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.

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.

Evidence of Anti-amyloid Characteristics of Plumbagin via Inhibition of Protein Aggregation and Disassembly of Protein Fibrils.

The biological consequences associated with the conversion of soluble proteins into insoluble toxic amyloids are not only limited to the onset of neurodegenerative diseases but also to the potential health risks associated with supplements of protein therapeutic agents as well. Hence, finding inhibitors against amyloid formation is important, and natural product-based anti-amyloid compounds have gained much interest because of their higher efficacy and biocompatibility. Plumbagin has been identified as a potential natural product with multiple medical benefits; however, it remains largely unclear whether plumbagin can act against amyloid formation of proteins. Here, we show that plumbagin can effectively inhibit the temperature-induced amyloid aggregation of important proteins (insulin and serum albumin). Both experimental and computational data revealed that the presence of plumbagin in protein solutions, under aggregating conditions, promotes a direct protein-plumbagin interaction, which is predominantly stabilized by stronger H-bonds and hydrophobic interactions. Plumbagin-mediated retention of the native structures of proteins appears to play a crucial role in preventing their conversion into insoluble ß-sheet-rich amyloid aggregates. More importantly, the addition of plumbagin into a suspension of protein fibrils triggered their spontaneous disassembly, promoting the release of soluble proteins. The results highlight that a possible synergistic effect via both the stabilization of protein structures and the restriction of the monomer recruitment at the fibril growth sites could be important for the mechanism of plumbagin's anti-aggregation effect. These findings may inspire the development of plumbagin-based formulations to benefit both the prevention and treatment of amyloid-related health complications.

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.

Osmoprotectant Coated Thermostable Gold Nanoparticles Efficiently Restrict Temperature-Induced Amyloid Aggregation of Insulin.

Naturally occurring osmoprotectants are known to prevent aggregation of proteins under various stress factors including extreme pH and elevated temperature conditions. Here, we synthesized gold nanoparticles coated with selected osmolytes (proline, hydroxyproline, and glycine) and examined their effect on temperature-induced amyloid-formation of insulin hormone. These uniform, thermostable, and hemocompatible gold nanoparticles were capable of inhibiting both spontaneous and seed-induced amyloid aggregation of insulin. Both quenching and docking experiments suggest a direct interaction between the osmoprotectant-coated nanoparticles and aggregation-prone hydrophobic stretches of insulin. Circular-dichroism results confirmed the retention of insulin's native structure in the presence of these nanoparticles. Unlike the indirect solvent-mediated effect of free osmolytes, the inhibition effect of osmolyte-coated gold nanoparticles was observed to be mediated through their direct interaction with insulin. The results signify the protection of the exposed aggregation-prone domains of insulin from temperature-induced self-assembly through osmoprotectant-coated nanoparticles, and such effect may inspire the development of osmolyte-based antiamyloid nanoformulations.

Myricetin inhibits amyloid fibril formation of globular proteins by stabilizing the native structures.

Myricetin has been identified as a naturally occurring flavonoid class of polyphenolic compound which shows multiple medical benefits including antidiabetic, anticancerous and antioxidant properties. Here, we report the protective effect of myricetin against in vitro amyloid fibril formation of selected globular proteins. The results reveal that myricetin is capable of inhibiting amyloid fibril formation of both insulin and serum albumin. Seed-induced aggregation of both proteins was also substantially suppressed in the presence of myricetin. Fluorescence quenching data indicated binding of myricetin with protein monomers as well as fibrils. The molecular docking studies revealed strong affinity of myricetin for both the native and partially unfolded conformation of proteins mediated by H-bonds and hydrophobic interactions. Myricetin was also observed to promote disassembly of mature amyloid fibrils. The results reveal that myricetin molecule has the potential for suppressing amyloid formation and such an inherent property may help in developing myricetin-based antiamyloid drugs.