Discovery of a Cell-Active SuTEx Ligand of Prostaglandin Reductase 2.

Sulfonyl-triazoles have emerged as a new reactive group for covalent modification of tyrosine sites on proteins through sulfur-triazole exchange (SuTEx) chemistry. The extent to which this sulfur electrophile can be tuned for developing ligands with cellular activity remains largely underexplored. Here, we performed fragment-based ligand discovery in live cells to identify SuTEx compounds capable of liganding tyrosine sites on diverse protein targets. We verified our quantitative chemical proteomic findings by demonstrating concentration-dependent activity of SuTEx ligands, but not inactive counterparts, against recombinant protein targets directly in live cells. Our structure-activity relationship studies identified the SuTEx ligand HHS-0701 as a cell-active inhibitor capable of blocking prostaglandin reductase 2 (PTGR2) biochemical activity.

Targeting the 15-keto-PGE2-PTGR2 axis modulates systemic inflammation and survival in experimental sepsis.

Sepsis is a systemic inflammation accompanied by multi-organ dysfunction due to microbial infection. Prostaglandins and their metabolites have long been studied for their importance in regulating the innate immune response. 15-keto-PGE2 (15k-PGE2) is a prostaglandin E2 (PGE2) metabolite, whose further processing is catalyzed by prostaglandin reductase 2 (PTGR2). We showed disruption of the Ptgr2 gene in mice improves the survival rate under both LPS- and cecum ligation/puncture (CLP)-induced experimental sepsis. Knockdown of PTGR2 showed significant accumulation of intracellular 15k-PGE2 in activated macrophages. Both PTGR2 knockdown and exogenous treatment with 15k-PGE2 resulted in reduced pro-inflammatory cytokines production in LPS-stimulated RAW264.7 cells or bone marrow-derived macrophages (BMDM). The same treatment in RAW264.7 and BMDM also led to increased levels of the anti-oxidative transcription factor, Nuclear factor (erythroid-2) related factor-2 (NRF2), augmented anti-oxidant response element (ARE)-mediated reporter activity and upregulated expression of the corresponding anti-oxidant genes. 15k-PGE2 further demonstrated modification to Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of Nrf2, at cysteine 288 (Cys288) site post-translationally. Finally, 15k-PGE2-treated mice were found to be more resistant to experimental sepsis. Taken together, our study affirms the significance of PTGR2 and 15k-PGE2 in mitigating inflammatory responses and suggests a novel anti-oxidative and anti-inflammatory therapy for sepsis through targeting PTGR2 and administering15k-PGE2.

Inhibition of Prostaglandin Reductase 2, a Putative Oncogene Overexpressed in Human Pancreatic Adenocarcinoma, Induces Oxidative Stress-Mediated Cell Death Involving xCT and CTH Gene Expressions through 15-Keto-PGE2.

Prostaglandin reductase 2 (PTGR2) is the enzyme that catalyzes 15-keto-PGE2, an endogenous PPARγ ligand, into 13,14-dihydro-15-keto-PGE2. Previously, we have reported a novel oncogenic role of PTGR2 in gastric cancer, where PTGR2 was discovered to modulate ROS-mediated cell death and tumor transformation. In the present study, we demonstrated the oncogenic potency of PTGR2 in pancreatic cancer. First, we observed that the majority of the human pancreatic ductal adenocarcinoma tissues was stained positive for PTGR2 expression but not in the adjacent normal parts. In vitro analyses showed that silencing of PTGR2 expression enhanced ROS production, suppressed pancreatic cell proliferation, and promoted cell death through increasing 15-keto-PGE2. Mechanistically, silencing of PTGR2 or addition of 15-keto-PGE2 suppressed the expressions of solute carrier family 7 member 11 (xCT) and cystathionine gamma-lyase (CTH), two important providers of intracellular cysteine for the generation of glutathione (GSH), which is widely accepted as the first-line antioxidative defense. The oxidative stress-mediated cell death after silencing of PTGR2 or addition of 15-keto-PGE2 was further abolished after restoring intracellular GSH concentrations and cysteine supply by N-acetyl-L-cysteine and 2-Mercaptomethanol. Our data highlight the therapeutic potential of targeting PTGR2/15-keto-PGE2 for pancreatic cancer.

Prostaglandin reductase 2 modulates ROS-mediated cell death and tumor transformation of gastric cancer cells and is associated with higher mortality in gastric cancer patients.

