Amyloid hexapeptide prevent dental caries by antibiofilm formation.

OBJECTIVES: Biofilm formed by cariogenic microbes is the direct cause of dental caries, therefore, prevention of dental caries should be anti-biofilm-based. Previously, we found the amyloid hexapeptides efficiently inhibited biofilm formation by aggregating into amyloid fibrils agglutinating microbes. This study aimed to select the most stable amyloid hexapeptide GIDLKI (GI6) and study its anti-caries effect. METHODS: Biofilms of multi-species bacteria, derived from mixed saliva, were cultured to evaluate the anti-biofilm formation effect of GI6. And then, the primary cariogenic bacterium Streptococcus mutans (S.mutans) was cultured in BHI with various pH, gradient concentrations of sucrose, glucose, and calcium ions to evaluate the anti-biofilm formation effects of GI6. Then models of human enamel block caries and twenty male SPF-SD rat caries induced by S. mutans biofilm were constructed, and confocal laser scanning microscopy, scanning electron microscopy, and micro-computed tomography were applied to investigate the anti-biofilm formation, anti-caries effects and use safety of GI6. RESULTS: GI6 could inhibit the multi-species bacteria biofilm formation and remained effective in anti-biofilm activity against S. mutans in environments closely related to caries. GI6 suppressed S. mutans biofilm formation and thus prevented or alleviated the development of caries in human tooth blocks and rat teeth. GI6 did not affect the intestinal flora, serum biochemical parameters, and the pathological changes of various organs. CONCLUSIONS: Amyloid hexapeptides, including but not limited to GI6, are novel effective anti-caries agents that can be used to prevent dental caries safely. CLINICAL SIGNIFICANCE: This study explored the anti-biofilm formation and anti-caries effect of GI6 in vitro, highlighting the anti-biofilm formation therapy for dental caries and setting a foundation for the practical application of GI6 for the treatment of dental caries.

Nordihydroguaiaretic Acid (NDGA) Inhibits CsgA Polymerization, Bacterial Amyloid Biogenesis, and Biofilm Formation.

Escherichia coli and other Enterobacteriaceae thrive in robust biofilm communities through the coproduction of curli amyloid fibers and phosphoethanolamine cellulose. Curli promote adhesion to abiotic surfaces and plant and human host tissues and are associated with pathogenesis in urinary tract infection and food-borne illness. The production of curli in the host has also been implicated in the pathogenesis of neurodegenerative diseases. We report that the natural product nordihydroguaiaretic acid (NDGA) is effective as a curlicide in E. coli. NDGA prevents CsgA polymerization in vitro in a dose-dependent manner. NDGA selectively inhibits cell-associated curli assembly and inhibits uropathogenic E. coli biofilm formation. More broadly, this work emphasizes the ability to evaluate and identify bioactive amyloid assembly inhibitors by using the powerful gene-directed amyloid biogenesis machinery in E. coli.

Designed peptides as nanomolar cross-amyloid inhibitors acting via supramolecular nanofiber co-assembly.

Amyloid self-assembly is linked to numerous devastating cell-degenerative diseases. However, designing inhibitors of this pathogenic process remains a major challenge. Cross-interactions between amyloid-ß peptide (Aß) and islet amyloid polypeptide (IAPP), key polypeptides of Alzheimer's disease (AD) and type 2 diabetes (T2D), have been suggested to link AD with T2D pathogenesis. Here, we show that constrained peptides designed to mimic the Aß amyloid core (ACMs) are nanomolar cross-amyloid inhibitors of both IAPP and Aß42 and effectively suppress reciprocal cross-seeding. Remarkably, ACMs act by co-assembling with IAPP or Aß42 into amyloid fibril-resembling but non-toxic nanofibers and their highly ordered superstructures. Co-assembled nanofibers exhibit various potentially beneficial features including thermolability, proteolytic degradability, and effective cellular clearance which are reminiscent of labile/reversible functional amyloids. ACMs are thus promising leads for potent anti-amyloid drugs in both T2D and AD while the supramolecular nanofiber co-assemblies should inform the design of novel functional (hetero-)amyloid-based nanomaterials for biomedical/biotechnological applications.

