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

Abstract

The skipping of exon 3 and/or 2 seen in transcripts for the IRF-1 tumor suppressor has been linked to the development of human hemopoietic malignancies, such as leukemia and myelodysplastic syndrome (1-3). More recently, the loss of exons 7-9 in various combinations has been reported in cervical cancer, and this leads to the generation of C-terminally truncated IRF-1 that can compete with the wild-type protein for DNA binding. The C-terminal mutants have longer halflives and, unlike wild-type IRF-1, can be expressed in a cell cycle independent manner (4). Understanding the function of the Mf1 domain of IRF-1, a regulatory subdomain that is located at the C terminus of the protein, could therefore increase our current knowledge of the role that truncated proteins play in tumor progression.The extreme C-terminal region of IRF-1 (Mf1 domain; amino acids 301-325) is a regulatory domain that plays a role in both positive and negative modulation of target gene expression (5). In addition, the Mf1 domain plays a role in determining the rate of IRF-1 protein degradation, with removal of the C terminus leading to a significant increase in the half-life of the protein (6, 7). Furthermore, binding of members of the Hsp70 family of molecular chaperones to the Mf1 domain appears to be critical for the normal function and regulation of IRF-1 (8). Thus previous studies have pointed to a potentially important role for the Mf1 domain of IRF-1 in its homeostatic regulation.The studies described above, by necessity, relied on the use of exogenous IRF-1 mutant proteins; therefore, we do not know whether the Mf1 domain is rate-limiting for IRF-1-mediated gene activation within the context of the endogenous protein.To address this issue, we have used antibody phage display to screen for scFv 3 antibody fragments (9, 10) to the Mf1 domain. The introduction of scFv nanobodies into a cell system showed that the Mf1 domain was rate-limiting for IRF-1-mediated transcription under normal cellular conditions, providing good evidence that IRF-1 transcriptional function is subject to posttranslational regulation. The data also highlight an unexpected role for the extreme C terminus in the negative regulation of IRF-1 turnover. MATERIALS AND METHODSReagents and Cells-Anti-IRF-1 (BD Biosciences), anti-IRF-1 C20 (Santa Cruz Biotechnology), anti-IRF-2 (Abcam), anti-His (Novagen), anti-HP1␣ (Millipore), anti-caspase3 (Santa Cruz Biotechnology), anti-ISG20 (Abnova), anti-PKR (Santa Cruz Biotechnology), anti-CDK2 (Santa Cruz Biotechnology), anti-GAPDH (Abcam), and anti-GFP (Clontech) were used at a 1:1000 dilution; and HRP-protein A (BD Biosciences) was used at 1 g/ml. Cycloheximide (Supelco) was dissolved in H 2 O to 5 mg/ml and used at a concentration of 30 g/ml. MG132 (Calbiochem) was dissolved in DMSO to 10 mM and used at 50 M. pcDNA3-IRF-1 and IRF-1 ⌬C25 were as described previously (5)

Advances and challenges in targeting IRF5, a key regulator of inflammation

“…There is overwhelming evidence that IRF5 plays a key role in physiology and disease, therefore dampening or enhancing IRF5 expression and activity provides new avenues for the development of therapeutic agents. Among the three strategies for modulation of IRF5 activity and expression discussed in this review, that is, (a) modulating IRF5 expression, (b) interfering with the post-translational modifications of IRF5, and (c) interfering with IRF5 association with protein partners, the modulation of IRF5 levels using siRNA, CRISPR/Cas9, LNAs or perhaps nanobodies, singledomain antibody fragments derived from camelid heavy-chain antibodies that have been successfully utilized to target transcription factors [96][97][98], and adenoviruses, may take a long path in the development of new therapies themselves, but provide excellent opportunities for the identification of new points for therapeutic interference.…”

The Role of Interferon Regulatory Factor-1 (IRF1) in Overcoming Antiestrogen Resistance in the Treatment of Breast Cancer

“…A single point mutation (P325A) in the C-terminal region of IRF1 (multifunctional-1; Mf1; residues 301–325) increases both IRF1's ability to regulate its own transcription and rate of degradation [ 22 ]. We have also reported a novel single nucleotide polymorphism in the IRF1 gene (A4396G).…”

A Multiprotein Binding Interface in an Intrinsically Disordered Region of the Tumor Suppressor Protein Interferon Regulatory Factor-1

“…Dual Luciferase Reporter Assay-Luciferase reporter assays were carried out in H1299 cells as described previously (31) using 120 ng of either p125-luc IFN␤ (which contains the human IFN-␤ promoter region Ϫ125 to ϩ19) or a control plasmid p55-luc IFN␤ (which lacks the interferon-stimulated response element or ISRE; promoter region Ϫ55 to ϩ19), TLR3-Luc (hTLR3-588) or hTLR3IRF (a mutant lacking the ISRE), TRAIL (pTRL3 or a mutant minus the ISRE/IRFE, pTRL3m6), and IL-7 (Ϫ609-Luc or a mutant Ϫ609-mtIRF-ELuc, which is missing the ISRE) (28,31). Reporter activity was determined 24 h post-transfection using luciferase luminescence as a read-out.…”