Various prostanoids and peroxisome proliferator-activated receptor γ (PPARγ) ligands play an important role in gastric cancer. Previously, we demonstrated that prostaglandin reductase 2 (PTGR2) catalyzes the reduction of the PPARγ ligand 15-keto-PGE(2) into 13,14-dihydro-15-keto-PGE(2). Here, we present functional data and clinical relevance for the role of PTGR2 in gastric cancer. Using lentiviral technology in AGS and SNU-16 gastric cancer cell lines, we either down-regulated or overexpressed PTGR2. In vitro analysis showed that PTGR2 knockdown resulted in decreased proliferation rate and colony formation, and in vivo xenograft models showed slower growth of tumors. Mechanistically, PTGR2 knockdown induced cell death, altered mitochondrial function, and increased reactive oxygen species production, which led to activation of ERK1/2 and caspase 3, with increased Bcl-2 and suppressed Bax expression. PTGR2 overexpression showed the opposite outcomes. Clinically, immunopathological staining showed strong PTGR2 expression in the gastric tumor portion, relative to nearby nontumor portions, and its expression negatively correlated with survival of patients with intestinal-type gastric cancer. Finally, in contrast to PTGR2-overexpressing cells, PTGR2-knockdown cells were more sensitive to cisplatin and 5-fluorouracil. Taken together, our findings not only provide functional and mechanistic evidence of the involvement of PTGR2 in gastric cancer, but also provide clinical observations affirming the significance of PTGR2 in gastric cancer and suggesting that PTGR2-target based therapy is worth further evaluation.

Up-regulation of human prostaglandin reductase 1 improves the efficacy of hydroxymethylacylfulvene, an antitumor chemotherapeutic agent.

Prostaglandin reductase 1 (PTGR1) is a highly inducible enzyme with enone reductase activity. Previous studies demonstrated the role of rat PTGR1 in the activation of acylfulvene analogs, a class of antitumor natural product derivatives. Of these, hydroxymethylacylfulvene (HMAF) was in advanced clinical development for the treatment of advanced solid tumors, including prostate, ovarian, and pancreatic cancers. However, the efficiency of human PTGR1 in activating acylfulvenes and its potential to enhance therapeutic efficacy have remained uncharacterized. In this study, human PTGR1 was polymerase chain reaction-cloned and purified. Conversion of HMAF to its cellular metabolite by the purified enzyme proceeded at a 20-fold higher rate than with the rat variant of the enzyme. The Km was 4.9 µM, which was 40-fold lower than for the rat variant and similar to the therapeutic dose. Human cell lines, including colon cancer lines, were transfected with a vector containing rat PTGR1 or human PTGR1, and cell viability was examined after dosing with HMAF. New data obtained in this study suggest that transfection with human PTGR1, or its induction in colon and liver cancer cell lines with 1,2-dithiol-3-thione, enhances susceptibility to the cytotoxic influences of HMAF by 2- to 10-fold. Furthermore, similar or enhanced enzyme induction and HMAF toxicity results from preconditioning cancer cells with the bioactive food components curcumin and resveratrol. The functional impact of PTGR1 induction in human cells and chemical-based strategies for its activation can provide important knowledge for the design of clinical strategies involving reductively activated cytotoxic chemotherapeutics.

Prostaglandin catabolic enzymes as tumor suppressors.

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is a key prostaglandin catabolic enzyme catalyzing the oxidation and inactivation of prostaglandin E(2) (PGE(2)) synthesized from the cyclooxygenase (COX) pathway. Accumulating evidence indicates that 15-PGDH may function as a tumor suppressor antagonizing the action of COX-2 oncogene. 15-PGDH has been found to be down-regulated contributing to elevated levels of PGE(2) in most tumors. The expression of 15-PGDH and COX-2 appears to be regulated reciprocally in cancer cells. Down-regulation of 15-PGDH in tumors is due, in part, to transcriptional repression and epigenetic silencing. Numerous agents have been found to up-regulate 15-PGDH by down-regulation of transcriptional repressors and by attenuation of the turnover of the enzyme. Up-regulation of 15-PGDH may provide a viable approach to cancer chemoprevention. Further catabolism of 15-keto-prostaglandin E(2) is catalyzed by 15-keto-prostaglandin-∆(13)-reductase (13-PGR), which also exhibits LTB(4)-12-hydroxydehydrogenase (LTB(4)-12-DH) activity. 13-PGR/LTB(4)-12-DH behaves as a tumor suppressor as well. This review summarizes current knowledge of the expression and function of 15-PGDH and 13-PGR/LTB(4)-12-DH in lung and other tissues during tumor progression. Future directions of research on these prostaglandin catabolic enzymes as tumor suppressors are also discussed.

Restoration of leukotriene B(4)-12-hydroxydehydrogenase/15- oxo-prostaglandin 13-reductase (LTBDH/PGR) expression inhibits lung cancer growth in vitro and in vivo.

