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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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Amylin is part of the endocrine pancreatic system that contributes to glycemic control, regulating blood glucose levels. However, human amylin has a high tendency to aggregate, forming isolated amylin deposits that are observed in patients with type 2 diabetes mellitus. In search of new inhibitors of amylin aggregation, we undertook the chemical analyses of five marine macroorganisms encountered in high populations in the Red Sea and selected a panel of 10 metabolites belonging to different chemical classes to evaluate their ability to inhibit the formation of amyloid deposits in the human amylin peptide. The thioflavin T assay was used to examine the kinetics of amyloid aggregation, and atomic force microscopy was employed to conduct a thorough morphological examination of the formed fibrils. The potential ability of these compounds to interact with the backbone of peptides and compete with ß-sheet formation was analyzed by quantum calculations, and the interactions with the amylin peptide were computationally examined using molecular docking. Despite their structural similarity, it could be observed that the hydrophobic and hydrogen bond interactions of pyrrolidinones 9 and 10 with the protein sheets result in one case in a stable aggregation, while in the other, they cause distortion from aggregation.

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The inhibition of amyloid-ß (Aß) fibrillation and clearance of Aß aggregates have emerged as a potential pharmacological strategy to alleviate Aß aggregate-induced neurotoxicity in Alzheimer's disease (AD). Maity et al. shortlisted ADH-353 from a small library of positively charged N-substituted oligopyrrolamides for its notable ability to inhibit Aß fibrillation, disintegrate intracellular cytotoxic Aß oligomers, and alleviate Aß-induced cytotoxicity in the SH-SY5Y and N2a cells. However, the molecular mechanism through which ADH-353 interacts with the Aß42 fibrils, leading to their disruption and subsequent clearance, remains unclear. Thus, a detailed molecular mechanism underlying the disruption of neurotoxic Aß42 fibrils (PDB ID 2NAO) by ADH-353 has been illuminated in this work using molecular dynamics simulations. Interestingly, conformational snapshots during simulation depicted the shortening and disappearance of ß-strands and the emergence of a helix conformation, indicating a loss of the well-organized ß-sheet-rich structure of the disease-relevant Aß42 fibril on the incorporation of ADH-353. ADH-353 binds strongly to the Aß42 fibril (ΔGbinding= -142.91 ± 1.61 kcal/mol) with a notable contribution from the electrostatic interactions between positively charged N-propylamine side chains of ADH-353 with the glutamic (Glu3, Glu11, and Glu22) and aspartic (Asp7 and Asp23) acid residues of the Aß42 fibril. This aligns well with heteronuclear single quantum coherence NMR studies, which depict that the binding of ADH-353 with the Aß peptide is driven by electrostatic and hydrophobic contacts. Furthermore, a noteworthy decrease in the binding affinity of Aß42 fibril chains on the incorporation of ADH-353 indicates the weakening of interchain interactions leading to the disruption of the double-horseshoe conformation of the Aß42 fibril. The illumination of key interactions responsible for the destabilization of the Aß42 fibril by ADH-353 in this work will greatly aid in designing new chemical scaffolds with enhanced efficacy for the clearance of Aß aggregates in AD.

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Quantifying the formation and decomposition of amyloid is a crucial issue in the development of new drugs and therapies for treating amyloidosis. The current technologies for grasping amyloid formation and decomposition include fluorescence analysis using thioflavin-T, secondary structure analysis using circular dichroism, and image analysis using atomic force microscopy or transmission electron microscopy. These technologies typically require spectroscopic devices or expensive nanoscale imaging equipment and involve lengthy analysis, which limits the rapid screening of amyloid-degrading drugs. In this study, we introduce a technology for rapidly assessing amyloid decomposition using capillary flow-based paper (CFP). Amyloid solutions exhibit gel-like physical properties due to insoluble denatured polymers, resulting in a shorter flow distance on CFP compared to pure water. Experimental conditions were established to consistently control the flow distance based on a hen-egg-white lysozyme amyloid solution. It was confirmed that as amyloid is decomposed by trypsin, the flow distance increases on the CFP. Our method is highly useful for detecting changes in the gel properties of amyloid solutions within a minute, and we anticipate its use in the rapid, large-scale screening of anti-amyloid agents in the future.

