Cellular models for characterisation of MINA53, a 2-oxoglutarate-dependent dioxygenase

Thesis

2-Oxoglutarate/FE(II)-dependent dioxygenases (2OG 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 thesis focuses on a 2OG Oxygenase termed Myc-Induced Nuclear Antigen (MINA53). This enzyme has been implicated in ribosome biogenesis and cell proliferation, and observed overexpressed in several tumour types, yet the identity of its substrate(s) and their role in cancer is unknown.

The aims of the research that has resulted in this thesis were to; (i) develop a cell model of MINA53 enzyme activity, (ii) apply this model to study the role of MINA53 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 MINA53 knockout mouse embryonic fibroblasts (MEFs) reconstituted with either wildtype or enzyme-inactive MINA53. Using this model I have shown that MINA53 activity maintains normal levels of the large ribosomal subunit (60S), and supresses anchorage-independent growth, authophagy 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 Rpl27a. Together we have shown that Rpl27a is abundantly hydroxylated, and that MINA53 is a histidinyl hydroxylase; this represents the first discovery of 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.

Authors: Zayer, A

• Publication date: 2012
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: ribosome biogenesis; MINA53; autophagy; 2-oxoglutarate/Fe(II)-dependent dioxygenases
• Subjects: Genetics (life sciences); Cell Biology (see also Plant sciences); Oncology; Medical Sciences; Life Sciences