Competition and allostery govern substrate selectivity of cyclooxygenase-2

Michelle Mitchener

Daniel Hermanson

Erin Shockley

H Brown

^{A. B. Hancock Memorial Laboratory for Cancer Research, Vanderbilt University School of Medicine, Nashville, TN, 37232;}

^{Department of Chemistry, Vanderbilt University School of Medicine, Nashville, TN, 37232;}

^{The Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN, 37232;}

^{Vanderbilt–Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN, 37232;}

^{Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN, 37232;}

^{Center for Quantitative Sciences, Vanderbilt University School of Medicine, Nashville, TN, 37232;}

^{Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, 37232;}

^{Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, 37232;}

^{Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN, 37232;}

^{Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, 37232;}

^{Department of Biomedical Informatics, Vanderbilt University School of Medicine, Nashville, TN, 37232;}

^{Department of Biomedical Engineering, Vanderbilt University School of Medicine, Nashville, TN, 37232;}

^{Vanderbilt University Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, TN, 37232}

^{To whom correspondence should be addressed. Email:}ude.tlibrednav@ttenram.yrral.

Edited by Michael A. Marletta, University of California, Berkeley, CA, and approved August 25, 2015 (received for review April 14, 2015)

Author contributions: M.M.M., D.J.H., C.A.R., and L.J.M. designed research; M.M.M., D.J.H., and C.A.R. performed research; E.M.S. and C.F.L. performed mechanistic modeling; H.A.B., C.W.L., and J.R. contributed new reagents/analytic tools; M.M.M., D.J.H., E.M.S., C.A.R., C.F.L., and L.J.M. analyzed data; and M.M.M., E.M.S., C.A.R., C.F.L., and L.J.M. wrote the paper.

Edited by Michael A. Marletta, University of California, Berkeley, CA, and approved August 25, 2015 (received for review April 14, 2015)

Significance

The cyclooxygenase enzymes perform the initial steps in the synthesis of prostaglandins, potent signaling molecules with diverse physiological functions. Recent data have suggested that these enzymes, although structural homodimers, act as functional heterodimers. Herein, we demonstrate that the heterodimeric nature of cyclooxygenase-2 leads to differential allosteric regulation of the enzyme by two of its substrates, arachidonic acid and the endocannabinoid 2-arachidonoylglycerol. We provide the first evidence (to our knowledge) that fatty acid-based modulation of COX-2 activity occurs in cellular settings. These findings demonstrate the complexity of the interaction of multiple substrates with COX-2, which involves competition for both the catalytic and allosteric sites in addition to differential allosteric regulation.

Keywords: cyclooxygenase, endocannabinoids, allosteric regulation, chemical kinetics, Bayesian inference

Significance

Abstract

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.

Abstract

The cyclooxygenase (COX) enzymes catalyze the dioxygenation and subsequent hydroperoxide reduction of arachidonic acid (AA). These reactions comprise the first two steps in the biosynthesis of prostaglandins (PGs), which play a role in a broad range of physiological and pathophysiological processes (1, 2). The COX enzymes are the primary targets of the pharmacologic action of the widely used nonsteroidal antiinflammatory drugs (e.g., aspirin, ibuprofen, and naproxen). There are two COX isoforms, COX-1 and COX-2, which are structurally and kinetically similar. COX-1 is constitutively expressed in most tissues, whereas COX-2 is highly inducible in response to inflammatory and mitogenic stimuli. Thus, it is generally accepted that the primary factor that differentiates the two isoforms is their distinct patterns of expression (1).

Although gene expression clearly is a factor in COX-related physiology, subtle structural differences between the two isoforms result in intrinsic functional distinctions. Specifically, the active site of COX-2 is larger than that of COX-1, allowing COX-2 to oxygenate bulkier amide and ester analogs of AA that are poor COX-1 substrates. Among these COX-2–selective substrates are the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (3 –5). COX-2–dependent oxygenation of these substrates leads to the biosynthesis of prostaglandin glyceryl esters (PG-Gs) and prostaglandin ethanolamides (prostamides), respectively (3, 4). 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.

