Inhibition of NF-kappaB DNA binding by nitric oxide.
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.
Matthews
J R
JR
School of Biological and Medical Sciences, Irvine Building, University of St. Andrews, Fife, UK.
Botting
C H
CH
Panico
M
M
Morris
H R
HR
Hay
R T
RT
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D052002:NF-kappa B p50 Subunit; D009569:Nitric Oxide / Q000494:pharmacology*; D009599:Nitroprusside / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000378:metabolism; D026423:S-Nitroso-N-Acetylpenicillamine; D051996:Transcription Factor RelA
D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D011993:Recombinant Fusion Proteins; D051996:Transcription Factor RelA; D009599:Nitroprusside; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D004247:DNA; D010396:Penicillamine