Identification of the interactive interface and phylogenic conservation of the Nrf2-Keap1 system.

Background:  The transcription factor Nrf2 and its negative regulator Keap1 play important roles in transcriptional induction of a set of detoxifying and anti-oxidant enzymes. To gain an insight into our present enigma as to how cells receive oxidative and electrophilic signals and transduce them to Nrf2, we have developed a zebrafish model system for molecular toxicological studies. Results: We systematically cloned zebrafish cytoprotective enzyme cDNAs and found their expression to be efficiently induced by electrophilic agents. We consequently identified the presence of Nrf2 and Keap1 in zebrafish. Both loss- and gain-of-function analyses demonstrated that Nrf2 is the primary regulator of a subset of cytoprotective enzyme genes, while Keap1 suppresses Nrf2 activity in zebrafish. An ETGE motif, critical for the Nrf2–Keap1 interaction, was identified in the Neh2 domain of Nrf2 by reverse two-hybrid screening and found to be indispensable for the regulation of Nrf2 activity in zebrafish. Conclusion: Taken together, these results indicate that the Nrf2-Keap1 system is highly conserved among vertebrates and that the interface between Nrf2 and Keap1 forms an important molecular basis of this regulatory system.