Pre-existent Asymmetry in the Human Cyclooxygenase-2 Sequence Homodimer

Sharma, Narayan P.; Jurban, Brice J.; Smith, William L.

doi:10.1074/jbc.m113.505503

Cited by 33 publications

(95 citation statements)

References 70 publications

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“…1B) was consistent with the addition of 3-hydroxyl groups (analyzed as acetyl esters) and reduction of one double bond. This was consistent with a PGF 2 -like structure generated from a PGH 2 for 2-AG (15); and V max of 43 units/mg and K m 10 M for AA (15). Thus, the catalytic efficiencies (V max /K m ) with huPGHS-2 are similar when each substrate is tested individually.…”

Section: Products Formed From 2-ag Ether By Hupghs-2supporting

confidence: 66%

“…4). PA was previously found to activate the oxygenation of 2-AG by huPGHS-2, although the magnitude of the effect was much less than that seen with AA (15). As shown in Fig.…”

Section: Comparison Of 2-ag and 2-ag Ether As Hupghs-2 Substratesmentioning

confidence: 80%

“…This variant has 90% of the activity of native huP-GHS-2, indicating that 90% of E allo monomers bear the Tyr385Phe mutation and 90% of E cat monomers are native monomers; moreover, once E allo and E cat of huPGHS-2 are formed, they do not interconvert (15). This variant can be used as a platform to determine the effect of amino acid substitutions in the COX binding site of E allo , the subunit with the Tyr385Phe substitution, as compared with the native E cat subunit (15). Figure 6 presents the results of studies of Arg-120 substitutions in the Y385F/Native huPGHS-2 platform on the effect of IBP on 2-AG ether oxygenation; supplementary Fig.…”

Section: Comparison Of 2-ag and 2-ag Ether As Hupghs-2 Substratesmentioning

confidence: 99%

“…We recently described a recombinant huPGHS-2 heterodimer variant, denoted as Y385F/Native huPGHS-2 (15). This variant has 90% of the activity of native huP-GHS-2, indicating that 90% of E allo monomers bear the Tyr385Phe mutation and 90% of E cat monomers are native monomers; moreover, once E allo and E cat of huPGHS-2 are formed, they do not interconvert (15).…”

Section: Comparison Of 2-ag and 2-ag Ether As Hupghs-2 Substratesmentioning

confidence: 99%

“…Procedures for the expression and purification of recombinant native huPGHS-2 and mutant huPGHS-2 heterodimer variants from insect cells were as described previously (7,15). The purity of the recombinant huPGHS-2 was determined by SDS-PAGE and Western blot analysis (9).…”

Section: Expression Purification and Assay Of Hupghs-2 Variantsmentioning

confidence: 99%

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Interactions of 2-O-arachidonylglycerol ether and ibuprofen with the allosteric and catalytic subunits of human COX-2

Zou

Yuan

Hong

et al. 2016

Journal of Lipid Research

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(1-4). PGHS-1 and PGHS-2 are considered to be the constitutive and inducible PGHS isoforms, respectively. PGHSs are often called cyclooxygenases (COXs). Both enzymes exhibit a bis-oxygenase or COX activity involved in the formation of the PG endoperoxide G 2 and a peroxidase activity that reduces PG endoperoxide G 2 to PGH 2 . COX activities of PGHSs are inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs) that include COX-2-specific inhibitors, sometimes referred to as coxibs (5).PGHSs are sequence homodimers composed of 72 kDa subunits. Despite the structural symmetry observed in crystal structures, both PGHS isoforms behave in solution as conformational heterodimers. One monomer (E allo ) acts as a regulatory allosteric monomer, and the other monomer (E cat ) binds heme and functions as the catalytic monomer (6-8). The COX activity of E cat of human (hu)PGHS-2 is allosterically modulated by many common FAs, including saturated and monounsaturated FAs that are not COX substrates [e.g., palmitic acid (PA)] (9, 10), and by some nonspecific NSAIDs, such as flurbiprofen and naproxen (7), that bind preferentially to E allo of huPGHS-2.Marnett and coworker (3) were the first to demonstrate that the endocannabinoid, 2-arachidonoylglycerol (2-AG), is an alternative substrate that is converted to 2-PGH 2 -glycerol by PGHS-2. This latter intermediate can, in turn, be converted to several different 2-prostanoyl-glycerol derivatives. A recent report has indicated that 2-AG binds Abstract Prostaglandin (PG) endoperoxide H synthase (PGHS)-2, also known as cyclooxygenase (COX)-2, can convert arachidonic acid (AA) to PGH 2 in the committed step of PG synthesis. PGHS-2 functions as a conformational heterodimer composed of an allosteric (E allo ) and a catalytic (E cat ) monomer. Here we investigated the interplay between human (hu)PGHS-2 and an alternative COX substrate, the endocannabinoid, 2-arachidonoylglycerol (2-AG), as well as a stable analog, 2-O-arachidonylglycerol ether (2-AG ether). We also compared the inhibition of huPGHS-2-mediated oxygenation of AA, 2-AG, and 2-AG ether by the well-known COX inhibitor, ibuprofen. When tested with huPGHS-2, 2-AG and 2-AG ether exhibit very similar kinetic parameters, responses to stimulation by FAs that are not COX substrates, and modes of inhibition by ibuprofen. The 2-AG ether binds E cat more tightly than E allo and, thus, can be used as a stable E cat -specific substrate to examine certain E allodependent responses.Ibuprofen binding to E allo of huPGHS-2 completely blocks 2-AG or 2-AG ether oxygenation; however, inhibition by ibuprofen of huPGHS-2-mediated oxygenation of AA engages a combination of both allosteric and competitive mechanisms.

