Terephthalates, particularly BHET and MHET, are partial hydrolysis products of poly(ethylene terephthalate) (PET), the most ubiquitous plastic waste, and considered major pollutants and health hazards. We demonstrate, for the first time, that amyloid fibrils, both de-novo designed fibrils comprising alternating phenylalanine-lysine motif, and physiological functional bacterial amyloids, efficiently catalyze BHET and MHET hydrolysis. Peptide variant screening, physicochemical experiments, and structural analyses shed light in the catalytic mechanism, facilitated by surface docking via pi-pi interactions between the terephthalates and phenylalanines, and hydrogen bonding with lysine sidechains. Hydrolysis subsequently 2 proceeds via nucleophilic attack by the lysines’ butylammonium sidechains. Catalytic, recyclable terephthalate degradation was also accomplished by coupling the amyloid fibrils to silica beads and placing in a conventional recyclable column setup, and by bacterially secreted PSMα amyloid fibrils, pointing to potential applicability in natural bacterial biofilms.

Light-mediated assembly of catalytic agents furnishes powerful means for spatiotemporal control of chemical reactions. Here, we demonstrate photo-switched hierarchical assembly of catalytic amyloid fibrils, through mixing an amphiphilic cross-ß peptide with a merocyanine photo-switch. The peptide monomers, comprising phenylalanine-lysine repeats, do not self-assemble in water because of the electrostatic repulsion between the positively charged lysine side chains; however, upon light-induced merocyanine-spiropyran transformation, amyloid fibrils were formed. Interestingly, increasing the mole ratio between the merocyanine photo-switch and the peptide gave rise to a remarkable assembly of aligned elongated fibrils. The process was fully reversible as light turn-off resulted in fibril disintegration. Notably, the photoinduced amyloid fibrils catalyzed hydrolysis of β-lactam antibiotics, offering a platform for light-mediated degradation of antibiotic pollutants in water. Microscopic and spectroscopic experiments reveal that the photoinduced transient catalytic amyloids were likely formed through π-π interactions between the spiropyran moieties and the aromatic residues of the phenylalanines, while electrostatic interactions between the negative sulfonates and lysine sidechains on the fibrils' surface were responsible for the macroscale alignment. The amyloid-merocyanine system represents a new concept for light-regulated assembly of catalytic amyloids, which may have also played a role in prebiotic evolution processes.

Brain-localized deposits of crystalline calcium phosphate, mainly comprising of hydroxyapatite, are a pathological hallmark of Alzheimer’s disease (AD). While calcium phosphate crystals are specifically identified in the neuronal amyloid plaques in AD, consisting mostly of beta amyloid (Aβ) fibrils, the mechanisms and factors affecting the biomineralization are unknown. Here, we present a novel mechanism for AD plaque-induced formation of crystalline calcium phosphate. Specifically, we show, for the first time, that Aβ amyloid fibrils catalyse dephosphorylation of adenosine triphosphate (ATP). Furthermore, incubating Aβ fibrils, ATP, and calcium ions gave rise to pronounced deposition of hydroxyapatite crystals upon the Aβ amyloid fibril matrix, originating through reaction between calcium ions and the monophosphate released through the catalytic dephosphorylation reaction. This pathway may explain amyloid plaque-associated calcification in AD, as elevated levels of both ATP and calcium are key features of the disease. ### Competing Interest Statement The authors have declared no competing interest.

Amyloid-mediated catalysis of key biological reactions has recently attracted significant interest as this phenomenon may portend new functions for physiological and synthetic amyloid proteins. Here, we report an allosteric mechanism of catalytic amyloids, mediated via an unconventional coiled-coil fibril organization, facilitating hydrolysis of β-lactam antibiotics. Specifically, the hydrolysis reaction was catalyzed by a fibrillar peptide comprising alternating lysine/phenylalanine β-sheet-forming sequence. Analysis of peptide variants, simulations, and cryogenic electron microscopy reveal that the β-lactam molecules attach electrostatically to the lysine sidechains on the fibrils' surfaces, generating a double-coiled fibril structure in which the anchored β-lactam molecules are nestled within twisted fibril strands. This organization facilitates the allosteric catalytic process in which hydrolytic β-lactam ring opening is induced via nucleophilic attacks by the lysine sidechains degradation. The allosteric catalytic activity of the phenylalanine/lysine amyloid fibrils highlights the functional versatility of amyloid fibrils and their potential applications in human health and environmental biotechnology.

