Identification of novel substrates of the Ubiquitin Protein Ligase CRL4Cdt2

JULIANA ENRIQUÉ STEINBERG; FABIANA ROSSI; LAURENCE FLORENS; MARIO ROSSI

Rosario

Congreso; L Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

E3 ubiquitin ligases represent the most important regulation level of the Ubiquitin-Proteasome System. The E3 ligase CRL4Cdt2 plays fundamental roles in the control of cell proliferation and 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. The purpose of this project was to identify and characterize novel CRL4Cdt2 substrates in an effort to contribute to the identification of potential molecular targets for the treatment of cancer. To this end, we performed Cdt2 immunoprecipitation followed by Tandem Mass Spectrometry (MS/MS) analysis. 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. We are in the process of validating and characterizing the functional interaction between Cdt2 and the most abundant putative binding partners identified in both fractions.