The homodimeric COX enzymes function as heterodimers, with one subunit that contains the required heme cofactor acting as a catalytic site and the second subunit, which lacks heme, acting as an allosteric site (8, 9). Evidence for allosteric regulation of COX is seen in the ability of various nonsubstrate fatty acids to modulate the activity and inhibitor sensitivity of the COXs, presumably through interaction with the allosteric site (8). A particularly striking example is the ability of 13-methylarachidonic acid to selectively and substantially increase the oxygenation of 2-AG while having no effect on the oxygenation of AA (10). Another example of allosteric regulation is substrate-selective inhibition, the ability of weak reversible inhibitors of AA oxygenation to strongly inhibit 2-AG oxygenation by COX-2 (11). These considerations led us to hypothesize that differential interactions of AA and 2-AG at the allosteric site of COX-2 might result in a complex interplay between the substrates when both are present. Here, we demonstrate that modulation of AA levels inversely affects cellular biosynthesis of PG-Gs. We go on to explore the interaction of the substrates AA and 2-AG during oxygenation by COX-2 in vitro and in silico. We build a mathematical model of COX-2 oxygenation kinetics with multiple ligands that explicitly incorporates the dynamics of all of the chemical intermediates in the reaction network, and we use a Bayesian parameter inference formalism to characterize the multiple reaction pathways that exist between the two substrates, the enzyme subunits, and their products. Our rigorous model calibration approach allows us to determine probability distributions, given our experimental data, for kinetic constants rather than single best-fit values, providing a systems-level understanding of multiple competing interactions in the reaction network and accurate confidence estimates for fitted values. The presented methods are generalizable to other kinetic systems that, like COX-2, can appear deceptively simple. Our findings support the hypothesis that a combination of competition and allosteric regulation controls the selective use of substrates by COX-2 and, to our knowledge, provide the first evidence of the physiologic relevance of this phenomenon in live cells.

Acknowledgments

This work was supported by the National Institutes of Health under Grants CA089450 and GM15431 (to L.J.M.), and K22CA151918 (to C.F.L.), the National Science Foundation under Grant MCB-1411482 (to C.F.L.), and the Edward P. Evans Foundation (C.F.L.). M.M.M. and E.M.S. were supported by US Public Health Services Grant T32 {"type":"entrez-nucleotide","attrs":{"text":"ES007028","term_id":"164011183","term_text":"ES007028"}}ES007028. C.W.L. was supported by William K. Warren, Jr., who funded the William K. Warren, Jr., Chair in Medicine at Vanderbilt University.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1507307112/-/DCSupplemental.

