Cobrotoxin inhibits NF-kappa B activation and target gene expression through reaction with NF-kappa B signal molecules.
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.
Park
Mi Hee
MH
College of Pharmacy, Chungbuk National University, 48, Gaesin-dong, Heungduk-gu, Cheongju, Chungbuk 361-763, Korea.
Song
Ho Seub
HS
Kim
Ki Hyun
KH
Son
Dong Ju
DJ
Lee
Seung Ho
SH
Yoon
Do Young
DY
Kim
Youngsoo
Y
Park
Il Young
IY
Song
Sukgil
S
Hwang
Bang Yeon
BY
Jung
Jae Kyung
JK
Hong
Jin Tae
JT
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D020868:Gene Silencing / Q000187:drug effects*; D051550:I-kappa B Kinase; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism* / Q000502:physiology; D052002:NF-kappa B p50 Subunit; D011485:Protein Binding / Q000187:drug effects; D011498:Protein Precursors / Q000037:antagonists & inhibitors* / Q000378:metabolism* / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics* / Q000502:physiology
D003039:Cobra Neurotoxin Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D011498:Protein Precursors; D008156:Luciferases; D017346:Protein Serine-Threonine Kinases; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse; D003545:Cysteine