25 %), arginine (15 %), phenylalanine (11 %), and lysine (7 %), in which half of them have hydrophobic side chains with no charge, and another half have hydrophilic side chains with balanced charges. This work may stimulate the development of highly permeable and selective protein membranes by carefully engineering their amino acid compositions with optimal charges and hydrophobicity for generation of numerous selective pores.

Surface-catalyzed liquid–liquid phase separation and amyloid-like assembly in microscale compartments

Liquid-liquid phase separation is a key phenomenon in the formation of membrane-less structures within the cell, appearing as liquid biomolecular condensates. Protein condensates are the most studied for their biological relevance, and their tendency to evolve, resulting in the formation of aggregates with a high level of order called amyloid.In this study, it is demonstrated that Human Insulin forms micrometric, round amyloid-like structures at room temperature within sub-microliter scale aqueous compartments. These distinctive particles feature a solid core enveloped by a fluid-like corona and form at the interface between the aqueous compartment and the glass coverslip upon which they are cast. Quantitative fluorescence microscopy is used to study in real-time the formation of amyloid-like superstructures. Their formation results driven by liquid–liquid phase separation process that arises from spatially heterogeneous distribution of nuclei at the glass-water interface. The proposed experimental setup allows modifying the surface-to-volume ratio of the aqueous compartments, which affects the aggregation rate and particle size, while also inducing fine alterations in the molecular structures of the final assemblies.These findings enhance the understanding of the factors governing amyloid structure formation, shedding light on the catalytic role of surfaces in this process.

The Characterization of Multifaceted PREP1 Peptides Provides Insights into Correlations between Spectroscopic and Structural Properties of Amyloid-like Assemblies.

The widespread ability of proteins and peptides to self-assemble by forming cross-β structure is one of the most significant discoveries in structural biology. Intriguingly, the cross-β association of proteins/peptides may generate intricate supramolecular architectures with uncommon spectroscopic properties. We have recently characterized self-assembled peptides extracted from the PREP1 protein that are endowed with interesting structural/spectroscopic properties. We here demonstrate that the green fluorescence emission of the peptide PREP1[117-132] (λem ~520 nm), can be induced by excitation with UV radiation. The associated unusually large Stokes shift (Δλ ~150 nm) represents, to the best of our knowledge, the first evidence of an internal resonance energy transfer in amyloid-like structures, where the blue emission of some assemblies becomes the excitation radiation for others. Moreover, the characterization of PREP1[117-132] variants provides insights into the sequence/structure and structure/spectroscopic properties relationships. Our data suggests that the green fluorescence is plausibly associated with antiparallel β-sheet states of the peptide whereas parallel β-sheet assemblies are only endowed with blue fluorescence. Notably, the different PREP1[117-132] variants also form assemblies characterized by distinct morphologies. Indeed, the parent peptide and single mutants form compact but structured aggregates whereas most of the double mutants exhibit elongated and highly extended fibers.

Metal Ions Can Modulate the Self-Assembly and Activity of Catalytic Peptide Amyloids.

Rational design of peptides has become a powerful tool to produce self-assembled nanostructures with the ability to catalyze different chemical reactions, paving the way to develop minimalistic enzyme-like nanomaterials. Catalytic amyloid-like assemblies have emerged among the most versatile and active, but they often require additional factors for activity. Elucidating how these factors influence the structure and activity is key for the design. Here, we showed that biologically relevant metal ions can guide and modulate the self-assembly of a small peptide into diverse amyloid architectures. The morphology and catalytic activity of the resulting fibrils were tuned by the specific metal ion decorating the surface, whereas X-ray structural analysis of the amyloids showed ion-dependent shape sizes. Molecular dynamics simulations showed that the metals can strongly affect the local conformational space, which can trigger major rearrangements of the fibrils. Our results demonstrate that the conformational landscape of catalytic amyloids is broad and tunable by external factors, which can be critical for future design strategies.

