Cu(II) Specifically Disassembles Insulin Amyloid Nanostructures via Direct Interaction with Cross-ß Fibrils.

In this work, we demonstrate direct evidence of the antiamyloid potential of Cu(II) ions against amyloid formation of insulin. The Cu(II) ions were found to efficiently disassemble the preformed amyloid nanostructures into soluble species and suppress monomer fibrillation under aggregation-prone conditions. The direct interaction of Cu(II) ions with the cross-ß structure of amyloid fibrils causes substantial disruption of both the interchain and intrachain interactions, predominantly the H-bonds and hydrophobic contacts. Further, the Cu(II) ions show a strong affinity for the aggregation-prone conformers of the protein and inhibit their spontaneous self-assembly. These results reveal the possible molecular mechanism for the antiamyloidogenic potential of Cu(II) which could be important for the development of metal-ion specific therapeutic strategies against amyloid linked complications.

Tailoring the Molecular Structure of 6-Gingerol for Targeting the Phase Separation in Human Lysozyme.

Human lysozyme undergoes a phase-separation process to form insoluble amyloid-architects that cause several pathologies including systemic amyloidosis. Here we have tailored 6-gingerol by extending its molecular framework with active functional groups to specifically target lysozyme phase-transition events. Aggregation assay revealed that tailored 6-gingerol with 4-aromatic moieties (MTV4) substantially suppressed the conversion of the lysozyme low-density liquid phase (LDLP) to solid-phase structured amyloids. The data obtained from biophysical, computational, and microscopic imaging tools suggest direct intervention of MTV4 with the liquid-liquid phase separation. The CD data suggest that MTV4 was able to retain the native conformation of lysozyme. Both biomolecular and computational data reveal the interference of MTV4 with the aggregation-prone hydrophobic stretches within the lysozyme, thereby retaining the native structure and reversing the misfolded intermediates to active monomers. Also, MTV4 was able to induce rapid dissolution of preformed-toxic amyloid fibrils. These results reinforce the importance of the aromatic-aromatic interaction in preventing human lysozyme phase separation.

Molecular insights into the phase transition of lysozyme into amyloid nanostructures: Implications of therapeutic strategies in diverse pathological conditions.

Lysozyme, a well-known bacteriolytic enzyme, exhibits a fascinating yet complex behavior when it comes to protein aggregation. Under certain conditions, this enzyme undergoes flexible transformation, transitioning from partially unfolded intermediate units of native conformers into complex cross-ß-rich nano fibrillar amyloid architectures. Formation of such lysozyme amyloids has been implicated in a multitude of pathological and medical severities, like hepatic dysfunction, hepatomegaly, splenic rupture as well as spleen dysfunction, nephropathy, sicca syndrome, renal dysfunction, renal amyloidosis, and systemic amyloidosis. In this comprehensive review, we have attempted to provide in-depth insights into the aggregating behavior of lysozyme across a spectrum of variables, including concentrations, temperatures, pH levels, and mutations. Our objective is to elucidate the underlying mechanisms that govern lysozyme's aggregation process and to unravel the complex interplay between its structural attributes. Moreover, this work has critically examined the latest advancements in the field, focusing specifically on novel strategies and systems, that have been implemented to delay or inhibit the lysozyme amyloidogenesis. Apart from this, we have tried to explore and advance our fundamental understanding of the complex processes involved in lysozyme aggregation. This will help the research community to lay a robust foundation for screening, designing, and formulating targeted anti-amyloid therapeutics offering improved treatment modalities and interventions not only for lysozyme-linked amyloidopathy but for a wide range of amyloid-related disorders.

Pleiotropic Nanostructures Built from l-Histidine Show Biologically Relevant Multicatalytic Activities.

The essential amino acid histidine plays a central role in the manifestation of several metabolic processes, including protein synthesis, enzyme-catalysis, and key biomolecular interactions. However, excess accumulation of histidine causes histidinemia, which shows brain-related medical complications, and the molecular mechanism of such histidine-linked complications is largely unknown. Here, we show that histidine undergoes a self-assembly process, leading to the formation of amyloid-like cytotoxic and catalytically active nanofibers. The kinetics of histidine self-assembly was favored in the presence of Mg(II) and Co(II) ions. Molecular dynamics data showed that preferential noncovalent interactions dominated by H-bonds between histidine molecules facilitate the formation of histidine nanofibers. The histidine nanofibers induced amyloid cross-seeding reactions in several proteins and peptides including pathogenic Aß1-42 and brain extract components. Further, the histidine nanofibers exhibited oxidase activity and enhanced the oxidation of neurotransmitters. Cell-based studies confirmed the cellular internalization of histidine nanofibers in SH-SY5Y cells and subsequent cytotoxic effects through necrosis and apoptosis-mediated cell death. Since several complications including behavioral abnormality, developmental delay, and neurological disabilities are directly linked to abnormal accumulation of histidine, our findings provide a foundational understanding of the mechanism of histidine-related complications. Further, the ability of histidine nanofibers to catalyze amyloid seeding and oxidation reactions is equally important for both biological and materials science research.

