Amyloid Mimicking Assemblies Formed by Glutamine, Glutamic Acid, and Aspartic Acid.

The aggregation of amino acids into amyloid-like structures is a critical phenomenon for understanding the pathophysiology of various diseases, including inborn errors of metabolism (IEMs) associated with amino acid imbalances. Previous studies have primarily focused on self-assembly of aromatic amino acids, leading to a limited understanding of nonaromatic, polar amino acids in this context. To bridge this gap, our study investigates the self-assembly and aggregation behavior of specific nonaromatic charged and uncharged polar amino acids l-glutamine (Gln), l-aspartic acid (Asp), and l-glutamic acid (Glu), which have not been reported widely in the context of amyloid aggregation. Upon aging these amino acids under controlled conditions, we observed the formation of uniform, distinct aggregates, with Gln forming fibrillar gel-like structures and Glu exhibiting fibrous globular morphologies. Computational simulations validated these findings, identifying Gln as the most potent in forming stable aggregates, followed by Glu and Asp. These simulations elucidated the driving forces behind the distinct morphologies and stabilities of the aggregates. Thioflavin T assays were employed to confirm the amyloid-like nature of these aggregates, suggesting their potential cytotoxic impact. To assess toxicity, we performed in vitro studies on neural cell lines and in vivo experiments in Caenorhabditis elegans (C. elegans), which demonstrated measurable cytotoxic effects, corroborated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and heat shock survival assays. Importantly, this study fills a critical gap in our understanding on the role of nonaromatic amino acids in amyloidogenesis and its implications for IEMs. Our findings provide a foundation for future investigations into the mechanisms of diseases associated with amino acid accumulation and offer potential avenues for the development of targeted therapeutic strategies.

Amyloidogenic Propensity of Metabolites in the Uric Acid Pathway and Urea Cycle Critically Impacts the Etiology of Metabolic Disorders.

Novel insights into the etiology of metabolic disorders have recently been uncovered through the study of metabolite amyloids. In particular, inborn errors of metabolism (IEMs), including gout, Lesch-Nyhan syndrome (LNS), xanthinuria, citrullinemia, and hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, are attributed to the dysfunction of the urea cycle and uric acid pathway. In this study, we endeavored to understand and mechanistically characterize the aggregative property exhibited by the principal metabolites of the urea cycle and uric acid pathway, specifically hypoxanthine, xanthine, citrulline, and ornithine. Employing scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM), we studied the aggregation profiles of the metabolites. Insights obtained through molecular dynamics (MD) simulation underscore the vital roles of π-π stacking and hydrogen bonding interactions in the self-assembly process, and thioflavin T (ThT) assays further corroborate the amyloid nature of these metabolites. The in vitro MTT assay revealed the cytotoxic trait of these assemblies, a finding that was substantiated by in vivo assays employing the Caenorhabditis elegans (C. elegans) model, which revealed that the toxic effects were more pronounced and dose-specific in the case of metabolites that had aged via longer preincubation. We hence report a compelling phenomenon wherein these metabolites not only aggregate but transform into a soft, ordered assembly over time, eventually crystallizing upon extended incubation, leading to pathological implications. Our study suggests that the amyloidogenic nature of the involved metabolites could be a common etiological link in IEMs, potentially providing a unified perspective to study their pathophysiology, thus offering exciting insights into the development of targeted interventions for these metabolic disorders.

Realization of Amyloid-like Aggregation as a Common Cause for Pathogenesis in Diseases.

Amyloids were conventionally referred to as extracellular and intracellular accumulation of Aß42 peptide, which causes the formation of plaques and neurofibrillary tangles inside the brain leading to the pathogenesis in Alzheimer's disease. Subsequently, amyloid-like deposition was found in the etiology of prion diseases, Parkinson's disease, type II diabetes, and cancer, which was attributed to the aggregation of prion protein, α-Synuclein, islet amyloid polypeptide protein, and p53 protein, respectively. Hence, traditionally amyloids were considered aggregates formed exclusively by proteins or peptides. However, since the last decade, it has been discovered that other metabolites, like single amino acids, nucleobases, lipids, glucose derivatives, etc., have a propensity to form amyloid-like toxic assemblies. Several studies suggest direct implications of these metabolite assemblies in the patho-physiology of various inborn errors of metabolisms like phenylketonuria, tyrosinemia, cystinuria, and Gaucher's disease, to name a few. In this review, we present a comprehensive literature overview that suggests amyloid-like structure formation as a common phenomenon for disease progression and pathogenesis in multiple syndromes. The review is devoted to providing readers with a broad knowledge of the structure, mode of formation, propagation, and transmission of different extracellular amyloids and their implications in the pathogenesis of diseases. We strongly believe a review on this topic is urgently required to create awareness about the understanding of the fundamental molecular mechanism behind the origin of diseases from an amyloid perspective and possibly look for a common therapeutic strategy for the treatment of these maladies by designing generic amyloid inhibitors.

