Abstract A58: Cul3-mediated Nrf2 ubiquitination and ARE activation are dependent on the partial molar volume at position 151 of Keap1

Abstract

Abstract Nrf2 is a transcription factor that activates transcription of a battery of cytoprotective genes by binding to the antioxidant response element (ARE). Nrf2 is repressed by the cysteine-rich Keap1 protein, which targets Nrf2 for ubiquitination and subsequent degradation by a Cul3-mediated ubiquitination complex. Several promising chemopreventive agents, including sulforaphane, activate Nrf2 in Keap1 C151-dependent manner. We find that modification of C151 of human Keap1 by mutation to a tryptophan relieves the repression by Keap1 and allows activation of the ARE by Nrf2. Keap1 C151W has a decreased affinity for Cul3, and can no longer serve to target Nrf2 for ubiquitination, though it retains its affinity for Nrf2. A series of 12 mutant Keap1 proteins, each containing a different residue at position 151, was constructed to explore the chemistry required for the effect. The series reveals that the extent to which Keap1 loses the ability to target Nrf2 for degradation, and hence the ability to repress ARE activation, correlates well with the partial molar volume of the residue. Other physico-chemical properties do not appear to contribute significantly to the effect. Based on this finding, a structural model of the Keap1-Cul3 binding interface was constructed to investigate the potential mechanism of action of a tryptophan or other group at position 151. Several residues surrounding position 151, K131, R135 and K150, were observed in the model to be close enough to be involved in a steric clash with the tryptophan. Keap1 proteins containing these residues mutated to alanine, along with C151W, were evaluated for their ability to repress Nrf2. We find that none of mutations were able to restore the ability of Keap1 C151W to repress Nrf2. Additional residues are being investigated in effort to determine which residue(s) might be responsible for translating the increase in partial molar volume at position 151 to a lack of Nrf2 repression. This work has significant implications for how chemopreventive electrophiles that modify C151 disrupt the repressive function of Keap1. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A58.