NEMO is an essential regulatory component of the IκB kinase (IKK) complex, which controls activation of the NF-κB signaling pathway. Herein, we show that NEMO exists as a disulfide-bonded dimer when isolated from several cell types and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. Treatment of cells with hydrogen peroxide (H 2 O 2 ) induces further formation of NEMO dimers. Disulfide bond-mediated formation of NEMO dimers requires Cys54 and Cys347. The ability of these residues to form disulfide bonds is consistent with their location in a NEMO dimer structure that we generated by molecular modeling. We also show that pretreatment with H 2 O 2 decreases TNFα-induced IKK activity in NEMO-reconstituted cells, and that TNFα has a diminished ability to activate NF-κB DNA binding in cells reconstituted with NEMO mutant C54/347A. This study implicates NEMO as a target of redox regulation and presents the first structural model for the NEMO protein.
-Carbolines are tricyclic nitrogen heterocycles formed in plants and animals as Maillard reaction products between amino acids and reducing sugars or aldehydes. They are being detected increasingly in human tissues, and their physiological roles need to be understood. Two -carboline carboxylates have been reported to accumulate in the human eye lens. We report here on the identification of another -carboline, namely 1-methyl-1-vinyl -2,3,4-trihydro--carboline-3-carboxylic acid, in the lenses of some cataract patients from India. Analysis of these three lenticular -carbolines using photodynamic and antioxidant assays shows all of them to be inert as sensitizers and effective as antioxidants; they quench singlet oxygen, superoxide and hydroxyl radicals and inhibit the oxidative formation of higher molecular weight aggregates of the test protein, eye lens ␥-crystallin. Such antioxidative ability of -carbolines is of particular relevance to the lens, which faces continual photic and oxidative stress. The -carboline diacid IV is also seen to display an unexpected ability of inhibiting the thermal coagulation of ␥-crystallin and the dithiothreitol-induced precipitation of insulin. These results offer experimental support to earlier suggestions that one of the roles that the -carbolines have is to offer protection against oxidative stress to the human tissues where they accumulate.
Chemicals and radiation can damage DNA leading to the formation of adducts/lesions, which -if not removed by DNA repair pathways -usually block replicative DNA polymerases (DNAPs). To overcome such potentially lethal blockage, cells have lesion bypass DNAPs, which are often in the Y-Family and include several classes. One class includes human DNAP κ and E. coli DNAP IV, and they insert dCTP in the non-mutagenic pathway opposite [+ta]-B[a]P-N 2 -dG, which is the major adduct formed by the environmental carcinogen benzo[a]pyrene. Another class includes hDNAP η and ecDNAP V, and they insert dATP opposite [+ta]-B[a]P-N 2 -dG in the dominant G → T mutagenic pathway. Herein we develop a hypothesis for why the IV/κ-class preferentially does cellular dCTP insertion. On the minor groove side of the active site, Y-Family DNAPs have a cleft/hole that can be analyzed based on an analogy to a "chimney." Our models of DNAP IV show a large chimney opening from which the pyrene of [+ta]-B[a]P-N 2 -dG can protrude, which allows canonical adductdG:dCTP pairing. In contrast, our models of DNAP V have small chimney openings that forces adduct-dG downward in the active site such that canonical adduct-dG:dCTP pairing is not possible. Based on X-ray structures, sequence alignment and our modeled structures of Y-Family DNAPs, chimney opening size seems primarily controlled by one amino acid ("flue-handle"), which dictates whether nearby amino acids ("flue") plug the chimney or not. Based on this analysis, a correlation is apparent: the flue is closed in V/η-class DNAPs giving small chimney openings, while the flue is open for the IV/κ-class giving large chimney openings. Secondarily, a hypothesis is developed for why the V/η-class might preferentially do cellular dATP insertion opposite [+ta]-B[a]P-N 2 -dG: the small chimney forces adduct-dG lower in the active site, possibly leading to catalysis using a noncanonical dNTP shape that permits syn-adenine:adduct-dG base pairing. In summary, a hypothesize is developed that the pyrene moiety of [+ta]-B[a]P-N 2 -dG protrudes from the large chimney opening of DNAP IV, thus permitting canonical dCTP:adduct-dG pairing, while the small chimney opening of DNAP V forces [+ta]-B[a]P-N 2 -dG lower down in the active site, in which syn-adenine can pair with adduct-dG via a non-canonical dNTP shape.
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