Prostaglandin H Synthase-2-catalyzed Oxygenation of 2-Arachidonoylglycerol Is More Sensitive to Peroxide Tone than Oxygenation of Arachidonic Acid

Musee, Joel; Marnett, Lawrence J.

doi:10.1074/jbc.m112.381202

Cited by 21 publications

(16 citation statements)

References 29 publications

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“…In vitro, COX-2 uses 2-AG and AA with similar kinetic efficiencies (3); however, PG-G production in intact cells is much lower than would be expected based on the relative amounts of cellular AA and 2-AG (6). This discrepancy is partially due to the fact that COX-2 requires activation by hydroperoxides, and higher concentrations of hydroperoxide are required to maintain 2-AG oxygenation than AA oxygenation (7). In addition, compartmentalization of the substrates within the cell might explain the poor utilization of 2-AG.…”

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confidence: 79%

Competition and allostery govern substrate selectivity of cyclooxygenase-2

Mitchener

Hermanson

Shockley

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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.

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confidence: 79%

Competition and allostery govern substrate selectivity of cyclooxygenase-2

Mitchener

Hermanson

Shockley

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Recent studies have shown that a higher concentration of peroxide substrate is required to activate and maintain the cyclooxygenase activity for oxygenation of endocannabinoid substrates compared with the concentration needed for AA oxygenation (17). The peroxidase activation efficiencies of prostaglandin G 2 and prostaglandin G 2 glycerol ester are likely similar.…”

Section: Discussionmentioning

confidence: 97%

“…The peroxidase activation efficiencies of prostaglandin G 2 and prostaglandin G 2 glycerol ester are likely similar. However, Musee and Marnett (17) suggest that release of a prostaglandin G 2 -G peroxyl radical before oxidation of Tyr-385 would result in inactive enzyme that would need to be activated by turnover of another molecule of hydroperoxide at the peroxidase active site. As a consequence, feedback activation of peroxidase catalysis by the hydroperoxide products generated during cyclooxygenase catalysis is more critical during the oxygenation of endocannabinoids versus AA.…”

Section: Discussionmentioning

confidence: 99%

“…Exogenous Lipid Hydroperoxide Alleviates Substrate-selective Inhibition-Compared with AA, the oxygenation of endocannabinoids by COX-2 is more sensitive to peroxide tone (17). Higher levels of peroxide are required to sustain maximal rates of endocannabinoid oxygenation compared with AA oxygenation.…”

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confidence: 99%

“…This is referred to as the branched chain mechanism of catalysis (14). Although endocannabinoid substrates bind in a similar fashion to AA in the cyclooxygenase active site (16), endocannabinoid oxygenation is sensitive to peroxide tone and requires more turnover of hydroperoxide to sustain cyclooxygenase catalysis, suggesting that the branched chain mechanism is less efficient with endocannabinoid substrates (17).…”

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confidence: 99%

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Substrate-selective Inhibition of Cyclooxygeanse-2 by Fenamic Acid Derivatives Is Dependent on Peroxide Tone

Orlando

Malkowski

2016

Journal of Biological Chemistry

161

128

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Cyclooxygenase-2 (COX-2) catalyzes the oxygenation of arachidonic acid (AA) and endocannabinoid substrates, placing the enzyme at a unique junction between the eicosanoid and endocannabinoid signaling pathways. COX-2 is a sequence homodimer, but the enzyme displays half-of-site reactivity, such that only one monomer of the dimer is active at a given time. Certain rapid reversible, competitive nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit COX-2 in a substrate-selective manner, with the binding of inhibitor to a single monomer sufficient to inhibit the oxygenation of endocannabinoids but not arachidonic acid. The underlying mechanism responsible for substrate-selective inhibition has remained elusive. We utilized structural and biophysical methods to evaluate flufenamic acid, meclofenamic acid, mefenamic acid, and tolfenamic acid for their ability to act as substrateselective inhibitors. Crystal structures of each drug in complex with human COX-2 revealed that the inhibitor binds within the cyclooxygenase channel in an inverted orientation, with the carboxylate group interacting with Tyr-385 and Ser-530 at the top of the channel. Tryptophan fluorescence quenching, continuous-wave electron spin resonance, and UV-visible spectroscopy demonstrate that flufenamic acid, mefenamic acid, and tolfenamic acid are substrate-selective inhibitors that bind rapidly to COX-2, quench tyrosyl radicals, and reduce higher oxidation states of the heme moiety. Substrate-selective inhibition was attenuated by the addition of the lipid peroxide 15-hydroperoxyeicosatertaenoic acid. Collectively, these studies implicate peroxide tone as an important mechanistic component of substrate-selective inhibition by flufenamic acid, mefenamic acid, and tolfenamic acid.The cyclooxygenases (COX-1 and COX-2) convert arachidonic acid (AA) 2 to prostaglandin H 2 (1). Prostaglandin H 2 is subsequently metabolized by downstream tissue-specific synthases into potent signaling molecules that play fundamental roles in both the regulation of physiological homeostasis as well as in disease states such as inflammation and cancer (2). COX-1 preferentially oxygenates AA, whereas COX-2 efficiently oxygenates a broad spectrum of fatty acid, ester, and amide substrates, including the endocannabinoids 1-arachidonoyl glycerol (1-AG), 2-arachidonoyl glycerol, and anandamide (3-8). 2-Arachidonoyl glycerol and anandamide are widely distributed in mammalian tissues and were the first characterized endogenous ligands for the cannabinoid receptors CB 1 and CB 2 (9). COX-2 oxygenates endocannabinoids using the same catalytic mechanism employed for AA, generating PG-glycerol esters and PG-ethanolamides (10 -12). Endocannabinoid signaling plays a significant role in various physiological processes and has been implicated in pathologies ranging from anxiety and depression, to multiple sclerosis, Parkinson disease, and cancer (13). The unique ability to oxygenate endocannabinoids places COX-2 at a critical junction between the eicosanoid and endocann...

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A guide to ferroptosis in cancer

Yapici,

Bebber,

von Karstedt

2024

Molecular Oncology

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Ferroptosis is a newly identified iron‐dependent type of regulated cell death that can also be regarded as death caused by the specific collapse of the lipid antioxidant defence machinery. Ferroptosis has gained increasing attention as a potential therapeutic strategy for therapy‐resistant cancer types. However, many ferroptosis‐inducing small molecules do not reach the pharmacokinetic requirements for their effective clinical use yet. Nevertheless, their clinical optimization is under development. In this review, we summarize the current understanding of molecular pathways regulating ferroptosis, how cells protect themselves from the induction of ferroptotic cell death, and how a better understanding of cancer cell metabolism can represent vulnerabilities for ferroptosis‐based therapies. Lastly, we discuss the context‐dependent effect of ferroptosis on various cell types within the tumor microenvironment and address controversies on how tissue ferroptosis might impact systemic cancer immunity in a paracrine manner.

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Prostaglandin H Synthase-2-catalyzed Oxygenation of 2-Arachidonoylglycerol Is More Sensitive to Peroxide Tone than Oxygenation of Arachidonic Acid

Cited by 21 publications

References 29 publications

Competition and allostery govern substrate selectivity of cyclooxygenase-2

Competition and allostery govern substrate selectivity of cyclooxygenase-2

Substrate-selective Inhibition of Cyclooxygeanse-2 by Fenamic Acid Derivatives Is Dependent on Peroxide Tone

A guide to ferroptosis in cancer

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