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Section: Products Formed From 2-ag Ether By Hupghs-2supporting

confidence: 66%

“…4). PA was previously found to activate the oxygenation of 2-AG by huPGHS-2, although the magnitude of the effect was much less than that seen with AA (15). As shown in Fig.…”

Section: Comparison Of 2-ag and 2-ag Ether As Hupghs-2 Substratesmentioning

confidence: 80%

Section: Comparison Of 2-ag and 2-ag Ether As Hupghs-2 Substratesmentioning

confidence: 99%

Section: Comparison Of 2-ag and 2-ag Ether As Hupghs-2 Substratesmentioning

confidence: 99%

Section: Expression Purification and Assay Of Hupghs-2 Variantsmentioning

confidence: 99%

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Interactions of 2-O-arachidonylglycerol ether and ibuprofen with the allosteric and catalytic subunits of human COX-2

Zou

Yuan

Hong

et al. 2016

Journal of Lipid Research

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The Cyclooxygenases

Malkowski

2017

Encyclopedia of Inorganic and Bioinorganic Chemistry

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Cyclooxygenases (COX‐1 and COX‐2) are membrane‐associated, heme‐containing enzymes that oxygenate arachidonic acid to generate prostaglandin H 2 in the committed step of prostanoid biosynthesis. The resulting prostanoids play a key role in the maintenance of numerous physiological processes within the body. Abnormal changes in COX‐mediated prostanoid production are associated with various disease pathologies, including inflammation, cardiovascular disease, and cancer. Aspirin, nonselective nonsterroidal anti‐inflammatory drugs, and COX‐2‐selective drugs target COX‐1 and COX‐2 to reduce acute and chronic inflammation. As such, the crystal structures of COX‐1 and COX‐2 have been extensively characterized in the presence of substrates and inhibitors in order to gain a deeper understanding at the molecular level of the mechanistic nuances that underlie catalysis and inhibition. This review serves to summarize the recent advances in COX structural biology.

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The Eicosanoids

Smith¹,

Murphy²

2016

Biochemistry of Lipids, Lipoproteins and Membranes

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ABBREVIATIONS 2-AG 2-Arachidonoylglycerol AA Arachidonic acid BLT (1 or 2) Leukotriene B4 receptor (subclass 1 or 2) COX Cyclooxygenase cPLA 2 Cytosolic phospholipase A 2 CRE cAMP response element cysLT (1 or 2) Cysteinyl leukotriene receptor (subclass 1 or 2) for which leukotriene C4, D4 and E4 are agonists CYP4F(x) Cytochrome P450 isozyme that carries out ω-oxidation of leukotriene B4 (subclass x) EET Epoxyeicosatetraenoic acid EP Prostaglandin E receptor ER Endoplasmic reticulum FLAP 5-Lipoxygenase-activating protein HETE Hydroxyeicosatetraenoic acid HpETE Hydroperoxyeicosatetraenoic acid LO Lipoxygenase LT Leukotriene (followed by letter to designate structural type) NSAID Nonsteroidal anti-inflammatory drug PG Prostaglandin (followed by letter to designate structural type) PGHS Prostaglandin endoperoxide H synthase PLA 2 Phospholipase A 2 sPLA 2 Secretory phospholipase A 2 TXAS TxA synthase 260 Biochemistry of Lipids, Lipoproteins and Membranes

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Pre-existent Asymmetry in the Human Cyclooxygenase-2 Sequence Homodimer

Cited by 33 publications

References 70 publications

Interactions of 2-O-arachidonylglycerol ether and ibuprofen with the allosteric and catalytic subunits of human COX-2

Interactions of 2-O-arachidonylglycerol ether and ibuprofen with the allosteric and catalytic subunits of human COX-2

The Cyclooxygenases

The Eicosanoids

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