Purpurin modulates Tau-derived VQIVYK fibrillization and ameliorates Alzheimer's disease-like symptoms in animal model.

Neurofibrillary tangles of the Tau protein and plaques of the amyloid ß peptide are hallmarks of Alzheimer's disease (AD), which is characterized by the conversion of monomeric proteins/peptides into misfolded ß-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 ß-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.

Membrane Determinants Affect Fibrillation Processes of ß-Sheet Charged Peptides.

Assembly of fibrillar peptide structures is dependent both upon their intrinsic propensities toward ß-structure formation, as well as on structural modulation by external molecular factors. ß-sheet structures may either be designed to form useful assemblies or be the undesired consequence of protein denaturation to toxic amyloid structures in several neurodegenerative diseases. Membrane bilayers have been implicated as primary initiators and modulators of amyloid fibrillation and the reasons for this effect are yet to be elucidated. Here, we employed a set of three charged peptides having the tendency to form ß-sheet fibrils, to investigate the effect of zwitterionic and negatively charged bilayer vesicles on their assembly structures. Microscopic and spectroscopic experiments revealed intimate relationship between peptide/membrane charges and fibrillation properties. Electrostatic attraction was apparent between oppositely charged peptides and vesicles; however, such interactions did not appear to significantly modulate fibril morphologies of either the net anionic peptide or the cationic one. Yet, a dramatic structural effect was observed when the nominal zwitterionic peptide underwent fibrillation in the presence of negatively charged vesicles. Assemblies of this peptide display a net positive charge, which facilitated the counterionic interactions with the vesicles. Furthermore, these interactions templated a unique twisted fiber morphology demonstrating the dramatic effect membrane-mediated interactions exert on fibril morphologies.

Inhibitory Effect of Naphthoquinone-Tryptophan Hybrid towards Aggregation of PAP f39 Semen Amyloid.

PAP248⁻286, a 39 amino acid peptide fragment, derived from the prostatic acid phosphatase secreted in human semen, forms amyloid fibrils and facilitates the attachment of retroviruses to host cells that results in the enhancement of viral infection. Therefore, the inhibition of amyloid formation by PAP248⁻286 (termed PAP f39) may likely reduce HIV transmission in AIDS. In this study, we show that the naphthoquinone tryptophan (NQTrp) hybrid molecule significantly inhibited PAP f39 aggregation in vitro in a dose-dependent manner as observed from the ThT assay, ANS assay, and transmission electron microscopy imaging. We found that even at a sub-molar concentration of 20:1 [PAP f39:NQTrp], NQTrp could reduce >50% amyloid formation. NQTrp inhibition of PAP f39 aggregation resulted in non-toxic intermediate species as determined by the vesicle leakage assay. Isothermal titration calorimetry and molecular docking revealed that the binding of NQTrp and PAP f39 is spontaneous, and NQTrp predominantly interacts with the polar and charged residues of the peptide by forming hydrogen bonds and hydrophobic contacts with a strong binding energy. Collectively, these findings indicate that NQTrp holds significant potential as a small molecule inhibitor of semen amyloids.

Catalytic physiological amyloids.

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.

Assessing the Catalytic Role of Native Glucagon Amyloid Fibrils.

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 the serine oxy anion on the carbonyl carbon of p-NPA. We computed the binding energy and free-energy profiles of this reaction using the protein-dipole Langevin-dipole (PDLD) within the linear response approximation (LRA) framework (PDLD/S-LRA-2000) and the empirical valence bond (EVB) methods. This included simulations of the reaction in an aqueous environment and in the fibril, enabling us to estimate the catalytic effect of the fibril. Our EVB 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 fibril provides supporting evidence to the catalytic role of glucagon amyloid fibrils. Moreover, 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-principles mechanistic investigation of fibrils using QM/MM methods. Therefore, our findings offer fruitful insights for future research into the mechanisms of related amyloid catalysis.

Staphylococcus aureus functional amyloids catalyze degradation of ß-lactam antibiotics.

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 ß-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 ß-lactam ring of nitrocefin, an antibiotic ß-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 ß-lactam association upon the fibril surface. Complementary experimental data underscore the generality of the functional amyloid-mediated catalytic phenomenon, demonstrating hydrolysis of clinically employed ß-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 ß-lactam antibiotics, underlying possible antibiotic resistance mechanisms associated with bacterial biofilms.

Amyloid fishing: ß-Amyloid adsorption using tailor-made coated titania nanoparticles.

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 ß-amyloid (Aß). Moreover, coated NPs incubated with Aß 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.

Native Glucagon Amyloids Catalyze Key Metabolic Reactions.

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.

Dual concentration-dependent effect of ascorbic acid on PAP(248-286) amyloid formation and SEVI-mediated HIV infection.

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 Aß, 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.

Inhibition of tau amyloid formation and disruption of its preformed fibrils by Naphthoquinone-Dopamine hybrid.

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 IC50 value (50% inhibition of fibril formation) of NQDA in inhibiting the aggregation of PHF6 (25 µm) was ~ 17 µm, which is lower than for other bona fide amyloid inhibitors, naphthoquinone-tryptophan, rosmarinic acid, epigallocatechin gallate, ~ 21, ~ 77, or ~ 19 µm, 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.

Revisiting thioflavin T (ThT) fluorescence as a marker of protein fibrillation - The prominent role of electrostatic interactions.

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 ß-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.

Deciphering the Rules for Amino Acid Co-Assembly Based on Interlayer Distances.

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.

Unravelling the role of amino acid sequence order in the assembly and function of the amyloid-ß core.

The amino acid sequence plays an essential role in amyloid formation. Here, using the central core recognition module of the Aß peptide and its reverse sequence, we show that although both peptides assemble into ß-sheets, their morphologies, kinetics and cell toxicities display marked differences. In addition, the native peptide, but not the reverse one, shows notable affinity towards bilayer lipid model membranes that modulates the aggregation pathways to stabilize the oligomeric intermediate states and function as the toxic agent responsible for neuronal dysfunction.

Tryptophan-glucosamine conjugates modulate tau-derived PHF6 aggregation at low concentrations.

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.

Chiral modulation of amyloid beta fibrillation and cytotoxicity by enantiomeric carbon dots.

Enantiomeric carbon dots (C-dots) synthesized from l-lysine or d-lysine, modulate aggregation and cytotoxicity of amyloid beta-42 (Aß42), the primary constituent of the amyloid plaques associated with Alzheimer's disease. In particular, l-Lys-C-dots dramatically remodeled Aß42 secondary structure and fibril morphologies, as well as inhibited Aß42 cytotoxicity and membrane interactions.