The involvement of the adaptor protein complex AP-3 in mast cell regulated secretion

Abstract

Mast cells (MCs) are multifunctional immune cells implicated in inflammation, allergy, tissue repair and angiogenesis. Along with neurons, melanocytes, endocrine and exocrine cells, as well as other immune cells, MCs are specialized secretory cells, which presents regulated secretion. The regulated secretory pathway requires the sorting of proteins to a specific route destined to secretory granules, which are secreted upon adequate physiological stimulation. MC involvement in various physiological and pathological processes occurs through the regulated secretion of its biological mediators, in particular, the preformed mediators stored in their secretory granules which are considered Lysosome-Related Organelles or LROs due to the several characteristics they shared with lysosomes. The biogenesis of LROs is strictly related to the endolisossomal system. In this system the sorting of proteins destined to organelles, relies on signals that direct their incorporation in transport vesicles. The AP protein complex adaptors are responsible for connecting coats to the donor membrane, selecting cargo, and recruiting accessory proteins that regulate vesicle budding and trafficking. The AP-3 complex adaptor is a stable heterotetramer formed by 4 different subunits (delta, beta3, mu3 e sigma3)and it is recognized for its role in the biogenesis of LROs, including melanosomes and platelet dense granules. It's believed that AP-3 acts on the traffic between the biosynthetic and endocytic pathway to late endosomes, lysosomes, and LROs. Genetic defects in the Beta3A subunit of AP-3 cause the type II Hermansky-Pudlak syndrome in humans, which is characterized by oculocutaneous albinism, prolonged bleeding and innate immune deficiency. These symptoms are associated to anomalies of secretory granule biogenesis and secretion in specialized cells including melanocytes, platelets, and cytotoxic T lymphocytes. MCs perform great part of their functions through the regulated secretion of mediators stored in their secretory granules. Hence, the aim of this project is to investigate the direct and indirect involvement of AP-3 in the biogenesis of mast cell secretory granules, more specifically in the sorting of proteins to these organelles. It will be also investigated the influence of AP-3 in the fusion of the granule with the plasma membrane, which we postulate to be dependent of the correct sorting of proteins important for exocytosis in MCs. The involvement of AP-3 in MC regulated secretion will be studied through RNAi in RBL-2H3 cells. The effects resulting of the absence of AP-3 on the traffic and secretion of granule proteins in MCs will be evaluated. Our findings will contribute to elucidate the role of AP-3 in MC regulated secretion. Also, it may help to shed light in the contribution of MCs for the symptoms observed in HPS2. The knowledge of the molecular mechanisms involved in the biogenesis and exocytosis of MC granules are of up-most importance because the mediators contained in these granules are involved in the inflammatory and pathological processes caused or exacerbated by these cells. The understanding of theses mechanisms can lead to therapeutic targets to aid in the control of these situations.