RESUMO
In recent years, the evaluation of the structural properties of food has become of crucial importance in the understanding of food-related disorders. One of the most exciting systems is gliadin, a protein in wheat gluten, that plays a protagonist role in gluten-related disorders with a worldwide prevalence of 5%, including autoimmune celiac disease (CeD) (1%) and non-celiac wheat sensitivity (0.5-13%). It is accepted that gliadin is not fully digested by humans, producing large peptides that reach the gut mucosa. The gliadin peptides cross the lamina propria eliciting different immune responses in susceptible patients. Many clinical and biomedical efforts aim to diagnose and understand gluten-related disorders; meanwhile, the early stages of the inflammatory events remain elusive. Interestingly, although the primary sequence of many gliadin peptides is well known, it was only recently revealed the self-assembly capability of two pathogenic gliadin fragments and their connection to the early stage of diseases. This review is dedicated to the most relevant biophysical characterization of the complex gliadin digest and the two most studied gliadin fragments, the immunodominant 33-mer peptide and the toxic p31-43 in connection with inflammation and innate immune response. Here, we want to emphasize that combining different biophysical methods with cellular and in vivo models is of key importance to get an integrative understanding of a complex biological problem, as discussed here.
RESUMO
The self-assembly of proteins is an essential process for a variety of cellular functions including cell respiration, mobility and division. On the other hand, protein or peptide misfolding and aggregation is related to the development of Parkinson's disease and Alzheimer's disease, among other aggregopathies. As a consequence, significant research efforts are directed towards the understanding of this process. In this review, we are focused on the use of UV-Visible Absorption Spectroscopy, Fluorescence Spectroscopy and Circular Dichroism to evaluate the self-organization of proteins and peptides in solution. These spectroscopic techniques are commonly available in most chemistry and biochemistry research laboratories, and together they are a powerful approach for initial as well as routine evaluation of protein and peptide self-assembly and aggregation under different environmental stimulus. Furthermore, these spectroscopic techniques are even suitable for studying complex systems like those in the food industry or pharmaceutical formulations, providing an overall idea of the folding, self-assembly, and aggregation processes, which is challenging to obtain with high-resolution methods. Here, we compiled and discussed selected examples, together with our results and those that helped us better to understand the process of protein and peptide aggregation. We put particular emphasis on the basic description of the methods as well as on the experimental considerations needed to obtain meaningful information, to help those who are just getting into this exciting area of research. Moreover, this review is particularly useful to those out of the field who would like to improve reproducibility in their cellular and biomedical experiments, especially while working with peptide and protein systems as an external stimulus. Our final aim is to show the power of these low-resolution techniques to improve our understanding of the self-assembly of peptides and proteins and translate this fundamental knowledge in biomedical research or food applications.
Assuntos
Peptídeos/química , Proteínas/química , Dicroísmo Circular , Peptídeos/síntese química , Agregados Proteicos , Proteínas/síntese química , Espectrometria de Fluorescência , Espectrofotometria UltravioletaRESUMO
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.
Assuntos
Doença Celíaca/tratamento farmacológico , Gliadina/farmacologia , Imunidade Inata/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Células CACO-2 , Doença Celíaca/genética , Doença Celíaca/imunologia , Doença Celíaca/patologia , Gliadina/genética , Gliadina/imunologia , Gliadina/ultraestrutura , Humanos , Imunidade Inata/imunologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Microscopia de Força Atômica , Conformação Molecular/efeitos dos fármacos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/ultraestrutura , Peptídeos/química , Peptídeos/imunologia , Peptídeos/farmacologia , Conformação Proteica em Folha beta , Soluções/química , Água/químicaRESUMO
Peptide structural transformation and aggregation is associated with a large number of outsider aetiology diseases, and it is intrinsically linked to amyloid peptide aggregation. Diphenylalanine self-assembled structures are used as robust minimalist beta amyloids not only to elucidate protein aggregation but also to generate hydrogels. Herein, we employed a neutral model peptide Ac-Phe-Phe-Cys-NH2 (Ac-FFC-NH2) to elucidate the role of intermolecular disulfide bonds in protein fibrillation. The Ac-FFC-NH2 peptide initially self-assembles into nanospheres that evolve to amyloid type fibrils under mild oxidative conditions. Incubation of the peptide in the presence of the chemical reduction agent TCEP inhibits the formation of the fibrils, detecting only spherical nanostructures with no secondary structure. Importantly, we triggered the transformation of the preformed linear straight amyloid fibrils to non-fibrillar structures by TCEP treatment. Under this condition, the amyloid bundles are transformed into rings, which evolve to a new spherical microstructure. We showed that the chemical reduction of intermolecular S-S in internal amyloid sequences might favour the off-path intermediates of amyloid fibril growth, even when the fibrils are formed. Our findings demonstrated that in internal amyloid sequences, the formation of intermolecular S-S promotes the formation of amyloid type fibrils; meanwhile, its reduction stabilises non-fibrillar structures. Altogether, this work provides fundamental understanding at the molecular and supramolecular level, thus facilitating the rational design of therapeutic tools for protein aggregation diseases.
Assuntos
Amiloide/química , Dissulfetos/química , Oligopeptídeos/química , Oligopeptídeos/síntese química , Oxirredução , Multimerização ProteicaRESUMO
In this review, we shall outline the basic principles of circular dichroism (CD) indicating the types of structural information relevant to the study of biomolecules, such as proteins or DNA. We are mainly interested to show the utility of this technique to study protein-ligand, DNA-ligand and protein-DNA interactions.