Rôle du système ubiquitine protéasome dans les séparations de phase nucléaires
The ubiquitin-proteasome system represents a major cell-signaling platform in eukaryotes and plays a pivotal role in the coordination of cellular processes. Recent studies provided evidence that ubiquitination plays a role in liquid-liquid phase separation (LLPS), a process that results in the localization of highly increased levels of a protein in a defined subcellular compartment, in order to achieve a biological function. Indeed, ubiquitination has been shown to play a central role in the mechanisms that govern LLPS and subsequent formation of stress granules in the cytoplasm or the DNA repair foci in the nucleus. On the other hand, several studies have shown that the proteasome itself is able to form granules in the cytoplasm following metabolic stress in yeasts. However, the mechanisms by which the ubiquitin-proteasome system and its regulators control LLPS processes remain to be determined. In the first study of this thesis, we investigated the mechanism of action of USP16 deubiquitinase, which has been suggested as a negative regulator of LLPS preventing the formation of DNA damage repair foci. However, our results demonstrate that USP16 is predominantly cytoplasmic and that only enforced nuclear entry of USP16 prevents the formation of repair foci after double strand breaks induced by ionizing radiation, and this by promoting the deubiquitination of histone H2A. In addition, no nuclear translocation of USP16 was observed during cell cycle or following DNA damage. Our study shows that USP16 is actively excluded from the nucleus via its nuclear export signal and would indirectly regulate LLPS that lead to DNA repair foci. In the second study, we describe the dynamic behavior of proteasome proteins during metabolic stress, a process that involves LLPS. Our results indicate that the proteasome forms distinct foci in the nucleus of human cells in response to nutrients deprivation. We found that these foci are enriched with conjugated ubiquitin and demonstrated that the ubiquitin receptor Rad23B as well as the absence of nonessential amino acids are the key elements necessary for the assembly of these proteasome foci. In addition, cell survival experiments show that the presence of these foci is associated with cell death by apoptosis. In conclusion, our work has shed new light on the importance of the ubiquitin-proteasome system in the formation and regulation of cell foci following LLPS. Likewise, this vi study will also help deepen our understanding of the mechanisms leading to protein homeostasis, cell survival and cancer development.
LLPS USP16 Rad23B Liquid-liquid phase separation double strand break repair nuclear export amino acids