Structural conformation and self-assembly process of p31-43 gliadin peptide in aqueous solution. Implications for celiac disease.

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 and using an oligomer/fibril-sensitive fluorescent probe, we showed the presence of oligomers of this peptide in solution. Furthermore, atomic force microscopy 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 CD, we observed that p31-43 self-organized in a polyproline II conformation in equilibrium with ß-sheets-like structures, whose pH remained stable in the range of 3-8. In addition, these findings were supported by molecular dynamics simulation. The formation of p31-43 nanostructures with increased ß-sheet structure may help to explain the molecular etiopathogenesis in the induction of proinflammatory effects and subsequent damage at the intestinal mucosa in CeD.

p31-43 Gliadin Peptide Forms Oligomers and Induces NLRP3 Inflammasome/Caspase 1- Dependent Mucosal Damage in Small Intestine.

Celiac disease (CD) is a chronic enteropathy elicited by a Th1 response to gluten peptides in the small intestine of genetically susceptible individuals. However, it remains unclear what drives the induction of inflammatory responses of this kind against harmless antigens in food. In a recent work, we have shown that the p31-43 peptide (p31-43) from α-gliadin can induce an innate immune response in the intestine and that this may initiate pathological adaptive immunity. The receptors and mechanisms responsible for the induction of innate immunity by p31-43 are unknown and here we present evidence that this may reflect conformational changes in the peptide that allow it to activate the NLRP3 inflammasome. Administration of p31-43, but not scrambled or inverted peptides, to normal mice induced enteropathy in the proximal small intestine, associated with increased production of type I interferon and mature IL-1ß. P31-43 showed a sequence-specific spontaneous ability to form structured oligomers and aggregates in vitro and induced activation of the ASC speck complex. In parallel, the enteropathy induced by p31-43 in vivo did not occur in the absence of NLRP3 or caspase 1 and was inhibited by administration of the caspase 1 inhibitor Ac-YVAD-cmk. Collectively, these findings show that p31-43 gliadin has an intrinsic propensity to form oligomers which trigger the NLRP3 inflammasome and that this pathway is required for intestinal inflammation and pathology when p31-43 is administered orally to mice. This innate activation of the inflammasome may have important implications in the initial stages of CD pathogenesis.

Food-grade gliadin microstructures obtained by electrohydrodynamic processing.

This paper presents a comprehensive study on the electrohydrodynamic processing of gliadin to develop food-grade delivery systems with different morphologies. The effects of biopolymer concentration, applied voltage and solution flow-rate on particle morphology, molecular organisation, crystallinity and thermal properties were investigated. Gliadin concentration influenced the apparent viscosity and conductivity of the solutions, giving raise to particle morphologies at 10 wt% gliadin and beaded-free fibers above 25 wt% gliadin. In general, increasing the voltage resulted in smaller average sizes of the obtained structures, while no significant differences in morphology were observed among the tested flow rates. Interestingly, the amide I position in the FTIR reflected changes in protein conformation which could be correlated with the final morphology attained. Moreover, the acetic acid used for solution preparation disrupted the original amino acid chain packing of the gliadin fraction, being the electrospun/electrosprayed samples amorphous. These gliadin-based microparticles and microfibers obtained could serve as food-grade delivery vehicles.

The In Vitro Effects of Enzymatic Digested Gliadin on the Functionality of the Autophagy Process.

Gliadin, the alcohol-soluble protein fraction of wheat, contains the factor toxic for celiac disease (CD), and its toxicity is not reduced by digestion with gastro-pancreatic enzymes. Importantly, it is proved that an innate immunity to gliadin plays a key role in the development of CD. The immune response induces epithelial stress and reprograms intraepithelial lymphocytes into natural killer (NK)-like cells, leading to enterocyte apoptosis and an increase in epithelium permeability. In this contribution, we have reported that in Caco-2 cells the administration of enzymatically digested gliadin (PT-gliadin) reduced significantly the expression of the autophagy-related marker LC3-II. Furthermore, electron and fluorescent microscope analysis suggested a compromised functionality of the autophagosome apparatus. The rescue of the dysregulated autophagy process, along with a reduction of PT-gliadin toxicity, was obtained with a starvation induction protocol and by 3-methyladenine administration, while rapamycin, a well-known autophagy inducer, did not produce a significant improvement in the clearance of extra- and intra-cellular fluorescent PT-gliadin amount. Altogether, our results highlighted the possible contribution of the autophagy process in the degradation and in the reduction of extra-cellular release of gliadin peptides and suggest novel molecular targets to counteract gliadin-induced toxicity in CD.

A novel globular protein electrospun fiber mat with the addition of polysilsesquioxane.

