RNF125 activated ubiquitin transfer by Ube2K
Fokkens, Thornton J.
The covalent attachment of ubiquitin to substrate proteins (ubiquitylation) is a post-translational modification involved in numerous cell signalling pathways, most notably the process of protein degradation. Ubiquitylation requires a cascade of enzymes, and the last step of this cascade is mediated by E3 ubiquitin-ligases (E3), which engage E2 ubiquitin-conjugating enzymes (E2) to transfer ubiquitin from the E2. Ubiquitin can be linked to another ubiquitin moiety, allowing the formation of poly-ubiquitin chains. The specific linkages of poly-ubiquitin chains result in different signalling responses, such as the degradative signalling by lysine-48 linked poly-ubiquitin chains. The most prevalent class of E3 ligases are the really interesting new gene (RING) E3s, which mediate the direct transfer of ubiquitin from an E2 to a substrate lysine. RING E3s engage the ubiquitin carrying E2 (E2~Ub) and position it into a conformation where the C-terminal tail of ubiquitin is primed for nucleophilic attack. The activity of RING E3 ligases are regulated to ensure ubiquitin transfer events only occur when appropriate. The regulatory mechanisms employed by RING E3s are complex and diverse, and this project aimed to understand the molecular mechanisms employed by the RING E3 ligase RNF125 to mediate ubiquitin transfer. RNF125 is a key regulator of the immune response, where it has been implicated in the termination of inflammatory signalling pathways. The role of RNF125 is best described with the anti-viral sensor RIG-I, where RNF125 facilitates the rapid degradation of activated RIG-I to quench immune signalling. RNF125 has been reported to mediate the synthesis of both lysine-63 and lysine-48 linked poly-ubiquitin chains, through interaction with several E2 enzymes, including the E2 enzyme Ube2K. Initially, the domains of RNF125 required for activity with Ube2K were investigated. Activity assays showed that the canonical RING domain of RNF125 is insufficient to mediate ubiquitin transfer, but rapid ubiquitin transfer with Ube2K occurs when the adjacent zinc finger (ZF) domain is included. Furthermore, RNF125 requires the RING domain and the ZF domain to bind Ube2K and form a complex. A stable 1:1 RNF125:Ube2K~Ub complex was confirmed by size exclusion chromatography coupled to multi-angle light scattering. A new model of the RNF125:Ube2K~Ub complex was generated based on these data, identifying a potential novel binding interaction between the RNF125 ZF and Ube2K. The residues predicted to interact with Ube2K were modified by mutagenesis for future assessment in activity and binding studies. A structure will be required to confirm this model, and progress was made towards crystallising a RNF125:Ube2K complex for structure determination by x-ray crystallography.
Advisor: Day, Catherine L.
Degree Name: Master of Science
Degree Discipline: Biochemistry
Publisher: University of Otago
Keywords: RNF125; Ubiquitin; Ube2K; Cell signalling; Protein biochemistry
Research Type: Thesis