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Cobrotoxin inhibits NF-kappa B activation and target gene expression through reaction with NF-kappa B signal molecules.
- Source :
-
Biochemistry [Biochemistry] 2005 Jun 14; Vol. 44 (23), pp. 8326-36. - Publication Year :
- 2005
-
Abstract
- Cobrotoxin is known to bind with cysteine residues of biological molecules such as nicotine acetylcholine receptor. Cobrotoxin may modify IKKs and p50 through protein-protein interaction since cysteine residues are present in the kinase domains of IKKalpha and IKKbeta and in the p50 of NF-kappaB. Our surface plasmon resonance analysis showed that cobrotoxin directly binds to p50 (K(d) = 1.54 x 10(-)(5) M), IKKalpha (K(d) = 3.94 x 10(-)(9) M) and IKKbeta (K(d) = 3.4 x 10(-)(8) M) with high binding affinity. Moreover, these protein-protein interactions suppressed the lipopolysaccharide (LPS, 1 microg/mL)- and the sodium nitroprusside (SNP, 200 microM)-induced DNA binding activity of NF-kappaB and NF-kappaB-dependent luciferase activity in astrocytes and Raw 264.7 macrophages. These inhibitory effects were correlated with the inhibition of IkappaB release and p50 translocation. Inhibition of NF-kappaB by cobrotoxin resulted in reductions in the LPS-induced expressions of COX-2, iNOS, cPLA(2), IL-4, and TNF-alpha in astrocytes and in COX-2 expression induced by SNP, LPS, and TNF-alpha in astrocytes. Moreover, these inhibitory effects of cobrotoxin were reversed by adding reducing agents, dithiothreitol and glutathione. In addition, cobrotoxin did not have any inhibitory effect on NF-kappaB activity in cells carrying mutant p50 (C62S), IKKalpha (C178A), and IKKbeta (C179A), with the exception of IKKbeta (K44A) mutant plasmid. Confocal microscopic analysis showed that cobrotoxin is uptaken into the nucleus of cells. These results demonstrate that cobrotoxin directly binds to the sulfhydryl groups of p50 and IKKs, and that this results in reduced IkappaB release and the translocation of p50, thereby inhibiting the activation of NF-kappaB.
- Subjects :
- Animals
Astrocytes drug effects
Astrocytes enzymology
Astrocytes metabolism
Binding, Competitive drug effects
Cell Line
Cells, Cultured
Cobra Neurotoxin Proteins metabolism
Cysteine chemistry
Cysteine metabolism
DNA-Binding Proteins antagonists & inhibitors
DNA-Binding Proteins genetics
DNA-Binding Proteins metabolism
DNA-Binding Proteins physiology
Enzyme Inhibitors chemistry
Enzyme Inhibitors metabolism
Gene Expression Regulation physiology
I-kappa B Kinase
Luciferases antagonists & inhibitors
Luciferases metabolism
Mice
NF-kappa B physiology
NF-kappa B p50 Subunit
Protein Binding drug effects
Protein Precursors physiology
Protein Serine-Threonine Kinases antagonists & inhibitors
Protein Serine-Threonine Kinases metabolism
Rats
Rats, Sprague-Dawley
Signal Transduction drug effects
Signal Transduction physiology
Cobra Neurotoxin Proteins chemistry
Gene Expression Regulation drug effects
Gene Silencing drug effects
NF-kappa B antagonists & inhibitors
NF-kappa B metabolism
Protein Precursors antagonists & inhibitors
Protein Precursors metabolism
Signal Transduction genetics
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 44
- Issue :
- 23
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 15938622
- Full Text :
- https://doi.org/10.1021/bi050156h