Solvent-controlled self-assembly of Fmoc protected aliphatic amino acids.

Self-assembly of modified amino acids facilitate the formation of various structures that have unique properties and therefore serve as excellent bio-organic scaffolds for diverse applications. Self-assembly of Fmoc protected single amino acids has attracted great interest owing to their ease of synthesis and applications as functional materials. Smaller assembly units enable synthetic convenience and potentially broader adoption. Herein, we demonstrate the ability to control the morphologies resulting from self-assembly of Fmoc modified aliphatic single amino acids (Fmoc-SAAs) namely, Alanine, Valine, Leucine, Isoleucine, and Proline. Controlled morphological transitions were observed through solvent variation and the mechanism that allows this control was investigated using coarse-grained molecular dynamics simulations. These show that FmocA can form well defined crystalline structures through uniform parallel Fmoc stacking and the optimization of ion concentrations, which is not observed for the other Fmoc-SAAs. We demonstrate that Fmoc protected aliphatic single amino acids are novel scaffolds for the design of distinct micro/nanostructures through a bottom-up approach that can be tuned by control of the environmental parameters.

An Isothiazolanthrone-Based Self-Assembling Anticancer Color-Changing Dye for Concurrent Imaging and Monitoring of Cell Viability.

We report the photophysical properties, self-assembly and biological evaluation of an isothiazolanthrone-based dye, 7-amino-6H-anthra[9,1-cd]isothiazol-6-one (AAT), which reveals anticancer properties and can be potentially used as dye for monitoring cell viability. The solvent-dependent photophysical studies suggest that the emission of AAT is sensitive to environment polarity due to which interesting changes in the colored emission may be observed owing to the charge transfer (CT) processes. AAT also self-assembles to tree-like branched morphologies and produce, a greenish emission inside the cells when imaged after short interval (15 mins) of incubation while a red fluorescence could be noted after 24 h. Interestingly, AAT also produce differential emission inside mouse normal cells as compared to its cancer cell lines since it possess anticancer activity. The experimental observations were also validated theoretically via computational modeling.

A new aggregation induced emission enhancement (AIEE) dye which self-assembles to panchromatic fluorescent flowers and has application in sensing dichromate ions.

We report for the very first time the crystal structure and self-assembly of a new aggregation-induced emission enhancement (AIEE) dye 4-(5-methoxythiazolo[4,5-b]pyridin-2-yl)-N,N-dimethylaniline (TPA) and its application in sensing dichromate ions. TPA reveals cyan blue emission under UV and visible light. The self-assembly properties of TPA were studied extensively by scanning electron microscopy (SEM) which revealed the formation of beautiful flower-like morphologies. These structures revealed both green and red fluorescence under FITC and rhodamine filters respectively when observed through fluorescence microscopy connoting the panchromatic emission properties of TPA from blue to red. The interactions which cause self-assembled structure formation in TPA were also validated theoretically using density functional theory (DFT) calculations. Crystal and molecular structure analysis of TPA was carried out via single-crystal X-ray diffraction to visualize the intermolecular interactions occurring in the solid-state and to study the structure-photophysical property relationship in the aggregated state. The photophysical properties of TPA were also studied extensively by UV-visible and fluorescence spectroscopy and its quantum yield and fluorescence lifetime were calculated by time-correlated single-photon counting (TCSPC). Interestingly, TPA could efficiently sense dichromate (Cr2O72-) ions in an acidic medium and an interesting morphological transition from a fluorescent flower to non-fluorescent disassembled structures could also be observed. The limit of detection of TPA for Cr2O72- ions was found to be as low as 5.5 nM, suggesting its exceptional sensitivity. More importantly, TPA could selectively sense Cr2O72- ions in real water samples even in the presence of other metal ions routinely present in polluted water, hence making it practically useful for water quality monitoring.

Metabolite assemblies: A surprising extension to the amyloid hypothesis.

