Estudio de la alergenicidad en Alt a 1, una proteína única de hongos

Résumé

ABSTRACT Allergy is a social problem that represents a serious decrease in quality of life of the patients, being considered the biggest chronic disease in Europe today. Since mid-twentieth century, a large increase in the prevalence of these diseases has occurred. However, the underlying causes for this increase and the factors that trigger allergy are still unknown. The knowledge of these factors would be very valuable to prevent and treat allergy disease. The main aim of this doctoral thesis was to study the molecular mechanisms involved in the allergic sensitization processes. With this purpose, the research was focused on Alt a 1, the major allergen from the fungus Alternaria alternata, which has been characterized from structural, physiological, and immunological standpoints. Alt a 1 protein is related to chronic asthma and pathogenesis in plants, although its biological function still remains unknown. However, this allergen has been characterized in this doctoral thesis as an effector which is released when the fungal spores reach the plant surface and is able to inhibit plant defense proteins such as PR5. On the other side, based upon the three-dimensional structure of Alt a 1 obtained in X-ray crystallography, this doctoral thesis also presents an in silico study of the behavior, stability, and properties of several aggregation states of this singular allergen. This study, conducted on salt aqueous solution by means of Molecular Dynamics simulations, has managed to elucidate the dynamical evolution of stabilities, properties, and interactions of the different oligomerization states of Alt a 1 that happened to depend on the presence of ligands and on the pH of the medium. By combining experimental assays in vitro and computational procedures for analyses in silico, the presence of a flavonoid-type ligand bound to Alt a 1 has been also identified in this doctoral thesis. Although it has not been possible to elucidate the precise chemical nature of this ligand, the experimental evidence reported has allowed to propose a plausible structure based on a flavone-like moiety. Finally, the immune response triggered by Alt a 1 in bronchial epithelium was studied. In a similar manner to what happens when Alternaria contacts plants, it has been observed that the presence of fungal spores produces an increase in secretion of defense proteins. Moreover, is has been also observed that Alt a 1 is able to interact with those proteins. The sequence of events that lead to this molecular recognition occur with release of the ligand and promote dimerization of Alt a 1. This dimeric structure is that which is ultimately recognized by antigen presenting cells, thus triggering the allergic response. Summarizing, data presented in this doctoral thesis demonstrate that the knowledge of the biological activity of an allergen provide essential information to better understand its participation in the complex mechanisms that lead to the allergic process.