Nuclear Oxidation of a Major Peroxidation DNA Adduct, M₁dG, in the Genome

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(3<i>H</i>)-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

Detection of Cyclooxygenase-2-Derived Oxygenation Products of the Endogenous Cannabinoid 2‑Arachidonoylglycerol in Mouse Brain

Cyclooxygenase-2 (COX-2) catalyzes the formation of prostaglandins, which are involved in immune regulation, vascular function, and synaptic signaling. COX-2 also inactivates the endogenous cannabinoid (eCB) 2-arachidonoylglycerol (2-AG) via oxygenation of its arachidonic acid backbone to form a variety of prostaglandin glyceryl esters (PG-Gs). Although this oxygenation reaction is readily observed in vitro and in intact cells, detection of COX-2-derived 2-AG oxygenation products has not been previously reported in neuronal tissue. Here we show that 2-AG is metabolized in the brain of transgenic COX-2-overexpressing mice and mice treated with lipopolysaccharide to form multiple species of PG-Gs that are detectable only when monoacylglycerol lipase is concomitantly blocked. Formation of these PG-Gs is prevented by acute pharmacological inhibition of COX-2. These data provide evidence that neuronal COX-2 is capable of oxygenating 2-AG to form a variety PG-Gs in vivo and support further investigation of the physiological functions of PG-Gs

Quantitative Analysis and Discovery of Lysine and Arginine Modifications

Post-translational modifications (PTMs) affect protein function, localization, and stability, yet very little is known about the ratios of these modifications. Here, we describe a novel method to quantitate and assess the relative stoichiometry of Lys and Arg modifications (QuARKMod) in complex biological settings. We demonstrate the versatility of this platform in monitoring recombinant protein modification of peptide substrates, PTMs of individual histones, and the relative abundance of these PTMs as a function of subcellular location. Lastly, we describe a product ion scanning technique that offers the potential to discover unexpected and possibly novel Lys and Arg modifications. In summary, this approach yields accurate quantitation and discovery of protein PTMs in complex biological systems without the requirement of high mass accuracy instrumentation

Protein Modification by Endogenously Generated Lipid Electrophiles: Mitochondria as the Source and Target

Determining the impact of lipid electrophile-mediated protein damage that occurs during oxidative stress requires a comprehensive analysis of electrophile targets adducted under pathophysiological conditions. Incorporation of ω-alkynyl linoleic acid into the phospholipids of macrophages prior to activation by Kdo₂-lipid A, followed by protein extraction, click chemistry, and streptavidin affinity capture, enabled a systems-level survey of proteins adducted by lipid electrophiles generated endogenously during the inflammatory response. Results revealed a dramatic enrichment for membrane and mitochondrial proteins as targets for adduction. A marked decrease in adduction in the presence of MitoTEMPO demonstrated a primary role for mitochondrial superoxide in electrophile generation and indicated an important role for mitochondria as both a source and target of lipid electrophiles, a finding that has not been revealed by prior studies using exogenously provided electrophiles