Exploration of Isoquinoline Alkaloids as Potential Inhibitors against Human Islet Amyloid Polypeptide.

Type-2 diabetes mellitus (T2DM) is one of the most concerning public health problems because of its high incidence, multiple complications, and difficult treatment. Human islet amyloid polypeptide (hIAPP) is closely linked to T2DM because its abnormal self-assembly causes membrane damage and cell dysfunction. The development of potential inhibitors to prevent hIAPP fibrillation is a promising strategy for the intervention and treatment of diabetes. Natural isoquinoline alkaloids are used as effective medication that targets different biomolecules. Although studies explored the efficacy of berberine, jatrorrhizine, and chelerythrine in diabetes, the underlying mechanism remains unclear. Herein, three isoquinoline alkaloids are selected to reveal their roles in hIAPP aggregation, disaggregation, and cell protection. All three compounds displayed good inhibitory effects on peptide fibrillation, scattered the preformed fibrils into small oligomers and most monomers, and upregulated cell viability by reducing hIAPP oligomerization. Moreover, combined biophysical analyses indicated that the compounds affected the ß-sheet structure and hydrophobicity of polypeptides significantly, and the benzo[c]phenanthridine structure of chelerythrine was beneficial to the inhibition of hIAPP aggregation and their hydrophobic interaction, compared with that of berberine and jatrorrhizine. Our work elaborated the effects of these alkaloids on hIAPP fibrillation and reveals a possible mechanism for these compounds against T2DM.

A study on the interaction of the amyloid fibrils of α-synuclein and hen egg white lysozyme with biological membranes.

Alpha-synuclein (α-syn) aggregation and mitochondrial dysfunction are considered as two of the main factors associated with Parkinson's disease (PD). In the present investigation, the effectiveness of the amyloid fibrils obtained from α-syn with those of hen egg white lysozyme (HEWL), as disease-related and-unrelated proteins, to damage rat brain and rat liver mitochondria have been investigated. This was extended by looking at SH-SY5Y human neuroblastoma cells and erythrocytes, thereby investigating the significance of structural characteristics of amyloid fibrils related to their interactions with biomembranes obtained from various sources. Results presented clearly demonstrate substantial differences in the response of tested biomembranes to toxicity induced by α-syn/HEWL amyloid fibrils, highlighting a structure-function relationship. We found that fibrillar aggregates of α-syn, but not HEWL, caused a significant increase in mitochondrial ROS, loss of membrane potential, and mitochondrial swelling, in a dose-dependent manner. Toxicity was found to be more pronounced in brain mitochondria, as compared to liver mitochondria. For SH-SY5Y cells and erythrocytes, however, both α-syn and HEWL amyloid fibrils showed the capacity to induce toxicity. Taken together, these results may suggest selective toxicity of α-syn amyloid fibrils to mitochondria mediated likely by their direct interaction with the outer mitochondrial membrane, indicating a correlation between specific structural characteristics of α-syn fibrils and an organelle strongly implicated in PD pathology.

An antiviral trap made of protein nanofibrils and iron oxyhydroxide nanoparticles.

Minimizing the spread of viruses in the environment is the first defence line when fighting outbreaks and pandemics, but the current COVID-19 pandemic demonstrates how difficult this is on a global scale, particularly in a sustainable and environmentally friendly way. Here we introduce and develop a sustainable and biodegradable antiviral filtration membrane composed of amyloid nanofibrils made from food-grade milk proteins and iron oxyhydroxide nanoparticles synthesized in situ from iron salts by simple pH tuning. Thus, all the membrane components are made of environmentally friendly, non-toxic and widely available materials. The membrane has outstanding efficacy against a broad range of viruses, which include enveloped, non-enveloped, airborne and waterborne viruses, such as SARS-CoV-2, H1N1 (the influenza A virus strain responsible for the swine flu pandemic in 2009) and enterovirus 71 (a non-enveloped virus resistant to harsh conditions, such as highly acidic pH), which highlights a possible role in fighting the current and future viral outbreaks and pandemics.

