Tesis doctoral de Caroline Mauvezin
Diabetes and obesity regulated protein (dor) is a nuclear protein previously reported as a co-activator transcriptional of thyroid hormone receptors. Recently, we have discovered and characterized a new function of dor in mammalian cells. First we observed that dor moves out of the nucleus under stressing conditions. Based on intracellular localization and live imaging we have determined that dor is a highly dynamic protein that shuttles between nucleus and cytosol. We have analyzed whether the movement of dor to the cytosol is associated to its degradation. In cells, proteins can be degraded through the ubiquitine-proteasome system or by macroautophagy (here after referred to autophagy) leading to lysosomal degradation. It has been previously determined in the lab that dor has a short half-life and is degraded through proteasome. As both protein breakdown systems have been recently described to be connected, we made sure that dor is only and specifically degraded through proteasome machinery. Interestingly, we determined that dor localizes to early autophagosomes by immunofluorescence and immunoprecipitation assays, thus, dor substantially co-localizes and physically interacts with atg8 mammalian homologues lc3 and gate-16. Both proteins participate as lipidated forms in the formation of autophagosomes. Lc3 is a widely well-recognized marker of autophagosomes. Therefore, dor associates only with a subset of lc3 structures and does not co-localize with lysosomal markers under any of the studied conditions which indicates that dor is a marker of early autophagosomes. Recently, a consensus lc3 interacting region (lir) has been described in the literature. We have mapped a lir motif in dor amino acids sequence. A specific mutant of this lir motif (w35a/i38a) was generated and characterized. Dor gain-of-function experiments revealed a statistically significant increase of long-lived protein degradation and an enhancement of autophagosome formation determined by both electron microscopy and immunofluorescence analysis. Reciprocally, studies of dor loss-of function in mammalian cells revealed a dramatic decrease of autophagosome formation and protein degradation. Dor repression effect on autophagy was further confirmed in vivo using drosophila melanogaster animals. The data indicated that the drosophila homologue of dor is required for autophagy activation at the onset of metamorphosis. together with our previous findings, our results suggest that dor is a scaffold and bi-functional protein operating both in the nucleus and in the cytosol. We proposed that autophagy in metazoans is not only modulated by cytosolic core machinery proteins but also by shuttling nuclear proteins such as dor.
Datos académicos de la tesis doctoral «A new dor to autophagy«
- Título de la tesis: A new dor to autophagy
- Autor: Caroline Mauvezin
- Universidad: Barcelona
- Fecha de lectura de la tesis: 29/04/2011
Dirección y tribunal
- Director de la tesis
- Antonio Zorzano Olarte
- Tribunal
- Presidente del tribunal: patrice Codogno
- patricia Boya tremoleda (vocal)
- (vocal)
- (vocal)