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Non celiac Gluten sensibility and protein aggregates

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Veronica I Dodero
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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. Coccidian parasites trigger zonulin upregulation and release via the binding of the parasite matching gliadin polyQ sequences to the CXCR‐type chemokine, thus representing an underappreciated mechanism of coccidial infection. The high sequence similarity between the PolyQs of gliadin and coccidia suggests that in humans, a coccidiosis episode may be an environmental trigger or predisposing factor for CeD and other gluten‐related disorders.
Veronica I Dodero
added 2 research items
Mol. Nutr. Food Res. 2021, 65, 202100200 DOI: 10.1002/mnfr.202100200 An integral physicochemical and functional approach demonstrated that pepsin gliadin digest forms spontaneously amyloid-type nanostructures whose interaction with the Caco-2 enterocyte cellular model induces the expression of selected mRNAs involved in the activation of immune cells. Therefore the pathogenic role of gliadin nanostructures may be relevant for biodemical research. This is reported by Veronica I. Dodero and co-workers in article number 2100200.
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.
Veronica I Dodero
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Veronica I Dodero
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Scope Proteolysis-resistant gliadin peptides are intensely investigated in biomedical research related 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 Caco-2 cell line. Methods and results First, we investigated gliadin degradation by pepsin at pH 3, and the physicochemical properties of p-gliadin were compared with gliadin. An integrated approach using interfacial, spectroscopic, and microscopic techniques revealed 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. Second, we stimulated CaCo-2 cells with the p-gliadin supramolecular system and screened the mRNA expression levels of a panel of genes involved in cellular inflammation, apoptosis, permeability, and chemoattraction of immune cells. Our findings suggest 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 the recruitment of specialized immune cells. This article is protected by copyright. All rights reserved
Veronica I Dodero
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Dear Colleagues,
As Guest Editor of the Special Issue of Molecules (IF 3.060) “Understanding Protein/Peptide Self-Assembly using Structural and Biophysical Chemistry”, it is my pleasure to invite you to submit an article on this topic. 
The article may be either a full paper or a communication based on your own research in this area or maybe a focused review article on some aspect of the subject. All submissions will be subject to peer review. If you plan to submit a review article, please provide me with a title and brief description at your earliest convenience, in order to avoid multiple reviews covering the same material.
Special Issue: Understanding Protein/Peptide Self-Assembly using Structural and Biophysical Chemistry 
Submission deadline: 30 April 2020.
Molecules is fully open access. Open access (unlimited and free access by readers) increases publicity and promotes more frequent citations, as indicated by several studies. Open access is supported by the authors and their institutes. An article-processing charge of CHF 1800 (APC) applies to each accepted paper. Note that for papers submitted after 31 December 2019, an APC of 2000 CHF will apply. You may be entitled to a discount if you have previously received a discount code. 
I would appreciate if you could let us know if you are interested in preparing a manuscript for this Special Issue and I will send you more detailed instructions for submission.
I look forward to hearing from you at the earliest opportunity.
With best wishes,
Veronica Dodero
 
Veronica I Dodero
added 2 research items
The proteolytical resistant 33-mer gliadin peptide is the immunodominant fragment in gluten and responsible for celiac disease, and other gluten-related disorders. Meanwhile, the primary structure of 33-mer is associated with the adaptive immune response in celiac patients, the structural transformation of 33-mer into protofilaments activates a primordial innate immune response in human macrophages. That means that accumulation, oligomerisation and structural transformation of 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 together with the presence of glutamine that favours H-bonds between oligomers are the driving forces that trigger oligomerisation. The high content of proline is critical to forming the flexible PPII secondary structure that let to a β structure transition on 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 triggers of disease as well as its inhibition may pose a novel therapeutic strategy for the treatment of gluten-related disorders
Celiac Disease (CeD) is a highly prevalent chronic immune‐mediated enteropathy developed in genetically predisposed individuals after ingestion of a group of wheat proteins (called gliadins and glutenins). The 13mer α‐gliadin peptide, p31‐43, induces proinflammatory responses, observed by in vitro assays and animal models, that may contribute to innate immune mechanisms of CeD pathogenesis. Since a cellular receptor for p31‐43 has not been identified, this raises the question of whether this peptide could mediate different biological effects. In this work, we aimed to characterize the p31‐43 secondary structure by different biophysical and in silico techniques. By Dynamic Light Scattering (DLS) and using an oligomer/fibril‐sensitive fluorescent probe, we showed the presence of oligomers of this peptide in solution. Furthermore, Atomic Force Microscopy (AFM) analysis showed p31‐43 oligomers with different height distribution. Also, peptide concentration had a very strong influence on peptide self‐organization process. Oligomers gradually increased their size at lower concentration. Whereas, at higher ones, oligomers increased their complexity, forming branched structures. By Circular Dichroism, we observed that p31‐43 self‐organized in a poly‐proline II conformation in equilibrium with β‐sheets‐like structures, whose pH remained stable in the range of 3 to 8. In addition, these findings were supported by Molecular Dynamics Simulation. The formation of p31‐43 nanostructures with increased β‐sheet structure may help to explain the molecular etiopathogenesis in the induction of pro‐inflammatory effects and subsequent damage at the intestinal mucosa in CeD.
