Abstract
Interferon Regulatory Factor-2 (IRF-2) belongs to IRF family, was identified as a mammalian transcription factor involved in Interferon beta (IFNβ) gene regulation. Besides that IRF-2 is involved in immunomodulation, hematopoietic differentiation, cell cycle regulation and oncogenesis. We have done molecular sub-cloning and expression of recombinant murine IRF-2 as GST (Glutathione-S-Transferase)- IRF-2 fusion protein in E. coli/XL-1blue cells. Recombinant IRF-2 with GST moiety at N-terminus expressed as GST-IRF-2 (~66 kd) in E. coli along with different low molecular mass degradation products revealed approximately 30, 42, 60 and 62 kd by SDS-PAGE and Western blot, respectively. We further confirm that degradation takes place at C-terminus of the fusion protein not at N-terminus as anti-GST antibody was detecting all bands in the immunoblot. The recombinant IRF-2 was biologically active along with their degradation products in terms of their DNA binding activity as assessed by Electrophoretically Mobility Shift Assay (EMSA). We observed three different molecular mass DNA/protein complexes (1 - 3) with Virus Response Element (VRE) derived from human Interferon IFNβ gene and five different molecular mass complexes (1 - 5) with IRF-E motif (GAAAGT)4 in EMSA gel. GST only expressed from empty vector did not bind to these DNA elements. To confirm that the binding is specific, all complexes were competed out completely when challenged with 100-X fold molar excess of IRF-E oligo under cold competition. It means degradation products along with full-length protein are able to interact with VREβ as well as IRF-E motif. This means degradation products may regulate the target gene (s) activation/repression via interacting with VRE/IRF-E.
Highlights
Interferon Regulatory Factor-2 (IRF-2) is a transcription regulator of virus- and IFN- induced signalling pathways, which plays a critical role in antiviral defence, immune response, cell growth regulation and oncogenesis [1]
Interferon Regulatory Factor-2 (IRF-2) belongs to IRF family, was identified as a mammalian transcription factor involved in Interferon beta (IFNβ) gene regulation
The diversity in function(s) among family members are due to presence of diverse C-terminus, for instance, IRF-1, IRF-3, IRF-7, IRF-9 and IRF-10 is transcriptional activator whereas IRF-2 and IRF-8 is transcriptional repressor/activator [1]
Summary
IRF-2 is a transcription regulator of virus- and IFN- induced signalling pathways, which plays a critical role in antiviral defence, immune response, cell growth regulation and oncogenesis [1]. The exact mechanism underlying this cell transformation is still unknown, it is possible that IRF-2 exerts its oncogenic function through mediation of IRF-1 and/or other IRF family members This possibility supported by the finding that NIH 3T3 cells expressing only the DBD of IRF-2 were transformed. IRF-2 makes the cancerous esophageal cells refractory to INF action via suppressing IFNGR1 in order to develop cancer In this condition, IRF-2 level goes up that down regulate the IFNGR1 expression and makes esophageal cancer cells resistance to antitumour cytokine IFN [10]. IRF-2 level goes up that down regulate the IFNGR1 expression and makes esophageal cancer cells resistance to antitumour cytokine IFN [10] This supports the conclusion that IRF-2 is an oncogene. This means degradation products are biologically active in terms of DNA binding and may influence the target gene(s) expression
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