Targeting phenylalanine assemblies as a prospective disease-modifying therapy for phenylketonuria

Phenylketonuria is characterized by the accumulation of phenylalanine, resulting in severe cognitive and neurological disorders if not treated by a remarkably strict diet. There are two approved drugs today, yet both provide only a partial solution. We have previously demonstrated the formation of amyloid-like toxic assemblies by aggregation of phenylalanine, suggesting a new therapeutic target to be further pursued. Moreover, we showed that compounds that halt the formation of these assemblies also prevent their resulting toxicity. Here, we performed high-throughput screening, searching for compounds with inhibitory effects on phenylalanine aggregation. Morin hydrate, one of the most promising hits revealed during the screen, was chosen to be tested in vivo using a phenylketonuria mouse model. Morin hydrate significantly improved cognitive and motor function with a reduction in the number of phenylalanine brain deposits. Moreover, while phenylalanine levels remained high, we observed a recovery in dopaminergic, adrenergic, and neuronal markers. To conclude, the ability of Morin hydrate to halt phenylalanine aggregation without reducing phenylalanine levels implies the toxic role of the phenylalanine assemblies in phenylketonuria and opens new avenues for disease-modifying treatment.

Characterization of amyloid-like metal-amino acid assemblies with remarkable catalytic activity.

While enzymes are potentially useful in various applications, their limited operational stability and production costs have led to an extensive search for stable catalytic agents that will retain the efficiency, specificity, and environmental-friendliness of natural enzymes. Despite extensive efforts, there is still an unmet need for improved enzyme mimics and novel concepts to discover and optimize such agents. Inspired by the catalytic activity of amyloids and the formation of amyloid-like assemblies by metabolites, our group pioneered the development of novel metabolite-metal co-assemblies (bio-nanozymes) that produce nanomaterials mimicking the catalytic function of common metalloenzymes that are being used for various technological applications. In addition to their notable activity, bio-nanozymes are remarkably safe as they are purely composed of amino acids and minerals that are harmless to the environment. The bio-nanozymes exhibit high efficiency and exceptional robustness, even under extreme conditions of temperature, pH, and salinity that are impractical for enzymes. Our group has recently also demonstrated the formation of ordered amino acid co-assemblies showing selective and preferential interactions comparable to the organization of residues in folded proteins. The identified bio-nanozymes can be used in various applications including environmental remediation, synthesis of new materials, and green energy.

Tuning Phase Transition of Molecular Self-Assembly by Artificial Chaperones through Aromatic-Aromatic Interactions.

The molecular chaperones are essential and play significant roles in controlling the protein phase transition and maintaining physiological homeostasis. However, manipulating phase transformation in biomimetic peptide self-assembly is still challenging. This work shows that an artificial chaperone modulates the energy landscape of supramolecular polymerization, thus controlling the phase transition of amyloid-like assemblies from crystals to hydrogels to solution. The absence of a chaperone allows the NapP to form crystals, while the presence of the chaperone biases the pathway to form nanofibrous hydrogels to soluble oligomers by adjusting the chaperone ratios. Mechanistic studies reveal that the aromatic-aromatic interaction is the key to trapping the molecules in a higher energy fold. Adding the chaperone relieves this restriction, lowers the energy barrier, and transforms the crystal into a hydrogel. This phase transformation can also be achieved in the macromolecular crowding environment, thus providing new insights into understanding molecular self-assembly in multiple component systems.

Hfq C-terminal region forms a β-rich amyloid-like motif without perturbing the N-terminal Sm-like structure

Hfq is a pleitropic actor that serves as stress response and virulence factor in the bacterial cell. To execute its multiple functions, Hfq assembles into symmetric torus-shaped hexamers. Extending outward from the hexameric core, Hfq presents a C-terminal region, described as intrinsically disordered in solution. Many aspects of the role and the structure of this region remain unclear. For instance, in its truncated form it can promote amyloid-like filament assembly. Here, we show that a minimal 11-residue motif at the C-terminal end of Hfq assembles into filaments with amyloid characteristics. Our data suggest that the full-length Hfq in its filamentous state contains a similar molecular fingerprint than that of the short β-strand peptide, and that the Sm-core structure is not affected by filament formation. Hfq proteins might thus co-exist in two forms in vivo, either as isolated, soluble hexamers or as self-assembled hexamers through amyloid-reminiscent interactions, modulating Hfq cellular functions.

Molecular characterization of the N-terminal half of TasA during amyloid-like assembly and its contribution to Bacillus subtilis biofilm formation

