Tesis doctoral de David Cruz García
The regulated secretory pathway is an intricate, multi-step process that involves generation of transport carriers, sorting of specific cargo proteins, delivery of transport carriers to the plasma membrane, and membrane fusion in a regulated manner. (Neuro)endocrine cells tightly control particular stages of this process by synthesizing a wide variety of proteins with specific functions within the regulated secretory pathway. Many of these proteins have been already identified; for instance, protein coats mediating vesicle budding and cargo selection, the family of rab proteins which play specific regulatory roles in controlling intracellular vesicle traffic, tethering factors which link adjacent membranes prior fusion, or the family of snare proteins, which control vesicle targeting, docking and fusion. In an attempt to identify new components of the regulated secretory pathway of (neuro)endocrine cells, we applied a differential genomic analysis on two distinct subtypes of frog pituitary melanotropes showing divergent secretory phenotypes of hypo- and hypersecretion. As a result of this analysis, we identified a sequence preferentially expressed in the hyposecretory melanotropes, which showed high identity with an uncharacterized human gene. This gene and a paralogue sequence which was identified by database searching code for proteins containing several coiled-coil domains and a hydrophobic region at their c-termini. On the basis of their predicted molecular structure and their high expression in the central nervous system and endocrine glands, the isolated sequence and its paralogue were referred by us as necc1 and necc2 (neuroendocrine long coiled-coil protein 1 and 2), respectively. Necc1 undergoes alternative tissue-dependent alternative splicing of the exon coding for the hydrophobic region. Our studies using an specific anti-necc1 antibody have shown that, in (neuro)endocrine cells, necc1 behaves as a peripheral membrane protein displaying high stability to detergent extraction and is located in the cis/medial cisternae of the golgi complex. These data suggest that necc1 can be a component of the golgi matrix and, thus, that it could act as a tethering factor in this organelle. Functional analysis revealed that necc1 silencing enhances k+-stimulated secretory activity in the (neuro)endocrine cell line pc12. All together, these results, as well as the increased expression levels of necc1 in melanotrope cells treated with npy (an inhibitor of the secretory activity in this cell type), suggest that necc1 may play a role in the regulated secretory pathway in (neuro)endocrine cells, likely by acting as a negative modulator of the membrane traffic in the golgi complex. In the central nervous system, necc1 expression is restricted to neurons. Specifically, in hippocampal neurons, necc1 is located at the golgi complex and presynaptic sites. This distribution is reminiscent of that observed in neuronal-differentiated pc12 cells (i.E. Golgi complex and vesicles along neurites). Finally, in both cell models, necc1 expression is increased throughout the process of neuronal differentiation. In sum, these results support a role of this protein in the maintenance of the structure and function of presynaptic zones and the golgi complex in neurons.
Datos académicos de la tesis doctoral «Identificacion y caracterizacion de nuevas proteinas involucradas en la ruta de secrecion regulada de celulas (neuro) endocrinas«
- Título de la tesis: Identificacion y caracterizacion de nuevas proteinas involucradas en la ruta de secrecion regulada de celulas (neuro) endocrinas
- Autor: David Cruz García
- Universidad: Córdoba
- Fecha de lectura de la tesis: 15/07/2009
Dirección y tribunal
- Director de la tesis
- María Del Mar Malagon Poyato
- Tribunal
- Presidente del tribunal: Francisco Gracia navarro
- hubert Vaudry (vocal)
- Rafael Fernández chacón (vocal)
- nicolas Vitale (vocal)