The realization of the ability of metabolites to form self-assembled amyloid-like nanostructures was a surprising phenomenon. This discovery paved the way towards understanding the pathophysiology of the inborn error of metabolism disorders from a new perspective, relating them to amyloid-associated diseases that are characterized by the aggregation of proteins and polypeptides. Hence, a 'generic amyloid hypothesis' can be proposed. This theory implies that the formation of amyloid-like structures is a general phenomenon not limited to proteins and reflects a common etiology for both age-related amyloid-associated diseases and inborn error of metabolism disorders. Here, we present a comprehensive survey of the recent research related to metabolite amyloids including their structure formation through self-association, propagation, interactions, transmission, and their role in metabolic disorders and neurodegenerative diseases and their applications for the fabrication of novel materials which implicate metabolite assemblies as a surprising extension to the amyloid scheme.

Unusual Aggregates Formed by the Self-Assembly of Proline, Hydroxyproline, and Lysine.

There is a plethora of significant research that illustrates toxic self-assemblies formed by the aggregation of single amino acids, such as phenylalanine, tyrosine, tryptophan, cysteine, and methionine, and their implication on the etiology of inborn errors of metabolisms (IEMs), such as phenylketonuria, tyrosinemia, hypertryptophanemia, cystinuria, and hypermethioninemia, respectively. Hence, studying the aggregation behavior of single amino acids is very crucial from the chemical neuroscience perspective to understanding the common etiology between single amino acid metabolite disorders and amyloid diseases like Alzheimer's and Parkinson's. Herein we report the aggregation properties of nonaromatic single amino acids l-proline (Pro), l-hydroxyproline (Hyp), and l-lysine hydrochloride (Lys). The morphologies of the self-assembled structures formed by Pro, Hyp, and Lys were extensively studied by various microscopic techniques, and controlled morphological transitions were observed under varied concentrations and aging times. The mechanism of structure formation was deciphered by concentration-dependent 1H NMR analysis, which revealed the crucial role of hydrogen bonding and hydrophobic interactions in the structure formation of Pro, Hyp, and Lys. MTT assays on neural (SHSY5Y) cell lines revealed that aggregates formed by Pro, Hyp, and Lys reduced cell viability in a dose-dependent manner. These results may have important implications in the understanding of the patho-physiology of disorders such as hyperprolinemia, hyperhydroxyprolinemia, and hyperlysinemia since all these IEMs are associated with severe neurodegenerative symptoms, including intellectual disability, seizures, and psychiatric problems. Our future studies will endeavor to study these biomolecular assemblies in greater detail by immuno-histochemical analysis and advanced biophysical assays.

Chemical Perspective of the Mechanism of Action of Antiamyloidogenic Compounds Using a Minimalistic Peptide as a Reductionist Model.

The diphenylalanine (FF) residue which is present at the 19 and 20 positions of the amyloid beta (1-42) (Aß42) peptide sequence is considered as a reductionist model for studying Aß42 aggregation. FF self-assembles into well-ordered tubular structures via aromatic π-π stacking. Herein the manuscript, we have presented a chemical perspective on the mechanism of action of antiamyloid compounds by assessing their interaction with FF. Therefore, we first coincubated FF fibers with single amino acids, since they are constituted of different R side chains yet have a common structural unit. This study revealed a crucial role of aromatic rings and functional groups like thiol (-SH) in causing destabilization of FF assembly via their interaction with π-electrons participating in π-π stacking present in FF. We further studied the interaction of different nonsteroidal anti-inflammatory drugs (NSAIDs), other known antiamyloidogenic compounds, and host-guest inclusion compounds like cyclodextrin (CD) to assess their mechanism of action and to decipher the functional moiety present in these compounds which could cause destabilization of π-π stacking. From the coincubation experiments, we could surmise a crucial role of aromatic rings present in these compounds for causing interference in aromatic stacking. We further consolidated our observations through microscopy analysis by various spectroscopic methods such as aggregation-induced emission enhancement (AIEE), fluorescence spectroscopy, solution-state 1H NMR, FTIR, and circular dichroism. The studies presented in the manuscript thus provide significant insights into the role of functional groups in imparting antiamyloid action and open new avenues for an efficient design of antiamyloid drugs in the future.

Sequential and cellular detection of copper and lactic acid by disaggregation and reaggregation of the fluorescent panchromatic fibres of an acylthiourea based sensor.