Biofilms are bacterial communities that result from a cell differentiation process leading to the secretion of an extracellular matrix (ECM) by part of the population. In Bacillus subtilis, the main protein component of the ECM is TasA, which forms a fiber-based scaffold that confers structure to the ECM. The N-terminal half of TasA is strongly conserved among Bacillus species and contains a protein domain, the rigid core (RcTasA), which is critical for the structural and functional properties of the recombinant protein. In this study, we demonstrate that recombinantly purified RcTasA in vitro retains biochemical properties previously observed for the entire protein. Further analysis of the RcTasA amino acid sequence revealed two aggregation-prone stretches and a region of imperfect amino acid repeats, which are known to contribute to functional amyloid assembly. Biochemical characterization of these stretches found in RcTasA revealed their amyloid-like capacity in vitro, contributing to the amyloid nature of RcTasA. Moreover, the study of the imperfect amino acid repeats revealed the critical role of residues D64, K68 and D69 in the structural function of TasA. Experiments with versions of TasA carrying the substitutions D64A and K68AD69A demonstrated a partial loss of function of the protein either in the assembly of the ECM or in the stability of the core and amyloid-like properties. Taken together, our findings allow us to better understand the polymerization process of TasA during biofilm formation and provide knowledge into the sequence determinants that promote the molecular behavior of protein filaments in bacteria.

Histidine modulates amyloid-like assembly of peptide nanomaterials and confers enzyme-like activity

Amyloid-like assembly is not only associated with pathological events, but also leads to the development of novel nanomaterials with unique properties. Herein, using Fmoc diphenylalanine peptide (Fmoc–F–F) as a minimalistic model, we found that histidine can modulate the assembly behavior of Fmoc–F–F and induce enzyme-like catalysis. Specifically, the presence of histidine rearranges the β structure of Fmoc–F–F to assemble nanofilaments, resulting in the formation of active site to mimic peroxidase-like activity that catalyzes ROS generation. A similar catalytic property is also observed in Aβ assembled filaments, which is correlated with the spatial proximity between intermolecular histidine and F-F. Notably, the assembled Aβ filaments are able to induce cellular ROS elevation and damage neuron cells, providing an insight into the pathological relationship between Aβ aggregation and Alzheimer’s disease. These findings highlight the potential of histidine as a modulator in amyloid-like assembly of peptide nanomaterials exerting enzyme-like catalysis.

Realization of Amyloid-like Aggregation as a Common Cause for Pathogenesis in Diseases.

Amyloids were conventionally referred to as extracellular and intracellular accumulation of Aβ42 peptide, which causes the formation of plaques and neurofibrillary tangles inside the brain leading to the pathogenesis in Alzheimer's disease. Subsequently, amyloid-like deposition was found in the etiology of prion diseases, Parkinson's disease, type II diabetes, and cancer, which was attributed to the aggregation of prion protein, α-Synuclein, islet amyloid polypeptide protein, and p53 protein, respectively. Hence, traditionally amyloids were considered aggregates formed exclusively by proteins or peptides. However, since the last decade, it has been discovered that other metabolites, like single amino acids, nucleobases, lipids, glucose derivatives, etc., have a propensity to form amyloid-like toxic assemblies. Several studies suggest direct implications of these metabolite assemblies in the patho-physiology of various inborn errors of metabolisms like phenylketonuria, tyrosinemia, cystinuria, and Gaucher's disease, to name a few. In this review, we present a comprehensive literature overview that suggests amyloid-like structure formation as a common phenomenon for disease progression and pathogenesis in multiple syndromes. The review is devoted to providing readers with a broad knowledge of the structure, mode of formation, propagation, and transmission of different extracellular amyloids and their implications in the pathogenesis of diseases. We strongly believe a review on this topic is urgently required to create awareness about the understanding of the fundamental molecular mechanism behind the origin of diseases from an amyloid perspective and possibly look for a common therapeutic strategy for the treatment of these maladies by designing generic amyloid inhibitors.

Amyloid-Like Assembly to Form Film at Interfaces: Structural Transformation and Application.

Protein-based biomaterials are attracting broad interest for their remarkable structural and functional properties. Disturbing the native protein's three-dimensional structural stability in vitro and controlling subsequent aggregation is an effective strategy to design and construct protein-based biomaterials. One of the recent developments in regulating protein structural transformation to ordered aggregation is amyloid assembly, which generates fibril-based 1D to 3D nanostructures as functional materials. Especially, the amyloid-like assembly to form films at interfaces has been reported, which is induced by the effective reduction of the intramolecular disulfide bond. The main contribution of this amyloid-like assembly is the large-scale formation of protein films at interfaces and excellent adhesion to target substrates. This review presents the research progress of the amyloid-like assembly to form films and related applications and thereby provides a guide to exploiting protein-based biomaterials. This article is protected by copyright. All rights reserved.

Electrostatic Modulation of Intramolecular and Intermolecular Interactions during the Formation of an Amyloid-like Assembly.

