FIG 1.

Novel Keap1 mutants that repress Nrf2 activity. (A) Cysteine residues of Keap1 are shown. Representative reactive cysteine residues against electrophiles are boxed. Keap1 domains: NTR (N-terminal region), BTB (broad complex, tramtrack, and bric-a-brac), IVR (intervening region), Kelch/DGR (double glycine repeat), and CTR (C-terminal region). (B and C) All 19 possible amino acid substitutions were introduced to Cys273 (B) and Cys288 (C) of human KEAP1. HEK293T cells were cotransfected with NRF2-degron LacZ (NRF2NT-LacZ) reporter plasmid (15 ng) and KEAP1 mutant expression vector (5, 15, or 45 ng) and then incubated for 48 h. The relative β-galactosidase activity was measured. Representative results from multiple independent experiments are shown. Circled and boxed terms indicate loss-of-function mutants and mutants that retain activity to repress Nrf2 accumulation, respectively. (D) HEK293T cells were cotransfected with ARE-luciferase reporter vector, Nrf2-overexpressing vector, and vector expressing 8 or 40 ng of Keap1 WT, C273S, C273W, C288S, C288E, or C273W&C288E. At 24 h after transfection, the relative luciferase activity was measured. Boxes indicate mutants that retain activity to repress Nrf2 accumulation.