The release of antibiotic compounds into wastewater constitutes a significant and growing health and environmental hazard, particularly contributing to the spread of antibiotic resistant bacterial strains. Here, we demonstrate that amyloid fibrils, consisting of an alternating lysine/phenylalanine -sheet forming short peptide, catalyze hydrolysis of β-lactam antibiotics, the most prominent family of antibiotic compounds, which is further widespread in wastewater. Peptide variant analysis, molecular dynamics (MD) simulations, and cryogenic electron microscopy (cryo-EM) reveal that the β-lactam molecules dock onto the fibrils’ surface via electrostatic interactions with the lysine sidechains. Importantly, catalytic hydrolysis occurs via an allosteric mechanism mediated by a unique coiled double fibril structure in which the anchored β-lactam molecules are embedded within twisted fiber strands, facilitating nucleophilic attacks by the lysine sidechains. Utilization of the catalytic lysine-displaying amyloid fibrils for hydrolytic degradation and removal of β-lactam antibiotics from water was accomplished through display of the fibrils on silica beads placed in a conventional column filtration setup. Amyloid fibrils displaying lysine arrays may furnish a versatile platform for hydrolysis and removal of β-lactam antibiotics in water, underscoring new avenues for addressing the considerable threat of antibiotics water contamination.

Glucagon stands out as a pivotal peptide hormone, instrumental in controlling blood glucose levels and lipid metabolism. While the formation of glucagon amyloid fibrils has been documented, their biological functions remain enigmatic. Recently, we demonstrated experimentally that glucagon amyloid fibrils can act as catalysts in several biological reactions, including esterolysis, lipid hydrolysis, and dephosphorylation. Herein we present a multiscale quantum mechanics/molecular mechanics (QM/MM) simulation of the acylation step in the esterolysis of para-nitrophenyl acetate (p-NPA), catalyzed by native glucagon amyloid fibrils, serving as a model system to elucidate their catalytic function. This step entails a concerted mechanism, involving proton transfer from serine to histidine, followed by the nucleophilic attack of serine oxy anion on the carbonyl carbon of p-NPA. We computed the binding energy and free-energy profiles of this reaction using the PDLD/S-LRA-2000 and the empirical valence bond (EVB) methods. This included simulations if the reaction in an aqueous environment and in the fibril, enabling us to estimate the catalytic effect of the fibril. Our calculations obtained a barrier of 23.4 kcal∙mol–1 for the enzyme-catalyzed reaction, compared to the experimental value of 21.9 kcal∙mol–1 (and a calculated catalytic effect of 3.2 kcal∙mol–1 compared to the observed effect of 4.7 kcal∙mol–1) This close agreement together with the barrier reduction when transitioning from the reference solution reaction to the amyloid fibrils provides supporting evidence to the catalytic role of glucagon amyloid fibrils. Moreover, by employing the PDLD/S-LRA-2000 approach further reinforced exclusively the enzyme's catalytic role. The results presented in this study contribute significantly to our understanding of the catalytic role of glucagon amyloid fibrils, marking, to the best of our knowledge, the first mechanistic investigation of fibrils using QM/MM methods. Therefore, our findings offer fruitful insights for future research on the mechanisms of related amyloid catalysis.

Amyloid fibrils have been identified in many protein systems, mostly linked to progression and cytotoxicity in neurodegenerative diseases and other pathologies, but have also been observed in normal physiological systems. A growing body of work has shown that amyloid fibrils can catalyze chemical reactions. Most studies have focused on catalysis by de-novo synthetic amyloid-like peptides; however, recent studies reveal that physiological, native amyloids are catalytic as well. Here, we discuss methodologies and major experimental aspects pertaining to physiological catalytic amyloids. We highlight analyzes of kinetic parameters related to the catalytic activities of amyloid fibrils, structure-function considerations, characterization of the catalytic active sites, and deciphering of catalytic mechanisms.

