Caroline Jill Rieke

Enzymes and Receptors of Prostaglandin Pathways with Arachidonic Acid-derived Versus Eicosapentaenoic Acid-derived Substrates and Products

Dietary fish oil containing ω3 highly unsaturated fatty acids has cardioprotective and anti-inflammatory effects. Prostaglandins (PGs) and thromboxanes are produced in vivo both from the ω6 fatty acid arachidonic acid (AA) and the ω3 fatty acid eicosapentaenoic acid (EPA). Certain beneficial effects of fish oil may result from altered PG metabolism resulting from increases in the EPA/AA ratios of precursor phospholipids. Here we report in vitro specificities of prostanoid enzymes and receptors toward EPA-derived, 3-series versus AA-derived, 2-series prostanoid substrates and products. The largest difference was seen with PG endoperoxide H synthase (PGHS)-1. Under optimal conditions purified PGHS-1 oxygenates EPA with only 10% of the efficiency of AA, and EPA significantly inhibits AA oxygenation by PGHS-1. Two- to 3-fold higher activities or potencies with 2-series versus 3-series compounds were observed with PGHS-2, PGD synthases, microsomal PGE synthase-1 and EP1, EP2, EP3, and FP receptors. Our most surprising observation was that AA oxygenation by PGHS-2 is only modestly inhibited by EPA (i.e. PGHS-2 exhibits a marked preference for AA when EPA and AA are tested together). Also unexpectedly, TxA3 is about equipotent to TxA2 at the TPα receptor. Our biochemical data predict that increasing phospholipid EPA/AA ratios in cells would dampen prostanoid signaling with the largest effects being on PGHS-1 pathways involving PGD, PGE, and PGF. Production of 2-series prostanoids from AA by PGHS-2 would be expected to decrease in proportion to the compensatory decrease in the AA content of phospholipids that would result from increased incorporation of ω3 fatty acids such as EPA.

The 19-amino acid cassette of cyclooxygenase-2 mediates entry of the protein into the endoplasmic reticulum-associated degradation system

Cyclooxygenase (COX) isoforms catalyze the committed step in prostaglandin biosynthesis. The primary structures of COX-1 and COX-2 are very similar except that COX-2 has a 19-amino acid (19-AA) segment of unknown function located just inside its C terminus. Here we provide evidence that the major role of the 19-AA cassette is to mediate entry of COX-2 into the ER-associated degradation system that transports ER proteins to the cytoplasm. COX-1 is constitutively expressed in many cells, whereas COX-2 is usually expressed inducibly and transiently. In murine NIH/3T3 fibroblasts, we find that COX-2 protein is degraded with a half-life (t½) of about 2 h, whereas COX-1 is reasonably stable (t½ >12 h); COX-2 degradation is retarded by 26 S proteasome inhibitors. Similarly, COX-1 expressed heterologously in HEK293 cells is quite stable (t½ > 24 h), whereas COX-2 expressed heterologously is degraded with a t½ of ∼5h, and its degradation is slowed by proteasome inhibitors. A deletion mutant of COX-2 was prepared lacking 18 residues of the 19-AA cassette. This mutant retains native COX-2 activity but, unlike native COX-2, is stable in HEK293 cells. Conversely, inserting the COX-2 19-AA cassette near the C terminus of COX-1 yields a mutant ins594–612 COX-1 that is unstable (t½ ∼3 h). Mutation of Asn-594, an N-glycosylation site at the beginning of the 19-AA cassette, stabilizes both COX-2 and ins594–612 COX-1; nonetheless, COX mutants that are glycosylated at Asn-594 but lack the remainder of the 19-amino acid cassette (i.e. del597–612 COX-2 and ins594–596 COX-1) are stable. Thus, although glycosylation of Asn-594 is necessary for COX-2 degradation, at least part of the remainder of the 19-AA insert is also required. Finally, kifunensine, a mannosidase inhibitor that can block entry of ER proteins into the ER-associated degradation system, retards COX-2 degradation.

The Role of Arginine 120 of Human Prostaglandin Endoperoxide H Synthase-2 in the Interaction with Fatty Acid Substrates and Inhibitors

