Differential effects of nitric oxide-mediated S-nitrosylation on p50 and c-jun DNA binding.
BACKGROUND
Nitric oxide (NO) regulates a variety of cellular functions. One mechanism by which NO may exert its influence is through formation of S-nitrosothiols at critical thiol residues in protein-active sites, including those of nuclear protein transcription factors.


METHODS
NF-kappa B p50 and AP-1 c-jun were S-nitrosylated in the presence of acidic NaNO2. Wild-type protein and protein subjected to nitrosylating conditions in the absence of NaNO2 served as controls. Confirmatory evidence for S-nitrosothiol bond formation was obtained by ultraviolet-visible spectrophotometry with the absorption maximum for S-NO bonds at approximately 320 to 360 nm. With consensus oligonucleotide probes, gel-shift analysis was used to examine DNA binding characteristics.


RESULTS
In the case of NF-kappa B p50, S-nitrosylation resulted in significantly decreased DNA binding. In contrast, S-nitrosylation did not alter c-jun DNA binding. The S-nitrosylating conditions themselves did not alter p50 or c-jun DNA binding. Quantitative analysis was performed according to the Scatchard plot technique to generate the respective dissociation constants. S-nitrosylated p50 was associated with a fourfold greater dissociation constant than that of the wild-type p50.


CONCLUSIONS
S-nitrosylation of transcription factors may be one mechanism by which NO may selectively regulate gene transcription.
delaTorre
A
A
Department of Surgery, University of Maryland, Baltimore, USA.
Schroeder
R A
RA
Bartlett
S T
ST
Kuo
P C
PC
D002273:Carcinogens / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D009566:Nitrates / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology*; D009603:Nitroso Compounds / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D002273:Carcinogens; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D009566:Nitrates; D009603:Nitroso Compounds; D016755:Proto-Oncogene Proteins c-jun; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D009569:Nitric Oxide; C031618:sodium nitrate