Initiation of Brain Extract Fibrillation and Effective Cellular Internalization of Tryptophan Fibrils Unveils Its Neurotoxicity Risk.

Recent discoveries on the self-assembly of aromatic amino acids into amyloid-like neurotoxic nanostructures have initiated a quest to decode the molecular mechanisms for the initiation of neurodegeneration. Moreover, the multicomponent nature of the amyloid deposits still questions the existing and well-defined amyloid cascade hypothesis. Hence, deciphering the neurotoxicity of amyloid-like nanostructures of aromatic amino acids becomes crucial for understanding the etiology of amyloidogenesis. Here, we demonstrate the cellular internalization and consequential damaging effects of self-assembled amyloid-like tryptophan nanostructures on human neuroblastoma cells. The cell-damaging potential of tryptophan nanostructure seems to be facilitated via ROS generation, necrosis and apoptosis mediated cell death. Further, tryptophan nanostructures were found to be seeding competent conformers, which triggered aggressive aggregation of brain extract components. The early stage intermediate nanostructures possess a higher cross-seeding efficacy than the seeding potential of the matured tryptophan fibrils. In addition to the cell-damaging and cross-seeding effects, tryptophan fibrils were found to catalyze oxidation of neuromodulator dopamine. These findings add more insights into the specific role of tryptophan self-assembly during the pathogenesis of hypertryptophanemia and other amyloid-associated neurodegenerative complications.

A robust yet simple method to generate fluorescent amyloid nanofibers.

Covalent tagging of fluorophores is central to the mechanistic understanding of important biological processes including protein-protein interaction and protein aggregation. Hence, studies on fluorophore-tagged peptides help in elucidating the molecular mechanism of amyloidogenesis, its cellular internalization, and crosstalk potential. Despite the many advantages the covalently tagged proteins offer, difficulties such as expensive and tedious synthesis and purification protocols have become a matter of concern. Importantly, covalently tagged fluorophores could introduce structural constraints, which may influence the conformation of the monomeric and aggregated forms of proteins. Here, we describe a robust-yet-simple method to make fluorescent-amyloid nanofibers through a coassembly-reaction route that does not alter the aggregation kinetics and the characteristic ß-sheet-conformers of resultant nanofibers. Fluorescent amyloid nanofibers derived from insulin, lysozyme, Aß1-42, and metabolites were successfully fabricated in our study. Importantly, the incorporated fluorophores exhibited remarkable stability, remaining intact without leaching even after undergoing serial dilutions and prolonged storage periods. This method enables monitoring of cellular internalization of the fluorescent-amyloid-nanofibers and the detection of FRET-signals during interfibrillar interactions. This simple and affordable protocol may significantly help amyloid researchers working on both in vitro and animal models.

Bioactivation of an orthodontic wire using multifunctional nanomaterials to prevent plaque accumulation.

Controlling the growth of biofilm on orthodontic material has become a difficult challenge in modern dentistry. The antibacterial efficacy of currently used orthodontic material becomes limited due to the higher affinity of oral microbial flora for plaque formation on the material surface. Thus it is crutial to device an efficient strategy to prevent plaque buildup caused by pathogenic microbiota. In this work, we have fabricated a bioactive orthodontic wire using titanium nanoparticles (TiO2NPs) and silver nanoparticles (AgNPs). AgNPs were synthesized from the extracts of Ocimum sanctum, Ocimum tenuiflorum, Solanum surattense, and Syzygium aromaticum, while the TiO2NPs were synthesized by the Sol-Gel method. The nanoparticles were characterized by various biophysical techniques. The surface of the dental wire was molded by functionalizing these AgNPs followed by an additional coating of TiO2NPs. Functionalized dental wires were found to counteract the formation of tenacious intraoral biofilm, and showed an enhanced anti-bacterial effect against Multi-Drug Resistant (MDR) bacteria isolated from patients with various dental ailments. Data revealed that such surface coating counteracts the bacterial pathogens by inducing the leakage of Ag ions which eventually disrupts the cell membrane as confirmed from TEM micrographs. The results offer a significant opportunity for innovations in developing nanoparticle-based formulations to modify or fabricate an effective orthodontic material.

