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Critical cysteine residues of Keap1 in suppression of Nrf2 basal activity and arsenic-sensing by regulating the ubiquitination-proteasomal degradation of Nrf2 protein.

He-X; Ma-Q
Toxicologist 2011 Mar; 120(Suppl 2):88
Nrf2 mediates the induction of antioxidant response element (ARE)-dependent drug metabolizing enzyme genes and antioxidative genes by a wide range of structurally divergent inducers including carcinogenic metals such as arsenic. Activation of Nrf2 involves modulation of Keap1/Cul3-mediated ubiquitination-proteasomal degradation of Nrf2. Here we analyzed arsenic-Keap1 cysteine thiol interactions for Nrf2 activation. Fluorescent arsenic FlAsH and phenylarsine oxide (PAO) were used to probe binding of arsenic to Keap1. FlAsH emitted strong fluorescence upon binding to purified Keap1, and arsenic, tert-butyhydroquinone (tBHQ), or the thiol reactive 2, 3-dimercaptopropanol inhibited FlAsH binding. Purified or endogenous Keap1 was effectively pulled down by PAO affinity beads in vitro and from hepa1c1c7 cells, and arsenic, tBHQ, free PAO, or cadmium blocked Keap1 pull down. Furthermore arsenic and free PAO significantly reduced the free thiol contents of purified or endogenous Keap1 in cells. Therefore, arsenic, FlAsH, PAO, tBHQ, and cadmium bind to Keap1 cysteine thiols competitively. All domains of Keap1 bound PAO, but the linker region exhibited highest binding activity. The function of arsenic-Keap1 interaction was evaluated in a reconstituted system that mimics endogenous Nrf2 regulation. Mutation of C273 or C288A in linker region resulted in higher level expression of Nrf2 protein in the absence of inducers. Mutation of C151A abolished Nrf2 activation by arsenic. Overexpression of C273A, C288A, or C151A altered the basal and arsenic-induced expression of Nqo1 consistent with regulated protein levels of Nrf2. The study demonstrates important roles of C273 and C288 in the suppression of Nrf2 by Keap1 under basal conditions and critical function of C151 in arsenic sensing and responsiveness. Our findings support the notion that arsenic binds to different sets of Keap1 cysteine residues to regulate divergent functions in Nrf2 signaling.
Biological-effects; Carcinogenesis; Carcinogenicity; Cell-biology; Cellular-reactions; Dose-response; Drug-interaction; Exposure-levels; Genes; Genetic-factors; Hepatocytes; Hepatotoxins; Inhalation-studies; Metal-fumes; Physiological-effects; Quantitative-analysis
7440-38-2; 7440-43-9; 7440-43-9
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The Toxicologist. Society of Toxicology 50th Annual Meeting and ToxExpo, March 6-10, 2011, Washington, DC