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.

Nanoscale Structural Characterization of Amyloid ß 1-42 Oligomers and Fibrils Grown in the Presence of Fatty Acids.

Mono- and polyunsaturated fatty acids (FAs) are broadly used as food supplements. However, their effect on the aggregation of amyloidogenic proteins remains unclear. In this study, we investigated the effect of a large number of mono- and polyunsaturated, as well as fully saturated FAs on the aggregation of amyloid ß1-42 (Aß1-42) peptide. A progressive aggregation of this peptide is the expected molecular cause of Alzheimer's disease (AD), one of the most common neurodegenerative pathologies in the world. We found that arachidonic and stearic acids delayed the aggregation of Aß1-42. Using Nano-Infrared spectroscopy, we found that FAs caused very little if any changes in the secondary structure of Aß1-42 oligomers and fibrils formed at different stages of protein aggregation. However, the analyzed mono- and polyunsaturated, as well as fully saturated FAs uniquely altered the toxicity of Aß1-42 fibrils. We found a direct relationship between the degree of FAs unsaturation and toxicity of Aß1-42 fibrils formed in their presence. Specifically, with an increase in the degree of unsaturation, the toxicity Aß1-42/FA fibrils increased. These results indicate that fully saturated or monounsaturated FAs could be used to decrease the toxicity of amyloid aggregates and, consequently, decelerate the development of AD.

Investigation of Structural Peculiarities of Smooth Muscle Titin Aggregates, Formed under Different In Vitro Conditions, by Small-Angle X-Ray Scattering and Fourier Transform Infrared Spectroscopy.

Small-angle X-ray scattering (SAXS) and Fourier transform infrared (FTIR) spectroscopy were used to investigate structural peculiarities of two types of amyloid aggregates of smooth muscle titin, which differed in their morphology and ability to disaggregate, and differently bound thioflavin T dye. SAXS showed that the structure/shape of the two titin aggregate types was close to a flat shape. FTIR spectroscopy revealed no differences in the secondary structure of the two types. These data suggest that both types of "flat-shape" titin aggregates are identical in their secondary structure and, as shown previously, have a quaternary cross-ß structure. An assumption was made that the most stable supramolecular complexes of a cross-ß structure, which do not differ in their secondary structure, formed first during the aggregation of smooth muscle titin. Then, depending on ambient conditions, these supramolecular structures could form titin aggregates of different morphology and properties.

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 ß-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.

Computational insights into the aggregation mechanism and amyloidogenic core of aortic amyloid medin polypeptide.

Medin amyloid, prevalent in the vessel walls of 97 % of individuals over 50, contributes to arterial stiffening and cerebrovascular dysfunction, yet our understanding of its aggregation mechanism remains limited. Dividing the full-length 50-amino-acid medin peptide into five 10-residue segments, we conducted individual investigations on each segment's self-assembly dynamics via microsecond-timescale atomistic discrete molecular dynamics (DMD) simulations. Our findings showed that medin1-10 and medin11-20 segments predominantly existed as isolated unstructured monomers, unable to form stable oligomers. Medin31-40 exhibited moderate aggregation, forming dynamic ß-sheet oligomers with frequent association and dissociation. Conversely, medin21-30 and medin41-50 segments demonstrated significant self-assembly capability, readily forming stable ß-sheet-rich oligomers. Residue pairwise contact frequency analysis highlighted the critical roles of residues 22-26 and 43-49 in driving the self-assembly of medin21-30 and medin41-50, acting as the ß-sheet core and facilitating ß-strand formation in other regions within medin monomers, expecting to extend to oligomers and fibrils. Regions containing residues 22-26 and 43-49, with substantial self-assembly abilities and assistance in ß-sheet formation, represent crucial targets for amyloid inhibitor drug design against aortic medial amyloidosis (AMA). In summary, our study not only offers deep insights into the mechanism of medin amyloid formation but also provides crucial theoretical and practical guidance for future treatments of AMA.

Study of Insulin Aggregation and Fibril Structure under Different Environmental Conditions.

