Excess accumulation of phenylalanine is the characteristic of untreated Phenylketonuria (PKU), a well-known genetic abnormality, which triggers several neurological, physical and developmental severities. However, the fundamental mechanism behind the origin of such diverse health problems, particularly the issue of how they are related to the build-up of phenylalanine molecules in the body, is largely unknown. Here, we show cross-seeding ability of phenylalanine fibrils that can effectively initiate an aggregation process in proteins under physiological conditions, converting native protein structures to β-sheet assembly. The resultant fibrils were found to cause severe hemolysis, yielding a plethora of deformed erythrocytes that is highly relevant to phenylketonuria. Unique arrangement of zwitterionic phenylalanine molecules in their amyloid-like higher order entities is predicted to promote both hydrophobic and electrostatic interaction, sufficient enough to trap proteins and to preferentially interact with the membrane components of RBCs. Since the prevalence of hemolysis and amyloid related psychoneurological severities are mostly observed in PKU patients, we propose that the inherent property of phenylalanine fibrils to trigger hemolysis and to induce protein aggregation may have direct relevance to the disease mechanism of PKU.

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 β-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, Aβ peptides, and aromatic metabolites that convert native structures to β-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.

Here, we have strategically synthesized stable gold (AuNPs(Tyr), AuNPs(Trp)) and silver (AgNPs(Tyr)) nanoparticles which are surface functionalized with either tyrosine or tryptophan residues and have examined their potential to inhibit amyloid aggregation of insulin. Inhibition of both spontaneous and seed-induced aggregation of insulin was observed in the presence of AuNPs(Tyr), AgNPs(Tyr), and AuNPs(Trp) nanoparticles. These nanoparticles also triggered the disassembly of insulin amyloid fibrils. Surface functionalization of amino acids appears to be important for the inhibition effect since isolated tryptophan and tyrosine molecules did not prevent insulin aggregation. Bioinformatics analysis predicts involvement of tyrosine in H-bonding interactions mediated by its C=O, -NH2, and aromatic moiety. These results offer significant opportunities for developing nanoparticle-based therapeutics against diseases related to protein aggregation.

Eugenol has attracted considerable attention because of its potential for many pharmaceutical applications including anti-inflammatory, anti-tumorigenic and anti-oxidant properties. Here, we have investigated the effect of eugenol on amyloid formation of selected globular proteins. We find that both spontaneous and seed-induced aggregation processes of insulin and serum albumin (BSA) are significantly suppressed in the presence of eugenol. Isothermal titration calorimetric data predict a single binding site for eugenol-insulin complex confirming the affinity of eugenol for native soluble insulin species. We also find that eugenol suppresses amyloid-induced hemolysis. Our findings reveal the inherent ability of eugenol to stabilize native proteins and to delay the conversion of protein species of native conformation into β-sheet assembled mature fibrils, which seems to be crucial for its inhibitory effect.

The question of how an aggregating protein can influence aggregation of other proteins located in its vicinity is particularly significant because many proteins coexist in cells. We demonstrate in vitro coaggregation and cross-seeding of lysozyme, bovine serum albumin, insulin, and cytochrome c during their amyloid formation. The coaggregation process seems to be more dependent on the temperature-induced intermediate species of these proteins and less dependent on their sequence identities. Because amyloid-linked inclusions and plaques are recognized as multicomponent entities originating from aggregation of the associated protein, these findings may add new insights into the mechanistic understanding of amyloid-related pathologies.