The Celiac-Disease Superantigen Oligomerizes and Increases Permeability in an Enterocyte Cell Model.

Celiac disease (CeD) is an autoimmune disorder triggered by gluten proteins, affecting approximately 1 % of the global population. The 33-mer deamidated gliadin peptide (DGP) is a metabolically modified wheat-gluten superantigen for CeD. Here, we demonstrate that the 33-mer DGP spontaneously assembles into oligomers with a diameter of approximately 24 nm. The 33-mer DGP oligomers present two main secondary structural motifs-a major polyproline II helix and a minor ß-sheet structure. Importantly, in the presence of 33-mer DGP oligomers, there is a statistically significant increase in the permeability in the gut epithelial cell model Caco-2, accompanied by the redistribution of zonula occludens-1, a master tight junction protein. These findings provide novel molecular and supramolecular insights into the impact of 33-mer DGP in CeD and highlight the relevance of gliadin peptide oligomerization.

IDP Force Fields Applied to Model PPII-Rich 33-mer Gliadin Peptides.

The 33-mer gliadin peptide and its deamidated metabolite, 33-mer DGP, are the immunodominant peptides responsible for the adaptive immune response in celiac disease (CD). CD is a complex autoimmune chronic disorder triggered by gluten ingestion that affects the small intestine and affects ∼1% of the global population. The 33-mers are polyproline II-rich (PPII) and intrinsically disordered peptides (IDPs), whose structures remain elusive. We sampled the conformational ensembles of both 33-mer peptides via molecular dynamics simulations employing two force fields (FFs) (Amber ff03ws and Amber ff99SB-disp) specifically validated for other IDPs. Our results show that both FFs allow the extensive exploration of the conformational landscape, which was not possible with the standard FF GROMOS53A6 reported before. Clustering analysis of the trajectories showed that the five largest clusters (78-88% of the total structures) present elongated, semielongated, and curved conformations in both FFs. Large average radius of gyration and solvent-exposed surfaces characterized these structures. While the structures sampled are similar, the Amber ff99SB-disp trajectories explored folded conformations with a higher probability. In addition, PPII secondary structure was preserved throughout the trajectories (58-73%) together with a non-negligible content of ß structures (11-23%), in agreement with previous experimental results. This work represents the initial step in studying further the interaction of these peptides with other biologically relevant molecules, which could lead to finally disclose the molecular events that lead to CD.

Celiac Disease and Possible Dietary Interventions: From Enzymes and Probiotics to Postbiotics and Viruses.

Celiac Disease (CeD) is a chronic small intestinal immune-mediated enteropathy caused by the ingestion of dietary gluten proteins in genetically susceptible individuals. CeD is one of the most common autoimmune diseases, affecting around 1.4% of the population globally. To date, the only acceptable treatment for CeD is strict, lifelong adherence to a gluten-free diet (GFD). However, in some cases, GFD does not alter gluten-induced symptoms. In addition, strict adherence to a GFD reduces patients' quality of life and is often a socio-economic burden. This narrative review offers an interdisciplinary overview of CeD pathomechanism and the limitations of GFD, focusing on current research on possible dietary interventions. It concentrates on the recent research on the degradation of gluten through enzymes, the modulation of the microbiome, and the different types of "biotics" strategies, from probiotics to the less explored "viromebiotics" as possible beneficial complementary interventions for CeD management. The final aim is to set the context for future research that may consider the role of gluten proteins and the microbiome in nutritional and non-pharmacological interventions for CeD beyond the sole use of the GFD.

From celiac disease to coccidia infection and vice-versa: The polyQ peptide CXCR3-interaction axis.

