Bases moleculares de la interacción, de proteínas de transporte vesicular

Abstract

Intracellular vesicles are transported from one organelle to another in response to various different stimuli. The direction and efficiency of vesicle delivery to their target membrane are mediated by cell signalling and carried out by numerous proteins and second messengers. Rabphilin3A (Rph3A) is a membrane trafficking protein that responds to two second messengers, calcium (Ca2+) and phosphatidyl inositol 4,5-bisphosphate (PIP2). PKCε is a protein kinase involved in signalling pathways by phosphorylating other proteins and responding to various lipids such as diacylglycerol (DAG) and phosphatidic acid (PA). The biomembranas research at the University of Murcia, in collaboratrion with the Institute of Molecular Biology in Barcelona have solved the 3D structure of the C2 domains of Rabphilin3A bound to Ca2+ and PIP2, and also the C2B domain bound to SNAP25. Moreover, PKCε has already been found to be sensitive to PA by researchers in my group. One of the objectives of this Ph.D. thesis was to characterize the function of Rabphilin3A and to determine novel proteins that modulate it. The second objective was to investigate the role that PA might have on PKCε during vesicle secretion in mast cells.To achieve these goals, molecular biology techniques were used to clone and mutate different genes of interest. Two cell lines were used as in vitro models: the PC12 cell line as an adrenal model, which was also differentiated with neuronal growth factor (NGF) as a neuronal model, and the RBL-2H3 cell line as a mast cell model. Proximity ligation assay (PLA) with confocal microscopy was used to study protein localization and interactions. In addition, "omics" techniques were used for high-throughput identification of proteins in the vicinity of Rabphilin3A, hence coupling proximity-dependent biotin identification (BioID2) with mass spectrometry. The results obtained show that the C2B domain of Rabphilin3A has a strong electropositive area in the SNAP25-interacting region that is not conserved in Synaptotagmin1 (Syt1). Rabphilin3A appears to interact with VAMP2, Sinaptotagmina1, and SNAP25 on vesicles, but only with SNAP25 at the plasma membrane. The interaction site of SNAP25 with Rabphilin3A and the interaction of Rabphilin3A with PIP2 appear to be key for the correct location of SNAP25 at the plasma membrane. Furthermore, overexpression of Rabphilin3A in a neuronal phenotype promotes the location of SNAP25 at the plasma membrane. The Rabphilin3A interactome shows that Rabphilin3A interacts with STIM1 and Tcp11l1. STIM1 is an endoplasmic reticulum (ER) protein involved in Ca2+ sensing in the ER. Tcp11l1 is a protein of unknown function, but its interactome shows that Tcp11l1 is a protein that interacts with cytoskeletal proteins such as tubulin and β-actin. Consequently, relating back to the first objective of this thesis, a model is proposed in which Rabphilin3A contributes to the transport of vesicles and SNAP25 to the plasma membrane as a result of the interaction with Tcp11l1, Rabphilin3A then returns to its initial state as a result of the interaction with STIM1. Moreover, with the regards to the second goal of this thesis, results suggest that the edges of the DAG-binding pocket of the C1B domain of PKCε are strongly electropositive, allowing its binding to PA. Activation of PKCε by PA together with Ca2+ triggers vesicle fusion in mast cells. This finding correlates with the increase in SNAP23 phosphorylation in response to the interaction of PKCε with PA. Therefore, a model is proposed here in which PKCε is activated by PA and phosphorylates SNAP23, facilitating vesicle fusion