Dissecting the role of CRLcdt2 in the maintenance of chromatin stability

JULIANA ENRIQUÉ STEINBERG; MICHELE PAGANO; FABIANA ALEJANDRA ROSSI; LAURENCE FLORENS; MARIO ROSSI

Congreso; Reunión Conjunta de Sociedades de Biociencias 2017; 2017

The formation and progression of cancer is ultimately regulated by the abundance and activity of both oncogenic and tumor suppressor proteins. The Ubiquitin-Proteasome System (UPS) consists of a complex network of enzymes where the E3 ligases are the final effectors and dictate the specificity of the UPS machinery. CRL4Cdt2ubiquitin 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 on how deregulation of CRL4Cdt2 might contribute to cancer development, we used an affinity purification and mass spectrometry approach to identify and characterize novel 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). All known CRL4Cdt2 substrates are ubiquitinated when they are bound to PCNA, therefore,we focused our attention on chromatin fraction and we identified several novel putative Cdt2 interactors. Amongthe most abundant putative Cdt2 interactors identified, we centered our work on different members of a multi-protein complex implicated in the maintenance of chromatin structure, gene transcription and DNA replication. These proteins play an important role as barriers of cancer stem cell self-renewal and thus the understanding of their functional interaction with CRL4Cdt2 might unveil new potential therapeutic point of intervention in cancer treatment.