The mechanism of protein aggregation can be broadly viewed as a shift from the native-state stabilizing intramolecular to the aggregated-phase sustaining intermolecular interactions. Understanding the role of electrostatic forces on the extent of modulation of this switch has recently evolved as a topic of monumental significance as protein aggregation has lately been connected to charge modifications of an aging proteome. To decipher the distinctive role of electrostatic forces on the extremely complicated phase separation landscape, we opted for a combined in vitro-in silico approach to ascertain the structure-dynamics-stability-aggregability relationship of the functional tandem RRM domains of the ALS-related protein TDP-43 (TDP-43tRRM), under a bivariate solution condition in terms of pH and salt concentration. Under acidic pH conditions, the native TDP-43tRRM protein creates an aggregation-prone entropically favorable partially unfolded conformational landscape due to enthalpic destabilization caused by the protonation of the buried ionizable residues and consequent overwhelming fluctuations of selective segments of the sequence leading to anti-correlated movements of the two domains of the protein. The evolved fluffy ensemble with a comparatively exposed backbone then easily interacts with incoming protein molecules in the presence of salt via typical amyloid-aggregate-like intermolecular backbone hydrogen bonds with a considerable contribution originating from the dispersion forces. Subsequent exposure to excess salt at low pH conditions expedites the aggregation process via an electrostatic screening mechanism where salt shows preferential binding to the positively charged side chain. The applied target observable-specific approach complementarity unveils the hidden information landscape of an otherwise complex process with unquestionable conviction.

The Action of Chemical Denaturants: From Globular to Intrinsically Disordered Proteins.

Proteins perform their many functions by adopting either a minimal number of strictly similar conformations, the native state, or a vast ensemble of highly flexible conformations. In both cases, their structural features are highly influenced by the chemical environment. Even though a plethora of experimental studies have demonstrated the impact of chemical denaturants on protein structure, the molecular mechanism underlying their action is still debated. In the present review, after a brief recapitulation of the main experimental data on protein denaturants, we survey both classical and more recent interpretations of the molecular basis of their action. In particular, we highlight the differences and similarities of the impact that denaturants have on different structural classes of proteins, i.e., globular, intrinsically disordered (IDP), and amyloid-like assemblies. Particular attention has been given to the IDPs, as recent studies are unraveling their fundamental importance in many physiological processes. The role that computation techniques are expected to play in the near future is illustrated.

A Comprehensive Analysis of the Intrinsic Visible Fluorescence Emitted by Peptide/Protein Amyloid-like Assemblies.

Amyloid aggregation is a widespread process that involves proteins and peptides with different molecular complexity and amino acid composition. The structural motif (cross-β) underlying this supramolecular organization generates aggregates endowed with special mechanical and spectroscopic properties with huge implications in biomedical and technological fields, including emerging precision medicine. The puzzling ability of these assemblies to emit intrinsic and label-free fluorescence in regions of the electromagnetic spectrum, such as visible and even infrared, usually considered to be forbidden in the polypeptide chain, has attracted interest for its many implications in both basic and applied science. Despite the interest in this phenomenon, the physical basis of its origin is still poorly understood. To gain a global view of the available information on this phenomenon, we here provide an exhaustive survey of the current literature in which original data on this fluorescence have been reported. The emitting systems have been classified in terms of their molecular complexity, amino acid composition, and physical state. Information about the wavelength of the radiation used for the excitation as well as the emission range/peak has also been retrieved. The data collected here provide a picture of the complexity of this multifaceted phenomenon that could be helpful for future studies aimed at defining its structural and electronic basis and/or stimulating new applications.

Cell scientist to watch – Elvan Böke

ABSTRACT Elvan Böke studied molecular biology and genetics at the Middle East Technical University in Ankara, Turkey. She then moved to the Cancer Research UK Manchester Institute (CRUK-MI) for a PhD with Iain Hagan, where she discovered that a PP1–PP2A phosphatase relay promotes mitotic exit in fission yeast. For her postdoc, she moved to the lab of Tim Mitchison at Harvard Medical School, Boston, USA, where she unraveled how the enigmatic Balbiani body is formed by an amyloid-like assembly mechanism in oocytes. Elvan established her group at the Centre for Genomic Regulation (CRG) Barcelona, Spain, in 2017, where her lab investigates oocyte biology and cellular dormancy. She was awarded an ERC Starting Grant in 2017 and an ERC Consolidator Grant in 2022. In 2021, Elvan became a member of the EMBO Young Investigator Network.

