Abstract
Ubiquitylation is a post-translational modification that modulates protein turnover, function and localisation. The attachment of ubiquitin is achieved by a sophisticated enzyme cascade. Specificity is provided by E3 ubiquitin ligases, many of which contain a RING domain. The RING E3s activate ubiquitin transfer by stabilising the closed conformation of the ubiquitin-conjugating E2 linked to a ubiquitin (E2~Ub), in which the ubiquitin is primed for nucleophilic attack by the incoming lysine. Despite the shared mechanism, it is not known how most RING E3 ligases promote the closed conformation. This study aimed to characterise two RING E3 ligases: RNF121 and RNF114; both ligases have a critical role in modulating the inflammatory response.
The study firstly characterised RNF121, which consists of a RING domain and six transmembrane helices. A library of human E2s was prepared to identify the E2 partner(s) of RNF121. Ubiquitylation assays showed that RNF121 may promote ubiquitylation with the Ubc13/Mms2 complex. Additionally, gel filtration calibration demonstrated that the RNF121 RING domain is in a monomer/dimer equilibrium.
The second part of the study focused on characterising RNF114, which is both a positive and negative regulator of the NF-kB signalling pathway. Biochemical analyses were performed to understand the mechanism that underpins ubiquitin transfer by RNF114. Ubiquitylation assays and size exclusion chromatography with multi-angle light scattering analysis showed that RNF114 is active with the E2s, UbcH5b, and Ube2K, as a monomeric RING E3 ligase. Notably, the recruitment of UbcH5b~Ub by RNF114 required both the RING domain and the ubiquitin binding motif (UIM). Remarkably, the UIM bound to polyubiquitin chains and this was a prerequisite for the ubiquitylation activity of RNF114. Mutation of S224 in the UIM disrupted binding to polyubiquitin chains and abolished the ubiquitylation activity. Moreover, the UIM recognised both K48 and K63 chains differently, possibly implying linkage selectivity.