Dual Antibody-Conjugated Amyloid Nanorods to Promote Selective Cell-Cell Interactions.

Grafting biomolecules on nanostructures' surfaces is an increasingly used strategy to target pathogenic cells, with both diagnostic and therapeutic applications. However, nanomaterials monofunctionalized by conjugating a single type of ligand find limited uses in pathologies/therapies that require two or more targets/receptors to be targeted and/or activated with a single molecular entity simultaneously. Therefore, multivalent nanomaterials for dual- or multitargeting are attracting significant interest. This study provides a proof of concept of such nanostructures. We have recently developed a modular methodology that allows obtaining amyloid-based materials decorated with active globular domains. Here, this approach is exploited to generate functional amyloid fibrils displaying antibody capture moieties. A high antibody binding affinity and capacity for the resulting nanofibrils, whose size can be manipulated to obtain homogeneous nanorods with high biocompatibility, are demonstrated. These nanorods are then used for specific antibody-mediated targeting of different cell types. Simultaneous conjugation of these nanorods with different antibodies allows obtaining a mimic of a bispecific antibody that redirects T lymphocytes to tumoral cells, holding high potential for immunotherapy. Overall, the work illustrates a modular and straightforward strategy to obtain preparative quantities of multivalent antibody-functionalized nanomaterials with multitargeting properties without the need for covalent modification.

Boosting the Photodynamic Degradation of Islet Amyloid Polypeptide Aggregates Via a "Bait-Hook-Devastate" Strategy.

Photosensitizers that can generate reactive oxygen species (ROS) upon irradiation have emerged as promising agents for photodynamic degradation of toxic amyloid aggregates that are linked to many amyloidogenic diseases. However, due to the ultrastable ß-sheet structure in amyloid aggregates and inefficient utilization of the generated ROS, it usually requires high stoichiometric concentration of the photosensitizer and/or intensive light irradiation to fully dissociate aggregates. In this work, we have developed a "bait-hook-devastate" strategy to boost the efficiency of the photodynamic degradation of amyloid aggregates. This strategy employs anionic polyacrylic acid as a bait to accumulate cationic human islet amyloid polypeptide (IAPP) aggregates and positively charged photosensitizer TPCI in a confined area through electronic interactions. Multiple characterization studies proved that the utilization rate of ROS generated by TPCI was remarkably improved via this strategy, which amplified the ability of TPCI to dissociate IAPP aggregates. Rapid and complete degradation of IAPP aggregates could be achieved by irradiating the system under very mild conditions for less than 30 min, and the IAPP-mediated cytotoxicity was also largely alleviated, providing a new paradigm to accelerate photodynamic degradation of amyloid aggregates for further practical applications.

Spearmint Extract Containing Rosmarinic Acid Suppresses Amyloid Fibril Formation of Proteins Associated with Dementia.

Neurological dementias such as Alzheimer's disease and Lewy body dementia are thought to be caused in part by the formation and deposition of characteristic insoluble fibrils of polypeptides such as amyloid beta (Aß), Tau, and/or α-synuclein (αSyn). In this context, it is critical to suppress and remove such aggregates in order to prevent and/or delay the progression of dementia in these ailments. In this report, we investigated the effects of spearmint extract (SME) and rosmarinic acid (RA; the major component of SME) on the amyloid fibril formation reactions of αSyn, Aß, and Tau proteins in vitro. SME or RA was added to soluble samples of each protein and the formation of fibrils was monitored by thioflavin T (ThioT) binding assays and transmission electron microscopy (TEM). We also evaluated whether preformed amyloid fibrils could be dissolved by the addition of RA. Our results reveal for the first time that SME and RA both suppress amyloid fibril formation, and that RA could disassemble preformed fibrils of αSyn, Aß, and Tau into non-toxic species. Our results suggest that SME and RA may potentially suppress amyloid fibrils implicated in the progression of Alzheimer's disease and Lewy body dementia in vivo, as well.

Internalisation and toxicity of amyloid-ß 1-42 are influenced by its conformation and assembly state rather than size.