Protein amyloid aggregation is linked with widespread and fatal neurodegenerative disorders as well as several amyloidoses. Insulin, a small polypeptide hormone, is associated with injection-site amyloidosis and is a popular model protein for in vitro studies of amyloid aggregation processes as well as in the search for potential anti-amyloid compounds. Despite hundreds of studies conducted with this specific protein, the procedures used have employed a vast array of different means of achieving fibril formation. These conditions include the use of different solution components, pH values, ionic strengths, and other additives. In turn, this variety of conditions results in the generation of fibrils with different structures, morphologies and stabilities, which severely limits the possibility of cross-study comparisons as well as result interpretations. In this work, we examine the condition-structure relationship of insulin amyloid aggregation under a range of commonly used pH and ionic strength conditions as well as solution components. We demonstrate the correlation between the reaction solution properties and the resulting aggregation kinetic parameters, aggregate secondary structures, morphologies, stabilities and dye-binding modes.

Multivalency drives interactions of alpha-synuclein fibrils with tau.

Age-related neurodegenerative disorders like Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by deposits of protein aggregates, or amyloid, in various regions of the brain. Historically, aggregation of a single protein was observed to be correlated with these different pathologies: tau in AD and α-synuclein (αS) in PD. However, there is increasing evidence that the pathologies of these two diseases overlap, and the individual proteins may even promote each other's aggregation. Both tau and αS are intrinsically disordered proteins (IDPs), lacking stable secondary and tertiary structure under physiological conditions. In this study we used a combination of biochemical and biophysical techniques to interrogate the interaction of tau with both soluble and fibrillar αS. Fluorescence correlation spectroscopy (FCS) was used to assess the interactions of specific domains of fluorescently labeled tau with full length and C-terminally truncated αS in both monomer and fibrillar forms. We found that full-length tau as well as individual tau domains interact with monomer αS weakly, but this interaction is much more pronounced with αS aggregates. αS aggregates also mildly slow the rate of tau aggregation, although not the final degree of aggregation. Our findings suggest that co-occurrence of tau and αS in disease are more likely to occur through monomer-fiber binding interactions, rather than monomer-monomer or co-aggregation.

Virtual birefringence imaging and histological staining of amyloid deposits in label-free tissue using autofluorescence microscopy and deep learning.

Systemic amyloidosis involves the deposition of misfolded proteins in organs/tissues, leading to progressive organ dysfunction and failure. Congo red is the gold-standard chemical stain for visualizing amyloid deposits in tissue, showing birefringence under polarization microscopy. However, Congo red staining is tedious and costly to perform, and prone to false diagnoses due to variations in amyloid amount, staining quality and manual examination of tissue under a polarization microscope. We report virtual birefringence imaging and virtual Congo red staining of label-free human tissue to show that a single neural network can transform autofluorescence images of label-free tissue into brightfield and polarized microscopy images, matching their histochemically stained versions. Blind testing with quantitative metrics and pathologist evaluations on cardiac tissue showed that our virtually stained polarization and brightfield images highlight amyloid patterns in a consistent manner, mitigating challenges due to variations in chemical staining quality and manual imaging processes in the clinical workflow.

HGA Triggers SAA Aggregation and Accelerates Fibril Formation in the C20/A4 Alkaptonuria Cell Model.

Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder caused by mutations in the homogentisate 1,2-dioxygenase (HGD) gene, leading to the accumulation of homogentisic acid (HGA), causing severe inflammatory conditions. Recently, the presence of serum amyloid A (SAA) has been reported in AKU tissues, classifying AKU as novel secondary amyloidosis; AA amyloidosis is characterized by the extracellular tissue deposition of fibrils composed of fragments of SAA. AA amyloidosis may complicate several chronic inflammatory conditions, like rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, chronic infections, neoplasms, etc. Treatments of AA amyloidosis relieve inflammatory disorders by reducing SAA concentrations; however, no definitive therapy is currently available. SAA regulation is a crucial step to improve AA secondary amyloidosis treatments. Here, applying a comprehensive in vitro and in silico approach, we provided evidence that HGA is a disruptor modulator of SAA, able to enhance its polymerization, fibril formation, and aggregation upon SAA/SAP colocalization. In silico studies deeply dissected the SAA misfolding molecular pathway and SAA/HGA binding, suggesting novel molecular insights about it. Our results could represent an important starting point for identifying novel therapeutic strategies in AKU and AA secondary amyloidosis-related diseases.

CsgA gatekeeper residues control nucleation but not stability of functional amyloid.