Zonulin is a physiological modulator of intercellular tight junctions, which upregulation is involved in several diseases like celiac disease (CeD). The polyQ gliadin fragment binds to the CXCR3 chemokine receptor that activates zonulin upregulation, leading to increased intestinal permeability in humans. Here, we report a general hypothesis based on the structural connection between the polyQ sequence of the immunogenic CeD protein, gliadin, and enteric coccidian parasites proteins. Firstly, a novel interaction pathway between the parasites and the host is described based on the structural similarities between polyQ gliadin fragments and the parasite proteins. Secondly, a potential connection between coccidial infections as a novel environmental trigger of CeD is hypothesized. Therefore, this report represents a promising breakthrough for coccidian research and points out the potential role of coccidian parasites as a novel trigger of CeD that might define a preventive strategy for gluten-related disorders in general. Also see the video abstract here: https://youtu.be/oMaQasStcFI.

Molecular and Structural Parallels between Gluten Pathogenic Peptides and Bacterial-Derived Proteins by Bioinformatics Analysis.

Gluten-related disorders (GRDs) are a group of diseases that involve the activation of the immune system triggered by the ingestion of gluten, with a worldwide prevalence of 5%. Among them, Celiac disease (CeD) is a T-cell-mediated autoimmune disease causing a plethora of symptoms from diarrhea and malabsorption to lymphoma. Even though GRDs have been intensively studied, the environmental triggers promoting the diverse reactions to gluten proteins in susceptible individuals remain elusive. It has been proposed that pathogens could act as disease-causing environmental triggers of CeD by molecular mimicry mechanisms. Additionally, it could also be possible that unrecognized molecular, structural, and physical parallels between gluten and pathogens have a relevant role. Herein, we report sequence, structural and physical similarities of the two most relevant gluten peptides, the 33-mer and p31-43 gliadin peptides, with bacterial pathogens using bioinformatics going beyond the molecular mimicry hypothesis. First, a stringent BLASTp search using the two gliadin peptides identified high sequence similarity regions within pathogen-derived proteins, e.g., extracellular proteins from Streptococcus pneumoniae and Granulicatella sp. Second, molecular dynamics calculations of an updated α-2-gliadin model revealed close spatial localization and solvent-exposure of the 33-mer and p31-43 peptide, which was compared with the pathogen-related proteins by homology models and localization predictors. We found putative functions of the identified pathogen-derived sequence by identifying T-cell epitopes and SH3/WW-binding domains. Finally, shape and size parallels between the pathogens and the superstructures of gliadin peptides gave rise to novel hypotheses about activation of innate immunity and dysbiosis. Based on our structural findings and the similarities with the bacterial pathogens, evidence emerges that these pathologically relevant gluten-derived peptides could behave as non-replicating pathogens opening new research questions in the interface of innate immunity, microbiome, and food research.

Pepsin Digest of Gliadin Forms Spontaneously Amyloid-Like Nanostructures Influencing the Expression of Selected Pro-Inflammatory, Chemoattractant, and Apoptotic Genes in Caco-2 Cells: Implications for Gluten-Related Disorders.

SCOPE: Proteolysis-resistant gliadin peptides are intensely investigated in biomedical research relates to celiac disease and gluten-related disorders. Herein, the first integrated supramolecular investigation of pepsin-digested gliadin peptides (p-gliadin) is presented in combination with its functional behavior in the Caco-2 cell line. METHODS AND RESULTS: First, gliadins are degraded by pepsin at pH 3, and the physicochemical properties of p-gliadin are compared with gliadin. An integrated approach using interfacial, spectroscopic, and microscopic techniques reveals that the p-gliadin forms spontaneously soluble large supramolecular structures, mainly oligomers and fibrils, capable of binding amyloid-sensitive dyes. The self-assembly of p-gliadin starts at a concentration of 0.40 µg mL-1 . Second, the stimulation of Caco-2 cells with the p-gliadin supramolecular system is performed, and the mRNA expression levels of a panel of genes are tested. The experiments show that p-gliadin composed of supramolecular structures triggers significant mRNA up-regulation (p < 0.05) of pro-apoptotic biomarkers (ratio Bcl2/Bak-1), chemokines (CCL2, CCL3, CCL4, CCL5, CXCL8), and the chemokine receptor CXCR3. CONCLUSIONS: This work demonstrates that p-gliadin is interfacial active, forming spontaneously amyloid-type structures that trigger genes in the Caco-2 cell line involved in recruiting specialized immune cells.

