Prokaryotic expression, purification and characterization of human cyclooxygenase-2.
ABSTRACT: Cyclooxygenase-2 (COX-2) is a key enzyme which catalyzes the conversion of arachidonic acid (AA) into prostaglandins (PGs). It plays an important role in pathophysiological processes, such as tumorigenesis, angiogenesis, inflammation and tumor cell drug resistance. Therefore, COX-2 has been viewed as an important target for cancer therapy. The preparation of COX-2 protein is an important initial step for the subsequent development of COX-2 inhibitors. In this study, we report a strategy to heterologously express truncated human COX-2 (trCOX-2) in Escherichia coli (E. coli) BL21(DE3) host cells. Following denaturation, purification and renaturation, we successfully obtained enzymatically active trCOX-2 containing 257 residues of the C-terminus. Homology modeling and molecular docking analyses revealed that trCOX-2 retained the predicted 3D catalytic domain structure and AA could still bind to its hydrophobic groove. Western blot analysis and ELISA indicated that the trCOX-2 still retained its characteristic antigenicity and binding activity, while COX assays revealed that trCOX-2 maintained its enzyme activity. On the whole, in this study, we provided a novel method to isolate trCOX-2 possessing AA binding and catalytic activities. This study thus lays a foundation to facilitate further investigations of COX-2 and offers a valuable method with which to achieve the prokaryotic expression of a eukaryotic membrane protein.
Project description:Prostaglandins (PGs) are powerful lipid mediators in many physiological and pathophysiological responses. They are produced by oxidation of arachidonic acid (AA) by cyclooxygenases (COX-1 and COX-2) followed by metabolism of endoperoxide intermediates by terminal PG synthases. PG biosynthesis is inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs). Specific inhibition of COX-2 has been extensively investigated, but relatively few COX-1-selective inhibitors have been described. Recent reports of a possible contribution of COX-1 in analgesia, neuroinflammation, or carcinogenesis suggest that COX-1 is a potential therapeutic target. We designed, synthesized, and evaluated a series of (E)-2'-des-methyl-sulindac sulfide (E-DMSS) analogues for inhibition of COX-1. Several potent and selective inhibitors were discovered, and the most promising compounds were active against COX-1 in intact ovarian carcinoma cells (OVCAR-3). The compounds inhibited tumor cell proliferation but only at concentrations >100-fold higher than the concentrations that inhibit COX-1 activity. E-DMSS analogues may be useful probes of COX-1 biology in vivo and promising leads for COX-1-targeted therapeutic agents.
Project description:The constitutively-expressed cyclooxygenase 1 (COX-1) and the inducible COX-2 are both involved in the conversion of arachidonic acid (AA) to prostaglandins (PGs). However, the functional roles of COX-1 at the cellular level remain unclear. We hypothesized that by comparing differential gene expression and eicosanoid metabolism in lung fibroblasts from wild-type (WT) mice and COX-2(-/-) or COX-1(-/-) mice may help address the functional roles of COX-1 in inflammation and other cellular functions. Compared to WT, the number of specifically-induced transcripts were altered descendingly as follows: COX-2(-/-)>COX-1(-/-)>WT+IL-1?. COX-1(-/-) or COX-2(-/-) cells shared about 50% of the induced transcripts with WT cells treated with IL-1?, respectively. An interactive "anti-inflammatory, proinflammatory, and redox-activated" signature in the protein-protein interactome map was observed in COX-2(-/-) cells. The augmented COX-1 mRNA (in COX-2(-/-) cells) was associated with the upregulation of mRNAs for glutathione S-transferase (GST), superoxide dismutase (SOD), NAD(P)H dehydrogenase quinone 1 (NQO1), aryl hydrocarbon receptor (AhR), peroxiredoxin, phospholipase, prostacyclin synthase, and prostaglandin E synthase, resulting in a significant increase in the levels of PGE2, PGD2, leukotriene B4 (LTB4), PGF1?, thromboxane B2 (TXB2), and PGF2?. The COX-1 plays a dominant role in shifting AA toward the LTB4 pathway and anti-inflammatory activities. Compared to WT, the upregulated COX-1 mRNA in COX-2(-/-) cells generated an "eicosanoid storm". The genomic characteristics of COX-2(-/-) is similar to that of proinflammatory cells as observed in IL-1? induced WT cells. COX-1(-/-) and COX-2(-/-) cells exhibited compensation of various eicosanoids at the genomic and metabolic levels.