Functional amyloids, beneficial to the organism producing them, are found throughout life, from bacteria to humans. While disease-related amyloids form by uncontrolled aggregation, the fibrillation of functional amyloid is regulated by complex cellular machinery and optimized sequences, including so-called gatekeeper residues such as Asp. However, the molecular basis for this regulation remains unclear. Here we investigate how the introduction of additional gatekeeper residues affects fibril formation and stability in the functional amyloid CsgA from E. coli. Step-wise introduction of additional Asp gatekeepers gradually eliminated fibrillation unless preformed fibrils were added, illustrating that gatekeepers mainly affect nucleus formation. Once formed, the mutant CsgA fibrils were just as stable as wild-type CsgA. HSQC NMR spectra confirmed that CsgA is intrinsically disordered, and that the introduction of gatekeeper residues does not alter this ensemble. NMR-based Dark-state Exchange Saturation Transfer (DEST) experiments on the different CsgA variants, however, show a decrease in transient interactions between monomeric states and the fibrils, highlighting a critical role for these interactions in the fibrillation process. We conclude that gatekeeper residues affect fibrillation kinetics without compromising structural integrity, making them useful and selective modulators of fibril properties.

Deciphering the Molecular Dance: Exploring the Dynamic Interplay Between Mouse Insulin B9-23 Peptides and their Variants.

Type 1 diabetes results from the autoimmune destruction of pancreatic insulin-producing ß-cells, primarily targeted by autoreactive T cells that recognize insulin B9-23 peptides as antigens. Using drift tube ion mobility spectrometry-mass spectrometry, transmission electron microscopy, and two-dimensional infrared spectroscopy, we characterized mouse insulin 1 B9-23 (Ins1 B9-23), insulin 2 B9-23 (Ins2 B9-23), along with two of their mutants, Ins2 B9-23 Y16A and Ins2 B9-23 C19S. Our findings indicate that Ins1 B9-23 and the Ins2 Y16A mutant exhibit rapid fibril formation, whereas Ins2 B9-23 and the Ins2 C19S mutant show slower fibrillization and a structural rearrangement from globular protofibrils to fibrillar aggregates. These differences in aggregation behaviors also manifest in interactions with (-)epigallocatechin gallate (EGCG), a canonical amyloid inhibitor. EGCG effectively disrupts the fibrils formed by Ins1 B9-23 and the Y16A mutant. However, it proves ineffective in preventing fibril formation of Ins2 B9-23 and the C19S mutant. These results establish a strong correlation between the aggregation behaviors of these peptides and their divergent effects on anti-islet autoimmunity.

Graphitic nanoflakes modulate the structure and binding of human amylin.

Human amylin is an inherently disordered protein whose ability to form amyloid fibrils is linked to the onset of type II diabetes. Graphitic nanomaterials have potential in managing amyloid diseases as they can disrupt protein aggregation processes in biological settings, but optimising these materials to prevent fibrillation is challenging. Here, we employ bias-exchange molecular dynamics simulations to systematically study the structure and adsorption preferences of amylin on graphitic nanoflakes that vary in their physical dimensions and surface functionalisation. Our findings reveal that nanoflake size and surface oxidation both influence the structure and adsorption preferences of amylin. The purely hydrophobic substrate of pristine graphene (PG) nanoflakes encourages non-specific protein adsorption, leading to unrestricted lateral mobility once amylin adheres to the surface. Particularly on larger PG nanoflakes, this induces structural changes in amylin that may promote fibril formation, such as the loss of native helical content and an increase in ß-sheet character. In contrast, oxidised graphene nanoflakes form hydrogen bonds between surface oxygen sites and amylin, and as such restricting protein mobility. Reduced graphene oxide (rGO) flakes, featuring lower amounts of surface oxidation, are amphiphilic and exhibit substantial regions of bare carbon which promote protein binding and reduced conformational flexibility, leading to conservation of the native structure of amylin. In comparison, graphene oxide (GO) nanoflakes, which are predominantly hydrophilic and have a high degree of surface oxidation, facilitate considerable protein structural variability, resulting in substantial contact area between the protein and GO, and subsequent protein unfolding. Our results indicate that tailoring the size, oxygen concentration and surface patterning of graphitic nanoflakes can lead to specific and robust protein binding, ultimately influencing the likelihood of fibril formation. These atomistic insights provide key design considerations for the development of graphitic nanoflakes that can modulate protein aggregation by sequestering protein monomers in the biological environment and inhibit conformational changes linked to amyloid fibril formation.

[Clinical Implementation of Amyloid PET: Latest Findings and Practical Approaches in the Diagnosis and Treatment of Alzheimer's Disease].

