Inhibition of nuclear factor-kappaB DNA binding by organoselenocyanates through covalent modification of the p50 subunit.
Most known chemopreventive agents including certain selenium compounds suppress the activation of the nuclear factor kappaB (NF-kappaB), but the mechanisms remain largely elusive. Toward this end, we initially showed that the inhibition of NF-kappaB DNA binding by benzyl selenocyanate (BSC) and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) was reversed by the addition of DTT; this suggests the formation of DTT-reducible selenium-sulfur bonds between selenocyanate moieties and cysteine residues in NF-kappaB (p50) protein. Furthermore, the inhibitory effect of selenocyanates on NF-kappaB was not altered in the presence of physiologic level of reduced glutathione (1 mmol/L), suggesting that selenocyanates can also inhibit NF-kappaB in vivo. Using both matrix-assisted laser desorption/ionization-time of flight and tandem mass spectrometry fragmentation, we showed for the first time that the Cys(62) residue in the active site of NF-kappaB (p50) protein was modified by BSC through the formation of a selenium-sulfur bond. In addition, p-XSC-bound NF-kappaB (p50) protein was also detected by a radiotracer method. To provide further support, molecular models of both BSC and p-XSC positioned in the DNA binding pocket of the p50 were constructed through the covalent modification of Cys(62); the models reveal that DNA substrate could be hindered to enter its DNA binding region. This study shows for the first time that BSC and p-XSC may exert their chemopreventive activity, at least in part, by inhibiting NF-kappaB through covalent modification of Cys(62) of the p50 subunit of NF-kappaB.
Chen
Kun-Ming
KM
Department of Biochemistry and Molecular Biology, Penn State College of Medicine, Hershey, PA 17033, USA.
Spratt
Thomas E
TE
Stanley
Bruce A
BA
De Cotiis
Dan A
DA
Bewley
Maria C
MC
Flanagan
John M
JM
Desai
Dhimant
D
Das
Arunangshu
A
Fiala
Emerich S
ES
Amin
Shantu
S
El-Bayoumy
Karam
K
D000595:Amino Acid Sequence; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D003485:Cyanates / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D052002:NF-kappa B p50 Subunit / Q000737:chemistry*; D016566:Organoselenium Compounds / Q000494:pharmacology*
D016588:Anticarcinogenic Agents; D003485:Cyanates; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D016566:Organoselenium Compounds; C054108:benzyl selenocyanate; C074153:1,4-phenylenebis(methylene)selenocyanate; D004247:DNA; D004229:Dithiothreitol