Thesis defended on May 21, 2025 to obtain the degree of Doctor of the Université Grenoble Alpes - Speciality: Cell Biology.​

Abstract :
The deubiquitinase USP8 plays a critical role in various cellular processes, including endocytosis, autophagy, apoptosis and DNA damage response. In particular, it regulates the stability of multiple targets by counteracting the activity of ubiquitin ligases and preventing their degradation, such as the well-characterized proto-oncoprotein EGFR. Deregulation of USP8, through mutations or altered expression, has been implicated in several pathologies, including cancers, neurodegenerative disorders, and Cushing’s disease, a rare endocrine disorder caused by pituitary microadenomas. USP8 has emerged as a promising therapeutic target. A comprehensive characterization of its interactome (targets, regulators….) is therefore essential to gain access to the extent of USP8 functions in eukaryotic cell biology and underlying signaling pathways, and to identify relevant USP8-partner interfaces that could be targeted in a therapeutic perspective. Specifically, I explored the interaction of USP8 with two potential partners, the nuclear mitotic apparatus protein NuMA1 and the end-binding protein EB1, identified through a yeast two-hybrid screen. A significant focus of my PhD work was on NuMA1. Using pull-down assays and truncated constructs of USP8 and NuMA1, I validated their direct interaction and mapped the binding interface to the rhodanese domain of USP8 and a small region within the central helical domain of NuMA1 responsible for its dimerization. Structural modeling with Alphafold suggested that USP8 binds both NuMA1 proteins within the dimer and identified key residues involved in the interaction, which we confirmed through mutagenesis. Functional assays in HEK293T cells indicated that USP8 silencing affects NuMA1 ubiquitination. In parallel to this work, I provided preliminary data confirming the direct binding of USP8 to EB1 in vitro, implicating USP8 rhodanese domain as well. These findings suggest a novel role for USP8 in microtubule-dependent processes that will need to be further investigated to fully apprehend the physiopathological consequences of USP8 deregulation and the impact of its drug targeting.​


Key words :  USP8, ubiquitination, NuMA1, cell signaling, EB1, microtubules