9 research outputs found
Competition and allostery govern substrate selectivity of cyclooxygenase-2
Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and its ester analog, 2-arachidonoylglycerol (2-AG), to prostaglandins (PGs) and prostaglandin glyceryl esters (PG-Gs), respectively. Although the efficiency of oxygenation of these substrates by COX-2 in vitro is similar, cellular biosynthesis of PGs far exceeds that of PG-Gs. Evidence that the COX enzymes are functional heterodimers suggests that competitive interaction of AA and 2-AG at the allosteric site of COX-2 might result in differential regulation of the oxygenation of the two substrates when both are present. Modulation of AA levels in RAW264.7 macrophages uncovered an inverse correlation between cellular AA levels and PG-G biosynthesis. In vitro kinetic analysis using purified protein demonstrated that the inhibition of 2-AG oxygenation by high concentrations of AA far exceeded the inhibition of AA oxygenation by high concentrations of 2-AG. An unbiased systems-based mechanistic model of the kinetic data revealed that binding of AA or 2-AG at the allosteric site of COX-2 results in a decreased catalytic efficiency of the enzyme toward 2-AG, whereas 2-AG binding at the allosteric site increases COX-2's efficiency toward AA. The results suggest that substrates interact with COX-2 via multiple potential complexes involving binding to both the catalytic and allosteric sites. Competition between AA and 2-AG for these sites, combined with differential allosteric modulation, gives rise to a complex interplay between the substrates, leading to preferential oxygenation of AA
Nuclear Oxidation of a Major Peroxidation DNA Adduct, M<sub>1</sub>dG, 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 (M<sub>1</sub>dG) is the most abundant DNA adduct
formed from the lipid peroxidation product, malondialdehyde, or the
DNA peroxidation product, base propenal. M<sub>1</sub>dG is mutagenic
in bacterial and mammalian cells and is repaired via the nucleotide
excision repair system. Here, we report that M<sub>1</sub>dG levels
in intact DNA were increased from basal levels of 1 adduct per 10<sup>8</sup> nucleotides to 2 adducts per 10<sup>6</sup> nucleotides following
adenine propenal treatment of RKO, HEK293, or HepG2 cells. We also
found that M<sub>1</sub>dG in genomic DNA was oxidized in a time-dependent
fashion to a single product, 6-oxo-M<sub>1</sub>dG (to ∼5 adducts
per 10<sup>7</sup> nucleotides), and that this oxidation correlated
with a decline in M<sub>1</sub>dG levels. Investigations in RAW264.7
macrophages indicate the presence of high basal levels of M<sub>1</sub>dG (1 adduct per 10<sup>6</sup> nucleotides) and the endogenous formation
of 6-oxo-M<sub>1</sub>dG. This is the first report of the production
of 6-oxo-M<sub>1</sub>dG in genomic DNA in intact cells, and it has
significant implications for understanding the role of inflammation
in DNA damage, mutagenesis, and repair
Competition and allostery govern substrate selectivity of cyclooxygenase-2
Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and its ester analog, 2-arachidonoylglycerol (2-AG), to prostaglandins (PGs) and prostaglandin glyceryl esters (PG-Gs), respectively. Although the efficiency of oxygenation of these substrates by COX-2 in vitro is similar, cellular biosynthesis of PGs far exceeds that of PG-Gs. Evidence that the COX enzymes are functional heterodimers suggests that competitive interaction of AA and 2-AG at the allosteric site of COX-2 might result in differential regulation of the oxygenation of the two substrates when both are present. Modulation of AA levels in RAW264.7 macrophages uncovered an inverse correlation between cellular AA levels and PG-G biosynthesis. In vitro kinetic analysis using purified protein demonstrated that the inhibition of 2-AG oxygenation by high concentrations of AA far exceeded the inhibition of AA oxygenation by high concentrations of 2-AG. An unbiased systems-based mechanistic model of the kinetic data revealed that binding of AA or 2-AG at the allosteric site of COX-2 results in a decreased catalytic efficiency of the enzyme toward 2-AG, whereas 2-AG binding at the allosteric site increases COX-2’s efficiency toward AA. The results suggest that substrates interact with COX-2 via multiple potential complexes involving binding to both the catalytic and allosteric sites. Competition between AA and 2-AG for these sites, combined with differential allosteric modulation, gives rise to a complex interplay between the substrates, leading to preferential oxygenation of AA
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 Kdo<sub>2</sub>-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