Show simple item record

dc.contributor.advisorDay, Catherine L.
dc.contributor.authorFokkens, Thornton J.
dc.date.available2019-03-07T03:10:18Z
dc.date.copyright2019
dc.identifier.citationFokkens, T. J. (2019). RNF125 activated ubiquitin transfer by Ube2K (Thesis, Master of Science). University of Otago. Retrieved from http://hdl.handle.net/10523/9036en
dc.identifier.urihttp://hdl.handle.net/10523/9036
dc.description.abstractThe 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.
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectRNF125
dc.subjectUbiquitin
dc.subjectUbe2K
dc.subjectCell signalling
dc.subjectProtein biochemistry
dc.titleRNF125 activated ubiquitin transfer by Ube2K
dc.typeThesis
dc.date.updated2019-03-07T02:19:57Z
dc.language.rfc3066en
thesis.degree.disciplineBiochemistry
thesis.degree.nameMaster of Science
thesis.degree.grantorUniversity of Otago
thesis.degree.levelMasters
otago.interloanno
otago.openaccessAbstract Only
 Find in your library

Files in this item

FilesSizeFormatView

There are no files associated with this item.

This item is not available in full-text via OUR Archive.

If you are the author of this item, please contact us if you wish to discuss making the full text publicly available.

This item appears in the following Collection(s)

Show simple item record