title: Intracellular activation of interferon regulatory factor-1 by nanobodies to the multifunctional (Mf1) domain.
abstract: IRF-1 is a tumor suppressor protein that activates gene expression from a range of promoters in response to stimuli spanning viral infection to DNA damage. Studies on the post-translational regulation of IRF-1 have been hampered by a lack of suitable biochemical tools capable of targeting the endogenous protein. In this study, phage display technology was used to develop a monoclonal nanobody targeting the C-terminal Mf1 domain (residues 301-325) of IRF-1. Intracellular expression of the nanobody demonstrated that the transcriptional activity of IRF-1 is constrained by the Mf1 domain as nanobody binding gave an increase in expression from IRF-1-responsive promoters of up to 8-fold. Furthermore, Mf1-directed nanobodies have revealed an unexpected function for this domain in limiting the rate at which the IRF-1 protein is degraded. Thus, the increase in IRF-1 transcriptional activity observed on nanobody binding is accompanied by a significant reduction in the half-life of the protein. In support of the data obtained using nanobodies, a single point mutation (P325A) involving the C-terminal residue of IRF-1 has been identified, which results in greater transcriptional activity and a significant increase in the rate of degradation. The results presented here support a role for the Mf1 domain in limiting both IRF-1-dependent transcription and the rate of IRF-1 turnover. In addition, the data highlight a route for activation of downstream genes in the IRF-1 tumor suppressor pathway using biologics.
authors.affiliation: Cell Signalling Unit, University of Edinburgh Cancer Research UK Centre, Crewe Road South, Edinburgh EH4 2XR, Scotland, United Kingdom.
authors.forename: Angeli
authors.initials: A
authors.lastname: Möller
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Pion
authors.forename: Vikram
authors.initials: V
authors.lastname: Narayan
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Ball
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D050856:Interferon Regulatory Factor-1 / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D057127:Single-Chain Antibodies / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; D057127:Single-Chain Antibodies; D025521:Tumor Suppressor Proteins
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