The aim of this work has been to elaborate well defined gliadin nanofibers with incorporation of inorganic molecules, such as polyhedral oligomeric silsesquioxane (POSS). Nanofibers were obtained by electrospinning processing, controlling the relevant parameters such as tip-to-collector distance, voltage and feed rate. The fiber mats were characterized by SEM, confocal images, DSC, viscosity, FTIR and conductivimetry analysis. FTIR spectra showed characteristic absorption bands related to the presence of POSS-NH(2) within the matrices. SEM micrographs showed that gliadin fibers decreased their dimensions as the amount of POSS-NH(2) increased in the spinning solution. The electrical conductivity of gliadin solutions diminished as the concentration of POSS-NH(2) was increased. Besides, confocal micrographs revealed that POSS-NH(2) might be dispersed as nanocrystals into gliadin and gluten fibers. The dimension of gluten nanofibers was also affected by the POSS-NH(2) concentration, but conversely, this dependence was not proportional to the POSS-NH(2) amount. Somehow, the interaction between gliadin and POSS-NH(2) in aqueous TFE affected the solution viscosity and, as a consequence, higher jet instabilities and thinner fiber dimensions were obtained.

A structural and immunological basis for the role of human leukocyte antigen DQ8 in celiac disease.

The risk of celiac disease is strongly associated with human leukocyte antigen (HLA) DQ2 and to a lesser extent with HLA DQ8. Although the pathogenesis of HLA-DQ2-mediated celiac disease is established, the underlying basis for HLA-DQ8-mediated celiac disease remains unclear. We showed that T helper 1 (Th1) responses in HLA-DQ8-associated celiac pathology were indeed HLA DQ8 restricted and that multiple, mostly deamidated peptides derived from protease-sensitive sites of gliadin were recognized. This pattern of reactivity contrasted with the more absolute deamidation dependence and relative protease resistance of the dominant gliadin peptide in DQ2-mediated disease. We provided a structural basis for the selection of HLA-DQ8-restricted, deamidated gliadin peptides. The data established that the molecular mechanisms underlying HLA-DQ8-mediated celiac disease differed markedly from the HLA-DQ2-mediated form of the disease. Accordingly, nondietary therapeutic interventions in celiac disease might need to be tailored to the genotype of the individual.

Gliadin characterization by SANS and gliadin nanoparticle growth modelization.

Nanosized colloidal carriers can ensure a controlled and targeted therapeutic substances delivery. The original contribution of this work was to use biopolymers of vegetable source, which are an interesting alternative to synthetic polymers. The aim of this study was to prepare submicronic particles from wheat proteins: Gliadins extracted from gluten. The carrier preparation was based on the desolvatation of the macromolecules by a couple solvent/non-solvent of the proteins. In a first step, it was of interest to elucidate the gliadin macromolecular conformation in order to understand the mechanism of nanoparticle formation. The experimental work was based on SANS experiments. Because the size of the colloidal particle suspension is an important parameter to monitor, the modelization of the particle growth was thoroughly studied. Furthermore, it was observed that the determination of the solubility parameters of the proteins allowed optimization of the size of the particles. From those previous experimental results it can be concluded that there is a correlation between the protein conformation in the solvent and the size of the nanoparticles (NP).

Microheterogeneity and microrheology of wheat gliadin suspensions studied by multiple-particle tracking.

By monitoring the thermally driven displacements of imbedded polystyrene microspheres via video fluorescence microscopy, we quantified the microstructural and micromechanical heterogeneities of wheat gliadin suspensions. We found that the degree of heterogeneity of the suspensions, as measured by the width and skewness of the microspheres' mean squared displacement (MSD) distribution, increased dramatically over a narrow range of gliadin concentrations. The ensemble-averaged MSD of a 250 mg/mL gliadin suspension exhibited a power-law behavior scaling linearly with time, a behavior similar to that observed for a homogeneous aqueous glycerol solution. However, the MSD distribution was wider and more asymmetric than for glycerol. With increasing concentration of gliadin, the ensemble-averaged MSD rapidly displayed a plateau at small time scales, the MSD distribution became wider and more asymmetric, and the local viscoelastic moduli extracted from multiple-particle-tracking measurements showed an increasingly wide range.

Molecular images of cereal proteins by STM.

Scanning tunnelling microscopy (STM) has been used to study a seed storage protein of wheat known as gamma-gliadin. The protein was deposited onto highly oriented pyrolytic graphite (HOPG) from solutions of trifluoroethanol (TFE) and 1% acetic acid. Samples were dried down and then scanned in air. Transmission electron microscopy (TEM) was also used to visualise the distribution of protein on the substrate. Small-angle X-ray scattering (SAXS) was used to compare the molecular size and shape obtained with those from the STM images.