Ab Hancock Jr Memorial Laboratory For Cancer Research

Nashville, TN, United States

Ab Hancock Jr Memorial Laboratory For Cancer Research

Nashville, TN, United States
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Wauchope O.R.,Ab Hancock Jr Memorial Laboratory For Cancer Research | Beavers W.N.,Chemistry and Pharmacology | Galligan J.J.,Ab Hancock Jr Memorial Laboratory For Cancer Research | Mitchener M.M.,Chemistry and Pharmacology | And 3 more authors.
Chemical Research in Toxicology | Year: 2015

Chronic inflammation results in increased production of reactive oxygen species (ROS), which can oxidize cellular molecules including lipids and DNA. Our laboratory has shown that 3-(2-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) is the most abundant DNA adduct formed from the lipid peroxidation product, malondialdehyde, or the DNA peroxidation product, base propenal. M1dG is mutagenic in bacterial and mammalian cells and is repaired via the nucleotide excision repair system. Here, we report that M1dG levels in intact DNA were increased from basal levels of 1 adduct per 108 nucleotides to 2 adducts per 106 nucleotides following adenine propenal treatment of RKO, HEK293, or HepG2 cells. We also found that M1dG in genomic DNA was oxidized in a time-dependent fashion to a single product, 6-oxo-M1dG (to-5 adducts per 107 nucleotides), and that this oxidation correlated with a decline in M1dG levels. Investigations in RAW264.7 macrophages indicate the presence of high basal levels of M1dG (1 adduct per 106 nucleotides) and the endogenous formation of 6-oxo-M1dG. This is the first report of the production of 6-oxo-M1dG in genomic DNA in intact cells, and it has significant implications for understanding the role of inflammation in DNA damage, mutagenesis, and repair. © 2015 American Chemical Society.


Aluise C.D.,Ab Hancock Jr Memorial Laboratory For Cancer Research | Camarillo J.M.,Ab Hancock Jr Memorial Laboratory For Cancer Research | Shimozu Y.,Ab Hancock Jr Memorial Laboratory For Cancer Research | Galligan J.J.,Ab Hancock Jr Memorial Laboratory For Cancer Research | And 3 more authors.
Chemical Research in Toxicology | Year: 2015

Products of oxidative damage to lipids include 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), both of which are cytotoxic electrophiles. ONE reacts more rapidly with nucleophilic amino acid side chains, resulting in covalent protein adducts, including residue-residue cross-links. Previously, we demonstrated that peptidylprolyl cis/trans isomerase A1 (Pin1) was highly susceptible to adduction by HNE and that the catalytic cysteine (Cys113) was the preferential site of modification. Here, we show that ONE also preferentially adducts Pin1 at the catalytic Cys but results in a profoundly different modification. Results from experiments using purified Pin1 incubated with ONE revealed the principal product to be a Cys-Lys pyrrole-containing cross-link between the side chains of Cys113 and Lys117. In vitro competition assays between HNE and ONE demonstrate that ONE reacts more rapidly than HNE with Cys113. Exposure of RKO cells to alkynyl-ONE (aONE) followed by copper-mediated click chemistry and streptavidin purification revealed that Pin1 is also modified by ONE in cells. Analysis of the Pin1 crystal structure reveals that Cys113 and Lys117 are oriented toward each other in the active site, facilitating formation of an ONE cross-link. © 2015 American Chemical Society.

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