Project description:We previously discovered that Caenorhabditis elegans synthesizes Cox-independent F-series prostaglandins (PGs). To delineate the Cox-independent prostaglandin pathways and evaluate their role in sperm motility in C. elegans, we developed a novel biochemical method for the rapid production of F-series PGs using arachidonic acid as the substrate and worm lysate as source of enzyme(s). Among the four F2-series PGs produced in the reaction, three of them were identified as 8-isoPGF2?, 5iPF2 VI, and PGF2? based on their retention times and MS/MS spectral comparison with standards using LC-MS/MS. PG production was not markedly affected by specific antioxidants, or Cox, Lox, and Cyp inhibitors, suggesting that these PGs are formed through a novel, biologically regulated mechanism in C. elegans. This study also assessed the ability of 8-isoPGF2?, 5iPF2 VI, PGF2?, and a mixture containing these PGs in a 0.5/0.08/1 ratio that reflects their synthetic composition to modulate sperm motility in fat-2 mutants. PGF2? and the PG mixture at 25??M concentration significantly stimulated sperm velocity by 28% and 38%, whereas 8-isoPGF2? and 5iPF2 VI reduced the velocity by 21% and 30%, respectively, compared to vehicle control. These results indicate that the sperm motility effects of PGs are structure- and composition-dependent in C. elegans.
Project description:Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-2 acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of 2-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments 2-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-2 complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-2 increases 2-AG oxygenation by increasing kcat and preventing inhibitory binding of 2-AG. AM-8138 restored the activity of COX-2 mutants that exhibited very poor 2-AG oxygenating activity and increased the activity of COX-1 toward 2-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-2-dependent 2-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or 2-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced 2-AG oxygenation in intact RAW264.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism.
Project description:Th17 cells comprise a distinct lineage of proinflammatory T helper cells that are major contributors to allergic responses. It is unknown whether cyclooxygenase (COX)-derived eicosanoids regulate Th17 cells during allergic lung inflammation.To determine the role of COX metabolites in regulating Th17 cell differentiation and function during allergic lung inflammation.COX-1(-/-), COX-2(-/-), and wild-type mice were studied in an in vivo model of ovalbumin-induced allergic inflammation and an in vitro model of Th17 differentiation using flow cytometry, cytokine assays, confocal microscopy, real-time polymerase chain reaction, and immunoblotting. In addition, the role of specific eicosanoids and their receptors was examined using synthetic prostaglandins (PGs), selective inhibitors, and siRNA knockdown.Th17 cell differentiation in lung, lymph nodes, and bronchoalveolar lavage fluid was significantly lower in COX-2(-/-) mice after ovalbumin sensitization and exposure in vivo. In vitro studies revealed significantly impaired Th17 cell differentiation of COX-2(-/-) naive CD4(+) T cells with decreased Stat3 phosphorylation and RORγt expression. Synthetic PGF(2α) and PGI(2) enhanced Th17 cell differentiation of COX-2(-/-) CD4(+) T cells in vitro. The selective COX-2 inhibitor, NS-398, and PGF(2α) receptor and PGI(2) receptor siRNA knockdown significantly decreased Th17 cell differentiation in vitro. Administration of synthetic PGs restored accumulation of Th17 cells in lungs of allergic COX-2(-/-) mice in vivo.COX-2 is a critical regulator of Th17 cell differentiation during allergic lung inflammation via autocrine signaling of PGI(2) and PGF(2α) through their respective cell surface receptors.
Project description:Pain associated with inflammation involves prostaglandins synthesized from arachidonic acid (AA) through cyclooxygenase-2 (COX-2) pathways while thromboxane A(2) formed by platelets from AA via cyclooxygenase-1 (COX-1) mediates thrombosis. COX-1 and COX-2 are both targets of nonselective nonsteroidal antiinflammatory drugs (nsNSAIDs) including aspirin whereas COX-2 activity is preferentially blocked by COX-2 inhibitors called coxibs. COXs are homodimers composed of identical subunits, but we have shown that only one subunit is active at a time during catalysis; moreover, many nsNSAIDS bind to a single subunit of a COX dimer to inhibit the COX activity of the entire dimer. Here, we report the surprising observation that celecoxib and other coxibs bind tightly to a subunit of COX-1. Although celecoxib binding to one monomer of COX-1 does not affect the normal catalytic processing of AA by the second, partner subunit, celecoxib does interfere with the inhibition of COX-1 by aspirin in vitro. X-ray crystallographic results obtained with a celecoxib/COX-1 complex show how celecoxib can bind to one of the two available COX sites of the COX-1 dimer. Finally, we find that administration of celecoxib to dogs interferes with the ability of a low dose of aspirin to inhibit AA-induced ex vivo platelet aggregation. COX-2 inhibitors such as celecoxib are widely used for pain relief. Because coxibs exhibit cardiovascular side effects, they are often prescribed in combination with low-dose aspirin to prevent thrombosis. Our studies predict that the cardioprotective effect of low-dose aspirin on COX-1 may be blunted when taken with coxibs.
