Regulation of Hdm2/HdmX-mediated ubiquitination and neddylation

Abstract

In response to stress (i.e. DNA damage, nucleolar or oxidative stress), mammalian cells stop their cell cycle and induce the transcription of a group of genes involved in repair of the damage. When the damage exceeds the natural barriers, the cells die by the induction of apoptosis genes. One of the key regulators of both responses is the tumor suppressor p53. According to its central role in the regulation of the cell cycle, the pathway of p53 is deregulated in almost all human tumors. The protein amount of p53 is maintained at very low levels by the ubiquitin E3 ligase Hdm2 and its related protein HdmX. The activity of p53 as a transcription factor is regulated by several proteins which directly bind to p53, (i.e Hdm2 and HdmX). In addition, several post-translational modifications as ubiquitination, neddylation, sumoylation, phosphorylation, methylation, and acetylation have been reported to regulate p53 activity. Neddylation of p53 has been shown to occur in an Hdm2-dependent manner inactivating p53 activity as a transcription factor.In the specific case of nucleolar stress, some ribosomal proteins such as L5, L11 and L23 are relocated into the nucleoplasm. They block the Hdm2-mediated ubiquitination of p53 by which it induces cells cycle arrest and apoptosis.This work is divided in four projects. i) The development of a technique which can provide a fast, reliable and homogenous knockdown in order to characterize the effect of downregulation of Hdm2 and HdmX in cells lacking p53 and in cells carrying wt p53. ii) The search of new interacting partners of HdmX which can regulate its enhancing effect on Hdm2-mediated ubiquitination and neddylation of p53. iii) In this work a new conjugating enzyme for Nedd8 (Nce2) is described. Here, Nce2 was characterized biochemically as a specific conjugating enzyme for Nedd8. In addition, it was discovered an auto-reactivity (auto-neddylation) of the Nedd8 conjugating enzymes Nce2 and Ubc12. Finally, the contribution of these enzymes to the neddylation of p53 was tested. iv) Using an in vitro system, a direct and specific inhibitory effect of L11 on the Hdm2-mediated ubiquitination of substrates was detected, which can be rescued partially by HdmX. This reveals an important buffering role of HdmX during the repression of Hdm2 induced by nucleolar stress.