Regolazione del fattore trascrizionale p73 da parte del complesso E3 ubiquitina ligasi CDL4a e della proteina N4BP1

p73 is a structural and functional homolog of the tumor suppressing transcriptional factor p53. p73 and p53 are modular proteins with a similar basic structure formed by a transactivation domain (TA) situated at N-terminus of the protein, a DNA binding domain (DBD) in the central part of the protein and an oligodimerization domain (OD) at C-terminus of the factor. The core DNA binding domain is the region which shares the highest homology between p73 and p53. Consequently p73 can bind to canonical p53 DNA-binding site and activate transcription from p53-responsive promoters, thus inducing cell cycle arrest and apoptosis. p73 activity is finely regulated by different post-translational modifications including phosphorylation, acetylation, sumoylation and ubiquitination. In this thesis we have focused our attention on the role of ubiquitin partners in the regulation of p73. Ubiquitination is a multi-step process which involves sequentially three different kind of enzyme: the E1-activating enzyme responsible for the activation of the ubiquitin through an ATP-dependent mechanism, the E2-coniugating enzyme which receives directly from the E1 the activated ubiquitin and the E3 ubiquitin ligase enzyme which physically binds specific substrates and transfers the activated ubiquitin from the E2 to the target proteins. So the specificity of the reaction of ubiquitination is conferred by the activity of the E3 ligase. The E3 ligase are divided into two major groups: the RING-finger ligase and the HECT ligase. The RING finger are also subdivided into two subgroups: the monomeric RING-ligase and the multimeric RING ligase. The HECT ligase are constituted by a C2 domain at N-terminus of the protein, which is a calcium-lipid domain responsible for the interaction with the membrane, different WW domains in the central part which serve for the binding of the different substrates and an HECT domain at the C-terminus involved in the recruitment of the E2 enzyme. Furthermore the HECT domain contains the Cys catalitical residue responsible for the binding with the activated ubiquitin. In our laboratory we previously identified Itch as the HECT-type E3 ubiquitin ligase of p73 responsible for the proteosomical-dependent degradation. Recently we identified the protein N4BP1, which is first identified as a novel substrate of the E3 HECT ligase Nedd4, as a new interactor of Itch. In the first part of the project we investigated the mechanism of action of N4BP1 and the physiological significance of its function. We found that N4BP1, as p73, binds to WW2 domain of Itch, a region involved in the interaction with its substrates. This suggest that N4BP1 could compete with Itch substrates for the binding to the ligase. By performing an in vitro binding assay, we indeed demonstrated that N4BP1 acts as a competitor of p73. As a consequence the overexpression of N4BP1 increases both the p73 half-life and the transcriptional activity. Conversely genetic and RNAi knockdown of N4BP1 diminish the steady-state protein levels of p73 and c-Jun, and significantly impair their transcriptional activity. These results demonstrate that N4BP1 functions as a negative regulator of Itch and this activity is reflected on stabilization of the cell death regulator p73. The second part of my project has been focused on the characterization of the significance of the interaction between p73 and CDL4a. CDL4a is a multimeric complex constituted by cul4a, ROC1 e DDB1. ROC1 is a RING-finger protein which recruits the E2 enzyme, cul4a interacts with ROC1 and acts as a scaffold protein orienting the E2 enzyme versus the different substrates. The substrates are recruited or directly by DDB1 or conversely the interaction could be mediated by a WD40 protein which binds DDB1. By GST pulldown assay in vitro and CO-IP experiments in vivo, we demonstrated that p73 binds to CDL4a through a direct interaction with DDB1. Moreover CDL4a mediates the multiubiquitilation of p73 on different residue of lysine. It’s well established that the multiubiquitination of a target protein, is not a signal for a proteosomical-dependent degradation. Therefore, we hypothesized that the multiubiquitilation of p73, CDL4a mediated, doesn’t effect its protein stability. To confirm this, we measured p73 protein levels in cells depleted for DDB1 or cul4a. We showed that genetic and RNAi knockdown of DDB1 or of Cul4a, obtained from different strategies, don’t exert any effect on stability of p73. In agreement with this datas p73 decay rate is not even affected by the overexpression of the different members of the CDL4a. Finally we demonstrated that CDL4a inhibits the transcriptional activity of p73. In particular RNA mediated silencing or genetic knockdown of the different members of CDL4a induce an activation of different target genes of p73. These results highlighted two different ubiquitin-mediated pathways which regulates p73 stability, function and activity. N4BP1 acts as an indirect positive regulator of p73 while CDL4a multiubiquitinates p73 and negatively regulates p73 trascriptional activity. Future studies will be necessary to understand the contribution of both CDL4a and N4BP1 in regulating cell cycle, apoptosis and cancer development.