La deltalactoferrine (ΔLf) est un facteur de transcription qui possède des propriétés anti-tumorales. Son activité et sa stabilité sont hautement contrôlées par des modifications post-traductionnelles comme la O-GlcNAcylation et la phosphorylation. Au cours de ma thèse, nous avons pu montrer que la ΔLf était modifiée par SUMO-1 et mettre en évidence 5 sites de SUMOylation. Nous avons produit un ensemble de mutants pour lesquels un seul site était préservé et un mutant M5S invalidé pour les cinq sites de SUMOylation. Nous avons pu montrer que cinq lysines étaient la cible de la machinerie de SUMOylation et que K13, K308 et K379 étaient les sites accepteurs majeurs. Nous avons ensuite étudié l’activité transcriptionnelle des différents mutants et montré que la SUMOylation réprime l’activité transcriptionnelle de la ΔLf. Dans un second temps nous avons étudié le dialogue entre différentes modifications post-traductionnelles. Nous avons pu démontrer qu’une compétition SUMO/ubiquitine existait et que sous la forme SUMOylée le mutant K379 avait une durée de ½ vie plus longue confirmant que la SUMOylation protégeait la ΔLf de la dégradation protéasomale. Nous avons également pu montrer que la ΔLf était acétylée principalement sur la lysine K13. En modulant le niveau de SUMOylation ou d’acétylation du mutant K13 nous avons pu montrer que la compétition SUMO/acétylation était impliquée dans la régulation de l’activité transcriptionnelle de la ΔLf. En effet, l’acétylation régule positivement l’activité transactivatrice de la ΔLf alors que la SUMOylation l’inhibe. En résumé, nous avons pu montrer que la ΔLf était multi-SUMOylée et que la SUMOylation était un nouveau mécanisme de régulation à la fois de l’activité transcriptionnelle et de la stabilité de la ΔLf. En parallèle, nous avons montré que la ΔLf transactivait le gène Bax déclenchant l’apoptose et que la balance O-GlcNAc/Phosphate modulait la transactivation de Bax. / Deltalactoferrin (ΔLf) is a transcription factor which possesses antitumoral activities. Posttranslational modifications such as O-GlcNAcylation and phosphorylation, efficiently modulate its transcription factor activity and stability. During my PhD thesis we first showed that ΔLf is modified by SUMO-1 and mapped the five SUMO sites. We produced a series of mutants for which only one site was preserved and a null-mutant in which all five SUMO sites were invalidated. We showed that all lysine residues were SUMO acceptors and that K13, K308 and K379 were the main SUMO sites. We next studied the impact of SUMOylation on ΔLf activity and showed that SUMOylation negatively regulated the transactivation function of ΔLf. During the second part of my PhD, we investigated the crosstalk between different posttranslational modifications. We showed that K379 which is either ubiquitinated or SUMOylated, is a pivotal site for the control of ΔLf stability. We also showed that SUMOylation competes with ubiquitination and protects ΔLf from proteosomal degradation by positively regulating its stability. We demonstrated that K13 is the main acetylation site and that favoring acetylation at K13 reduced SUMOylation and increased ΔLf transcriptional activity. Collectively, our results indicate that multi-SUMOylation occurs on ΔLf to repress its transcriptional activity. Reciprocal occupancy of K13 by either SUMO-1 or an acetyl group may contribute to the establishment of finely regulated mechanisms to control ΔLf transcriptional activity. Moreover, competition between SUMOylation and ubiquitination at K379 coordinately regulates the stability of ΔLf toward proteolysis. Therefore SUMOylation of ΔLf is a novel mechanism controlling both its activity and stability. In parallel, we demonstrated that ΔLf transactivates the Bax promoter leading to the triggering of apoptosis and that the O-GlcNAc/Phosphate interplay controls Bax transactivation. Lf) is a transcription factor which possesses antitumoral activities. Posttranslational modifications such as O-GlcNAcylation and phosphorylation, efficiently modulate its transcription factor activity and stability. During my PhD thesis we first showed that ΔLf is modified by SUMO-1 and mapped the five SUMO sites. We produced a series of mutants for which only one site was preserved and a null-mutant in which all five SUMO sites were invalidated. We showed that all lysine residues were SUMO acceptors and that K13, K308 and K379 were the main SUMO sites. We next studied the impact of SUMOylation on ΔLf activity and showed that SUMOylation negatively regulated the transactivation function of ΔLf. During the second part of my PhD, we investigated the crosstalk between different posttranslational modifications. We showed that K379 which is either ubiquitinated or SUMOylated, is a pivotal site for the control of ΔLf stability. We also showed that SUMOylation competes with ubiquitination and protects ΔLf from proteosomal degradation by positively regulating its stability. We demonstrated that K13 is the main acetylation site and that favoring acetylation at K13 reduced SUMOylation and increased ΔLf transcriptional activity. Collectively, our results indicate that multi-SUMOylation occurs on ΔLf to repress its transcriptional activity. Reciprocal occupancy of K13 by either SUMO-1 or an acetyl group may contribute to the establishment of finely regulated mechanisms to control ΔLf transcriptional activity. Moreover, competition between SUMOylation and ubiquitination at K379 coordinately regulates the stability of ΔLf toward proteolysis. Therefore SUMOylation of ΔLf is a novel mechanism controlling both its activity and stability. In parallel, we demonstrated that ΔLf transactivates the Bax promoter leading to the triggering of apoptosis and that the O-GlcNAc/Phosphate interplay controls Bax transactivation.
| Identifer | oai:union.ndltd.org:theses.fr/2014LIL10145 |
| Date | 19 December 2014 |
| Creators | Escobar-Ramirez, Adelma |
| Contributors | Lille 1, Pierce-Crétel, Annick |
| Source Sets | Dépôt national des thèses électroniques françaises |
| Language | French, English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation, Text |
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