Peptidotriazolamers Inhibit Aß(1-42) Oligomerization and Cross a Blood-Brain-Barrier Model.

In peptidotriazolamers every second peptide bond is replaced by a 1H-1,2,3-triazole. Such foldamers are expected to bridge the gap in molecular weight between small-molecule drugs and protein-based drugs. Amyloid ß (Aß) aggregates play an important role in Alzheimer's disease. We studied the impact of amide bond replacements by 1,4-disubstituted 1H-1,2,3-triazoles on the inhibitory activity of the aggregation "hot spots" K16 LVFF20 and G39 VVIA42 in Aß(1-42). We found that peptidotriazolamers act as modulators of the Aß(1-42) oligomerization. Some peptidotriazolamers are able to interfere with the formation of toxic early Aß oligomers, depending on the position of the triazoles, which is also supported by computational studies. Preliminary in vitro results demonstrate that a highly active peptidotriazolamer is also able to cross the blood-brain-barrier.

Novel Arylindigoids by Late-Stage Derivatization of Biocatalytically Synthesized Dibromoindigo.

Indigoids represent natural product-based compounds applicable as organic semiconductors and photoresponsive materials. Yet modified indigo derivatives are difficult to access by chemical synthesis. A biocatalytic approach applying several consecutive selective C-H functionalizations was developed that selectively provides access to various indigoids: Enzymatic halogenation of l-tryptophan followed by indole generation with tryptophanase yields 5-, 6- and 7-bromoindoles. Subsequent hydroxylation using a flavin monooxygenase furnishes dibromoindigo that is derivatized by acylation. This four-step one-pot cascade gives dibromoindigo in good isolated yields. Moreover, the halogen substituent allows for late-stage diversification by cross-coupling directly performed in the crude mixture, thus enabling synthesis of a small set of 6,6'-diarylindigo derivatives. This chemoenzymatic approach provides a modular platform towards novel indigoids with attractive spectral properties.

Implementing Complementary Approaches to Shape the Mechanism of α-Synuclein Oligomerization as a Model of Amyloid Aggregation.

The aggregation of proteins into amyloid fibers is linked to more than forty still incurable cellular and neurodegenerative diseases such as Parkinson's disease (PD), multiple system atrophy, Alzheimer's disease and type 2 diabetes, among others. The process of amyloid formation is a main feature of cell degeneration and disease pathogenesis. Despite being methodologically challenging, a complete understanding of the molecular mechanism of aggregation, especially in the early stages, is essential to find new biological targets for innovative therapies. Here, we reviewed selected examples on α-syn showing how complementary approaches, which employ different biophysical techniques and models, can better deal with a comprehensive study of amyloid aggregation. In addition to the monomer aggregation and conformational transition hypothesis, we reported new emerging theories regarding the self-aggregation of α-syn, such as the alpha-helix rich tetramer hypothesis, whose destabilization induce monomer aggregation; and the liquid-liquid phase separation hypothesis, which considers a phase separation of α-syn into liquid droplets as a primary event towards the evolution to aggregates. The final aim of this review is to show how multimodal methodologies provide a complete portrait of α-syn oligomerization and can be successfully extended to other protein aggregation diseases.

Phenothiazine-Biaryl-Containing Fluorescent RGD Peptides.

Cyclic RGD peptides are well-known ligands of integrins. The integrins αV ß3 and α5 ß1 are involved in angiogenesis, and integrin αV ß3 is abundantly present on cancer cells, thus representing a therapeutic target. Hence, synthetic and biophysical studies continuously are being directed towards the understanding of ligand-integrin interaction. In this context, the development of versatile synthetic strategies to obtain fluorescent building blocks that can add molecular diversity and modular spectral characteristics while not compromising binding affinity or selectivity is a relevant task. An on-resin intramolecular Suzuki-Miyaura cross-coupling (SMC) between l- or d-7-bromotryptophan (7BrTrp) and a phenothiazine (Ptz) boronic acid affords fluorescent cyclic RGD pseudopeptides, c(RGD(W/w)Ptz). Ring closure by SMC establishes a phenothiazine-indole moiety with axial chirality. An array of eight novel compounds has been synthesized, among them one fluorescent compound with good affinity to integrin αV ß3 . The fluorescence properties of the analogues can be efficiently tuned depending on the substituents in Ptz moiety even for fluorescence emission in the visible (red) spectral range.

