title: Mapping of NRF binding motifs of NF-kappaB p65 subunit.
abstract: NF-kappaB repressing factor (NRF) is a nuclear transcription factor that binds to a specific DNA sequence in NF-kappaB target promoters. Previous reports suggested that NRF interferes with the transcriptional activity of NF-kappaB binding sites through a direct interaction with NF-kappaB subunits. The aim of this study was to map specific NRF binding domains in the NF-kappaB proteins, p65 and p50. Our data demonstrate that NRF is able to interact with the p65 subunit and inhibit its transcription enhancing activity in reporter gene experiments. Using tandem affinity purifications (TAP), we show that NRF protein significantly binds to the endogenous p65, subunit but not to the p50 subunit. The selective binding activity of the NRF protein is consistently mediated by the N-terminal domain of NRF (Amino acids 1-380). Moreover, the Rel homology domain (RHD) of p65 is sufficient for binding to the N-terminal domain of NRF. Using detailed peptide mapping studies, we finally identify three peptide motifs in p65 RHD showing distinctive binding specificities for the NRF protein. According to the predicted structure of p65, all three peptide motifs align within an exposed region of p65 and might hint at promising targets for inhibitors.
authors.affiliation: Department of Chemical Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Marc R
authors.initials: MR
authors.lastname: Reboll
authors.forename: Aike T
authors.initials: AT
authors.lastname: Schweda
authors.forename: Myriam
authors.initials: M
authors.lastname: Bartels
authors.forename: Raimo
authors.initials: R
authors.lastname: Franke
authors.forename: Ronald
authors.initials: R
authors.lastname: Frank
authors.forename: Mahtab
authors.initials: M
authors.lastname: Nourbakhsh
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D000595:Amino Acid Sequence; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D012097:Repressor Proteins; D051996:Transcription Factor RelA