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.

Deglycosylated RBD produced in Pichia pastoris as a low-cost sera COVID-19 diagnosis tool and a vaccine candidate.

During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.

The Role of Ubiquitin in Regulating Stress Granule Dynamics.

Stress granules (SGs) are dynamic, reversible biomolecular condensates, which assemble in the cytoplasm of eukaryotic cells under various stress conditions. Formation of SGs typically occurs upon stress-induced translational arrest and polysome disassembly. The increase in cytoplasmic mRNAs triggers the formation of a protein-RNA network that undergoes liquid-liquid phase separation when a critical interaction threshold has been reached. This adaptive stress response allows a transient shutdown of several cellular processes until the stress is removed. During the recovery from stress, SGs disassemble to re-establish cellular activities. Persistent stress and disease-related mutations in SG components favor the formation of aberrant SGs that are impaired in disassembly and prone to aggregation. Recently, posttranslational modifications of SG components have been identified as major regulators of SG dynamics. Here, we summarize new insights into the role of ubiquitination in affecting SG dynamics and clearance and discuss implications for neurodegenerative diseases linked to aberrant SG formation.

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.

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.

Circular dichroism and electron microscopy studies in vitro of 33-mer gliadin peptide revealed secondary structure transition and supramolecular organization.

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 polyproline II secondary structure but also depending on concentration resulted in conformational transition and self-assembly under aqueous condition, pH 7.0. A 33-mer dimer is presented as one initial possible step in the self-assembling 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, 1 mM sodium borate, 1 mM sodium phosphate buffer, 15 mM NaCl, the nanospheres were stabilized, whereas in water, a linear organization and formation of fibrils were observed. It is hypothesized that the self-assembling process could be the result of the combination of hydrophobic effect, intramolecular hydrogen bonding, and electrostatic complementarity due to 33-mer's high content of proline and glutamine amino acids and its calculated nonionic amphiphilic character. Although, performed in vitro, these 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.