Photocontrolled DNA minor groove interactions of imidazole/pyrrole polyamides.

Azobenzenes are photoswitchable molecules capable of generating significant structural changes upon E-to-Z photoisomerization in peptides or small molecules, thereby controlling geometry and functionality. E-to-Z photoisomerization usually is achieved upon irradiation at 350 nm (π-π* transition), while the Z-to-E isomerization proceeds photochemically upon irradiation at >400 nm (n-π* transition) or thermally. Photoswitchable compounds have frequently been employed as modules, e.g., to control protein-DNA interactions. However, their use in conjunction with minor groove-binding imidazole/pyrrole (Im/Py) polyamides is yet unprecedented. Dervan-type Im/Py polyamides were equipped with an azobenzene unit, i.e., 3-(3-(aminomethyl)phenyl)azophenylacetic acid, as the linker between two Im/Py polyamide strands. Only the (Z)-azobenzene-containing polyamides bound to the minor groove of double-stranded DNA hairpins. Photoisomerization was exemplarily evaluated by 1H NMR experiments, while minor groove binding of the (Z)-azobenzene derivatives was proven by CD titration experiments. The resulting induced circular dichroism (ICD) bands of the bound ligands, together with the photometric determination of the dsDNA melting temperature, revealed a significant stabilization of the DNA upon association with the ligand. The (Z)-azobenzene acted as a building block inducing a reverse turn, which favored hydrogen bonds between the pyrrole/imidazole amide and the DNA bases. In contrast, the E-configured polyamides did not induce any ICD characteristic for minor groove binding. The incorporation of the photoswitchable azobenzene unit is a promising strategy to obtain photoswitchable Im/Py hairpin polyamides capable of interacting with the dsDNA minor groove only in the Z-configuration.

Real-Time BODIPY-Binding Assay To Screen Inhibitors of the Early Oligomerization Process of Aß1-42 Peptide.

Misfolding and aggregation of amyloid ß1-42 peptide (Aß1-42) play a central role in the pathogenesis of Alzheimer's disease (AD). Targeting the highly cytotoxic oligomeric species formed during the early stages of the aggregation process represents a promising therapeutic strategy to reduce the toxicity associated with Aß1-42. Currently, the thioflavin T (ThT) assay is the only established spectrofluorometric method to screen aggregation inhibitors. The success of the ThT assay is that it can detect Aß1-42 aggregates with high ß-sheet content, such as protofibrils or fibrils, which appear in the late aggregation steps. Unfortunately, by using the ThT assay, the detection of inhibitors of early soluble oligomers that present a low ß-sheet character is challenging. Herein, a new, facile, and robust boron-dipyrromethene (BODIPY) real-time assay suitable for 96-well plate format, which allows screening of compounds as selective inhibitors of the formation of Aß1-42 oligomers, is reported. These inhibitors decrease the cellular toxicity of Aß1-42, although they fail in the ThT assay. The findings have been confirmed and validated by structural analysis and cell viability assays under comparable experimental conditions. It is demonstrated that the BODIPY assay is a convenient method to screen and discover new candidate compounds that slow down or stop the pathological early oligomerization process and are active in the cellular assay. Therefore, it is a suitable complementary screening method of the current ThT assay.

Molecular mechanisms of 33-mer gliadin peptide oligomerisation.

