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.
"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 . We have also reported a novel single nucleotide polymorphism in the IRF1 gene (A4396G). "
[Show abstract][Hide abstract] ABSTRACT: Resistance to endocrine therapy is common among breast cancer patients with estrogen receptor alpha-positive (ER+) tumors and limits the success of this therapeutic strategy. While the mechanisms that regulate endocrine responsiveness and cell fate are not fully understood, interferon regulatory factor-1 (IRF1) is strongly implicated as a key regulatory node in the underlying signaling network. IRF1 is a tumor suppressor that mediates cell fate by facilitating apoptosis and can do so with or without functional p53. Expression of IRF1 is downregulated in endocrine-resistant breast cancer cells, protecting these cells from IRF1-induced inhibition of proliferation and/or induction of cell death. Nonetheless, when IRF1 expression is induced following IFNγ treatment, antiestrogen sensitivity is restored by a process that includes the inhibition of prosurvival BCL2 family members and caspase activation. These data suggest that a combination of endocrine therapy and compounds that effectively induce IRF1 expression may be useful for the treatment of many ER+ breast cancers. By understanding IRF1 signaling in the context of endocrine responsiveness, we may be able to develop novel therapeutic strategies and better predict how patients will respond to endocrine therapy.
"The strategy aimed at inhibiting the HIV-1 infection using VHHs that recognize envelope proteins and neutralize the virions by competing with the main cellular receptor CD4 [77-79] is conceptually similar. Finally, nanobodies have been also used to bind intracellularly the multifunctional Mf1 domain of the IRF-1 tumor suppressor with the idea of regulating this viral infection-stimulated regulative protein . "
[Show abstract][Hide abstract] ABSTRACT: Single-domain antibody fragments possess structural features, such as a small dimension, an elevated stability, and the singularity of recognizing epitopes non-accessible for conventional antibodies that make them interesting for several research and biotechnological applications.
The discovery of the single-domain antibody's potentials has stimulated their use in an increasing variety of fields. The rapid accumulation of articles describing new applications and further developments of established approaches has made it, therefore, necessary to update the previous reviews with a new and more complete summary of the topic.
Beside the necessary task of updating, this work analyses in detail some applicative aspects of the single-domain antibodies that have been overseen in the past, such as their efficacy in affinity chromatography, as co-crystallization chaperones, protein aggregation controllers, enzyme activity tuners, and the specificities of the unconventional single-domain fragments.
[Show abstract][Hide abstract] ABSTRACT: The interferon-regulated transcription factor and tumor suppressor protein IRF-1 is predicted to be largely disordered outside of the DNA-binding domain. One of the advantages of intrinsically disordered protein domains is thought to be their ability to take part in multiple, specific but low affinity protein interactions; however, relatively few IRF-1-interacting proteins have been described. The recent identification of a functional binding interface for the E3-ubiquitin ligase CHIP within the major disordered domain of IRF-1 led us to ask whether this region might be employed more widely by regulators of IRF-1 function. Here we describe the use of peptide aptamer-based affinity chromatography coupled with mass spectrometry to define a multiprotein binding interface on IRF-1 (Mf2 domain; amino acids 106-140) and to identify Mf2-binding proteins from A375 cells. Based on their function as known transcriptional regulators, a selection of the Mf2 domain-binding proteins (NPM1, TRIM28, and YB-1) have been validated using in vitro and cell-based assays. Interestingly, although NPM1, TRIM28, and YB-1 all bind to the Mf2 domain, they have differing amino acid specificities, demonstrating the degree of combinatorial diversity and specificity available through linear interaction motifs.
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