Physiological Significance of Reactive Cysteine Residues of Keap1 in Determining Nrf2 Activity

Center for Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan; Environmental Response Project, Exploratory Research for Advanced Technology-Japan Science and Technology Corporation, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan; Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan

^* To whom correspondence should be addressed. Email: masi{at}mail.tains.tohoku.ac.jp.

	Abstract

Keap1 and Cul3 constitute a unique ubiquitin E3 ligase that degrades Nrf2, a key activator of cytoprotective genes. On exposure to oxidants/electrophiles, the enzymatic activity of this ligase complex is abrogated and fails to degrade Nrf2, resulting in transcriptional activation of Nrf2 target genes. Keap1 possesses several reactive cysteine residues that covalently bond with electrophiles in vitro. To clarify the functional significance of each Keap1 cysteine residue under physiological conditions, we established a transgenic complementation rescue model. Transgenic expression of mutant Keap1-C273A and/or Keap1-C288A in Keap1-null mice failed to reverse constitutive Nrf2 activation, indicating that cysteine residues 273 and 288 are essential for Keap1 to repress Nrf2 activity in vivo. In contrast, Keap1-C151S retained repressor activity and mice expressing this molecule were viable. Mouse embryonic fibroblasts from Keap1-C151S transgenic mice displayed decreased expression of Nrf2 target genes both before and after an electrophilic challenge, suggesting that Cys151 is important in facilitating Nrf2 activation. These results demonstrate critical roles of the cysteine residues in vivo in maintaining Keap1 function, such that Nrf2 is repressed under quiescent conditions and active in response to oxidants/electrophiles.