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Identification of critical cysteine residues of Keap1 in arsenic-sensing and suppression of Nrf2.
FASEB J 2010 Apr; 24(Meeting Abstracts):759.2
Arsenic activates Nrf2 to induce phase II and antioxidative genes. Here we analyzed arsenic-Keap1 cysteine thiol interactions for Nrf2 activation. Arsenic-based Nrf2 activators 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 C288 in linker region resulted in higher level expression of Nrf2 protein in the absence of inducers. Mutation of C151 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.
Cell-biology; Cell-metabolism; Cellular-reactions; Chemical-analysis; Chemical-reactions; Chemical-synthesis; Exposure-assessment; Occupational-exposure; Oxidative-metabolism; Oxidative-processes; Sensitivity-testing; Statistical-analysis
Issue of Publication
The FASEB Journal. Experimental Biology 2010, April 24 - 28, 2010, Anaheim, California
Page last reviewed: September 2, 2020
Content source: National Institute for Occupational Safety and Health Education and Information Division