Cells respond to oxidants and electrophiles by activating receptor/transcription factor Nrf2 to coordinate induction of cytoprotective genes critical for defense against oxidative and other stresses. Activation involves blocking the ubiquitination-proteasomal degradation of Nrf2. Modification of cysteine thiol groups by inducers in the linker region of Keap1, which congregates Nrf2 into the Keap1/Cul-3-based E3 complex for ubiquitination, is important but not sufficient for activation of Nrf2. Here we show that evolutionally conserved cysteine residues of Nrf2 are critical for Nrf2 regulation. FlAsH (an arsenic-based fluorophore) and phenylarsine oxide (PAO) potently induce Nrf2 target genes and bind to Nrf2 in vitro and in vivo. Binding is inhibited by prototypical inducers arsenic and tBHQ. PAO affin- ity pulldown and mutation of individual cysteine to alanine reveal that C235, C311, C316, C414 and C506 are critical for binding and binding is modulated by intra-molecular interactions. To corroborate the functions of cysteine residues, Nrf2 wild-type or mutants are expressed in Nrf2 knockout cells to reconstitute Nrf2 regulation. Nrf2 mutants have reduced t1/2 values that inversely correlate with increased binding to Keap1 and polyubiquitination of mutant proteins. Remarkably, the mutants fail to respond to arsenic for Nrf2 activation and gene in- duction. Furthermore, mutations at C119, C235, and C506 impede binding of Nrf2 to endogenous antioxidant response element and to coactivator CBP/p300. The findings demonstrate for the first time that Nrf2 cysteine residues critically regulate oxidant/eletrophile sensing, repress Keap1-dependent ubiquitination-proteasomal degradation, and promote recruitment of co-activators, such that chemical sensing, receptor activation, and transcription activation are integrated at the receptor molecule.
Biological-effects; Cell-biology; Chemical-hypersensitivity; Epidemiology; Exposure-assessment; Exposure-levels; Exposure-methods; Genetic-factors; Genetic-factors; Laboratory-testing; Molecular-biology; Occupational-exposure; Oxidative-metabolism; Particulates; Respiratory-hypersensitivity; Stress; Toxic-effects
The Toxicologist. Society of Toxicology 49th Annual Meeting and ToxExpo, March 7-11, 2010, Salt Lake City, Utah