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The aggregation of the protein α-synuclein is closely associated with several neurodegenerative disorders and as such the structures of the amyloid fibril aggregates have high scientific and medical significance. However, there are dozens of unique atomic-resolution structures of these aggregates, and such a highly polymorphic nature of the α-synuclein fibrils hampers efforts in disease-relevant in vitro studies on α-synuclein amyloid aggregation. In order to better understand the factors that affect polymorph selection, we studied the structures of α-synuclein fibrils in vitro as a function of pH and buffer using cryo-EM helical reconstruction. We find that in the physiological range of pH 5.8-7.4, a pH-dependent selection between Type 1, 2, and 3 polymorphs occurs. Our results indicate that even in the presence of seeds, the polymorph selection during aggregation is highly dependent on the buffer conditions, attributed to the non-polymorph-specific nature of secondary nucleation. We also uncovered two new polymorphs that occur at pH 7.0 in phosphate-buffered saline. The first is a monofilament Type 1 fibril that highly resembles the structure of the juvenile-onset synucleinopathy polymorph found in patient-derived material. The second is a new Type 5 polymorph that resembles a polymorph that has been recently reported in a study that used diseased tissues to seed aggregation. Taken together, our results highlight the shallow amyloid energy hypersurface that can be altered by subtle changes in the environment, including the pH which is shown to play a major role in polymorph selection and in many cases appears to be the determining factor in seeded aggregation. The results also suggest the possibility of producing disease-relevant structure in vitro.

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Amyloid formation by α-synuclein (αSyn) occurs in Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies. Deciphering the residues that regulate αSyn amyloid fibril formation will not only provide mechanistic insight but may also reveal targets to prevent and treat disease. Previous investigations have identified several regions of αSyn to be important in the regulation of amyloid formation, including the non-amyloid-ß component (NAC), P1 region (residues 36 to 42), and residues in the C-terminal domain. Recent studies have also indicated the importance of the N-terminal region of αSyn for both its physiological and pathological roles. Here, the role of residues 2 to 7 in the N-terminal region of αSyn is investigated in terms of their ability to regulate amyloid fibril formation in vitro and in vivo. Deletion of these residues (αSynΔN7) slows the rate of fibril formation in vitro and reduces the capacity of the protein to be recruited by wild-type (αSynWT) fibril seeds, despite cryo-EM showing a fibril structure consistent with those of full-length αSyn. Strikingly, fibril formation of αSynΔN7 is not induced by liposomes, despite the protein binding to liposomes with similar affinity to αSynWT. A Caenorhabditis elegans model also showed that αSynΔN7::YFP forms few puncta and lacks motility and lifespan defects typified by expression of αSynWT::YFP. Together, the results demonstrate the involvement of residues 2 to 7 of αSyn in amyloid formation, revealing a target for the design of amyloid inhibitors that may leave the functional role of the protein in membrane binding unperturbed.

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Mechanical energy, specifically in the form of ultrasound, can induce pressure variations and temperature fluctuations when applied to an aqueous media. These conditions can both positively and negatively affect protein complexes, consequently altering their stability, folding patterns, and self-assembling behavior. Despite much scientific progress, our current understanding of the effects of ultrasound on the self-assembly of amyloidogenic proteins remains limited. In the present study, we demonstrate that when the amplitude of the delivered ultrasonic energy is sufficiently low, it can induce refolding of specific motifs in protein monomers, which is sufficient for primary nucleation; this has been revealed by MD. These ultrasound-induced structural changes are initiated by pressure perturbations and are accelerated by a temperature factor. Furthermore, the prolonged action of low-amplitude ultrasound enables the elongation of amyloid protein nanofibrils directly from natively folded monomeric lysozyme protein, in a controlled manner, until it reaches a critical length. Using solution X-ray scattering, we determined that nanofibrillar assemblies, formed either under the action of sound or from natively fibrillated lysozyme, share identical structural characteristics. Thus, these results provide insights into the effects of ultrasound on fibrillar protein self-assembly and lay the foundation for the potential use of sound energy in protein chemistry.

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Functional amyloids formed by the protein FapC in Pseudomonas bacteria are key structural components of Pseudomonas biofilms, which mediate chronic infections and also contribute to antimicrobial resistance. Here, we combine kinetic experiments with mechanistic modelling to probe the role of surfaces in FapC functional amyloid formation. We find that nucleation of new fibrils is predominantly heterogeneous in vitro, being catalysed by reaction vessel walls but not by the air/water interface. Removal of such interfaces by using microdroplets greatly slows heterogeneous nucleation and reveals a hitherto undetected fibril surface-catalysed "secondary nucleation" reaction step. We tune the degree of catalysis by varying the interface chemistry of the reaction vessel and by adding nanoparticles with tailored surface properties that catalyse fibril nucleation. In so doing, we discover that the rate of nucleation is controlled predominantly by the strength with which FapC binds to the catalytic sites on the interface, and by its surface area. Surprisingly, neither primary nucleation rate nor catalytic site binding strength appear closely correlated to the charge and hydrophilicity of the interface. This indicates the importance of considering experimental design in terms of surface chemistry of the reaction container while also highlighting the notion that fibril nucleation during protein aggregation is a heterogeneous process.