Leukotriene B(4)-12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase (LTBDH/PGR) is a bifunctional enzyme capable of inactivating leukotriene B(4) (LTB(4)) and 15-oxo-prostaglandins (15-PGs). Its role in growth suppressive functions in lung cancer was studied in in vitro and in vivo systems. The LTBDH/PGR gene was expressed in lung cancer cell lines through recombinant adenovirus infection, and through a tetracycline-inducible expression system. After restoration of LTBDH/PGR expression in LTBDH/PGR-negative (H1299) or -low (A549) lung cancer cell lines, the restored enzyme induced apoptosis and growth inhibition in vitro. Ectopic expression of LTBDH/PGR caused also suppression of tumorigenicity of A549 cells in nude mice. In contrast, LTBDH/PGR over-expression in LTBDH/PGR-positive (H157) lung cancer cell line induced little apoptosis and growth inhibition. This study indicates that restoration of LTBDH/PGR expression is effective in preventing lung cancer growth in vitro and in vivo.

Structural basis for catalytic and inhibitory mechanisms of human prostaglandin reductase PTGR2.

PTGR2 catalyzes an NADPH-dependent reduction of the conjugated alpha,beta-unsaturated double bond of 15-keto-PGE(2), a key step in terminal inactivation of prostaglandins and suppression of PPARgamma-mediated adipocyte differentiation. Selective inhibition of PTGR2 may contribute to the improvement of insulin sensitivity with fewer side effects. PTGR2 belongs to the medium-chain dehydrogenase/reductase superfamily. The crystal structures reported here reveal features of the NADPH binding-induced conformational change in a LID motif and a polyproline type II helix which are critical for the reaction. Mutation of Tyr64 and Tyr259 significantly reduces the rate of catalysis but increases the affinity to substrate, confirming the structural observations. Besides targeting cyclooxygenase, indomethacin also inhibits PTGR2 with a binding mode similar to that of 15-keto-PGE(2). The LID motif becomes highly disordered upon the binding of indomethacin, indicating plasticity of the active site. This study has implications for the rational design of inhibitors of PTGR2.

Leukotriene B4 12-hydroxydehydrogenase/15-ketoprostaglandin Delta 13-reductase (LTB4 12-HD/PGR) responsible for the reduction of a double-bond of the alpha,beta-unsaturated ketone of an aryl propionic acid non-steroidal anti-inflammatory agent CS-670.

CS-670 is a non-steroidal anti-inflammatory agent with an alpha,beta-unsaturated ketone structure. It exerts its pharmacological activity after being transformed to the active metabolite (2S,1'R,2'S)-trans-alcohol. Two consecutive reductions are needed for the formation of the active metabolite, reduction of the double-bond of the alpha,beta-unsaturated ketone moiety, followed by reduction of the resulting saturated ketone. The objective of the current study was to identify the enzyme responsible for reduction of the double-bond. An enzyme purified from rat liver cytosol as a single band on sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was analysed by a Mascot database search of nano-LC tandem mass spectrometry (MS/MS) data and the enzyme was identified as 2-alkenal reductase (EC 1.3.1.74), which is known as an beta-nicotinamide adenine dinucleotide phosphate (NADPH)-dependent alkenal/one oxidoreductase and has a role for leukotriene B(4) 12-hydroxydehydrogenase/15-ketoprostaglandinDelta13-reductase (LTB(4) 12-HD/PGR). The identification was confirmed by cloning LTB(4) 12-HD/PGR cDNA from rat liver, expressing it in Escherichia coli, and characterizing the properties of the enzyme. The identity was further supported by the subcellular localization in cytosol, a cofactor requirement for NADPH, substrate specificity, and substantial inhibition by 15-ketoPGF(2alpha), benzylideneacetophenone, indomethacin, and quercitrin. In addition to catalysing the biological reduction of eicosanoids, including prostaglandins, leukotrienes, and lipoxins, LTB(4) 12-HD/PGR was also determined to function as a xenobiotic-metabolizing enzyme.

Identification of a novel prostaglandin reductase reveals the involvement of prostaglandin E2 catabolism in regulation of peroxisome proliferator-activated receptor gamma activation.