Veronica I Dodero
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A new hypothesis about the relation of gliadin and its immunodominant 33-mer fragment aggregates as triggers of gluten-related disorders was presented.
Veronica I Dodero
added a research item
Gliadin, an immunogenic protein present in wheat, is not fully degraded by humans and after the normal gastric and pancreatic digestion, the immunodominant 33-mer gliadin peptide remains unprocessed. The 33-mer gliadin peptide is found in human faeces and urine, proving not only its proteolytic resistance in vivo but more importantly its transport through the entire human body. Here, we demonstrate that 33-mer supramolecular structures larger than 220nm induce the overexpression of nuclear factor kappa B (NF-κB) via a specific Toll-like Receptor (TLR) 2 and (TLR) 4 dependent pathway and the secretion of pro-inflammatory cytokines such as IP-10/CXCL10 and TNF-α. Using helium ion microscopy, we elucidated the initial stages of oligomerisation of 33-mer gliadin peptide, showing that rod-like oligomers are nucleation sites for protofilament formation. The relevance of the 33-mer supramolecular structures in the early stages of the disease is paving new perspectives in the understanding of gluten-related disorders.
Veronica I Dodero
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Veronica I Dodero
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Alpha-gliadin is a highly immunogenic protein from wheat, which is associated with many human diseases, like celiac disease and non-celiac gluten sensitivity. Because of that, gliadin solution is subject to intense biomedical research. However, the physicochemical nature of the employed gliadin solution at physiological pH is not understood. Herein, we present a supramolecular evaluation of the alpha-gliadin protein in water at pH 3.0 by dynamic light scattering (DLS), cryo-transmission electron microscopy (cryo-TEM) and small-angle-.X-ray scattering (SAXS). We report that at 0.5 wt% concentration (0.1 mg/ml), gliadin is already a colloidal polydisperse system with an average hydrodynamic radius of 30 ± 10 nm. By cryo-TEM, we detected mainly large clusters. However, it was possible to visualise for the first time prolate oligomers of around 68 nm and 103 nm, minor and major axis, respectively. SAXS experiments support the existence of prolate/rod-like structures. At 1.5 wt% concentration gliadin dimers, small oligomers and large clusters coexist. The radius of gyration (Rg1) of gliadin dimer is 5.72 ± 0.23 nm with a dimer cross-section (Rc) of 1.63 nm, and an average length of around 19 nm, this suggests that gliadin dimers are formed longitudinally. Finally, our alpha-gliadin 3D model, obtained by ab initio prediction and analysed by molecular dynamics (MD), predicts that two surfaces prone to aggregation are exposed to the solvent, at the C-terminus. We hypothesise that this region may be involved in the dimerisation process of alpha-gliadin.
Veronica I Dodero
added a research item
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.
Veronica I Dodero
added 3 research items
Gliadin, a protein present in wheat, rye and barley is not fully degraded by humans, leading to allergies, gluten sensitivity and celiac disease.(1,2,3).It has been hypothesized that increased intestinal permeability is an early event in celiac disease pathogenesis (4) but it is completely unknown what endows gliadin the unusual proteolytic resistance. From the physicochemical point of view, this protein is known to be soluble in alcohol solutions in a low percentage and it could be isolated from flour by dilute acetic acid extraction. By this procedure is have been demonstrated the formation of fibrils depending on ionic strength (5). Under different aqueous solutions conditions much less is known. Herein, it is present the evaluation of gliadin protein under different pHs and ionic strength in water. We obtained the solubility of a mix of commercial gliadin proteins under basic and acid conditions varying the temperature. It has been observed the importance of the pH and the temperature in order to form different self-assembly structures. At physiological pH spherical particules of 1-2μm of diameter were detected by electron microscopy. We hypothesize that the observed self-assembly process and the formation of supramolecular structures depending on pH could a be a special feature in order to understand the unusual proteolytic resistances. Taking into account that gastrointestinal environment is highly dynamic(6) in health and disease , recent insights on the potential of intestinal bacteria to influence human health, our results may help to understand Gliadin intolerance. A supramolecular evaluation by different Spectroscopies (UV-Vis and Fluorescence) and Electron Microscopy (SEM, TEM) is presented. References: 1).Hadjivassiliou, M., Williamson, C. A., Woodroofe, N. ,Trends Immunol.,25, 578-582 (2004). 2).Rubio-Tapia, A. , Murray, J. A. ,Curr. Opin. Gastroenterol., 26, 116-122 (2010). 3).Shan, L. et. al. ,Science, 297, 2275-2279 (2002). 4).Fassano, A. , Shea-Donohue, T., Nat. Clin. Pract. Gastroenterol. & Hepatol., 2, 416-422 (2005) 5).Kasarda, D., Bernardin, J.E., Thomas, R., Nature, 203-205 (1967). 6).Possemiers, S., Grootaert, C., Curr Pharm Des.,15(18):2051-65 (2009).
