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Title: Bioinformatics, phylogenetic and biochemical analyses of the proteins of the muskelin/RanBP9/CTLH complex
Author: Francis, Ore
ISNI:       0000 0004 5347 0882
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2014
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Ubiquitination is an essential post-translational modification that regulates signalling and protein turnover in eukaryotic cells. However, many ubiquitin E3 ligases remain poorly understood. The mammalian muskelin/RanBP9/CTLH complex contains eight proteins, five of which, RanBP9 (RanBPM), TWA1, Maea, Rmnd5a and muskelin, share striking similarities of domain organisation. In Saccharomyces cerevisiae, the related GID complex includes the Rmnd5a homologue GID2 which has E3 ubiquitin ligase activity and down-regulates gluconeogenesis. E3 ubiquitin ligase activity of mammalian Rmnd5a has not been reported. To better understand the large mammalian complex a major goal of this thesis was to analyse its evolution as a multi-protein system. Bioinformatic studies identify that TWA1, Rmnd5 and Maea are conserved throughout five eukaryotic supergroups. RanBPM is absent from excavates and from some lineages within other super-groups, and muskelin is present only in opisthokonts. Phylogenetic analysis based on the shared sequence regions that correspond to the lissencephaly-l homology (LisH) and C-terminal to LisH (CTLH) domains revealed closer relationships between Rmnd5 and MAEA, and TWAl and RanBPM, respectively. In-depth sequence analyses confirmed the greater similarity of the LisH/ CTLH domains of Rmnd5 and MAEA vs. TWAl and RanBPM, respectively, and id~ntified unique signatures of conserved residues within the LisH and CTLH domains of each protein. ~ further goal was to purify and express Rmnd5a and TWAl for laboratory experiments. Bacterially expressed Rmnd5a exhibits E3 ubiquitin ligase activity in Escherichia coli BL21lysates but not as a purified protein. Bacterial expression and purification of TWAl enabled biophysical characterisation of TWAl as an all a-helical, natively-dimerised protein. TWAl crystals were produced. When optimized, crystals diffracted to 3.5A, though a 3D structure was not resolved. Threaded structure predictions of Rmnd5a and TWAl agreed with secondary structure prediction algorithms. These studies advance knowledge of structural! functional relationships of proteins in this poorly-understood complex.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available