Coassembly of peptide biomaterials offers a compelling avenue to broaden the spectrum of hierarchically ordered supramolecular nanoscale structures that may be relevant for biomedical and biotechnological applications. In this study we present a comprehensive exploration of binary coassembly leveraging amphiphilic and oppositely charged, anionic and cationic, -sheet peptides, which may give rise to a diverse range of coassembled forms. Mixtures of the peptides exhibit a notably diminished critical assembly concentration (CAC), in comparison to the corresponding values of the pure peptides. Intriguingly, the sweet spot for coassembled fibril formation was found to require excess of the cationic peptide whereas equimolar mixtures of the peptides exhibited the maximum folding into β-sheet structures. Mixtures of the peptides coassembled sequentially from solutions at concentrations surpassing each peptide's intrinsic CAC, were also found to require a higher portion of the cationic peptide to stabilize hydrogels. This study illuminates a systematic exploration of complementary charged -sheet peptides. The results may be relevant to the fundamental understanding of such intricate assembly systems and to the formulation of peptide-based nanostructures with diverse functionalities.

Antibiotic resistance of bacteria is considered one of the most alarming developments in modern medicine. While varied pathways for bacteria acquiring antibiotic resistance have been identified, there still are open questions concerning the mechanisms underlying resistance. Here, we show that alpha phenol-soluble modulins (PSMαs), functional bacterial amyloids secreted by Staphylococcus aureus , catalyze hydrolysis of β-lactams, a prominent class of antibiotic compounds. Specifically, we show that PSMα2 and, particularly, PSMα3 catalyze hydrolysis of the amide-like bond of the four membered β-lactam ring of nitrocefin, an antibiotic β-lactam surrogate. Examination of the catalytic activities of several PSMα3 variants allowed mapping of the active sites on the amyloid fibrils’ surface, specifically underscoring the key roles of the cross-α fibril organization, and the combined electrostatic and nucleophilic functions of the lysine arrays. Molecular dynamics simulations further illuminate the structural features of β-lactam association upon the fibril surface. Complementary experimental data underscore the generality of the functional amyloid-mediated catalytic phenomenon, demonstrating hydrolysis of clinically employed β-lactams by PSMα3 fibrils, and illustrating antibiotic degradation in actual S. aureus biofilms and live bacteria environments. Overall, this study unveils functional amyloids as catalytic agents inducing degradation of β-lactam antibiotics, underlying possible antibiotic resistance mechanisms associated with bacterial biofilms.

Protein amyloids generally constitute beta-sheet rich fibrillar assemblies. Amyloid fibrils have been identified in varied diseases, formed by bacterially secreted proteins, and generated in de novo designed peptides. This review article summarizes the burgeoning body of work reporting catalytic properties of amy-loid fibrils. We highlight representative studies focusing on catalytic amyloid peptides, both synthetic and naturally occurring. We discuss the structural features associated with catalysis and putative catalytic sites on amyloid fibrils' surfaces. We also highlight studies demonstrating catalytic functions of short amyloid-like sequences and their possible involvement in early-life reactions, acting as primitive enzymes. Finally, we discuss recent reports of the catalytic activities of native amyloids, pointing to possible roles of amyloid catalysis in disease progression and pathologies.

Amyloidoses are a family of diseases characterized by abnormal protein folding that leads to fibril aggregates, amyloids. Extensive research efforts are devoted to developing inhibitors to amyloid aggregates. Here we set to explore functionalized titania (TiO2) nanoparticles (NPs) as potential amyloid inhibiting agents. TiO2 NPs were coated by a catechol derivative, dihydroxy-phenylalanine propanoic acid (DPA), and further conjugated to the amyloids' specific dye Congo-Red (CR). TiO2-DPA-CR NPs were found to target mature fibrils of β-amyloid (Aβ). Moreover, coated NPs incubated with Aβ proteins suppressed amyloid fibrillation. TiO2-DPA-CR were found to target amyloids in solution and induce their sedimentation upon centrifugation. This work demonstrates the potential utilization of TiO2-DPA NPs for labeling and facilely separating from solution mature amyloid fibrils.

Glucagon is a prominent peptide hormone, playing central roles in the regulation of glucose blood-level and lipid metabolism. Formation of glucagon amyloid fibrils has been previously reported, although no biological functions of such fibrils are known. Here, we demonstrate that glucagon amyloid fibrils catalyze biologically important reactions, including esterolysis, lipid hydrolysis, and dephosphorylation. In particular, we found that glucagon fibrils catalyze dephosphorylation of adenosine triphosphate (ATP), a core metabolic reaction in cell biology. Comparative analysis of several glucagon variants allowed mapping the catalytic activity to an enzymatic pocket-like triad formed at the glucagon fibril surface, comprising the histidyl-serine domain at the N-terminus of the peptide. This study may point to previously unknown physiological roles and pathological consequences of glucagon fibrillation and supports the hypothesis that catalytic activities of native amyloid fibrils play functional roles in human physiology and disease.