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Left-to-right differences in the histopathologic patterns of transthyretin-derived amyloid (ATTR) deposition in the atria of older adults have not yet been investigated. Hence, this study evaluated heart specimens from 325 serial autopsy subjects. The amount of ATTR deposits in the seven cardiac regions, including both sides of atria and atrial appendages, was evaluated semiquantitatively. Using digital pathology, we quantitatively evaluated the immunohistochemical deposition burden of ATTR in the myocardium. We identified 20 sporadic ATTR cardiac amyloidosis cases (nine males). All patients had ATTR deposition in the left atrial regions of the myocardium. In the semiquantitative analysis, 14 of the 20 cases showed more severe ATTR deposition on the left atrial regions than on the right side, with statistically significant differences in the pathology grading (p < 0.01 for both the atrium and atrial appendage). Quantitative analysis further supported the difference. Moreover, six had ATTR deposition in the epineurium and/or neural fibers of the atria. Cluster analysis revealed that ATTR deposition in the myocardium was significantly more severe in males than in females. The heterogeneous distribution of amyloid deposits between atria revealed in this study may impair the orderly transmission of the cardiac conduction system and induce arrhythmias, which may be further aggravated by additional neuropathy in the advanced phase. This impairment could be more severe among males. These findings emphasize that atrial evaluation is important for individuals with sporadic ATTR cardiac amyloidosis, particularly for early detection.

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The formation and analysis of amyloid fibers by two ß-glucosidases, BglA and BglB, belonging to the GH1 enzyme family, are reported. Both proteins have the (ß/α)8 TIM-barrel fold, which is characteristic of this family and is also the most common protein structure. BglA is an octamer, whereas BglB is a monomer. Amyloid fibrillation using pH and temperature as perturbing agents was investigated using fluorescence spectroscopy as a preliminary approach and corroborated using wide-field optical microscopy, confocal microscopy, and field-emission scanning electron microscopy. These analyses showed that both enzymes fibrillate at a wide range of acidic and alkaline conditions and at several temperature conditions, particularly at acidic pH (3-4) and at temperatures between 45 and 65 °C. Circular dichroism spectroscopy corroborated the transition from an α-helix to a ß-sheet secondary structure of both proteins in conditions where fibrillation was observed. Overall, our results suggest that fibrillation is a rather common phenomenon caused by protein misfolding, driven by a transition from an α-helix to a ß-sheet secondary structure, that many proteins can undergo if subjected to conditions that disturb their native conformation.

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Protein phase transitions (PPTs) from the soluble state to a dense liquid phase (forming droplets via liquid-liquid phase separation) or to solid aggregates (such as amyloids) play key roles in pathological processes associated with age-related diseases such as Alzheimer's disease. Several computational frameworks are capable of separately predicting the formation of droplets or amyloid aggregates based on protein sequences, yet none have tackled the prediction of both within a unified framework. Recently, large language models (LLMs) have exhibited great success in protein structure prediction; however, they have not yet been used for PPTs. Here, we fine-tune a LLM for predicting PPTs and demonstrate its usage in evaluating how sequence variants affect PPTs, an operation useful for protein design. In addition, we show its superior performance compared to suitable classical benchmarks. Due to the "black-box" nature of the LLM, we also employ a classical random forest model along with biophysical features to facilitate interpretation. Finally, focusing on Alzheimer's disease-related proteins, we demonstrate that greater aggregation is associated with reduced gene expression in Alzheimer's disease, suggesting a natural defense mechanism.

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Biofilm-protected pathogenic Staphylococcus aureus causes chronic infections that are difficult to treat. An essential building block of these biofilms are functional amyloid fibrils that assemble from phenol-soluble modulins (PSMs). PSMα1 cross-seeds other PSMs into cross-ß amyloid folds and is therefore a key element in initiating biofilm formation. However, the paucity of high-resolution structures hinders efforts to prevent amyloid assembly and biofilm formation. Here, we present a 3.5 Å resolution density map of the major PSMα1 fibril form revealing a left-handed cross-ß fibril composed of two C2-symmetric U-shaped protofilaments whose subunits are unusually tilted out-of-plane. Monomeric α-helical PSMα1 is extremely cytotoxic to cells, despite the moderate toxicity of the cross-ß fibril. We suggest mechanistic insights into the PSM functional amyloid formation and conformation transformation on the path from monomer-to-fibril formation. Details of PSMα1 assembly and fibril polymorphism suggest how S. aureus utilizes functional amyloids to form biofilms and establish a framework for developing therapeutics against infection and antimicrobial resistance.

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