CONICET | Buscador de Institutos y Recursos Humanos

The Ubiquitin-Proteasome System is a major coordinator of cellular physiology through the regulation of protein homeostasis. The UPS substrate specificity is tightly regulated by the family of E3 ubiquitin ligases and thus they represent a critical control point of this post-translational modification pathway. CRL4Cdt2 ubiquitin ligase is emerging as a master regulator of cellular proliferation involved in multiple DNA repair processes. Different lines of evidence demonstrate that alterations in the expression and activity of CRL4Cdt2 induce genomic instability. Therefore, it is not surprising that Cdt2 protein levels are increased in many tumor cells and its expression correlates with tumor grade, metastasis and poor survival. In order to broaden our understanding how deregulation of CRL4Cdt2 might contribute to cancer development, we used an affinity purification and mass spectrometry approach to identify and characterize new CRL4Cdt2 substrates. Hek293t cells were transfected with Cdt2 containing vector (Cdt2) or empty vector (PCDNA) and were treated with different DNA damage agents (Hydroxyurea, Camptothecin and UVC), alone or in combination with the proteasome inhibitor MG132 and an inhibitor of NAE1 (MLN4924). From each sample both the CSK buffer soluble and chromatin fraction were analyzed and several novel putative Cdt2 interactors were identified. Among the most abundant putative Cdt2 protein interacting factors identified, we focused our attention on different members of a multi-protein complex implicated in the maintenance of chromatin structure and gene transcription. These proteins play an important role as barriers of cancer stem cell self-renewal and thus the characterization of their functional interaction with CRL4Cdt2 might unveil new potential therapeutic point of intervention in cancer treatment.