Amyloid fibrils found in plaques in Alzheimer's disease (AD) brains are composed of amyloid-ß peptides. Oligomeric amyloid-ß 1-42 (Aß42) is thought to play a critical role in neurodegeneration in AD. Here, we determine how size and conformation affect neurotoxicity and internalisation of Aß42 assemblies using biophysical methods, immunoblotting, toxicity assays and live-cell imaging. We report significant cytotoxicity of Aß42 oligomers and their internalisation into neurons. In contrast, Aß42 fibrils show reduced internalisation and no toxicity. Sonicating Aß42 fibrils generates species similar in size to oligomers but remains nontoxic. The results suggest that Aß42 oligomers have unique properties that underlie their neurotoxic potential. Furthermore, we show that incubating cells with Aß42 oligomers for 24 h is sufficient to trigger irreversible neurotoxicity.

Reverse engineering synthetic antiviral amyloids.

Human amyloids have been shown to interact with viruses and interfere with viral replication. Based on this observation, we employed a synthetic biology approach in which we engineered virus-specific amyloids against influenza A and Zika proteins. Each amyloid shares a homologous aggregation-prone fragment with a specific viral target protein. For influenza we demonstrate that a designer amyloid against PB2 accumulates in influenza A-infected tissue in vivo. Moreover, this amyloid acts specifically against influenza A and its common PB2 polymorphisms, but not influenza B, which lacks the homologous fragment. Our model amyloid demonstrates that the sequence specificity of amyloid interactions has the capacity to tune amyloid-virus interactions while allowing for the flexibility to maintain activity on evolutionary diverging variants.

Discovery of Chemicals to Either Clear or Indicate Amyloid Aggregates by Targeting Memory-Impairing Anti-Parallel Aß Dimers.

Amyloid-ß (Aß) oligomers are implicated in Alzheimer disease (AD). However, their unstable nature and heterogeneous state disrupts elucidation of their explicit role in AD progression, impeding the development of tools targeting soluble Aß oligomers. Herein parallel and anti-parallel variants of Aß(1-40) dimers were designed and synthesized, and their pathogenic properties in AD models characterized. Anti-parallel dimers induced cognitive impairments with increased amyloidogenesis and cytotoxicity, and this dimer was then used in a screening platform. Through screening, two FDA-approved drugs, Oxytetracycline and Sunitinib, were identified to dissociate Aß oligomers and plaques to monomers in 5XFAD transgenic mice. In addition, fluorescent Astrophloxine was shown to detect aggregated Aß in brain tissue and cerebrospinal fluid samples of AD mice. This screening platform provides a stable and homogeneous environment for observing Aß interactions with dimer-specific molecules.

Neutrophil extracellular trap-enriched supernatants carry microRNAs able to modulate TNF-α production by macrophages.

Neutrophil extracellular traps (NETs) emerge from the cell as a DNA scaffold associated with cytoplasmic and granular proteins, able to immobilize and kill pathogens. This association occurs following nuclear and granular membrane disintegration, allowing contact with the decondensed chromatin. Thus, it is reasonable to speculate that the DNA can also mix with miRNAs and carry them in NETs. Here, we report for the first time the presence of the miRNA carriers associated with NETs and miRNAs present in NET-enriched supernatants (NET-miRs), thus adding a novel class of molecules and new proteins that can be released and transported in the NET platform. We observed that the majority of NET-miRs were common to all four stimuli used (PMA, interleukin-8, amyloid fibrils and Leishmania), and that miRNA-142-3p carried by NETs down-modulates protein kinase Cα and regulates TNF-α production in macrophages upon NET interaction with these cells. Our findings unveil a novel role for NETs in the cell communication processes, allowing the conveyance of miRNA from neutrophils to neighboring cells.

Terminal Alkynes as Raman Probes of α-Synuclein in Solution and in Cells.