Footnotes

References

1. Rouzer CA, Marnett LJEndocannabinoid oxygenation by cyclooxygenases, lipoxygenases, and cytochromes P450: Cross-talk between the eicosanoid and endocannabinoid signaling pathways. Chem Rev. 2011;111(10):5899–5921.[Google Scholar]
2. Funk CDProstaglandins and leukotrienes: Advances in eicosanoid biology. Science. 2001;294(5548):1871–1875.[PubMed][Google Scholar]
3. Kozak KR, Rowlinson SW, Marnett LJOxygenation of the endocannabinoid, 2-arachidonylglycerol, to glyceryl prostaglandins by cyclooxygenase-2. J Biol Chem. 2000;275(43):33744–33749.[PubMed][Google Scholar]
4. Yu M, Ives D, Ramesha CSSynthesis of prostaglandin E2 ethanolamide from anandamide by cyclooxygenase-2. J Biol Chem. 1997;272(34):21181–21186.[PubMed][Google Scholar]
5. Kozak KR, et al Metabolism of the endocannabinoids, 2-arachidonylglycerol and anandamide, into prostaglandin, thromboxane, and prostacyclin glycerol esters and ethanolamides. J Biol Chem. 2002;277(47):44877–44885.[PubMed][Google Scholar]
6. Rouzer CA, et al Zymosan-induced glycerylprostaglandin and prostaglandin synthesis in resident peritoneal macrophages: Roles of cyclo-oxygenase-1 and -2. Biochem J. 2006;399(1):91–99.[Google Scholar]
7. Musée J, Marnett LJProstaglandin H synthase-2-catalyzed oxygenation of 2-arachidonoylglycerol is more sensitive to peroxide tone than oxygenation of arachidonic acid. J Biol Chem. 2012;287(44):37383–37394.[Google Scholar]
8. Dong L, et al Human cyclooxygenase-2 is a sequence homodimer that functions as a conformational heterodimer. J Biol Chem. 2011;286(21):19035–19046.[Google Scholar]
9. Zou H, et al Human cyclooxygenase-1 activity and its responses to COX inhibitors are allosterically regulated by nonsubstrate fatty acids. J Lipid Res. 2012;53(7):1336–1347.[Google Scholar]
10. Kudalkar SN, et al 13-Methylarachidonic acid is a positive allosteric modulator of endocannabinoid oxygenation by cyclooxygenase. J Biol Chem. 2015;290(12):7897–7909.[Google Scholar]
11. Duggan KC, et al (R)-Profens are substrate-selective inhibitors of endocannabinoid oxygenation by COX-2. Nat Chem Biol. 2011;7(11):803–809.[Google Scholar]
12. Rouzer CA, et al Lipid profiling reveals arachidonate deficiency in RAW264.7 cells: Structural and functional implications. Biochemistry. 2006;45(49):14795–14808.[Google Scholar]
13. Gijón MA, Leslie CCRegulation of arachidonic acid release and cytosolic phospholipase A2 activation. J Leukoc Biol. 1999;65(3):330–336.[PubMed][Google Scholar]
14. Gijón MA, Spencer DM, Siddiqi AR, Bonventre JV, Leslie CC. Cytosolic phospholipase A2 is required for macrophage arachidonic acid release by agonists that do and do not mobilize calcium. Novel role of mitogen-activated protein kinase pathways in cytosolic phospholipase A2 regulation. J Biol Chem. 2000;275(26):20146–20156.[PubMed]
15. Hirabayashi T, Shimizu TLocalization and regulation of cytosolic phospholipase A₂. Biochim Biophys Acta. 2000;1488(1-2):124–138.[PubMed][Google Scholar]
16. Fujishima H, et al Cytosolic phospholipase A2 is essential for both the immediate and the delayed phases of eicosanoid generation in mouse bone marrow-derived mast cells. Proc Natl Acad Sci USA. 1999;96(9):4803–4807.[Google Scholar]
17. Magrioti V, Kokotos GPhospholipase A2 inhibitors as potential therapeutic agents for the treatment of inflammatory diseases. Expert Opin Ther Pat. 2010;20(1):1–18.[PubMed][Google Scholar]
18. Rouzer CA, Marnett LJMechanism of free radical oxygenation of polyunsaturated fatty acids by cyclooxygenases. Chem Rev. 2003;103(6):2239–2304.[PubMed][Google Scholar]