This report identifies a novel gene encoding 15-oxoprostaglandin-Delta13-reductase (PGR-2), which catalyzes the reaction converting 15-keto-PGE2 to 13,14-dihydro-15-keto-PGE2. The expression of PGR-2 is up-regulated in the late phase of 3T3-L1 adipocyte differentiation and predominantly distributed in adipose tissue. Overexpression of PGR-2 in cells decreases peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent transcription and prohibits 3T3-L1 adipocyte differentiation without affecting expression of PPARgamma. Interestingly, we found that 15-keto-PGE2 can act as a ligand of PPARgamma to increase co-activator recruitment, thus activating PPARgamma-mediated transcription and enhancing adipogenesis of 3T3-L1 cells. Overexpression of 15-hydroxyprostaglandin dehydrogenase, which catalyzes the oxidation reaction of PGE2 to form 15-keto-PGE2, significantly increased PPARgamma-mediated transcription in a PGE2-dependent manner. Reciprocally, overexpression of wild-type PGR-2, but not the catalytically defective mutant, abolished the effect of 15-keto-PGE2 on PPARgamma activation. These results demonstrate a novel link between catabolism of PGE2 and regulation of ligand-induced PPARgamma activation.

Crystal structure of anti-configuration of indomethacin and leukotriene B4 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase complex reveals the structural basis of broad spectrum indomethacin efficacy.

The crystal structure of the ternary complex of leukotriene B4 12-hydroxydehydrogenase/15-oxo-prostaglandin (15-oxo-PG) 13-reductase (LTB4 12HD/PGR), an essential enzyme for eicosanoid inactivation pathways, with indomethacin and NADP+ has been solved. An indomethacin molecule bound in the anti-configuration at one of the two active site clefts of the homo-dimer interface in the LTB4 12HD/PGR and was confirmed by a binding calorimetry. The chlorobenzene ring is buried in the hydrophobic pore used as a binding site by the omega-chain of 15-oxo-PGE2. The carboxyl group interacts with the guanidino group of Arg56 and the phenolic hydroxyl group of Tyr262. Indomethacin shows a broad spectrum of efficacy against lipid-mediator related proteins including cyclooxygenase-2, phospholipase A2, PGF synthase and PGE synthase-2 but in the syn-configuration as well as LTB4 12HD/PGR in the anti-configuration. Indomethacin does not necessarily mimic the binding mode of the lipid-mediator substrates in the active sites of these complex structures. Thus, the broad spectrum of indomethacin efficacy can be attributed to its ability to adopt a range of different stable conformations. This allows the indomethacin to adapt to the distinct binding site features of each protein whilst maintaining favorable interactions between the carboxyl group and a counter charged functional group.

Structural basis of leukotriene B4 12-hydroxydehydrogenase/15-Oxo-prostaglandin 13-reductase catalytic mechanism and a possible Src homology 3 domain binding loop.

The bifunctional leukotriene B(4) 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase (LTB(4) 12-HD/PGR) is an essential enzyme for eicosanoid inactivation. It is involved in the metabolism of the E and F series of 15-oxo-prostaglandins (15-oxo-PGs), leukotriene B(4) (LTB(4)), and 15-oxo-lipoxin A(4) (15-oxo-LXA(4)). Some nonsteroidal anti-inflammatory drugs (NSAIDs), which primarily act as cyclooxygenase inhibitors also inhibit LTB(4) 12-HD/PGR activity. Here we report the crystal structure of the LTB(4) 12-HD/PGR, the binary complex structure with NADP(+), and the ternary complex structure with NADP(+) and 15-oxo-PGE(2). In the ternary complex, both in the crystalline form and in solution, the enolate anion intermediate accumulates as a brown chromophore. PGE(2) contains two chains, but only the omega-chain of 15-oxo-PGE(2) was defined in the electron density map in the ternary complex structure. The omega-chain was identified at the hydrophobic pore on the dimer interface. The structure showed that the 15-oxo group forms hydrogen bonds with the 2'-hydroxyl group of nicotine amide ribose of NADP(+) and a bound water molecule to stabilize the enolate intermediate during the reductase reaction. The electron-deficient C13 atom of the conjugated enolate may be directly attacked by a hydride from the NADPH nicotine amide in a stereospecific manner. The moderate recognition of 15-oxo-PGE(2) is consistent with a broad substrate specificity of LTB(4) 12-HD/PGR. The structure also implies that a Src homology domain 3 may interact with the left-handed proline-rich helix at the dimer interface and regulate LTB(4) 12-HD/PGR activity by disruption of the substrate binding pore to accommodate the omega-chain.

Cloning and characterization of a novel splicing variant of the ZADH1 gene.

We report here the cloning and characterization of a novel splicing variant of the human zinc binding alcohol dehydrogenase, domain containing 1 (ZADH1) gene. ZADH1 is localized on chromosome 14q24.2. The cDNA of this splicing variant is 1613 base pairs in length, and encodes a 351-amino acid protein with a putative molecular weight of 38.5 kDa. We named the novel splicing variant ZADH1b. By MTC- panel PCR analysis, it was found that ZADH1b was widely expressed in human tissues. Computer analysis revealed ZADH1 had a potential ADH_zinc_N domain and it had considerable homology with some dehydrogenases. It was speculated that ZADH1 may have definite metabolic roles in vivo as a dehydrogenase.

