Development of an intrabody capable of activating interferon regulatory factor-1 [IRF-1] and identification of IRF-1-binding peptide motifs
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 o f cancer. This thesis details the development o f an intrabody, an intracellular antibody, that binds specifically to endogenous IRF-1. The binding o f the intrabody to ERF-1 enhanced transcription from ERF-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 o f 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 invitro technique enables the identification o f peptides able to bind a specific target protein. The sequence o f 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 o f an IRF-1-binding peptide that held sequence similar to a region o f Zinc Finger 350 (ZNF350), a transcription factor involved in regulating the D N A damage response. Subsequently, endogenous ZNF350 and IRF-1 were co- immunoprecipitated from a human cancer cell line. The extreme C-terminus o f ERF-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 o f the extreme C-terminus o f IRF-1 in modulating the protein’s activity. This study also provides new and IRF-1-specific molecular tools, in the form o f intrabodies and IRF-1-binding peptides, which could be used in the future to further characterise the activity and regulation o f this tumour suppressor protein.