Experiments were carried out to detect cysteine residues on human Keap1 protein that may be sensors of oxidative stress that gives rise to changes in the GSH/GSSG redox couple. Human Keap1 protein, at a final concentration of 6 μM, was incubated for two hours in aqueous buffer containing 0.010 M GSH, pH 8, in an argon atmosphere. Subsequently, excess iodoacetamide and trypsin were added to generate a peptide map effected by LCMS analysis. Peptides containing all 27 carboxamidomethylated cysteines were identified. Replacement of GSH by 0.010 M GSSG yielded a map in which 13 of the original carboxamidomethylated peptides were unperturbed, while other caboxamidomethylated cysteine-containing peptides were undetected, and a number of new cysteine-containing peptide peaks were observed. By mass analysis, and in some cases, by isolation, reduction, carboxamidomethylation, and reanalysis, these were identified as S-glutathionylated (Type 1) or Cys-Cys (Type 2) disulfides. Such peptides derived from the N-terminal, dimerization, central linker, Kelch repeat and C-terminal domains of Keap1. Experiments were carried out in which Keap1 was incubated similarly but in the presence of various GSH/GSSG ratios between 100 and 1 ([GSH + GSSG] = 0.010 M), with subsequent caraboxamidomethylation and trypsinolysis to determine differences in sensitivities of the different cysteines to the type 1 and type 2 modifications. Cysteines most sensitive to S-glutathionylation include Cys77, Cys297, Cys319, Cys368, and Cys434, while cysteine disulfides most readily formed are Cys23-Cys38 and Cys257-Cys297. The most reducing conditions at which these modifications are at GSH/GSSG = 10, which computes to an oxidation potential of Eh = −268.5 mV, a physiologically relevant value. Under somewhat more oxidizing, but still physiologically relevant, conditions, GSH/GSSG = 1 (Eh = −231.1 mV), a Cys319-Cys319 disulfide is detected far from the dimerization domain of the Keap1 homodimer. The potential impact on protein structure of the glutathionylation of Cys434 and Cys368, the two modified residues in the Kelch repeat domain, was analyzed by docking and energy minimizations of glutathione residues attached to the Kelch repeat domain, whose coordinates are known. The energy minimizations indicated marked alterations in structure with a substantial constriction of Neh2 binding domain of the Keap1 Kelch repeat domain. This alteration appears to be enforced by an extended hydrogen-bonding network between residues on the glutathione moiety attached to Cys434 and amino acid side chains that have been shown to be essential for repression of Nrf2 by Keap1. The modifications of Keap1 detected in the present study are discussed in the context of previous work of others who have examined the sensitivity of cysteines on Keap1 to electrophile assault.

The protein Kelch-like ECH-associated protein 1 (Keap1) is a cysteine-rich regulatory and scaffold protein. Human Keap1 contains 27 cysteines. Some of these cysteines are believed to mediate derepression of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which subsequently upregulates phase 2 enzymes, in response to electrophilic=oxidative assault. Some current models depict a highly select group of two and possibly a few more cysteine residues as key sensors. The assumptions and approaches undergirding these models are commented upon. The chemical reactivity of the cysteines of Keap1 toward an array of electrophiles and one oxidant is reviewed. A number of reports in the recent literature of molecules that putatively modify cysteines of Keap1 are also included. Insights into the current molecular basis of electrophile=oxidant activation of the Nrf2 pathway via reaction at cysteines of Keap1 are discussed. Finally, important knowns and unknowns are summarized. Antioxid. Redox Signal. 13, 1749-1761.