Amyloid PET plays a crucial role in the early diagnosis of Alzheimer's disease and the determination of the feasibility of disease-modifying therapies. It offers several advantages, including high sensitivity and specificity, minimal invasiveness, and the ability to provide spatial evaluation, all of which contribute to the optimization of dementia care. However, proper use and interpretation of the results require a thorough understanding of their limitations. Although careful consideration is necessary when using scans on asymptomatic individuals, clinical applications could broaden if preemptive treatments and high-precision individual risk assessments for the preclinical stage are developed.

Controlled preparation of tannic acid-derived carbonized dots and their use to inhibit amyloid aggregation and promote aggregate disaggregation.

Tannic acid (TA)-derived carbon dots (TACDs) were synthesized for the first time via a solvothermal method using TA as one of the raw materials, which may effectively inhibit amyloid fibril aggregation and disaggregate mature fibril. The fluorescent property of TACDs were modulated by adjusting the ratio of TA to o-phenylenediamine (oPD), and TACDs fabricated with the precursor ratio as 1:1 showed the best fluorescent property. Circular dichroism spectra (CD) showed that the structure of ß-sheet decreased as the concentration of TACDs increased. The inhibition efficiency, as confirmed by thioflavin T (ThT) and transmission electron microscopy (TEM), is extraordinary at 98.16%, whereas disaggregation efficiency is noteworthy at 97.97%, and the disaggregated lysozyme fibrils did not reaggregate after 7 days. More critically, TACDs can also alleviate the cellular toxicity caused by Aß fibrils and improve cell viability. This work offers a new perspective on the design of scavengers for amyloid plaques.

Converting the Amyloidogenic Islet Amyloid Polypeptide into a Potent Nonaggregating Peptide Ligand by Side Chain-to-Side Chain Macrocyclization.

The islet amyloid polypeptide (IAPP), also known as amylin, is a hormone playing key physiological roles. However, its aggregation and deposition in the pancreatic islets are associated with type 2 diabetes. While this peptide adopts mainly a random coil structure in solution, its secondary conformational conversion into α-helix represents a critical step for receptor activation and contributes to amyloid formation and associated cytotoxicity. Considering the large conformational landscape and high amyloidogenicity of the peptide, as well as the complexity of the self-assembly process, it is challenging to delineate the delicate interplay between helical folding, peptide aggregation, and receptor activation. In the present study, we probed the roles of helical folding on the function-toxicity duality of IAPP by restricting its conformational ensemble through side chain-to-side chain stapling via azide-alkyne cycloaddition. Intramolecular macrocyclization (i; i + 4) constrained IAPP into α-helix and inhibited its aggregation into amyloid fibrils. These helical derivatives slowed down the self-assembly of unmodified IAPP. Site-specific macrocyclization modulated the capacity of IAPP to perturb lipid bilayers and cell plasma membrane and reduced, or even fully inhibited, the cytotoxicity associated with aggregation. Furthermore, the α-helical IAPP analogs showed moderate to high potency toward cognate G protein-coupled receptors. Overall, these results indicate that macrocyclization represents a promising strategy to protect an amyloidogenic peptide hormone from aggregation and associated toxicity, while maintaining high receptor activity.

Hybrid Amyloid Quantum Dot Nano-Bio Assemblies to Probe Neuroinflammatory Damage.

Various oligomeric species of amyloid-beta have been proposed to play different immunogenic roles in the cellular pathology of Alzheimer's Disease. The dynamic interconversion between various amyloid oligomers and fibrillar assemblies makes it difficult to elucidate the role each potential aggregation state may play in driving neuroinflammatory and neurodegenerative pathology. The ability to identify the amyloid species that are key and essential drivers of these pathological hallmarks of Alzheimer's Disease is of fundamental importance for also understanding downstream events including tauopathies that mediate neuroinflammation with neurologic deficits. Here, we report the design and construction of a quantum dot mimetic for larger spherical oligomeric amyloid species as an "endogenously" fluorescent proxy for this cytotoxic assembly of amyloid to investigate its role in inducing inflammatory and stress response states in neuronal and glial cell types. The design parameters and construction protocol developed here may be adapted for developing quantum dot nano-bio assemblies for other biological systems of interest, particularly neurodegenerative diseases involving other protein aggregates.

Antioxidant amyloid fibril derived from rice protein hydrolysate as stabilizer towards preparing high-stable emulsion.

