Intracellular Activation of Interferon Regulatory Factor-1 by Nanobodies to the Multifunctional (Mf1) Domain

Angeli Moeller, Emmanuelle Pion, Vikram Narayan, Kathryn L. Ball

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)38348-38361
Number of pages14
JournalJournal of Biological Chemistry
Volume285
Issue number49
DOIs
Publication statusPublished - 3 Dec 2010

Keywords

  • Antibodies
  • Interferon
  • Protein Domains
  • Transcription Factors
  • Tumor Suppressor
  • IRF-1
  • Intracellular Activation
  • Nanobody
  • Negative Regulation
  • Epitope

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