Nucleotide incorporation and extension opposite N 2 -ethylGua by DNA polymerase was measured and structures of the DNA polymerase -N 2 -ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase opposite N 2 -ethyl-Gua was determined by steady state kinetic analysis with Mg 2؉ or Mn 2؉ as the activating metal. DNA polymerase incorporates dCMP opposite N 2 -ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg 2؉ and with greater efficiencies in the presence of Mn 2؉ . However, the fidelity of nucleotide incorporation by DNA polymerase opposite N 2 -ethyl-Gua and Gua using Mn 2؉ is lower relative to that using Mg 2؉ indicating a metal-dependent effect. DNA polymerase extends from the N 2 -ethyl-Gua:Cyt 3 terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase catalyzed reaction is well suited for N 2 -ethyl-Gua bypass. The structure of DNA polymerase with N 2 -ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase complexed with N 2 -ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.2 is an acetaldehyde-derived DNA adduct generated from the reduction of acetaldehyde with 2Ј-deoxyguanosine-3Ј-monophosphate (1). Humans are exposed to acetaldehyde from the environment and through the formation of acetaldehyde by the oxidation of ethanol (2). N 2 -Ethyl-Gua has been detected in the DNA of both alcoholic and nonalcohol drinkers (2, 3). Ethanol is classified as a human carcinogen, and acetaldehyde is known to contribute to the formation of malignant tumors (4). The formation of N 2 -ethylGua during the reduction of acetaldehyde could cause ethanolrelated cancers (5).The ethyl moiety of N 2 -ethyl-Gua is predicted to project into the minor groove of duplex DNA. The N 2 -ethyl-Gua adduct is a strong block to DNA replication by replicative DNA polymerases in vitro and in cells (6, 7). Structures of bacteriophage DNA polymerase (pol) RB69, a homolog of human DNA pol ␣, indicate a possible mechanism of N 2 -ethyl-Gua blocked DNA replication. The structures reveal a DNA-binding motif that contacts the DNA minor groove and functions as an important safeguard to replication fidelity (8). The blocking of replicative DNA pols by N 2 -ethyl-Gua could arise when the ethyl group, protruding into the minor groove, disrupts protein:DNA contacts involved in the proposed "checking mechanism" (8). N 2 -Ethyl-Gua also has a high mis-coding potential during DNA replication with the Klenow fragment of Escherichia coli DNA po...

The effects of N(2)-ethylGua, O(6)-ethylGua, and O(6)-methylGua adducts in template DNA on polymerization by mammalian DNA polymerases alpha and eta have been investigated. The N(2)-ethylGua adduct blocks polymerization by the replicative DNA polymerase alpha to a much greater extent than does the O(6)-ethyl- or the O(6)-methylGua adducts. The DNA polymerase eta efficiently and accurately bypasses the N(2)-ethylGua lesion but like DNA polymerase alpha is similarly blocked by the O(6)-ethyl- or the O(6)-methylGua adducts. A steady state kinetic analysis of nucleotide insertion opposite the N(2)-ethylGua and the O(6)-ethylGua adducts by the DNA polymerases alpha and eta and extension from 3'-termini positioned opposite these adducts was performed to measure the efficiency and the accuracy of DNA synthesis past these lesions. This analysis showed that insertion of Cyt opposite the N(2)-ethylGua adduct by DNA polymerase alpha is approximately 10(4)-fold less efficient than insertion of Cyt opposite an unadducted Gua residue at the same position. Extension from the N(2)-ethylGua:Cyt 3'-terminus by DNA polymerase alpha is approximately 10(3)-fold less efficient than extension from a Cyt opposite the unadducted Gua. Insertion of Cyt opposite the N(2)-ethylGua lesion by the DNA polymerase eta is about 370-fold more efficient than by the DNA polymerase alpha, and extension from the N(2)-ethylGua:Cyt 3'-terminus by the DNA polymerase eta is about 3-fold more efficient than by the DNA polymerase alpha. Furthermore, the DNA polymerase eta preferably inserts the correct nucleotide Cyt opposite the N(2)-ethylGua lesion with nearly the same level of accuracy as opposite an unadducted Gua, thus minimizing the mutagentic potential of this lesion. This result contrasts with the relatively high misincorporation efficiency of Thy opposite the O(6)-ethylGua adduct by the DNA polymerases alpha and eta. In reactions containing both DNA polymerases alpha and eta, synthesis past the N(2)-ethylGua adduct is detected to permit completed replication of the adducted oligonucleotide template. These results suggest that accurate replication past the N(2)-ethylGua adduct might be facilitated in cells by pausing of replication catalyzed by DNA polymerase alpha and lesion bypass catalyzed by DNA polymerase eta.