Project description:Cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid (AA) to generate prostaglandins. The enzymes associate with one leaflet of the membrane bilayer. We utilized nanodisc technology to investigate the function of human (hu) COX-2 and murine (mu) COX-2 in a lipid bilayer environment. huCOX-2 and muCOX-2 were incorporated into nanodiscs composed of POPC, POPS, DOPC, or DOPS phospholipids. Size-exclusion chromatography and negative stain electron microscopy confirm that a single COX-2 homodimer is incorporated into the nanodisc scaffold. Nanodisc-reconstituted COX-2 exhibited similar kinetic profiles for the oxygenation of AA, eicosapentaenoic acid, and 1-arachidonoyl glycerol compared to those derived using detergent solubilized enzyme. Moreover, changing the phospholipid composition of the nanodisc did not alter the ability of COX-2 to oxygenate AA or to be inhibited by various nonselective NSAIDs or celecoxib. The cyclooxygenase activity of nanodisc-reconstituted COX-2 was reduced by aspirin acetylation and potentiated by the nonsubstrate fatty acid palmitic acid to the same extent as detergent solubilized enzyme, independent of phospholipid composition. The stabilization and maintenance of activity afforded by the incorporation of the enzyme into nanodiscs generates a native-like lipid bilayer environment to pursue studies of COX utilizing solution-based techniques that are otherwise not tractable in the presence of detergents.
Project description:Cyclooxygenases (COX)-1 and -2 are key enzymes required for the conversion of arachidonic acid to eicosanoids, potent mediators of inflammation. In patients with multiple sclerosis, COX-2 derived prostaglandins (PGs) are elevated in the CSF and COX-2 is up-regulated in demyelinating plaques. However, it is not known whether COX-2 activity contributes to oligodendrocyte death. In cuprizone-induced demyelination, oligodendrocyte apoptosis and a concomitant increase in the gene expression of COX-2 and PGE?-EP2 receptor precede histological demyelination. COX-2 and EP2 receptor were expressed by oligodendrocytes, suggesting a causative role for the COX-2/EP2 pathway in the initiation of oligodendrocyte death and demyelination. COX-2 gene deletion, chronic treatment with the COX-2 selective inhibitor celecoxib, or with the EP2 receptor antagonist AH6809 reduced cuprizone-induced oligodendrocyte apoptosis, the degree of demyelination and motor dysfunction. These data indicate that the PGE? EP2 receptor contributes to oligodendrocyte apoptosis and open possible new therapeutic approaches for multiple sclerosis.
Project description:Arachidonic acid (AA), a representative ω6-polyunsaturated fatty acid (PUFA), is a precursor of 2-series prostaglandins (PGs) that play important roles in inflammation, pain, fever, and related disorders including cardiovascular diseases. Eating fish or supplementation with the ω3-PUFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is widely assumed to be beneficial in preventing cardiovascular diseases. A proposed mechanism for a cardio-protective role of ω3-PUFAs assumes competition between AA and ω3-PUFAs for cyclooxygenases (COX), leading to reduced production of 2-series PGs. In this study, we have used a systems biology approach to integrate existing knowledge and novel high-throughput data that facilitates a quantitative understanding of the molecular mechanism of ω3- and ω6-PUFA metabolism in mammalian cells. We have developed a quantitative computational model of the competitive metabolism of AA and EPA via the COX pathway through a two-step matrix-based approach to estimate the rate constants. This model was developed by using lipidomic data sets that were experimentally obtained from EPA-supplemented ATP-stimulated RAW264.7 macrophages. The resulting model fits the experimental data well for all metabolites and demonstrates that the integrated metabolic and signaling networks and the experimental data are consistent with one another. The robustness of the model was validated through parametric sensitivity and uncertainty analysis. We also validated the model by predicting the results from other independent experiments involving AA- and DHA-supplemented ATP-stimulated RAW264.7 cells using the parameters estimated with EPA. Furthermore, we showed that the higher affinity of EPA binding to COX compared with AA was able to inhibit AA metabolism effectively. Thus, our model captures the essential features of competitive metabolism of ω3- and ω6-PUFAs.
Project description:Cyclooxygenases (COXs), including COX-1 and -2, are enzymes essential for lipid mediator (LMs) syntheses from arachidonic acid (AA), such as prostaglandins (PGs). Furthermore, COXs could interplay with other enzymes such as lipoxygenases (LOXs) and cytochrome P450s (CYPs) to regulate the signaling of LMs. In this study, to comprehensively analyze the function of COX-1 and -2 in regulating the signaling of bioactive LMs in skeletal muscle, mouse primary myoblasts and C2C12 cells were transfected with specific COX-1 and -2 siRNAs, followed by targeted lipidomic analysis and customized quantitative PCR gene array analysis. Knocking down COXs, particularly COX-1, significantly reduced the release of PGs from muscle cells, especially PGE2 and PGF2α, as well as oleoylethanolamide (OEA) and arachidonoylethanolamine (AEA). Moreover, COXs could interplay with LOXs to regulate the signaling of hydroxyeicosatetraenoic acids (HETEs). The changes in LMs are associated with the expression of genes, such as Itrp1 (calcium signaling) and Myh7 (myogenic differentiation), in skeletal muscle. In conclusion, both COX-1 and -2 contribute to LMs production during myogenesis in vitro, and COXs could interact with LOXs during this process. These interactions and the fine-tuning of the levels of these LMs are most likely important for skeletal muscle myogenesis, and potentially, muscle repair and regeneration.