The proteolytic resistant 33-mer gliadin peptide is an immunodominant fragment in gluten and responsible for the celiac disease and other gluten-related disorders. Meanwhile, the primary structure of the 33-mer is associated with the adaptive immune response in celiac patients, and the structural transformation of the 33-mer into protofilaments activates a primordial innate immune response in human macrophages. This means that accumulation, oligomerisation and structural transformation of the 33-mer could be the unknown first event that triggers the disease. Herein, we reveal the early stepwise mechanism of 33-mer oligomerisation by combining multiple computational simulations, tyrosine cross-linking, fluorescence spectroscopy and circular dichroism experiments. Our theoretical findings demonstrated that the partial charge distribution along the 33-mer molecule and the presence of glutamine that favours H-bonds between the oligomers are the driving forces that trigger oligomerisation. The high content of proline is critical for the formation of the flexible PPII secondary structure that led to a ß structure transition upon oligomerisation. Experimentally, we stabilised the 33-mer small oligomers by dityrosine cross-linking, detecting from dimers to higher molecular weight oligomers, which confirmed our simulations. The relevance of 33-mer oligomers as a trigger of the disease as well as its inhibition may be a novel therapeutic strategy for the treatment of gluten-related disorders.

Sequence-Specific DNA Binding by Noncovalent Peptide-Azocyclodextrin Dimer Complex as a Suitable Model for Conformational Fuzziness.

Transcription factors are proteins lying at the endpoint of signaling pathways that control the complex process of DNA transcription. Typically, they are structurally disordered in the inactive state, but in response to an external stimulus, like a suitable ligand, they change their conformation, thereby activating DNA transcription in a spatiotemporal fashion. The observed disorder or fuzziness is functionally beneficial because it can add adaptability, versatility, and reversibility to the interaction. In this context, mimetics of the basic region of the GCN4 transcription factor (Tf) and their interaction with dsDNA sequences would be suitable models to explore the concept of conformational fuzziness experimentally. Herein, we present the first example of a system that mimics the DNA sequence-specific recognition by the GCN4 Tf through the formation of a non- covalent tetra-component complex: peptide-azoß-CyD(dimer)-peptide-DNA. The non-covalent complex is constructed on the one hand by a 30 amino acid peptide corresponding to the basic region of GCN4 and functionalized with an adamantane moiety, and on the other hand an allosteric receptor, the azoCyDdimer, that has an azobenzene linker connecting two ß-cyclodextrin units. The azoCyDdimer responds to light stimulus, existing as two photo-states: the first thermodynamically stable with an E:Z isomer ratio of 95:5 and the second obtained after irradiation with ultraviolet light, resulting in a photostationary state with a 60:40 E:Z ratio. Through electrophoretic shift assays and circular dichroism spectroscopy, we demonstrate that the E isomer is responsible for dimerization and recognition. The formation of the non-covalent tetra component complex occurs in the presence of the GCN4 cognate dsDNA sequence ('5-..ATGA cg TCAT..-3') but not with ('5-..ATGA c TCAT..-3') that differs in only one spacing nucleotide. Thus, we demonstrated that the tetra-component complex is formed in a specific manner that depends on the geometry of the ligand, the peptide length, and the ds DNA sequence. We hypothesized that the mechanism of interaction is sequential, and it can be described by the polymorphism model of static fuzziness. We argue that chemically modified peptides of the GCN4 Tf are suitable minimalist experimental models to investigate conformational fuzziness in protein-DNA interactions.

Translational Chemistry Meets Gluten-Related Disorders.

Gluten-related disorders are a complex group of diseases that involve the activation of the immune system triggered by the ingestion of gluten. Among these, celiac disease, with a prevalence of 1 %, is the most investigated, but recently, a new pathology, named nonceliac gluten sensitivity, was reported with a general prevalence of 7 %. Finally, there other less-prevalent gluten-related diseases such as wheat allergy, gluten ataxia, and dermatitis herpetiformis (with an overall prevalence of less than 0.1 %). As mentioned, the common molecular trigger is gluten, a complex mixture of storage proteins present in wheat, barley, and a variety of oats that are not fully degraded by humans. The most-studied protein related to disease is gliadin, present in wheat, which possesses in its sequence many pathological fragments. Despite a lot of effort to treat these disorders, the only effective method is a long-life gluten-free diet. This Review summarizes the actual knowledge of gluten-related disorders from a translational chemistry point of view. We discuss what is currently known from the literature about the interaction of gluten with the gut and the critical host responses it evokes and, finally, connect them to our current and novel molecular understanding of the supramolecular organization of gliadin and the 33-mer gliadin peptide fragment under physiological conditions.

