The functional characterisation of human RMND5 proteins in normal physiology and prostate cancer

Alison Louw

    Research output: ThesisDoctoral Thesis

    547 Downloads (Pure)


    [Truncated abstract] RMND5A and RMND5B are highly homologous uncharacterised proteins named after their yeast orthologue, Required for Meiotic Nuclear Division 5 (RMD5), a RING domain-containing E3 ubiquitin ligase. RMND5B was originally identified in our laboratory to interact with the prostatic tumour suppressor, NKX3.1, expression of which is reduced or undetectable in up to 80% of metastatic prostate tumours. Bioinformatics analyses of the cDNA and translated sequences of RMND5A and RMND5B identified four protein-protein interaction domains, a Lissencephaly 1 homology (LisH), a C-terminal to LisH (CTLH), a CT11-RanBPM (CRA) and a Really Interesting New Gene (RING) domain. Alignment of the RING domains of RMND5 proteins with that of yeast RMD5 identified that all eight amino acid residues essential for RING domain folding and therefore activity were identical between the proteins, suggesting that RMND5A and RMND5B function as E3 ubiquitin ligases. In vitro ubiquitination assays carried out using the RING domains of RMND5A and RMND5B and a panel of 11 E2 conjugating enzymes identified that RMND5A interacted with the E2 enzymes UbcH2, UbcH5b and UbcH5c, whilst RMND5B associated with UbcH5b and UbcH5c to mediate ubiquitin transfer to substrate lysine residues. Consistent with this finding, full length RMND5 proteins were associated with ubiquitinated proteins in vivo in LNCaP prostate cancer cells and this effect was augmented by proteasome inhibition. Site-directed mutagenesis reduced the in vitro autoubiquitination activity of the RMND5A (C356A/H358A) and RMND5B (C358A/H360A) RING domain mutants, while in vivo, interaction of RMND5B (C358A/H360A) with ubiquitinated proteins was decreased.
    Original languageEnglish
    QualificationDoctor of Philosophy
    Publication statusUnpublished - 2013


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