The role and regulation of ubiquitin-specific protease 4 in nuclear factor kappa B signalling

Abstract

Deubiquitinating enzymes (DUBs) constitute a diverse family of regulators of ubiquitin signalling. Aberrations in this can underlie pathologies including osteoarthritis, cancers, inflammatory diseases, and even ageing. Understanding the biology of ubiquitin signalling components and how they regulate cell signalling pathways is therefore a widening area of research. The aim of this thesis was to further understanding on the function of the DUB, ubiquitin-specific protease 4 (USP4), and the mechanism of Usp4 regulation by its post-translational modification (PTM) at two serine residues, focusing on the nuclear factor of κB (NF-κB) pathway. Initial observations in Usp4 null (-/-) mice suggested a role for Usp4 in regulating circulating levels of specific inflammatory cytokines and, perhaps consequentially, a role in viral infection clearance. Thus, in vitro, the role of USP4 in IL-1- and TNFα-mediated NF-κB signalling was examined. Overexpression of Usp4 significantly increased NF-κB signalling, and using an RNAi approach to deplete USP4 resulted in suppressed NF-κB activity and significantly reduced expression of NF-κB-regulated genes (MMP13, IL-6 and IL-8). Depletion of USP4 also significantly reduced ubiquitination and degradation of IκBα, and phosphorylation of p65 and IKK. Together, data were indicative of USP4 as a positive regulator of NF-κB signalling by functioning upstream of the IKK complex. Importantly, aspects of these findings were confirmed in mouse embryonic fibroblasts (MEFs) isolated from the Usp4-/- mice. Further, identification of potential USP4 substrates were assessed. Reductions in K48- and K63-linked polyubiquitination were observed with depletion of USP4. To further understanding of the regulation of USP4, the PTM at serines 675 and 680 was examined. Lentiviral-mediated delivery of Usp4 phosphorylation variants revealed that a complex mechanism of phosphorylation/dephosphorylation of USP4 may regulate its function in NF-κB signalling. In conclusion, this thesis identifies USP4 as a positive regulator of IL-1-induced NF-κB signalling, potentially regulating itself through its PTM.