Arg-120 is located near the mouth of the hydrophobic channel that forms the cyclooxygenase active site of prostaglandin endoperoxide H synthases (PGHSs)-1 and -2. Replacement of Arg-120 of ovine PGHS-1 with a glutamine increases the apparentK m of PGHS-1 for arachidonate by 1,000-fold (Bhattacharyya, D. K., Lecomte, M., Rieke, C. J., Garavito, R. M., and Smith, W. L. (1996) J. Biol. Chem. 271, 2179–2184). This and other evidence indicate that the guanido group of Arg-120 forms an ionic bond with the carboxylate group of arachidonate and that this interaction is an important contributor to the overall strength of arachidonate binding to PGHS-1. In contrast, we report here that R120Q human PGHS-2 (hPGHS-2) and native hPGHS-2 have very similar kinetic properties, but R120L hPGHS-2 catalyzes the oxygenation of arachidonate inefficiently. Our data indicate that the guanido group of Arg-120 of hPGHS-2 interacts with arachidonate through a hydrogen bond rather than an ionic bond and that this interaction is much less important for arachidonate binding to PGHS-2 than to PGHS-1. The K m values of PGHS-1 and -2 for arachidonate are the same, and all but one of the core residues of the active sites of the two isozymes are identical. Thus, the results of our studies of Arg-120 of PGHS-1 and -2 imply that interactions involved in the binding of arachidonate to PGHS-1 and -2 are quite different and that residues within the hydrophobic cyclooxygenase channel must contribute more significantly to arachidonate binding to PGHS-2 than to PGHS-1. As observed previously with R120Q PGHS-1, flurbiprofen was an ineffective inhibitor of R120Q hPGHS-2. PGHS-2-specific inhibitors including NS398, DuP-697, and SC58125 had IC50 values for the R120Q mutant that were up to 1,000-fold less than those observed for native hPGHS-2; thus, the positively charged guanido group of Arg-120 interferes with the binding of these compounds. NS398 did not cause time-dependent inhibition of R120Q hPGHS-2, whereas DuP-697 and SC58125 were time-dependent inhibitors. Thus, Arg-120 is important for the time-dependent inhibition of hPGHS-2 by NS398 but not by DuP-697 or SC58125.

Prostaglandin Endoperoxide H Synthases PEROXIDASE HYDROPEROXIDE SPECIFICITY AND CYCLOOXYGENASE ACTIVATION

The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic “dome” over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site.

Crystal Structure of Arachidonic Acid Bound to a Mutant of Prostaglandin Endoperoxide H Synthase-1 That Forms Predominantly 11-Hydroperoxyeicosatetraenoic Acid

Kinetic studies and analysis of the products formed by native and mutant forms of ovine prostaglandin endoperoxide H synthase-1 (oPGHS-1) have suggested that arachidonic acid (AA) can exist in the cyclooxygenase active site of the enzyme in three different, catalytically competent conformations that lead to prostaglandin G2 (PGG2), 11R-hydroperoxyeicosatetraenoic acid (HPETE), and 15R,S-HPETE, respectively. We have identified an oPGHS-1 mutant (V349A/W387F) that forms predominantly 11R-HPETE. Thus, the preferred catalytically competent arrangement of AA in the cyclooxygenase site of this double mutant must be one that leads to 11-HPETE. The crystal structure of Co3+-protoporphyrin IX V349A/W387F oPGHS-1 in a complex with AA was determined to 3.1 Å. Significant differences are observed in the positions of atoms C-3, C-4, C-5, C-6, C-10, C-11, and C-12 of bound AA between native and V349A/W387F oPGHS-1; in comparison, the positions of the side chains of cyclooxygenase active site residues are unchanged. The structure of the double mutant presented here provides structural insight as to how Val349 and Trp387 help position C-9 and C-11 of AA so that the incipient 11-peroxyl radical intermediate is able to add to C-9 to form the 9,11 endoperoxide group of PGG2. In the V349A/W387F oPGHS-1·AA complex the locations of C-9 and C-11 of AA with respect to one another make it difficult to form the endoperoxide group from the 11-hydroperoxyl radical. Therefore, the reaction apparently aborts yielding 11R-HPETE instead of PGG2. In addition, the observed differences in the positions of carbon atoms of AA bound to this mutant provides indirect support for the concept that the conformer of AA shown previously to be bound within the cyclooxygenase active site of native oPGHS-1 is the one that leads to PGG2.

Fatty-Acid Substrate Interactions with Cyclo-oxygenases

Prostaglandin endoperoxide H synthases 1 and -2 (PGHS-1 and -2) convert arachidonic acid and O2 (along with two reducing equivalents) to PGH2 — the committing step in the formation of prostanoids (Smith and DeWitt 1996; Smith et al. 1996). PGHS-1 is often referred to as the constitutive enzyme, whereas PGHS-2 is known as the inducible isoform. They differ from one another mainly with respect to their temporal patterns of expression. The reason for the existence of the two PGHS isozymes is still unknown. One possibility is that PGHS-2 is induced and then functions at relatively low fatty-acid substrate and hydroperoxide-activator concentrations to generate prostanoid products during early stages of cell replication or differentiation, whereas PGHS-1 forms products that are involved in “housekeeping” functions when circulating hormones act on cells acutely to cause the release of higher concentrations of arachidonate (Capdevila et al. 1995; Kulmacz and Wang 1995; Kulmacz 1998; So et al. 1998).

Fatty acid binding to cyclooxygenases

Abstract Prostaglandin endoperoxide H synthase-1 and -2 (PGHS-1 and -2) convert arachidonic acid (AA) to prostaglandin H 2 (PGH 2 ) in the committed step in prostaglandin biosynthesis. Although the cyclooxygenase activity favors AA as the substrate, both isoforms will oxygenate a variety of 18–22 carbon fatty acids with reduced efficiencies. In this review, we discuss how the fatty acid substrates AA and dihomo-γ-linolenic acid (DHLA; 20:3 ω−6) are bound in the cyclooxygenase active site of ovine (o) PGHS-1. Based on the conformations of the fatty acids within the active site, we describe the key roles that Val349 and Ser530 play as determinants of fatty acid substrate specificity for oPGHS-1.