Homocysteine fibrillar assemblies display cross-talk with Alzheimer's disease ß-amyloid polypeptide.

High levels of homocysteine are reported as a risk factor for Alzheimer's disease (AD). Correspondingly, inborn hyperhomocysteinemia is associated with an increased predisposition to the development of dementia in later stages of life. Yet, the mechanistic link between homocysteine accumulation and the pathological neurodegenerative processes is still elusive. Furthermore, despite the clear association between protein aggregation and AD, attempts to develop therapy that specifically targets this process have not been successful. It is envisioned that the failure in the development of efficacious therapeutic intervention may lie in the metabolomic state of affected individuals. We recently demonstrated the ability of metabolites to self-assemble and cross-seed the aggregation of pathological proteins, suggesting a role for metabolite structures in the initiation of neurodegenerative diseases. Here, we provide a report of homocysteine crystal structure and self-assembly into amyloid-like toxic fibrils, their inhibition by polyphenols, and their ability to seed the aggregation of the AD-associated ß-amyloid polypeptide. A yeast model of hyperhomocysteinemia indicates a toxic effect, correlated with increased intracellular amyloid staining that could be rescued by polyphenol treatment. Analysis of AD mouse model brain sections indicates the presence of homocysteine assemblies and the interplay between ß-amyloid and homocysteine. This work implies a molecular basis for the association between homocysteine accumulation and AD pathology, potentially leading to a paradigm shift in the understanding of AD initial pathological processes.

Branched-Chain Amino Acid Assembly into Amyloid-like Fibrils Provides a New Paradigm for Maple Syrup Urine Disease Pathology.

Inborn error of metabolism disorders (IEMs) are a family of diseases resulting from single-gene mutations that lead to the accumulation of metabolites that are usually toxic or interfere with normal cell function. The etiological link between metabolic alteration and the symptoms of IEMs is still elusive. Several metabolites, which accumulate in IEMs, were shown to self-assemble to form ordered structures. These structures display the same biophysical, biochemical, and biological characteristics as proteinaceous amyloid fibrils. Here, we have demonstrated, for the first time, the ability of each of the branched-chain amino acids (BCAAs) that accumulate in maple syrup urine disease (MSUD) to self-assemble into amyloid-like fibrils depicted by characteristic morphology, binding to indicative amyloid-specific dyes and dose-dependent cytotoxicity by a late apoptosis mechanism. We could also detect the presence of the assemblies in living cells. In addition, by employing several in vitro techniques, we demonstrated the ability of known polyphenols to inhibit the formation of the BCAA fibrils. Our study implies that BCAAs possess a pathological role in MSUD, extends the paradigm-shifting concept regarding the toxicity of metabolite amyloid-like structures, and suggests new pathological targets that may lead to highly needed novel therapeutic opportunities for this orphan disease.

ASL expression in ALDH1A1+ neurons in the substantia nigra metabolically contributes to neurodegenerative phenotype.

Argininosuccinate lyase (ASL) is essential for the NO-dependent regulation of tyrosine hydroxylase (TH) and thus for catecholamine production. Using a conditional mouse model with loss of ASL in catecholamine neurons, we demonstrate that ASL is expressed in dopaminergic neurons in the substantia nigra pars compacta, including the ALDH1A1 + subpopulation that is pivotal for the pathogenesis of Parkinson disease (PD). Neuronal loss of ASL results in catecholamine deficiency, in accumulation and formation of tyrosine aggregates, in elevation of α-synuclein, and phenotypically in motor and cognitive deficits. NO supplementation rescues the formation of aggregates as well as the motor deficiencies. Our data point to a potential metabolic link between accumulations of tyrosine and seeding of pathological aggregates in neurons as initiators for the pathological processes involved in neurodegeneration. Hence, interventions in tyrosine metabolism via regulation of NO levels may be therapeutic beneficial for the treatment of catecholamine-related neurodegenerative disorders.

Chemical Chaperones Modulate the Formation of Metabolite Assemblies.

The formation of amyloid-like structures by metabolites is associated with several inborn errors of metabolism (IEMs). These structures display most of the biological, chemical and physical properties of protein amyloids. However, the molecular interactions underlying the assembly remain elusive, and so far, no modulating therapeutic agents are available for clinical use. Chemical chaperones are known to inhibit protein and peptide amyloid formation and stabilize misfolded enzymes. Here, we provide an in-depth characterization of the inhibitory effect of osmolytes and hydrophobic chemical chaperones on metabolite assemblies, thus extending their functional repertoire. We applied a combined in vivo-in vitro-in silico approach and show their ability to inhibit metabolite amyloid-induced toxicity and reduce cellular amyloid content in yeast. We further used various biophysical techniques demonstrating direct inhibition of adenine self-assembly and alteration of fibril morphology by chemical chaperones. Using a scaffold-based approach, we analyzed the physiochemical properties of various dimethyl sulfoxide derivatives and their role in inhibiting metabolite self-assembly. Lastly, we employed whole-atom molecular dynamics simulations to elucidate the role of hydrogen bonds in osmolyte inhibition. Our results imply a dual mode of action of chemical chaperones as IEMs therapeutics, that could be implemented in the rational design of novel lead-like molecules.

