2-0xoglutarate/Fe(II)-dependent dioxygenases (ZOG Oxygenases) are a relatively poorly characterised enzyme family that hydroxylate biological macromolecules to regulate a variety of essential cellular processes in mammals, including; chromatin remodeling, extra-cellular matrix formation and oxygen sensing. The work in this th esis focuses on a ZOG Oxygenase termed Myc-Induced Nuclear Antigen (MINAS3). This enzyme has been implicated in ribosome biogenesis and cell proliferation, and observed overexpressed in several tumour types, yet the identity afits substrate(s) and their role in cancer is unknown. The aims of the resea rch that has resulted in this thesis were to; (i) develop a cell model of MINAS3 enzyme activity, (ii) apply this model to study the role of MINAS3 activity in cell transformation and cancer, and (iii) discover novel cellular processes regulated by MINA53 activity. As such, I have created an isogenic cell model consisting of K-Ras-transformed MINAS3 knockout mouse embryonic fibroblasts (MEFs) reconstituted with either wildtype or enzyme-inactive MINAS3. Using this model I have shown that MINAS3 activity maintains normal levels of the large ribosomal subunit (60S), and suppresses anchorage-independent growth, autophagy and gene expression. These observations suggest the existence and involvement of one or more substrates. Indeed, proteomic and biochemical analyses in collaboration with the Schofield laboratory (Chemistry, Oxford) confirmed the identity of a MINA53 substrate, the 60S ribosomal protein Rp127a. Together we have shown that Rpl27a is abundantly hydroxylated, and that MINA53 is a histidinyJ hydroxylase; this represents the first discovery of a ribosomal oxygenase. The model developed here did not support a positive role for MINA53 in the transformation of MEFs. Rather it suggested that MINA53 can suppress transformation in some contexts, This prompted a wider investigation that demonstrated underexpression of MINA53 in several tumour types, and the presence of inactivating mutations in breast. ovarian and colon cancer. This thesis provides data supporting further research to understand the role of Rpl27a hydroxylation in the regulation of 60S biogenesis, autophagy and cancer. 2
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:599914 |
| Date | January 2012 |
| Creators | Zayer, Adam |
| Contributors | Ratcliffe, Peter ; Coleman, Mathew |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:ebd1dfcd-0c8e-4c87-9644-8ddfd9208456 |
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