Autooxidation of curcumin in physiological buffer causes an enhanced synergistic anti-amyloid effect.

Curcumin is an important food additive that shows multiple medical-benefits including anticarcinogenic, anti-inflammatory, antibiotic and antiamyloid properties. However, understanding the mechanism of curcumin-mediated effects becomes rather complicated since it has low bio-viability and it undergoes autooxidation, influenced by temperature, pH and buffer. We find that curcumin's antiamyloid-potential is not primarily due to curcumin alone, rather due to a synergistic action of curcumin and its autooxidized-products generated during inhibition reactions. In physiological buffer curcumin undergoes thermally induced autooxidation and yields stable compounds which can synergistically work for both inhibition of amyloid aggregation and promotion of amyloid-disaggregation into soluble protein species. Curcumin also showed substantial inhibition effect against coaggregation of different food proteins. Curcumin's strong affinity for the hydrophobic moieties of the aggregation-prone partially-folded insulin structures seems crucial for the inhibition mechanism. Further, autooxidized curcumin products were found to protect UV-induced protein damage. The results provide conceptual foundations highlighting the link between chemistry and antiamyloid-activity of curcumin and may inspire curcumin-based therapeutics against amyloidogenesis.

Tryptophan self-assembly yields cytotoxic nanofibers containing amyloid-mimicking and cross-seeding competent conformers.

Dietary consumption of Trp via protein-based foods is essential for the maintenance of crucial metabolic processes including the synthesis of proteins and several vital metabolites such as serotonin, melatonin, acetyl CoA, and NADP. However, the abnormal build-up of Trp is known to cause familial hypertryptophanemia and several brain-related medical complications. The molecular mechanism of the onset of such Trp-driven health issues is largely unknown. Here, we show that Trp, under the physiologically mimicked conditions of temperature and buffer, undergoes a concentration driven self-assembly process, yielding amyloid-mimicking nanofibers. Viable H-bonds, π-π interactions and hydrophobic contacts between optimally coordinated Trp molecules become important factors for the formation of a Trp nanoassembly that displays a hydrophobic exterior and a hydrophilic interior. Importantly, Trp nanofibers were found to possess high affinity for native proteins, and they act as cross-seeding competent conformers capable of nucleating amyloid formation in globular proteins including whey protein ß-lactoglobulin and type II diabetes linked insulin hormone. Moreover, these amyloid mimicking Trp nanostructures showed toxic effects on neuroblastoma cells. Since the key symptoms in hypertryptophanemia such as behavioural defects and brain-damaging oxidative stress are also observed in amyloid related disorders, our findings on amyloid-like Trp-nanofibers may help in the mechanistic understanding of Trp-related complications and these findings are equally important for innovation in applied nanomaterials design and strategies.

Amyloid-mimicking toxic nanofibers generated via self-assembly of dopamine.

Molecular self-assembly of biologically relevant aromatic metabolites is known to generate cytotoxic nanostructures and this unique property has opened up new concepts in the molecular mechanisms of metabolite-linked disorders. Because aromaticity is intrinsic to the chemical structure of some important neuromodulators, the question of whether this property can promote their self-assembly into toxic higher order structures is highly relevant to the advancement of both fundamental and applied research. We show here that dopamine, an aromatic neuromodulator of high significance, undergoes self-assembly, under physiological buffer conditions, yielding cytotoxic supramolecular nanostructures. The oxidation of dopamine seems crucial in driving the self-assembly, and substantial inhibition effect was observed in the presence of antioxidants and acidic buffers. Strong H-bonds and π-π interactions between optimally-oriented dopamine molecules were found to stabilize the dopamine nanostructure which displayed characteristic ß-structure-patterns with hydrophobic exterior and hydrophilic interior moieties. Furthermore, dopamine nanostructures were found to be highly toxic to human neuroblastoma cells, revealing apoptosis and necrosis-mediated cytotoxicity. Abnormal fluctuation in the dopamine concentration is known to predispose a multitude of neuronal complications, hence, the new findings of this study on oxidation-driven buildup of amyloid-mimicking neurotoxic dopamine assemblies may have direct relevance to the molecular origin of several dopamine related disorders.