We report, for the first time, the self-assembly of an acyl-thiourea based sensor, N-{(6-methoxy-pyridine-2-yl) carbamothioyl}benzamide (NG1), with panchromatic fluorescent fibres and its dual-sensing properties for the sequential detection of Cu2+ ions and lactic acid. The panchromatic fibres formed by NG1 were disrupted in the presence of Cu2+ ions and this was accompanied by a visible colour change in the solution from colourless to yellow. The addition of lactic acid to the NG1 + Cu2+ solution, on the other hand, induced re-aggregation to fibrillar structures and the colour of the solution again changed to colourless. Hence, it may be surmised that the disaggregation and re-aggregation impart unique dual-sensing properties to NG1 for the sequential detection of Cu2+ ions and lactic acid. The application of NG1 as a selective sensor for Cu2+ ions and lactic acid has been assessed in detail by UV-visible and fluorescence spectroscopy. Furthermore, two structural variants of NG1, namely, NG2 and NG3, were synthesized, which suggest the crucial role of pyridine in imparting panchromatic emission properties and of both pyridine and acyl-thiourea side chain in the binding of Cu2+ ions. The O-methoxy group plays an important part in making NG1 the most sensitive probe of its structural analogs. Finally, the utility of NG1 for the sequential and cellular detection of Cu2+ ions and lactic acid was studied in human RPE cells. The experimental results of the interaction of NG1 with Cu2+ ions and lactic acid have also been validated theoretically by using quantum chemical calculations based on density functional theory (DFT). To the best of our knowledge, this is the first report wherein a dual sensor for Cu2+ ions and lactate ions is synthesized. More importantly, the aggregation properties of the sensor have been studied extensively and an interesting correlation of the photophysical properties of the probe with its self-assembling behavior has been elucidated.

Synthesis and Aggregation Studies of a Pyridothiazole-Based AIEE Probe and Its Application in Sensing Amyloid Fibrillation.

We report the aggregation and photophysical properties of a pyridothiazole-based, aggregation-induced, emission-enhancement (AIEE) luminogen 4-(5-methoxy-thiazolo[4,5-b]pyridin-2-yl)benzoic acid (PTC1) and its application for the sensitive detection and monitoring of amyloid fibrillation. The aggregation properties of the AIEE probe were extensively studied by atomic force microscopy (AFM) and dynamic light scattering (DLS), and it was noted that as aggregation increases the fluorescence of PTC1 also was increased. The fluorescence of PTC1 was quenched upon the addition of cupric (Cu2+) ions, while the fluorescence is regenerated in the presence of amyloid fibers. AFM studies reveal that the PTC1 molecules self-associate/aggregate to hairy micelle-like structures, which dissociate or disrupt in the presence of the Cu2+ ions and again reassemble in the presence of amyloid fibers. Hence, the quenching and regeneration of PTC1 fluorescence may be attributed to the disaggregation and aggregation-induced emission (AIE), respectively. Further, a comparative analysis of the performance of PTC1 was done with conventional Thioflavin T, which confirms it to be a more sensitive probe for the detection of the amyloid, both in the presence and absence of Cu2+ ions. The experimental results were also validated theoretically via molecular docking and simulation studies. Of note, a very simple, facile, and cost-effective methodology for the detection of the amyloid fibers is presented, wherein fluorescence quenching/enhancement can be visualized under the UV light without the use of sophisticated instrumentation techniques. The AIEE probe was designed using an unusual pyridothiazole scaffold unlike commonly used archetypal AIE scaffolds based on tetraphenylethene (TPE) and hexaphenylsilole (HPS). Hence, the work also has implications in designing future AIEE dyes based on the pyridothiazole scaffold reported.

Amyloid-like Structures Formed by Single Amino Acid Self-Assemblies of Cysteine and Methionine.

