Nabi, Naomi (2020) Characterisation of the potential lymphoma biomarker MORC4. Doctoral thesis, University of Huddersfield.

The microrchidia (MORC) protein family is a set of proteins comprising a conserved GHKL-type ATPase domain and a Zf-CW domain, which is associated with methylated histone binding in chromatin. This suggests an involvement in chromatin remodelling. There are 4 members of the MORC protein family in humans, MORC1, MORC2, MORC3 and MORC4. MORC2 and MORC3 are relatively well structurally and biochemically characterised with some cell biological characterisation. However, MORC4 is poorly characterised, although some evidence suggests that MORC4 could be a potential diffuse large B-cell lymphoma (DLBCL) biomarker. MORC4 mRNA is overexpressed in activated B-cell (ABC)-DLBCL cells compared with normal B cells, suggesting that MORC4 is an ABC-DLBCL biomarker. This study aims to take a broad focus to the characterisation of MORC4, both from structural and biochemical perspectives. This project also involves the investigation of RNA and protein expression analysis in several cancer cell types, in an attempt to gain an insight into the structure and function of MORC4 and further potential as a cancer biomarker.

This study involved recombinant protein design, expression and purification to produce high levels of pure MORC4 protein. This involved screening of multiple single and tandem domain MORC4 protein fragments in Escherichia coli, which were then purified to milligram quantities and homogeneity. Purified ATPase-Zf-CW tandem domain MORC429-480 protein was used for structural and biophysical analysis methods such protein crystallisation, analytical size exclusion (SEC) and SEC-multiangle laser light scattering (SEC-MALLS). Biochemical and mutagenesis approaches such as chemical crosslinking and site-directed mutagenesis (SDM), respectively, indicated that MORC4 forms ATP (AMP-PNP)-induced dimers. MORC2 and MORC3 structural analysis also indicated dimerisation, which was supported by homology modelling approaches. To investigate MORC4 protein expression in a number of cancer cell lines, novel monoclonal antibody reagent conditions were optimised in western blotting. High levels of MORC4 protein expression was identified in colorectal, breast and pancreatic cancer cell lines. This high level of MORC4 expression was supported by RNA analysis methods such as quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and RT-PCR isoform analysis.

Following extensive protein crystallisation trials, optimisation and X-ray diffraction screening, the high yielding and pure MORC429-480 ATPase-Zf-CW tandem domain protein produced crystals diffracting to 20 Å. However, a higher resolution dataset was required for structural resolution, hence, homology modelling was instead used to predict the structure of MORC4 using MORC2 and MORC3 as templates. Homology modelling and sequence alignments suggested that the ATPase domain of MORC4 was structurally homologous to MORC3. However, the C-terminus was less conserved in comparison to other MORC proteins. This suggests that it is more difficult to predict the C-terminal structure of MORC4 and that perhaps there may be a different function between MORC paralogues. RNA and protein expression analysis identified that MORC4 RNA and protein expression was relatively high in colorectal, breast and pancreatic cancer cell lines. This indicates that MORC4 could play a role in these specific types of cancers. However, further future cellular analysis is required to investigate MORC4 function such as siRNA/CRISPR approaches to assess MORC4 knockout on apoptosis and survival. Future work is also required to determine MORC4 as an ATPase and chromatin remodeller such as ATPase assays, isothermal titration calorimetry (ITC) and chromatin association assays. Overall, this study gives preliminary insights into the structure and function of MORC4.

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FINAL THESIS PART 1 - Nabi.pdf - Accepted Version
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