Molecular evolution of Keap1. Two Keap1 molecules with distinctive intervening region structures are conserved among fish.

Keap1 is a BTB-Kelch-type substrate adaptor protein of the Cul3-dependent ubiquitin ligase complex. Keap1 facilitates the degradation of Nrf2, a transcription factor regulating the inducible expression of many cytoprotective genes. Through comparative genome analyses, we found that amino acid residues composing the pocket of Keap1 that interacts with Nrf2 are highly conserved among Keap1 orthologs and related proteins in all vertebrates and in certain invertebrates, including flies and mosquitoes. The interaction between Nrf2 and Keap1 appears to be widely preserved in vertebrates. Similarly, cysteine residues corresponding to Cys-273 and Cys-288 in the intervening region of mouse Keap1, which are essential for the repression of Nrf2 activity in cultured cells, are conserved among Keap1 orthologs in vertebrates and invertebrates, except fish. We found that fish have two types of Keap1, Keap1a and Keap1b. To our surprise, Keap1a and Keap1b contain the cysteine residue corresponding to Cys-288 and Cys-273, respectively. In our analysis of zebrafish Keap1a and Keap1b activities, both Keap1a and Keap1b were able to facilitate the degradation of Nrf2 protein and repress Nrf2-mediated target gene activation. Individual mutation of either residual cysteine residue in Keap1a and Keap1b disrupted the ability of Keap1 to repress Nrf2, indicating that the presence of either Cys-273 or Cys-288 is sufficient for fish Keap1 molecules to fully function. These results provide an important insight into the means by which Keap1 cysteines act as sensors of electrophiles and oxidants.