In mammals, Maml1 belongs to a family of proteins, which act as transcriptional coactivators for Notch signalling, an evolutionarily conserved pathway. Maml1 has been recently shown to act as a coactivator in other cell signalling pathways, including p53, MEF2C, ß-catenin, EGR1, NF-¿B, Runx2, Gli1 and YAP/TAZ in a Notch-independent manner. The E3 protein ubiquitin ligase (E3) Itch, belonging to the HECT family, binds different proteins diverting most of them to a proteasome/lysosome-dependent degradative pathway. In Hedgehog context, Itch activity is enhanced by the protein Numb, that binds the WW2 site of Itch, which is released from its self-inhibited conformation and induces Gli1 ubiquitination and proteasome degradation. Moreover, Numb interacts with Itch HECT domain to promote degradation of Notch intracellular domain. Preliminary data suggest a potential role of Maml1 in the post-transcriptional regulation of Gli1 and Notch1, preventing their degradation mediated by Itch. Besides a direct role as transcriptional coactivator, the observation suggests that Maml1 may play an important role in controlling the stability of the proteins through its C-terminal domain, still poorly characterized. So far, scientific research on Maml1 has been generally focused on its activity as a transcriptional coactivator, while overlooking its role in the post-transcriptional regulation. A thorough understanding of the molecular mechanism mediated by Maml1 to regulate Itch activity through Maml1 C-terminal domain, might lead to novel future therapeutic approaches directed against cancer. Maml1 and Itch are both key molecules that connect different signalling pathways. Therefore, the ability of Maml1 to regulate Itch activity could have an impact in controlling the force of several pathways, as Shh and Notch, in deregulated pathological contexts. This could set out new therapeutic approach based on the dual role of Maml1 and adding a piece in the understanding of tumour biology.
It is known that Maml1 protein is able to induce post-translational modification in target proteins, as p53, NF-¿B and EGR1, but the molecular mechanism has not been demonstrated yet. Here, we suggest that Maml1 is able to play a dual role: as a transcriptional coactivator and a post-translational regulator. Moreover, for the first time, we suggest the molecular mechanism that underlies the stability of Gli1 target protein, which impinges on Itch functional role. Indeed, Maml1 shows a role in Gli1 post-translational regulation preventing Itch-mediated ubiquitylation, able to restore the normal ubiquitylated levels in a dose dependent manner. It is known that Itch function is controlled through phosphorylation mechanisms, binding with adaptor proteins, and autocatalytic processes to regulate ubiquitylation events inside the cells. Our preliminary results strongly suggested a novel role for Maml1, able to induce post-translational modifications that could alter Itch activity, impinging on the regulation of canonical target proteins and triggering a different function of the E3 ubiquitin ligase, controlling in this way the function of several downstream signalling pathway. The numerous evidence that points out the role of Maml1 in supporting tumour progression and metastasis need a careful characterization of the molecular mechanism that binds Maml1, in a dual role of transcriptional and post-translational cofactor, which could lay the foundations for future antitumor therapeutic approaches. We are confident that our planned studies will be performed without important deviations. The in vitro studies described in WP1 will be carried out to elucidate the mechanism that underlie the effects of Maml1 on Itch regulation. We can hypothesize that Maml1 binding could impinge on Itch/ Numb interaction and/or recruiting other factors able to induce a conformational change in Itch protein folding, resulting in auto-catalytic events. Finally, we propose to study Maml1/Itch interaction on different tumoral backgrounds (Task 2.1). Maml1 and Itch are both two proteins involved in several pathway and these observations could set out a new molecular mechanism that applies to different signalling, characterised by Maml1 and Itch activity. Therefore, the ability of Maml1 in controlling the activity of Itch/E3 ubiquitin ligase could have an impact in controlling the force of several signalling pathways inside the cell, as Shh and Notch, in deregulated pathological contexts. The results obtained from mass spectrometry experiments described in the Task 2.2 will allow us to spotlight the analysis of Itch regulation Maml1-mediated (Task 2.1). Therefore, the possible lack in achieving the aims of this proposal, due to temporal overlap or experimental setbacks, can be overcome, since we are confident of confirming at least the inverse relationship between Maml1 and Itch in CRC and BC.
Our aim is to demonstrate a novel mechanism triggered by Maml1, able to control the activity of E3 ubiquitin ligase Itch, with a broad effect on the stability of target proteins. Maml1 and Itch are both involved in controlling the activity of several pathways, some of which overlap. Therefore, the ability of Maml1 in controlling the activity of Itch/E3 ubiquitin ligase could have an impact in controlling the force of several signalling pathways inside the cell, as Shh and Notch, both in physiological and pathological contexts. The balance between Maml1 and Itch could shed the light on the molecular mechanisms that underlie the onset of numerous diseases. This accurate characterization will open a variety of possibilities toward new therapeutic approaches in the treatment of cancer.