The role of OTUD5-dependent deubiquitination on the maintenance of nuclear homeostasis

Abstract

Ubiquitin is a 76 residues polypeptide, which works as a post-translational modification that can be attached to proteinaceous substrates, either as monomers or as protein polymers by the sequential attachment of ubiquitin monomers on top of each other. Modification of substrates by ubiquitin leads to diverse outcomes, such as alterations in their stability, shuttling to specific cellular compartments or working as a molecular switch to control the substrate activity. The attachment of ubiquitin is mediated by a three-step process, which can be reversed by the employment of a dedicated set of enzymes to perform the removal of ubiquitin signal. OTUD5 is a deubiquitinating enzyme that removes the modification from its substrates. Recently, OTUD5 has emerged as a prominent regulator of nuclear processes and linked to a severe developmental syndrome, based on the absence of its catalytic activity. Although its impact on some nuclear homeostasis events has been described, its full spectrum of bona fide substrates has not been systematically explored in order to explain by which means it controls the associated processes. We employed unbiased mass spectrometry-based approaches to determine the endogenous proteins that have their ubiquitination status altered by OTUD5 knockout, showing novel highconfidence substrates of OTUD5 involved in chromosome spatial organization. By analysis of the proteome, we quantified elevated levels of OTUD5 new substrates and their elevated chromatinbound status. Furthermore, we established a new nuclear interaction between OTUD5 and VCP, mediated by NPLOC4. Altogether, we uncovered novel pathways by which cells control their nuclear homeostasis, using the deubiquitinase OTUD5 to regulate the abundance of the modification on specific factors. Ultimately, this leads to a tight modulation of substrate levels and chromatin binding that, in turn, modulates essential processes, such as cell cycle and cellular growth, demonstrating the central role OTUD5 has in these events.