19. DeGroot MH, Schervish MJ Probability and Statistics. 3rd Ed Addison-Wesley; Boston: 2002. [PubMed][Google Scholar]
20. Eydgahi H, et al Properties of cell death models calibrated and compared using Bayesian approaches. Mol Syst Biol. 2013;9:644.[Google Scholar]
21. Swinney DC, Mak AY, Barnett J, Ramesha CSDifferential allosteric regulation of prostaglandin H synthase 1 and 2 by arachidonic acid. J Biol Chem. 1997;272(19):12393–12398.[PubMed][Google Scholar]
22. Chen W, Pawelek TR, Kulmacz RJHydroperoxide dependence and cooperative cyclooxygenase kinetics in prostaglandin H synthase-1 and -2. J Biol Chem. 1999;274(29):20301–20306.[PubMed][Google Scholar]
23. Vecchio AJ, Malkowski MGThe structural basis of endocannabinoid oxygenation by cyclooxygenase-2. J Biol Chem. 2011;286(23):20736–20745.[Google Scholar]
24. Vecchio AJ, Simmons DM, Malkowski MGStructural basis of fatty acid substrate binding to cyclooxygenase-2. J Biol Chem. 2010;285(29):22152–22163.[Google Scholar]
25. Prusakiewicz JJ, Duggan KC, Rouzer CA, Marnett LJDifferential sensitivity and mechanism of inhibition of COX-2 oxygenation of arachidonic acid and 2-arachidonoylglycerol by ibuprofen and mefenamic acid. Biochemistry. 2009;48(31):7353–7355.[Google Scholar]
26. Rouzer CA, Marnett LJGlycerylprostaglandin synthesis by resident peritoneal macrophages in response to a zymosan stimulus. J Biol Chem. 2005;280(29):26690–26700.[PubMed][Google Scholar]
27. Lopez CF, Muhlich JL, Bachman JA, Sorger PKProgramming biological models in Python using PySB. Mol Syst Biol. 2013;9:646.[Google Scholar]
28. Patil A, Huard D, Fonnesbeck CJPyMC: Bayesian stochastic modelling in Python. J Stat Softw. 2010;35(4):1–81.[Google Scholar]
29. ter Braak C, Vrugt JADifferential evolution Markov Chain with snooker updater and fewer chains. Stat Comput. 2008;18(4):435–446.[PubMed][Google Scholar]
30. Rowlinson SW, Crews BC, Lanzo CA, Marnett LJ. The binding of arachidonic acid in the cyclooxygenase active site of mouse prostaglandin endoperoxide synthase-2 (COX-2). A putative L-shaped binding conformation utilizing the top channel region. J Biol Chem. 1999;274(33):23305–23310.[PubMed]
31. McKew JC, et al Indole cytosolic phospholipase A2 alpha inhibitors: Discovery and in vitro and in vivo characterization of 4-3-[5-chloro-2-(2-[(3,4-dichlorobenzyl)sulfonyl]aminoethyl)-1-(diphenylmethyl)-1H-indol-3-yl]propylbenzoic acid, efipladib. J Med Chem. 2008;51(12):3388–3413.[PubMed][Google Scholar]
32. Johnson KA, Simpson ZB, Blom TGlobal kinetic explorer: A new computer program for dynamic simulation and fitting of kinetic data. Anal Biochem. 2009;387(1):20–29.[PubMed][Google Scholar]
33. Johnson KA, Simpson ZB, Blom TFitSpace explorer: An algorithm to evaluate multidimensional parameter space in fitting kinetic data. Anal Biochem. 2009;387(1):30–41.[PubMed][Google Scholar]
34. Johnson KAFitting enzyme kinetic data with KinTek Global Kinetic Explorer. Methods Enzymol. 2009;467:601–626.[PubMed][Google Scholar]
35. Geweke J Evaluating the Accuracy of Sampling-Based Approaches to Calculating Posterior Moments. Clarendon; Oxford: 1992. [PubMed][Google Scholar]
36. Gelman AR, Rubin DB A Single Series from the Gibbs Sampler Provides a False Sense of Security. Clarendon; Oxford: 1992. [PubMed][Google Scholar]
37. Kingsley PJ, Marnett LJAnalysis of endocannabinoids by Ag^{coordination tandem mass spectrometry.}Anal Biochem. 2003;314(1):8–15.[PubMed][Google Scholar]
38. Kingsley PJ, Rouzer CA, Saleh S, Marnett LJSimultaneous analysis of prostaglandin glyceryl esters and prostaglandins by electrospray tandem mass spectrometry. Anal Biochem. 2005;343(2):203–211.[PubMed][Google Scholar]