Antibodies towards Tyrosine Amyloid-Like Fibrils Allow Toxicity Modulation and Cellular Imaging of the Assemblies.

The amino acid tyrosine forms cytotoxic amyloid-like fibrils by molecular self-assembly. However, the production of antibodies towards tyrosine assemblies, reflecting their presentation to the immune system, was not demonstrated yet. Here, we describe the production of antibodies that specifically recognize tyrosine in its fibrillated form. The antibodies were demonstrated to specifically bind self-assembled tyrosine, in contrast to its non-aggregated form or disintegrated fibrils. The antibodies could be used for immunostaining of tyrosine fibrils in cultured cells. Furthermore, confocal microscopy allowed a demonstration of the intracellular presence of the metabolite amyloids in a neuroblastoma cell model. Finally, pre-incubation of tyrosine fibrils with the antibodies resulted in significant reduction in their cytotoxicity. Taken together, we provide an experimental proof for the immunogenicity of tyrosine amyloid fibrillary assemblies. These specific antibodies against tyrosine structures could be further used as a research tool to study the dynamics, toxicity and cellular localization of the assemblies.

Inhibition of Respiratory RNA Viruses by a Composition of Ionophoric Polyphenols with Metal Ions.

Controlling the infectivity of respiratory RNA viruses is critical, especially during the current SARS-CoV-2 pandemic. There is an unmet need for therapeutic agents that can reduce viral replication, preferably independent of the accumulation of viral mutations. Zinc ions have an apparent activity as modulators of intracellular viral RNA replication and thus, appear attractive in reducing viral RNA load and infectivity. However, the intracellular concentration of zinc is usually too low for achieving an optimal inhibitory effect. Various herbal polyphenols serve as excellent zinc ionophores with known antiviral properties. Here, we combined zinc picolinate with a collection of flavonoids, representing commonly used polyphenols. Copper was added to avoid ionic imbalance during treatment and to improve efficacy. Each component separately, as well as their combinations, did not interfere with the viability of cultured A549, H1299, or Vero cells in vitro as determined by MTT assay. The safe combinations were further evaluated to determine antiviral activity. Fluorescence-activated cell sorting and quantitative polymerase chain reaction were used to evaluate antiviral activity of the combinations. They revealed a remarkable (50-95%) decrease, in genome replication levels of a diverse group of respiratory RNA viruses, including the human coronavirus OC43 (HCoV-OC43; a betacoronavirus that causes the common cold), influenza A virus (IAV, strain A/Puerto Rico/8/34 H1N1), and human metapneumovirus (hMPV). Collectively, our results offer an orally bioavailable therapeutic approach that is non-toxic, naturally sourced, applicable to numerous RNA viruses, and potentially insensitive to new mutations and variants.

On-off transition and ultrafast decay of amino acid luminescence driven by modulation of supramolecular packing.

Luminescence of biomolecules in the visible range of the spectrum has been experimentally observed upon aggregation, contrary to their monomeric state. However, the physical basis for this phenomenon is still elusive. Here, we systematically examine all coded amino acids to provide non-biased empirical insights. Several amino acids, including non-aromatic, show intense visible luminescence. Lysine crystals display the highest signal, whereas the very chemically similar non-coded ornithine does not, implying a role for molecular packing rather than the chemical characteristics. Furthermore, cysteine shows luminescence that is indeed crystal packing dependent as repeated rearrangements between two crystal structures result in a reversible on-off optical transition. In addition, ultrafast lifetime decay is experimentally validated, corroborating a recently raised hypothesis regarding the governing role of nπ∗ states in the emission formation. Collectively, our study supports that electronic interactions between non-fluorescent, non-absorbing molecules at the monomeric state may result in reversible optically active states by the formation of supramolecular fluorophores.

Peptide Self-Assembly Is Linked to Antibacterial, but Not Antifungal, Activity of Histatin 5 Derivatives.

