Covalent modification at Cys151 dissociates the electrophile sensor Keap1 from Cullin3, the ubiquitin E3 ligase scaffold

A148

Induction of antioxidant and Phase II detoxification proteins by small molecules and natural products represents an integral component of chemoprevention strategies designed to inhibit the initiation phase of carcinogenesis. Antioxidant and Phase II gene expression is regulated by Nuclear factor E2-related factor 2 (Nrf2) heterodimeric binding to the proximal promoter Antioxidant Response Element. Under basal conditions Nrf2 associates with the CUL3 adaptor protein, Keap1, and undergoes constitutive CUL3-ligase-mediated ubiquitylation. Inhibition of CUL3-based E3 ubiquitination of Nrf2 by small molecule and natural product chemoprevention agents allows robust Nrf2-directed gene expression. Here we used the model electrophile N-iodoacetyl-N-biotinylhexylenediamine (IAB) to directly test the hypothesis that adduction of Cys151 in Keap1 alters structure and function. Adduction of recombinant hKeap1 was accompanied by progressive loss of protein secondary structure, as monitored by CD spectroscopy, and loss of the ability to associate with recombinant hCUL3, determined in an in vitro binding assay. IAB-mediated dissociation of Keap1 from CUL3 in vitro was shown to be dependent upon Cys151 in Keap1. A quantitative mass spectrometry-based kinetic analyses of FLAG-Keap1 adduction in vivo revealed a propensity for Cys151 adduction. Immunoprecipitation experiments demonstrated that Cys151 was required for IAB-mediated dissociation of the Keap1-CUL3 complex. These results demonstrate that Cys151 adduction confers a critical alkylation sensor function upon Keap1, regulating Nrf2-mediated expression of antioxidant and Phase II detoxification proteins. This work was supported in part by NIH/NCI RO-1 CA 104590.