APAP overdose of Keap1 W to A mice
We discovered that the interaction of Keap1 to Cul3 is weaker than that of other KLHL proteins. The BTB domain of Keap1 has an unique Cul3-interacting region (designated C3IR) compared with the other Cul3-binding BTB domains, and replacement of this region in Keap1 with the corresponding sequence of other KLHL proteins restored stronger binding to Cul3. Evolutionary tracing suggests that Keap1 was duplicated during whole genomic duplication (WGD) in fish into Keap1A and Keap1B, each of which binds to Cul3 strongly or weakly, respectively. Keap1A was lost in reptiles and later, and Keap1B evolved into mammalian Keap1. To investigate the reason why terrestrial Keap1 has weak affinity to Cul3, we generated mice in which the C3IR of wild-type (WT) mKeap1 was replaced with that of zKeap1A (hereafter referred to as W to A mutation) to increase its affinity to Cul3, and intraperitoneally administered 300 mg/kg APAP and dissected 24 h later.