Conformational changes in α-synuclein (α-syn) are central to its biological function and Parkinson's disease pathology. Here, terminal alkynes (homopropargylglycine) were employed as environmentally sensitive Raman probes at residues 1, 5, 116, and 127 to characterize soluble (disordered), micelle-bound (α-helical), and fibrillar (ß-sheet) α-syn. Along with the full-length protein, a disease-related C-terminal truncation (1-115) was also studied. For the first time, ß-sheet α-syn amyloid structure was detected by the amide-I band in N27 dopaminergic rat cells, where a reciprocal relationship between levels of fibrils and lipids was seen. Site-specific spectral features of the terminal alkynes also revealed the heterogeneity of the cellular environment. This work shows the versatility of Raman microspectroscopy and the power of unnatural amino acids in providing structural and residue-level insights in solution and in cells.

Amyloid-Polyphenol Hybrid Nanofilaments Mitigate Colitis and Regulate Gut Microbial Dysbiosis.

It is a desirable and powerful strategy to precisely fabricate functional soft matter through self-assembly of molecular building blocks across a range of length scales. Proteins, nucleic acids, and polyphenols are the self-assemblers ubiquitous in nature. Assembly of proteins into flexible biocolloids, amyloid fibrils with high aspect ratio, has emerged as an unchallenged templating strategy for high-end technological materials and bio-nanotechnologies. We demonstrate the ability of these fibrils to support the deposition and self-assembly of polyphenols into hybrid nanofilaments and functional macroscopic hydrogels made thereof. The length scale of the substance that amyloid fibrils can attach with acting as the building templates was extended from nanometer down to sub-nanometer. Significantly increased loading capacities of polyphenols (up to 4.0 wt %) compared to that of other delivery systems and improved stability were realized. After oral administration, the hydrogels could transport from the stomach to the small intestine and finally to the gut (cecum, colon, rectum), with a long retention time in the colon. Oral administration of the hydrogels significantly ameliorated colitis in a mouse model, promoted intestinal barrier function, suppressed the pro-inflammatory mRNA expression, and very significantly (P < 0.01) regulated gut microbial dysbiosis. Specifically, it reduced the abundance of normally enriched operational taxonomic units related to colitis, especially targeting facultative anaerobes of the phylum Proteobacteria, such as Aestuariispira and Escherichia. The short-chain fatty acid metabolites were enriched. Combined with their nontoxic nature observed in this long-term study in mice, the obtained amyloid-polyphenol gels have high application potentials for gastrointestinal diseases by "drugging the microbiome".

Designer Amyloid Cell-Penetrating Peptides for Potential Use as Gene Transfer Vehicles.

Cell-penetrating peptides are used extensively to deliver molecules into cells due to their unique characteristics such as rapid internalization, charge, and non-cytotoxicity. Amyloid fibril biomaterials were reported as gene transfer or retroviral infection enhancers; no cell internalization of the peptides themselves is reported so far. In this study, we focus on two rationally and computationally designed peptides comprised of ß-sheet cores derived from naturally occurring protein sequences and designed positively charged and aromatic residues exposed at key residue positions. The ß-sheet cores bestow the designed peptides with the ability to self-assemble into amyloid fibrils. The introduction of positively charged and aromatic residues additionally promotes DNA condensation and cell internalization by the self-assembled material formed by the designed peptides. Our results demonstrate that these designer peptide fibrils can efficiently enter mammalian cells while carrying packaged luciferase-encoding plasmid DNA, and they can act as a protein expression enhancer. Interestingly, the peptides additionally exhibited strong antimicrobial activity against the enterobacterium Escherichia coli.

Amyloid-Like Rapid Surface Modification for Antifouling and In-Depth Remineralization of Dentine Tubules to Treat Dental Hypersensitivity.

