Chen, Lina (2017) Structural and functional studies of the cell cycle regulator RGC-32. Doctoral thesis (PhD), University of Sussex.

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Abstract

Epstein-Barr virus (EBV) immortalises resting B-lymphocytes to lymphoblastoid cell lines (LCLs) and is associated with many cancers. The cell-cycle regulator response gene to complement 32 (RGC-32) is upregulated in EBV-infected cells, binds the mitotic kinases CDK1 and PLK1 and disrupted cell cycle checkpoints. RGC-32 may therefore play a role in EBV-mediated cell-cycle deregulation. RGC-32 has no homology with any other known proteins, so affinity-tagged forms of RGC-32 were expressed in E.coli for structure-function studies. Replacing a polyhistidine tag with a glutathione S-Transferase (GST) tag and optimising expression conditions improved RGC-32 solubility. Purified soluble RGC-32 was produced for structural studies, but no crystals were obtained. Using the GST-RGC-32 fusion protein I showed that RGC-32 interacts with CDK1, Plk1 and the kinetochore component Spc24 from B-cell lysates. Interestingly, RGC-32 did not interact with cyclin B1, suggesting that it may activate CDK1 in a cyclin-independent manner. Mapping the regions of interaction between RGC-32 and CDK1 and Plk1 revealed these kinases bind to different but adjacent regions of RGC-32. To investigate the role of RGC-32 in cell cycle disruption by EBV, I made a series of cell-lines stably expressing inducible RGC-32 constructs and RGC-32 disrupted G2/M checkpoint in an additional B-cell line. Investigating the mechanism of RGC-32 transcription control in EBV-infected cells demonstrated that although EBV transcription factors bind to intronic regions of the RGC-32 gene, no regulation was detected in reporter assay. These data reveal a novel aspect of CDK1 activation by RGC-32, identify the sites of protein-protein interactions and provide new cell-lines for further investigation of RGC-32 function.

Item Type:	Thesis (Doctoral)
Schools and Departments:	School of Life Sciences > Biochemistry
Subjects:	Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry R Medicine > RC Internal medicine > RC0109 Infectious and parasitic diseases > RC0141.5 Epstein-Barr virus disease
Depositing User:	Library Cataloguing
Date Deposited:	09 Jun 2017 08:27
Last Modified:	27 Aug 2019 09:24
URI:	http://sro.sussex.ac.uk/id/eprint/68451

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