We report for the very first time the discovery of amyloid-like self-assemblies formed by the nonaromatic single amino acids cysteine (Cys) and methionine (Met) under neutral aqueous conditions. The structure formation was assessed and characterized by various microscopic and spectroscopic techniques such as optical microscopy, phase contrast microscopy, scanning electron microscopy, and transmission electron microscopy. The mechanism of self-assembly and the role of hydrogen bonding and thiol interactions of Cys and Met were assessed by Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray diffraction, and solid state NMR along with various control experiments. In addition, molecular dynamics simulations were carried out to gain insight into assembly initiation. Further, Thioflavin T and Congo red binding assays with Cys and Met structures indicated that these single amino acid assemblies may have amyloid-like characteristics. To understand the biological significance of the Cys and Met structures, cytotoxicity assays of the assemblies were performed on human neuroblastoma IMR-32 cells and monkey kidney cells (COS-7). The results revealed that both Cys and Met fibers were cytotoxic. The cell viability assay further supported the hypothesis that aggregation of single amino acid may contribute to the etiology of metabolic disorders like cystinuria and hypermethioninemia. The results presented in this study are striking, and to the best of our knowledge this is the first report which demonstrates that nonaromatic amino acids like Cys and Met can undergo spontaneous self-assembly to form amyloidogenic aggregates. The results presented are also consistent with the established generic amyloid hypothesis and support a new paradigm for the study of the etiology of single amino acid initiated metabolic disorders in amyloid related diseases.

Formation of DNA-copolymer fibrils through an amyloid-like nucleation polymerization mechanism.

Conjugation of a hydrophobic poly(2-oxazoline) bearing tertiary amide groups along its backbone with a short single stranded nucleotide sequence results in an amphiphilic comb/graft copolymer, which organizes in fibrils upon direct dissolution in water. Supported by circular dichroism, atomic force microscopy, transmission electron microscopy, and scattering data, fibrils are formed through inter- and intramolecular hydrogen bonding between hydrogen accepting amide groups along the polymer backbone and hydrogen donating nucleic acid grafts leading to the formation of hollow tubes.

Label-free, optical sensing of the supramolecular assembly into fibrils of a ditryptophan-DNA hybrid.

The grafting of a short nucleic acid strand to ditryptophan dipeptide (WW) results in a peptide-DNA hybrid, which assembles into fibrils under controlled aggregation conditions as evidenced by label free optical sensing owing to the intrinsic fluorescence of the dipeptide.

Controlling morphology of peptide-based soft structures by covalent modifications.

Control of gross morphology of soft matter remains an area of continued interest. Towards this goal, this paper describes conjugation of mannose residues and introduction of thiol functionalities to diphenylalanine (FF) dipeptide, a fibrillating motif from amyloid-ß peptide, as covalent modifiers of its solution-phase self-assembly process. It was found that covalent attachment of a single mannose residue to FF leads to the retention of tubular structures, whereas the conjugation of two mannose units, linked through a Lys residue, resulted in a dramatic change from tubular morphology to spherical structures. However, a similar switch to spherical objects could be achieved by introducing a thiol residue in the mono-mannosylated FF dipeptide. Interestingly, these glycopeptides also exhibited interaction with concanavalin A, thereby providing an indirect evidence for the availability of mannose units for the process of lectin-carbohydrate interaction in the self-organized state.

Self-assembling DNA-peptide hybrids: morphological consequences of oligonucleotide grafting to a pathogenic amyloid fibrils forming dipeptide.

For the very first time, highly efficient synthesis of DNA-peptide hybrids to scaffold self-assembled nanostructures is described. Oligonucleotide conjugation to the diphenylalanine dipeptide triggers a morphological transition from fibrillar to vesicular structures which may potentially be used as delivery vehicles, since they exhibit pH triggered release.

Synthesis and self-assembly of a neoglycopeptide: morphological studies and ultrasound-mediated DNA encapsulation.

Self-assembly in peptides and proteins is an often encountered concept, where constituent building blocks are recruited and stabilized, via carefully orchestrated hydrophobic interactions, hydrogen bonding and other non-covalent interactions, to eventually reveal an array of supramolecular aggregates, with defined structural features. This study presents synthesis and self-assembly of a mannosylated peptide in aqueous medium. Turbidimetric assay with Concanavalin A (Con A), a mannose binding protein, was conducted to confirm the presence of hydrophilic mannose group on the exterior surface of self-assembled structures. DNA encapsulation in these soft structures was achieved by ultrasonication of soft spherical structures in the presence of plasmid DNA.

Mannosylated self-assembled structures for molecular confinement and gene delivery applications.

This paper reports self-assembly of a lysine conjugated with a biantennary mannose to form spherical structures. These supramolecular structures are found to be hollow in nature and they afford effective encapsulation of alkaline phosphatase enzyme, plasmid DNA and a GFP reporter gene, which was transfected in COS-7 cells. Loaded hollow structures also get disrupted upon mild sonication, releasing encapsulated molecules thereby illustrating their potential for confinement and delivery applications.