Genesis of Neurotoxic Hybrid Nanofibers from the Coassembly of Aromatic Amino Acids.

Considering the relevance of accumulation and self-assembly of metabolites and aftermath of biological consequences, it is important to know whether they undergo coassembly and what properties the resultant hybrid higher-order structures would exhibit. This work reveals the inherent tendency of aromatic amino acids to undergo a spontaneous coassembly process under physiologically mimicked conditions, which yields neurotoxic hybrid nanofibers. Resultant hybrid nanostructures resembled the ß-structured conformers stabilized by H-bonds and π-π stacking interactions, which were highly toxic to human neuroblastoma cells. The hybrid nanofibers also showed strong cross-seeding potential that triggered in vitro aggregation of diverse globular proteins and brain extract components, converting the native structures into cross-ß-rich amyloid aggregates. The heterogenic nature of the hybrid nanofibers seems crucial for their higher toxicity and faster cross-seeding potential as compared to the homogeneous amino acid nanofibers. Our findings reveal the importance of aromaticity-driven optimized intermolecular arrangements for the coassembly of aromatic amino acids, and the results may provide important clues to the fundamental understanding of metabolite accumulation-related complications.

Self-Assembly of Artificial Sweetener Aspartame Yields Amyloid-like Cytotoxic Nanostructures.

Recent reports have revealed the intrinsic propensity of single aromatic metabolites to undergo self-assembly and form nanostructures of amyloid nature. Hence, identifying whether aspartame, a universally consumed artificial sweetener, is inherently aggregation prone becomes an important area of investigation. Although the reports on aspartame-linked side effects describe a multitude of metabolic disorders, the mechanistic understanding of such destructive effects is largely mysterious. Since aromaticity, an aggregation-promoting factor, is intrinsic to aspartame's chemistry, it is important to know whether aspartame can undergo self-association and if such a property can predispose any cytotoxicity to biological systems. Our study finds that aspartame molecules, under mimicked physiological conditions, undergo a spontaneous self-assembly process yielding regular ß-sheet-like cytotoxic nanofibrils of amyloid nature. The resultant aspartame fibrils were found to trigger amyloid cross-seeding and become a toxic aggregation trap for globular proteins, Aß peptides, and aromatic metabolites that convert native structures to ß-sheet-like fibrils. Aspartame fibrils were also found to induce hemolysis, causing DNA damage resulting in both apoptosis and necrosis-mediated cell death. Specific spatial arrangement between aspartame molecules is predicted to form a regular amyloid-like architecture with a sticky exterior that is capable of promoting viable H-bonds, electrostatic interactions, and hydrophobic contacts with biomolecules, leading to the onset of protein aggregation and cell death. Results reveal that the aspartame molecule is inherently amyloidogenic, and the self-assembly of aspartame becomes a toxic trap for proteins and cells, exposing the bitter side of such a ubiquitously used artificial sweetener.

Strategically Designed Antifibrotic Gold Nanoparticles to Prevent Collagen Fibril Formation.

Because uncontrolled accumulation of collagen fibrils has been implicated in a series of pathologies, inhibition of collagen fibril formation has become one of the necessary strategies to target such collagen-linked complications. The presence of hydroxyproline (Hyp) at the Y position in (Gly-X-Y)n sequence pattern of collagen is known to facilitate crucial hydrophobic and hydration-linked interactions that promote collagen fibril formation. Here, to target such Hyp-mediated interactions, we have synthesized uniform, thermostable, and hemocompatible Hyp coated gold nanoparticles (AuNPsHYP) and have examined their inhibition effect on the fibril formation of type I collagen. We found that collagen fibril formation is strongly suppressed in the presence of AuNPsHYP and no such suppression effect was observed in the presence of free Hyp and control Gly-coated nanoparticles at similar concentrations. Both isothermal titration calorimetric studies and bioinformatics analysis reveal possible interaction between Hyp and (Gly-Pro-Hyp) stretches of collagen triple-helical model peptides. Further, gold nanoparticles coated with proline (AuNPsPRO) and tryptophan (AuNPsTRP) also suppressed collagen fibril formation, suggesting their ability to interfere with aromatic-proline as well as hydrophobic interactions between collagen molecules. The Hyp molecules, when surface functionalized, are predicted to interfere with the Hyp-mediated forces that drive collagen self-assembly, and such inhibition effect may help in targeting collagen linked pathologies.