Inhibition of NF- B DNA Binding by Nitric Oxide

Abstract

It has been suggested that the NF-kappaB transcription factor family may mediate expression of the gene encoding the cytokine-inducible form of nitric oxide synthase (iNOS). To establish if nitric oxide (NO) could in turn affect activity of NF-kappaB, the ability of NO-donor compounds to influence NF-kappaB DNA binding activity in vitro was investigated. NO-donor compounds sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) both inhibited the DNA binding activity of recombinant NF-kappaB p50 and p65 homodimers and of p50-p65 heterodimers. Inhibition of NF-kappaB p50 DNA binding by NO-donor compounds involved modification of the conserved redox-sensitive C62 residue, as a C62S p50 mutant was significantly more resistant to SNP-mediated inactivation. Non-reducing SDS-polyacrylamide gel electrophoresis demonstrated that SNP could inhibit p50 DNA binding by mechanisms other than the formation of intersubunit disulphide bonds involving p50 residue C62. Electrospray ionization mass spectrometry of a synthetic NF-kappaB p5O peptide containing the C62 residue suggested that NO gas can modify C62 by S-nitrosylation. This study indicates that NO-donors can directly inhibit the DNA binding activity of NF-kappaB family proteins, suggesting that cellular NO provides another control mechanism for modulating the expression of NF-kappaB-responsive genes.

Osteopontin Induces Ubiquitin-Dependent Degradation of STAT1 in RAW264.7 Murine Macrophages

“…The cellular and biochemical consequences of NO production in the setting of sepsis are exemplified by DNA single-strand breakage, ADP ribosylation of nuclear proteins, nitrosative stressmediated alteration in gene transcription, and inhibition of mitochondrial respiration. In experimental endotoxemia, NO production results in cytotoxicity, inhibition of mitochondrial respiration, platelet aggregation, neutrophil adhesion, hypotension, and vasoplegia (22,23). However, evidence indicates that LPS-mediated iNOS gene transcription is an exceedingly complex and redundant signal transduction pathway with varying signal-and cell-dependent responses.…”

Nitric oxide modulates murine yolk sac vasculogenesis and rescues glucose induced vasculopathy

“…NO has been demonstrated to interact with several intracellular signaling cascades, including mitogenactivated protein kinase (MAPK), janus kinase (JAK) and Jun N-terminal kinase (JNK) (Lander, 1997;Kim et al, 1997;So et al, 1998). Additionally, transcription of several gene classes, including cytokines, matrix proteins and hormones, are modulated by NO regulation of nuclear factor κB (NFκB), hypoxia inducible factor 1 (HIF1) and zinc-finger transcription factors (Huang et al, 1999b;Kroncke and Carlberg, 2000;Matthews et al, 1996;Tabuchi et al, 1996;Torres and Forman, 2000). ROS, however, regulates transcription of protein kinases, growth factors and transcription factors, and plays a role in signaling in the vasculature (Burdon, 1996;Giaccia and Kastan, 1998;Maulik, 2002;Meyer et al, 1994;Nose, 2000).…”

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…Examples of transcription factors that are inhibited following cysteine oxidation include the p50 and RelA (p65) subunits of NF-κB, and the c-Jun member of the AP-1 family. Further, both S-nitrosylation and S-glutathionylation of those proteins has been shown to interfere with their DNA binding [197][198][199][200][201], and S-nitrosylation is coupled to iNOS function in vivo. Multiple cysteines in murine p53 were found to be functionally important, and their selective mutation afftected various aspects of its function [202].…”