Amyloid fibrils are one of the hallmarks of Alzheimer's disease (AD), although a causative link between plaque-forming amyloid fibrils and AD pathology remains to be clarified. This study demonstrates, for the first time for a naturally occurring amyloid, that fibrils comprising the 42-residue amyloid-β peptide (Aβ42) exhibit significant catalytic properties. Aβ42 fibrils catalyzed the hydrolysis of the model ester para-nitrophenyl acetate (pNPA) and of acetylthiocholine, a surrogate for the neurotransmitter acetylcholine. Aβ42 fibrils also catalyzed oxidation of the prominent neurotransmitters dopamine and adrenaline. Importantly, the catalytic activity was specifically manifested by mature Aβ42 fibrils and not the peptide monomers or oligomeric Aβ42, the putative neurotoxic species. Furthermore, maximal catalytic activity was recorded by the full-length Aβ42 fibrils, whereas fibrillar assemblies comprising Aβ42 subdomains were significantly less catalytic. The catalytic activity of Aβ fibrils could exhibit insidious roles in AD pathophysiology.

Misfolding and aggregation of tau protein, into pathological amyloids, are hallmarks of a group of neurodegenerative diseases collectively termed tauopathies and their modulation may be therapeutically valuable. Herein, we describe the synthesis and characterization of a dopamine‐based hybrid molecule, naphthoquinone–dopamine (NQDA). Using thioflavin S assay, CD, transmission electron microscopy, dynamic light scattering, Congo Red birefringence, and large unilamellar vesicle leakage assays, we demonstrated its efficacy in inhibiting the in vitro aggregation of key tau‐derived amyloidogenic fragments, PHF6 (VQIVYK) and PHF6* (VQIINK), prime drivers of aggregation of full‐length tau in disease pathology. Isothermal titration calorimetry analysis revealed that the interaction between NQDA and PHF6 is spontaneous and has significant binding efficiency driven by both entropic and enthalpic processes. Furthermore, NQDA efficiently disassembled preformed fibrils of PHF6 and PHF6* into nontoxic species. Molecular dynamic simulations supported the in vitro results and provided a plausible mode of binding of NQDA with PHF6 fibril. NQDA was also capable of inhibiting the aggregation of full‐length tau protein and disrupting its preformed fibrils in vitro in a dose‐dependent manner. In a comparative study, the IC 50 value (50% inhibition of fibril formation) of NQDA in inhibiting the aggregation of PHF6 (25 µ m ) was ~ 17 µ m , which is lower than for other bona fide amyloid inhibitors, naphthoquinone‐tryptophan, rosmarinic acid, epigallocatechin gallate, ~ 21, ~ 77, or ~ 19 µ m , respectively. Comparable superiority of NQDA was observed for inhibition of PHF6*. These findings suggest that NQDA can be a useful scaffold for designing new therapeutics for Alzheimer's disease and other tauopathies.

Human semen contains various amyloidogenic peptides derived from Prostatic Acid Phosphatase (PAP) and Semenogelin proteins that are capable of enhancing HIV-1 infection when assembled into fibrils. The best characterized among them is a 39 amino acid peptide PAP(248-286), which forms amyloid fibrils termed SEVI (semen-derived enhancer of viral infection) that increase the infectivity of HIV-1 by orders of magnitude. Inhibiting amyloid formation by PAP(248-286) may mitigate the sexual transmission of HIV-1. Several vitamins have been shown to reduce the aggregation of amyloids such as Aβ, α-Synuclein, and Tau, which are associated with neurodegenerative diseases. Since ascorbic acid (AA, vitamin C) is the most abundant vitamin in semen with average concentrations of 0.4 mM, we here examined how AA affects PAP(248-286) aggregation in vitro. Using ThT binding assays, transmission electron microscopy, and circular dichroism spectroscopy, a dual and concentration-dependent behavior of AA in modulating PAP(248-286) fibril formation was observed. We found that low molar ratios of AA:PAP(248-286) promoted whereas high molar ratios inhibited PAP(248-286) fibril formation. Accordingly, PAP(248-286) aggregated in the presence of low amounts of AA enhanced HIV-1 infection, whereas excess amounts of AA during aggregation reduced the infectivity enhancing effect in cell culture. Collectively, this work provides a biophysical insight into the effect of AA, an important seminal component, on SEVI fibrillation which might impact amyloid formation kinetics, thereby modulating the biological activity of semen amyloids.

