PRAME (preferentially expressed antigen in melanoma) is a cancer-testis antigen that is expressed in normal testis tissue and is detected at high levels in a number of haematological and solid organ malignancies. Little is known about the physiological function of PRAME, although a role in the repression of retinoic acid receptor signalling and modulation of cell proliferation has been proposed. PRAME has been shown to promote leukaemogenesis; therefore it is important to understand its expression is regulated in normal and cancerous cells. The aim of this thesis was to investigate PRAME gene regulation and to discover clues to its function by affinity purification of interacting proteins. Using sequence similarity searches, PRAME is defined here as a leucine-rich repeat protein, and it is shown to undergo both nuclear and cytoplasmic localisation. Transcription of the PRAME gene is shown to be induced by exposure of malignant cell lines to bacterial pathogen-associated molecular patterns (PAMPs) in combination with interferon-y. Evidence is presented that PAMP/interferon-y treatment also upregulates the translation of PRAME, and alters the subcellular localisation of PRAME protein, resulting in its association with the Golgi apparatus. These results suggest that PRAME may have a role in the innate immune response. Using affinity purification and mass spectrometry, novel PRAME- interacting proteins were identified. PRAME is shown to associate with the Cullin-2-E3 ubiquitin ligase complex via interaction with elongin C, as validated by in vitro binding studies, co-immunoprecipitation and immunofluorescence staining. Cytoplasmic PRAME is shown to colocalise with elongins in Golgi-like vesicles. In addition, PRAME is shown to interact with histones. Based on these results, it is proposed that PRAME is a BC-box protein that may function as a substrate targeting component of Cullin-2-E3 ubiquitin ligase complexes, which may aid in trafficking of intracellular proteins to endosomes and Golgi for degradation or modification. The interaction with histones suggests that nuclear PRAME may perform some role in gene regulation, and future studies will explore its potential role in histone ubiquitination.
Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:574661 |
Date | January 2012 |
Creators | Wadelin, Frances Roswyn |
Publisher | University of Nottingham |
Source Sets | Ethos UK |
Detected Language | English |
Type | Electronic Thesis or Dissertation |
Page generated in 0.0031 seconds