Medicinal chemistry is intimately connected with basic science such as organic synthesis, chemical biology and biophysical chemistry among other disciplines. The reason of such connections is due to the power of organic synthesis to provide designed molecules; chemical biology to give tools to discover biological and/or pathological pathways and biophysical chemistry which provides the techniques to characterize and the theoretical background to understand molecular behaviour. The present review provides some selective examples of these research areas. Initially, template dsDNA organic synthesis and the spatio-temporal control of transcription are presenting following by the supramolecular entities used in drug delivery, such as liposomes and liquid crystal among others. Finally, peptides and protein self-assembly is connected with biomaterials and as an important event in the balance between health and disease. The final aim of the present review is to show the power of chemical tools not only for the synthesis of new molecules but also to improve our understanding of recognition and self-assembly in the biological context.
This chapter provides a general overview of several spectroscopic techniques currently available for studying protein secondary structure in solution and assessing its changes in response to ligand and/or environmental interaction. These techniques include ultraviolet—visible (UV—vis) absorption spectroscopy, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, Fourier transform infrared (FT-IR) and Raman spectroscopy, and photon correlation spectroscopy.
Veronica I Dodero
added 4 research items
Gliadin, a protein present in wheat, rye and barley, undergoes incomplete enzymatic degradation during digestion, producing an immunogenic 33-mer peptide, LQLQPF(PQPQLPY)3 PQPQPF. The special features of 33-mer that provoke a break in its tolerance leading to gliadin sensitivity and celiac disease remains elusive. Herein it is reported that 33-mer gliadin peptide was not only able to fold into PPII secondary structure, but also depending on concentration conformational transition and self-assembly proceeded under aqueous condition, pH 7.0. A 33-mer dimer is presented as one initial possible step in the self-assembly process obtained by partial electrostatics charge distribution calculation and molecular dynamics. In addition, electron microscopy experiments revealed supramolecular organization of 33-mer into colloidal nanospheres. In the presence of 1 mM sodium citrate, 1mM sodium borate, 1 mM sodium phosphate buffer, 15 mM NaCl the nanospheres were stabilized, while in water a linear organization and formation of fibrils were observed. It is hypothesized that the self-assembly process could be the result of the combination of hydrophobic effect, intramolecular hydrogen bonding and electrostatic complementarity due to 33-mer high content of proline and glutamine amino acids and its calculated non-ionic amphiphilic character. Although, performed in vitro, our experiments have revealed new features of the 33-mer gliadin peptide that could represent an important and unprecedented event in the early stage of 33-mer interaction with the gut mucosa prior to onset of inflammation. Moreover, these findings may open new perspectives for the understanding and treatment of gliadin intolerance disorders.
The 33-mer gliadin peptide, LQLQPF(PQPQLPY)3PQPQPF, is a highly immunogenic peptide involved in celiac disease and probably in other immunopathologies associated with gliadin. Herein, dynamic light scattering measurements showed that 33-mer, in the micromolar concentration range, forms polydisperse nano- and micrometer range particles in aqueous media. This behaviour is reminiscent of classical association of colloids and we hypothesized that the 33-mer peptide self-assembles into micelles that could be the precursors of 33-mer oligomers in water. Deposition of 33-mer peptide aqueous solution on bare mica generated nano- and microstructures with different morphologies as revealed by atomic force microscopy. At 6 μM, the 33-mer is organised in isolated and clusters of spherical nanostructures. In the 60 to 250 μM concentration range, the spherical oligomers associated mainly in linear and annular arrangements and structures adopting a "sheet" type morphology appeared. At higher concentrations (610 μM), mainly filaments and plaques immersed in a background of nanospherical structures were detected. The occurrence of different morphologies of oligomers and finally the filaments suggests that the unique specific geometry of the 33-mer oligomers has a crucial role in the subsequent condensation and organization of their fractal structures into the final filaments. The self-assembly process on mica is described qualitatively and quantitatively by a fractal diffusion limited aggregation (DLA) behaviour with the fractal dimension in the range of 1.62 ± 0.02 to 1.73 ± 0.03. Secondary structure evaluation of the oligomers by Attenuated Total Reflection FTIR spectroscopy (ATR-FTIR) revealed the existence of a conformational equilibrium of self-assembled structures, from an extended conformation to a more folded parallel beta elongated structures. Altogether, these findings provide structural and morphological information about supramolecular organization of the 33-mer peptide, which might offer new perspectives for the understanding and treatment of gliadin intolerance disorders.