Characterisation of the ubiquitin E3 ligase COP1
Curry, Jack Richard

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Curry, J. R. (2018). Characterisation of the ubiquitin E3 ligase COP1 (Thesis, Master of Science). University of Otago. Retrieved from http://hdl.handle.net/10523/8170
Permanent link to OUR Archive version:
http://hdl.handle.net/10523/8170
Abstract:
Constitutive photomorphogenic 1 (COP1) is a ubiquitin E3 ligase that has been implicated in development of a variety cancers. The goal was to characterise the ubiquitin E3 ligase activity of COP1 and the relationship between COP1 and its interaction partners—especially the substrate adapters, TRIB1 and TRIB2 in order to better understand their role in tumourigenesis.COP1 consists of a N-terminal RING domain and a C-terminal seven bladed β-propeller fold (WD40 domain), connected by a central coiled-coil. As well as direct substrates, the COP1 WD40 domain binds to the C-terminal tails of TRIB1/2, which recruit further substrates through their pseudo-kinase domains. An expression system for the COP1 WD40 domain in insect cell lines from Spodoptera frugiperda and Trichoplusia ni was developed. The ternary interactions between COP1, TRIB1, and their substrate C/EBPα were characterised using fluorescence polarisation assays. The evidence presented indicates that the interaction of C/EBPα with TRIB1 induces conformational change in TRIB1, which releases the TRIB1 C-terminal tail for interaction with COP1. The relationship between oligomerisation of the COP1 and its ubiquitin E3 ligase activity, was investigated using multiple-angle light scattering and in vitro activity assays. These analyses have revealed that the oligomerisation of COP1 is crucial for its ubiquitin E3 ligase activity. Specifically, dimerisation mediated by the coiled-coil domain significantly enhances the ubiquitin E3 ligase activity of COP1—a common feature of RING-type ubiquitin E3 ligases. Together, these experiments will help elucidate the mechanism through which COP1 functions as a ubiquitin E3 ligase and the interaction with substrates recruited by TRIB1/2, which will guide our understanding of COP1 in the pathogenesis of disease.
Date:
2018
Advisor:
Mace, Peter
Degree Name:
Master of Science
Degree Discipline:
Biochemistry
Publisher:
University of Otago
Keywords:
protein; ubiquitin; COP1
Research Type:
Thesis
Languages:
English
Collections
- Biochemistry collection [228]
- Thesis - Masters [3378]