The rise of multidrug-resistant pathogens has awakened interest in new drug candidates such as antimicrobial peptides and their derivatives. Recent work suggests that some antimicrobial peptides have the ability to self-assemble into ordered amyloid-like nanostructures which facilitate their antibacterial activity. Here, we evaluate a histatin-based antimicrobial peptide, and its self-assembling derivative, in the interplay between self-assembly, membrane interactions, and antibacterial and antifungal activities. We demonstrate substantial membrane targeting by both peptides, as well as mechanistic insights into this mode of action, which correlates to their antifungal activity and is not affected by their self-assembling state. The ability to self-assemble does, however, significantly affect peptide antibacterial activity against both Gram-negative and Gram-positive bacteria. These results are surprising and hint at important distinctions between antifungal and antibacterial peptide activities in prokaryotes and eukaryotic microbes.IMPORTANCE Antimicrobial peptides are important modulators of host defense against bacterial, fungal, and viral pathogens in humans and other multicellular organisms. Two converging paradigms point to a link between antimicrobial peptides that self-assemble into amyloid-like nanoassemblies and classical amyloidogenic peptides that often have potent broad-spectrum antimicrobial activity, suggesting that antimicrobial and amyloidogenic peptides may represent two sides of the same coin. Here, we asked if the ability of an antifungal peptide to self-assemble affects its antifungal or antibacterial activity. We found that modifications of classical antifungal peptide derivative allowed it to self-assemble and did not alter its antifungal activity, and yet self-assembly substantially increased the antibacterial activity of the peptide. These results support the idea that peptide self-assembly can enhance antibacterial activities and emphasize a distinction between the action of antifungal peptides and that of antibacterial peptides. Accordingly, we suggest that the possible generality of this distinction should be widely tested.

Ultrashort Cell-Penetrating Peptides for Enhanced Sonophoresis-Mediated Transdermal Transport.

The skin is a key site for drug administration because of its large surface area and noninvasive accessibility. However, the dermal architecture serves as an excellent barrier, protecting from external mechanical, chemical, microbial, and physical perturbations. Most drugs display poor permeability through this barrier, thus making dermal and subdermal delivery challenging. Cell-penetrating peptides (CPPs), a diverse group of relatively short cationic and amphipathic membrane-interacting peptides, are fast becoming an important class of drug carriers and could potentially be developed for the dermal delivery of active molecules. However, the mechanism of CPP transdermal delivery is not fully understood, and there is a genuine need for a minimal model to understand this important phenomenon. Here, we demonstrate the potent membrane interactions of a minimal four-amino-acid-long CPP as well as the significance of guanidinium patterning and cationic nature of this palindromic peptide on its bioactivity. Furthermore, we demonstrate the biocompatibility of this peptide as well as its rapid cellular uptake and endosomal distribution. Finally, by utilizing a porcine full-thickness skin model, we demonstrate the substantial independent dermal and sonophoresis-based transdermal penetration of this minimal model. These results provide a minimal model for CPPs which can be easily manipulated for further biophysical and biochemical evaluations as well as a potent functional CPP with excellent skin permeability, which can be utilized for a wide variety of cosmetic and medical applications.

Induction of retinopathy by fibrillar oxalate assemblies.

The formation of metabolite fibrillar assemblies represents a paradigm shift in the study of human metabolic disorders. Yet, direct clinical relevance has been attributed only to metabolite crystals. A notable example for metabolite crystallization is calcium oxalate crystals observed in various diseases, including primary hyperoxaluria. We unexpectedly observed retinal damage among young hyperoxaluria patients in the absence of crystals. Exploring the possible formation of alternative supramolecular organizations and their biological role, here we show that oxalate can form ordered fibrils with no associated calcium. These fibrils inflict intense retinal cytotoxicity in cultured cells. A rat model injected with oxalate fibrils recaptures patterns of retinal dysfunction observed in patients. Antibodies purified from hyperoxaluria patient sera recognize oxalate fibrils regardless of the presence of calcium. These findings highlight a new molecular basis for oxalate-associated disease, and to our knowledge provide the first direct clinical indication for the pathogenic role of metabolite fibrillar assemblies.

Single cell imaging and quantification of TDP-43 and α-synuclein intercellular propagation.

The intercellular spreading of protein assemblies is a major factor in the progression of neurodegenerative disorders. The quantitative study and visualization of cell-to-cell propagation using tagged-proteins is challenging due to the steric effect of relatively large fluorescence tags and the risk of 'false positive' identification when analyzing these rare transmission events. Here, we established a cell culture model to characterize the cell-to-cell transmission of TAR DNA-binding protein and α-synuclein, involved in amyotrophic lateral sclerosis and Parkinson's disease, respectively, using the small nine amino acid influenza hemagglutinin tag. The novel use of single cell resolution imaging flow cytometry allowed the visualization and quantification of all individual transmission events. Cell-level analysis of these events indicated that the degree of transfer is lower than previously reported based on conventional flow cytometry. Furthermore, our analysis can exclude 'false positive' events of cellular overlap and extracellular debris attachment. The results were corroborated by high-resolution confocal microscopy mapping of protein localization.