Interferon regulatory factor 1 (IRF-1) is a tumour suppressor protein and transcription factor. It has been shown to modulate target gene expression in response to stimuli, which include viral infection and DNA damage, and to be down-regulated in several forms of cancer. This thesis details the development of an intrabody, an intracellular antibody, that binds specifically to endogenous IRF-1. The binding of the intrabody to IRF-1 enhanced transcription from IRF-1-responsive reporter gene constructs and endogenous promoters, thus it was shown to activate IRF-1. Intrabody binding also increased the rate at which IRF-1 was degraded, suggesting that the intrabody epitope may be regulating both IRF-1 activity and turnover. These results were supported point mutation within the intrabody epitope (P325 to A) as the resultant mutant also displayed both a higher transcriptional activity and increased rate of degradation. In an effort to understand the mechanisms which regulate IRF-1 activity a search for novel IRF-1-interacting proteins was carried out using phage peptide display. This in vitro technique enables the identification of peptides able to bind a specific target protein. The sequence of these peptides can then be used to search protein databases for homologous, full-length proteins that could also bind the target protein. This led to the identification of an IRF-1-binding peptide that held sequence similar to a region of Zinc Finger 350 (ZNF350), a transcription factor involved in regulating the DNA damage response. Subsequently, endogenous ZNF350 and IRF-1 were co-immunoprecipitated from a human cancer cell line. The extreme C-terminus of IRF-1 was shown to be sufficient for an interaction with ZNF350, although a second, more N-terminal site was also shown to be essential for a stable intracellular interaction. This data sheds new light on the role of the extreme C-terminus of IRF-1 in modulating the protein‟s activity. This study also provides new and IRF-1-specific molecular tools, in the form of intrabodies and IRF-1-binding peptides, which could be used in the future to further characterise the activity and regulation of this tumour suppressor protein.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:563544 |
| Date | January 2011 |
| Creators | Möller, Angeli |
| Contributors | Moeller, Angeli Kishor; Ball, Kathryn. ; Patton, Elizabeth. ; Wallace, Maura |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/5590 |
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