Multistep molecular mechanism of amyloid-like aggregation of nucleic acid-binding domain of TDP-43.

TDP-43 protein is associated with many neurodegenerative diseases and has been shown to adopt various oligomeric and fibrillar states. However, a detailed kinetic understanding of the structural transformation of the native form of the protein to the fibrillar state is missing. In this study, we delineate the temporal sequence of structural events during the amyloid-like assembly of the functional nucleic acid-binding domain of TDP-43. We kinetically mapped the aggregation process using multiple probes such as tryptophan and thioflavin T (ThT) fluorescence, circular dichroism (CD), and dynamic light scattering (DLS) targeting different structural events. Our data reveal that aggregation occurs in four distinct steps-very fast, fast, slow, and very slow. The "very fast" change results in partially unfolded forms that undergo conformational conversion, oligomerization and bind to ThT in the "fast step" to form higher order intermediates (HOI). The temporal sequence of the formation of ThT binding sites and conformational conversion depends upon the protein concentration. The HOI further undergoes structural rearrangement to form protofibrils in the "slow" step, which, consequently, assembles in the "very slow" step to form an amyloid-like assembly. The spectroscopic properties of the amyloid-like assembly across the protein concentration remain similar. Additionally, we observe no lag phase across protein concentration for all the probes studied, suggesting that the aggregation process follows a linear polymerization reaction. Overall, our study demonstrates that the amyloid-like assembly forms in multiple steps, which is also supported by the temperature dependence of the kinetics.

Lysozyme@Fe3O4 composite adsorbents with enhanced adsorption rates and uranium/vanadium selectivity by photothermal property

Herein, magnetic lysozyme@Fe3O4 composites have been fabricated via the amyloid-like assembly for the uranium extraction. The Fe3O4 nanoparticles and lysozyme coatings endow the composite adsorbents with magnetism for facile recovery and excellent binding affinity towards uranium, respectively. In addition, the composite adsorbents also possess outstanding photothermal properties derived from the Fe3O4 nanoparticles. The photothermal property benefits not only adsorption rates but also selectivity. The solution temperatures would increase under the sunlight, resulting in faster diffusion rates of uranyl ions and more ions could be contacted with the composite adsorbents. Moreover, the adsorption enthalpies of uranium are much higher than that of vanadium. Therefore, higher solution temperatures could generate higher uranium/vanadium selectivity.

Amyloid-like coatings decorated electrodes boost the uranium electro-adsorption from seawater

Electro-adsorption has been regarded as one of the most effective methods for uranium extraction from seawater since this method could gather ultralow-concentration uranyl ions at the adsorbent surfaces for higher adsorption capacity and adsorption rates. However, the marine microorganisms and pollutants would also migrate to the adsorbent surfaces, causing serious inhibiting effects. Herein, the pristine electrodes were modified by the convenient amyloid-like assembly to solve the above problem. The unfolded bovine serum albumin (BSA) coatings not only offer adsorption sites toward uranium but also enable the electrodes with excellent anti-fouling and anti-adhesion properties. The modified electrodes achieved superior reusability and long-term stability even with the interferences of various pollutants and microorganisms. To the best of our knowledge, the overlooked but pernicious influences of the fouling phenomenon are firstly investigated in electro-adsorption. This finding not only removes the barriers in the practical applications of electro-adsorption, but also guides the design strategy of adsorbents for uranium extraction.

Clearance of an amyloid-like translational repressor is governed by 14-3-3 proteins.

SUMMARYAmyloids are fibrous protein aggregates associated with age-related diseases. While these aggregates are typically described as irreversible and pathogenic, some cells use reversible amyloid-like structures that serve important functions. The RNA-binding protein Rim4 forms amyloid-like assemblies that are essential for translational control during Saccharomyces cerevisiae meiosis. Rim4 amyloid-like assemblies are disassembled in a phosphorylation-dependent manner at meiosis II onset. By investigating Rim4 clearance, we elucidate co-factors that mediate clearance of amyloid-like assemblies in a physiological setting. We demonstrate that yeast 14-3-3 proteins bind to Rim4 assemblies and facilitate their subsequent phosphorylation and timely clearance. Furthermore, distinct 14-3-3 proteins play non-redundant roles in facilitating phosphorylation and clearance of amyloid-like Rim4. Additionally, we find that 14-3-3 proteins contribute to global protein aggregate homeostasis. Based on the role of 14-3-3 proteins in aggregate homeostasis and their interactions with disease-associated assemblies, we propose that these proteins may protect against pathological protein aggregates.