Development of a Nonionic Azobenzene Amphiphile for Remote Photocontrol of a Model Biomembrane.

We report the synthesis and characterization of a simple nonionic azoamphiphile, C12OazoE3OH, which behaves as an optically controlled molecule alone and in a biomembrane environment. First, Langmuir monolayer and Brewster angle microscopy (BAM) experiments showed that pure C12OazoE3OH enriched in the (E) isomer was able to form solidlike mesophase even at low surface pressure associated with supramolecular organization of the azobenzene derivative at the interface. On the other hand, pure C12OazoE3OH enriched in the (Z) isomer formed a less solidlike monolayer due to the bent geometry around the azobenzene moiety. Second, C12OazoE3OH is well-mixed in a biological membrane model, Lipoid s75 (up to 20%mol), and photoisomerization among the lipids proceeded smoothly depending on light conditions. It is proposed that the cross-sectional area of the hydroxyl triethylenglycol head of C12OazoE3OH inhibits azobenzenes H-aggregation in the model membrane; thus, the tails conformation change due to photoisomerization is transferred efficiently to the lipid membrane. We showed that the lipid membrane effectively senses the azobenzene geometrical change photomodulating some properties, like compressibility modulus, transition temperature, and morphology. In addition, photomodulation proceeds with a color change from yellow to orange, providing the possibility to externally monitor the system. Finally, Gibbs monolayers showed that C12OazoE3OH is able to penetrate the highly packing biomembrane model; thus, C12OazoE3OH might be used as photoswitchable molecular probe in real systems.

Self-organization of gliadin in aqueous media under physiological digestive pHs.

Here we showed that gliadin, a complex protein system related to celiac disease and other human diseases, is spontaneously self-organized in a very dilute solution at pH 3.0 and 7.0 in water under low ionic strength (10mM NaCl). The spontaneous self-organization at pH 3.0 increases the apparent solubility due to the formation of finite sized aggregates, such as those formed in the micellization of amphiphilic molecules. Switching the pH from 3.0 to 7.0 lead to a phase separation, however part of the nano-particles are stable remaining disperse in water after centrifugation. Also, beside the pH change led to changes in protein composition and concentration, we determined that the secondary structure of both system is the same. Moreover, Tyrs are slightly more buried and Trps are slightly more exposed to water at pH 7.0 than those at pH 3.0. Electron microscopy techniques showed that both gliadin systems are composed of nanostructures and in the case of pH 7.0 amorphous microaggregates were found, too. Only nanostructures at pH 3.0 showed a micromolar binding affinity to Nile red probe, suggesting the presence of accessible hydrophobic patches which are not more accessible at pH 7.0. All our results suggest that gliadin is able to self-organized at pH 3.0 forming protein micelles type nanostructures (ζ=+13, 42 ± 1.55 mV), meanwhile at 7.0 the decrease of superficial charge to ζ of +4, 78 ± 0.48 mV led to the formation of stable colloidal nanoparticles, unable to interact with Nile red probe. Our findings may open new perspectives for the understanding of gliadin ability to avoid proteolysis, to reach and cross the intestinal lumen and to trigger different immunological disorders.

Dyes and stains: from molecular structure to histological application.

In the present review, the chemistry of dyes as well as the interaction mechanisms between tissue and dye has been detailed, and also some of the key factors affecting the selectivity of dyes by certain cellular structures have been mentioned. Moreover, due to the relevance that histological stains have acquired in biomedical research, some of the most common stains have been described, pointing out previous and current applications in basic and applied research.