Exposure of dentinal tubules (DTs) leads to the transmission of external stimuli within the DTs, causing dental hypersensitivity (DH). To treat DH, various desensitizers have been developed for occluding DTs. However, most desensitizers commercially available or in development are only able to seal the orifices, rather than the deep regions of the DTs, thus lacking long-term stability. Herein, it is shown that the fast amyloid-like aggregation of lysozyme (lyso) conjugated with poly(ethylene glycol) (PEG) (lyso-PEG) can afford a robust ultrathin nanofilm on the deep walls of DTs through a rapid one-step aqueous coating process (in 2 min). The resultant nanofilm provides a highly effective antifouling platform for resisting the attachment of oral bacteria such as Streptococcus mutans and induces remineralization in the DTs to seal both the orifices and depths of the DTs by forming hydroxyapatite (HAp) minerals in situ. Both in vitro and in vivo animal experiments prove that the nanofilm-coated DTs are occluded with a depth of over 60 ± 5 µ m, which is at least 6 times deeper than that reported in the literature. This approach thus demonstrates the concept that an amyloid-like proteinaceous nanofilm can offer an inexpensive, rapid, and efficient therapy for treating DH with long-term effect.

Impact of semen-derived amyloid (SEVI) on sperm viability and motility: its implication in male reproductive fitness.

Human semen contains a large number of macromolecules, including proteins/enzymes and carbohydrates, regulating and protecting sperm cells. Proteomic analysis of human seminal fluid led to the discovery of semen amyloids derived from short peptide fragments of the proteins prostatic acid phosphatase (PAP) and semenogelin (SG) which are known to play a crucial role in enhancing HIV infection. However, the relevance of their existence in human semen and role in maintaining sperm behavior remains unclear. Distinct physiological, biochemical, and biophysical attributes might cause these amyloids to influence sperm behavior positively or negatively, affecting fertilization or other reproductive processes. We assessed the direct effect of amyloids derived from a PAP248-286 fragment, on sperm motility and viability, which are crucial parameters for assessment of sperm quality in semen. Co-incubation of human sperm with PAP248-286 amyloids at normal physiological concentrations formed in buffer led to significant reduction in sperm viability, though approximately a 10× higher concentration was needed to show a similar effect with amyloid formed in seminal fluid. Both forms of PAP248-286 amyloid also had a significant impact on sperm motility at physiological levels, in agreement with a previous report. Our study suggests that PAP248-286 amyloids can directly influence sperm motility and viability in a concentration-dependent manner. We hypothesise that the direct toxic effect of PAP248-286 amyloid is normally mitigated by other seminal fluid ingredients, but that in pathological conditions, where PAP248-286 concentrations are elevated and it plays a role in determining sperm health and viability, with relevance for male fertility as well as sterility.

Blocking Inflammasome Activation Caused by ß-Amyloid Peptide (Aß) and Islet Amyloid Polypeptide (IAPP) through an IAPP Mimic.

Inflammation in the brain and pancreas is linked to cell degeneration and pathogenesis of both Alzheimer's disease (AD) and type 2 diabetes (T2D). Inflammatory cascades in both tissues are triggered by the uptake of ß-amyloid peptide (Aß) or islet amyloid polypeptide (IAPP) aggregates by microglial cells (AD) or macrophages (T2D) and their insufficient lysosomal degradation. This results in lysosomal damage, caspase-1/NLRP3 inflammasome activation and release of interleukin-1ß (IL-1ß), a key proinflammatory cytokine in both diseases. Here we show that the inflammatory processes mediated by Aß and IAPP aggregates in microglial cells and macrophages are blocked by IAPP-GI, a nonamyloidogenic IAPP mimic, which forms high-affinity soluble and nonfibrillar hetero-oligomers with both polypeptides. In contrast to fibrillar Aß aggregates, nonfibrillar Aß/IAPP-GI or Aß/IAPP hetero-oligomers become rapidly internalized by microglial cells and targeted to lysosomes where Aß is fully degraded. Internalization occurs via IAPP receptor-mediated endocytosis. Moreover, in contrast to IAPP aggregates, IAPP/IAPP-GI hetero-oligomers become rapidly internalized and degraded in the lysosomal compartments of macrophages. Our findings uncover a previously unknown function for the IAPP/Aß cross-amyloid interaction and suggest that conversion of Aß or IAPP into lysosome-targeted and easily degradable hetero-oligomers by heteroassociation with IAPP mimics could become a promising approach to specifically prevent amyloid-mediated inflammation in AD, T2D, or both diseases.