An antioxidant amyloid fibril was prepared as an emulsifier by fibrillating limited enzymatic hydrolysis-modified rice protein (HRP). The purpose of this study was to investigate the feasibility of using fibrillated HRP to stabilize oil-in-water emulsion. A free radical scavenging assay revealed that the antioxidant activity of fibrillated HRP was 2.09 times higher than that of native rice protein. Fibrillated HRP demonstrated a marked reduction in interfacial tension, increased surface hydrophobicity and contact angle (> 80°), and rapid adsorption to the interface, with 35.34 ± 2.43% interfacial adsorbed protein content. The fibrillated HRP barriers resisted environment stresses such as NaCl, pH variations, long-term storage, while reducing lipid oxidation degree. Additionally, fibrillated HRP-based emulsion was more effective in protecting ß-carotene from degradation compared to other samples. These findings provide theoretical support for the development of rice protein-based antioxidant emulsifiers and modification of emulsifying properties of plant proteins.

Amyloid transformations of phenol soluble modulin α1 in Staphylococcus aureus and their modulation deploying a prenylated chalcone.

Phenol soluble modulins (PSMs) are small amphipathic peptides involved in a series of biological functions governing staphylococcal pathogenesis, primarily by facilitating the formation of an extracellular fibril structure with amyloid-like properties. This fibrillar architecture stabilizes the staphylococcal biofilm making it resilient to antibiotic treatment. Our study aims to abrogate the amyloid fibrillation of PSM α1 with novel insights on the amyloid modulatory potential of a prenylated chalcone, Isobavachalcone (IBC). A combination of biophysical and computational assays to address the amyloid modulatory effect of IBC has been undertaken to arrive at a model for the inhibition of PSM α1 fibrillation. ThT kinetics studies indicated that IBC must be stably interacting with the amyloidogenic core of PSM α1 monomers or it may be inhibiting the pre-fibrillar aggregates populated at the early stages of amyloid transformation kinetics. This heteromolecular association further inhibits the amyloid transformation corroborated by a ∼ 94% and ∼ 91% reduction in the ThT maxima, even at sub-stoichiometric concentrations. Transmission electron microscopy (TEM) of end-stage aggregates (∼ 55 h) depict mature, inter-twined, laterally stacked amyloid fibrils in untreated PSM α1 samples while this fibrillar load is remarkably reduced in the presence of IBC. The inhibitory effect of IBC on the ß-sheet transitions of PSM α1 were also validated using far-UV CD spectra. Molecular dynamics simulation studies with PSM aggregates (PSM-A) have also suggested that IBC disrupts the hydrogen bonding interactions and corroborates the inhibition of alpha to beta transitions of PSM-A. Collectively, our data proposes a novel structural motif for the rational discovery of non-toxic therapeutic agents targeting the functional amyloids which have slowly emerged as potent factors, consolidating the antibiotic resistant staphylococcal biofilm assembly.

Quinoline yellow acts as a novel amyloid fibrillation probe by using surface-enhanced Raman spectroscopy.

Protein amyloid fibrillation is linked to a wide range of neurodegenerative diseases. Protein oligomer is an intermediate substance in the process of fibrillation, which is neurotoxic and formed by the aggregation of protein molecules under physiological stress. Early detection of protein oligomers could make timely intervention of protein fibrillation related diseases. Therefore, it is crucial to develop efficient inhibitors and probes for monitoring amyloid fibril formation. In this study, we developed a novel amyloid inhibitor quinoline yellow (QY), which was proved to be effective in inhibiting insulin protein fibrillation as demonstrated by fluorescence, morphology characterization and circular dichroism. When QY binds to insulin, it exerts inhibitory effects on the nucleation process and effectively impedes the formation of fibrillar fibrils. In addition, we present the application of surface-enhanced Raman spectroscopy (SERS) as an extremely sensitive technique for identifying amyloid oligomers. The investigation employed the probe QY, which demonstrated a linear reaction for identifying oligomers in the concentration range of 1.0-58.0 µM. Impressively, it showcased an exceptionally sensitive detection threshold of 0.2 µM. And also illustrating the binding sites and interaction mechanisms between small molecules of QY and insulin by SERS. The aforementioned methodology was also employed for the identification of insulin oligomers in human serum samples. Thereby, the proposed approach presenting a promising avenue with extensive implications in the realms of pharmaceutical exploration and disease diagnosis.