ABSTRACT
We previously discovered (-)-DHMEQ as a selective inhibitor of NF-κB, and it was shown to suppress many cancer and inflammation models in animals. (-)-DHMEQ directly binds to NF-κB components to inhibit DNA binding, and moreover, it often inhibits nuclear translocation of NF-κB. The mechanism of inhibiting nuclear translocation has been elucidated for RelB, a main noncanonical NF-κB component. However, it was not elucidated for p65, a main canonical NF-κB component. In the present research, we studied how (-)-DHMEQ inhibits nuclear localization of p65. First, (-)-DHMEQ inhibited p65 nuclear accumulation in adult T-cell leukemia MT-2 cells in which canonical p65 is constitutively activated. But there was no change in the stability and importin-α3 affinity of p65. Then, we prepared a p65 mutant protein with Arg35Ala and Tyr36Ala (AA) mutations having no DNA-binding ability in HeLa cells. The p65 AA mutant showed reduced nuclear localization without changing the stability and importin affinity. Taken together, the mechanism of inhibition is different between RelB and p65, and inhibition of p65 nuclear localization is likely to be due to the inhibition of DNA binding changing the equilibrium between the nuclear and cytoplasmic amounts of p65.
Subject(s)
Benzamides/pharmacology , Cyclohexanones/pharmacology , DNA/metabolism , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Breast Neoplasms , Cell Line, Tumor , Cell Nucleus , Enzyme Activation , Gene Expression , HeLa Cells , Humans , Leukemia-Lymphoma, Adult T-Cell/metabolism , Models, Biological , Mutation , NF-kappa B/genetics , Protein Binding/drug effects , Protein Transport/drug effects , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolismABSTRACT
NF-κB is a transcription factor for the immune activation and tissue stability, but excess activation of NF-κB often causes inflammation and cancer. An NF-κB component RelB is involved in B-cell maturation and autoimmunity. In the present research we studied the role of the RelB DNA binding domain on cellular stability and importin affinity. We prepared a RelB protein mutated at Arg141 to Ala and Tyr142 to Ala (AA mutant) having no DNA binding activity. The stability of this mutant protein was greatly reduced compared with that of the wild-type protein. We also constructed a nuclear localization signal-inactivated mutant of RelB, and found that this mutant was also unstable in the cells. Thus, RelB destabilization was caused by the loss of DNA binding possibly because of the change in cellular localization. The mutation also decreased the affinity to importin-α5 decreasing the nuclear localization. Our newly discovered NF-κB inhibitor (-)-DHMEQ binds to a specific Cys residue in RelB to inhibit DNA binding and also decreased the stability and importin affinity. These findings would indicate that the DNA binding activity of this transcription factor is a crucial for its stability and intracellular localization.