Neurofibrillary tangles of the Tau protein and plaques of the amyloid β peptide are hallmarks of Alzheimer’s disease (AD), which is characterized by the conversion of monomeric proteins/peptides into misfolded β-sheet rich fibrils. Halting the fibrillation process and disrupting the existing aggregates are key challenges for AD drug development. Previously, we performed in vitro high-throughput screening for the identification of potent inhibitors of Tau aggregation using a proxy model, a highly aggregation-prone hexapeptide fragment 306VQIVYK311 (termed PHF6) derived from Tau. Here we have characterized a hit molecule from that screen as a modulator of Tau aggregation using in vitro, in silico, and in vivo techniques. This molecule, an anthraquinone derivative named Purpurin, inhibited ~ 50% of PHF6 fibrillization in vitro at equimolar concentration and disassembled pre-formed PHF6 fibrils. In silico studies showed that Purpurin interacted with key residues of PHF6, which are responsible for maintaining its β-sheets conformation. Isothermal titration calorimetry and surface plasmon resonance experiments with PHF6 and full-length Tau (FL-Tau), respectively, indicated that Purpurin interacted with PHF6 predominantly via hydrophobic contacts and displayed a dose-dependent complexation with FL-Tau. Purpurin was non-toxic when fed to Drosophila and it significantly ameliorated the AD-related neurotoxic symptoms of transgenic flies expressing WT-FL human Tau (hTau) plausibly by inhibiting Tau accumulation and reducing Tau phosphorylation. Purpurin also reduced hTau accumulation in cell culture overexpressing hTau. Importantly, Purpurin efficiently crossed an in vitro human blood–brain barrier model. Our findings suggest that Purpurin could be a potential lead molecule for AD therapeutics.

Thioflavin T (ThT), a benzothiazole-based fluorophore, is a prominent dye widely employed for monitoring amyloid fibril assembly. Despite the near-universal presumption that ThT binds to β-sheet domains upon fibrillar surface via hydrophobic forces, the contribution of the positive charge of ThT to fibril binding and concomitant fluorescence enhancement have not been thoroughly assessed. Here we demonstrate a considerable interdependence between ThT fluorescence and electrostatic charges of peptide fibrils. Specifically, by analyzing both fibril-forming synthetic peptides and prominent natural fibrillar peptides, we demonstrate pronounced modulations of ThT fluorescence signal that were solely dependent upon electrostatic interactions between ThT and peptide surface. The results further attest to the fact that fibril ζ-potential rather than pH-dependent assembly of the fibrils constitute the primary factor affecting ThT binding and fluorescence. This study provides the first quantitative assessment of electrostatically driven ThT fluorescence upon adsorption to amyloid fibrils.

Metabolite materials are extremely useful to obtain functional bioinspired assemblies with unique physical properties for various applications in the fields of material science, engineering, and medicine by self-assembly of the simplest biological building blocks. Supramolecular co-assembly has recently emerged as a promising extended approach to further expand the conformational space of metabolite assemblies in terms of structural and functional complexity. Yet, the design of synergistically co-assembled amino acids to produce tailor-made functional architectures is still challenging. Herein, we propose a design rule to predict the supramolecular co-assembly of naturally occurring amino acids based on their interlayer separation distances observed in single crystals. Using diverse experimental techniques, we demonstrate that amino acids with comparable interlayer separation strongly interact and co-assemble to produce structural composites distinctly different from their individual properties. However, such an interaction is hampered in a mixture of differentially layer-separated amino acids, which self-sort to generate individual characteristic structures. This study provides a different paradigm for the modular design of supramolecular assemblies based on amino acids with predictable properties.

Glycosylation of amyloidogenic proteins enhances their solubility and reduces propensity for aggregation. We therefore, prepared tryptophan-glucosamine conjugates to modulate aggregation of tau-derived PHF6-peptide. Combined in vitro and in silico approaches indicated that these conjugates inhibited oligomerization and fibril formation of PHF6 and disrupted its preformed fibrils at very low concentration. These effects mainly arise from the glucopyranoside moiety.