Prostaglandin catabolizing enzymes.

The primary catabolic pathway of prostaglandins and related eicosanoids is initiated by the oxidation of 15(S)-hydroxyl group catalyzed by NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) followed by the reduction of delta13 double bond catalyzed by NADPH/NADH dependent delta13-15-ketoprostaglandin reductase (13-PGR). 13-PGR was also found to exhibit NADP+-dependent leukotriene B4 12-hydroxydehydrogenase (12-LTB4DH) activity. These enzymes are considered to be the key enzymes responsible for biological inactivation of prostaglandins and related eicosanoids. A separate catabolic pathway of thromboxane involves the oxidation of thromboxane B2 (TXB2) at C-11 catalyzed by NAD+-dependent 11-hydroxythromboxane B2 dehydrogenase (11-TXB2DH). The product of this reaction, 11-dehydro-TXB2, has been considered to be a more reliable quantitative index of thromboxane formation in the circulation. Recent biochemical and molecular biological studies have revealed interesting catalytic properties, structure, and activity relationship, and regulation of gene expression of these three enzymes. Future investigation may shed more light on the roles of these enzymes in health and diseases.

Immunohistochemical localization of guinea-pig leukotriene B4 12-hydroxydehydrogenase/15-ketoprostaglandin 13-reductase.

We have cloned cDNA for leukotriene B4 12-hydroxydehydrogenase (LTB4 12-HD)/15-ketoprostaglandin 13-reductase (PGR) from guinea-pig liver. LTB4 12-HD catalyzes the conversion of LTB4 into 12-keto-LTB4 in the presence of NADP+, and plays an important role in inactivating LTB4. The cDNA contained an ORF of 987 bp that encodes a protein of 329 amino-acid residues with a 78% identity with porcine LTB4 12-HD. The amino acids in the putative NAD+/NADP+ binding domain are well conserved among the pig, guinea-pig, human, rat, and rabbit enzymes. The guinea-pig LTB4 12-HD (gpLTB4 12-HD) was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli, which exhibited similar enzyme activities to porcine LTB4 12-HD. We examined the 15-ketoprostaglandin 13-reductase (PGR) activity of recombinant gpLTB4 12-HD, and confirmed that the Kcat of the PGR activity is higher than that of LTB4 12-HD activity by 200-fold. Northern and Western blot analyses revealed that gpLTB4 12-HD/PGR is widely expressed in guinea-pig tissues such as liver, kidney, small intestine, spleen, and stomach. We carried out immunohistochemical analyses of this enzyme in various guinea-pig tissues. Epithelial cells of calyx and collecting tubules in kidney, epithelial cells of airway, alveoli, epithelial cells in small intestine and stomach, and hepatocytes were found to express the enzyme. These findings will lead to the identification of the unrevealed roles of PGs and LTs in these tissues.

Identification of dual cyclooxygenase-eicosanoid oxidoreductase inhibitors: NSAIDs that inhibit PG-LX reductase/LTB(4) dehydrogenase.

Eicosanoids play key roles in many physiologic and disease processes, and their regulation by nonsteroidal anti-inflammatory drugs (NSAIDs) is critical to many therapeutic approaches. These autacoids are rapidly inactivated by specific enzymes such as 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and 15-oxoprostaglandin 13-reductase/leukotriene B(4) 12-hydroxydehydrogenase (PGR/LTB(4)DH) that act on main series of eicosanoids (i.e., leukotrienes, prostaglandins), and recently found to act in lipoxin inactivation. Here, a panel of NSAIDs was assessed to determine each compound's ability to inhibit eicosanoid-directed activities of either the recombinant 15-PGDH or the PG-LXR/LTB(4)DH. The recombinant 15-PGDH that acts on both prostaglandin E(2) (PGE(2)) and lipoxin A(4) (LXA(4)) was not significantly inhibited by the NSAIDs tested. In contrast, several of the widely used NSAIDs were potent inhibitors of the PG-LXR/LTB(4)DH that metabolizes 15-oxo-PGE(2), and LTB(4) as well as 15-oxo-LXA(4). Diclofenac and indomethacin each inhibited PG-LXR/LTB(4)DH-catalyzed conversion of 15-oxo-PGE(2) to 13,14-dihydro-15-oxo-PGE(2) by 70 and 95%, respectively. Also, a COX-2 inhibitor, niflumic acid, inhibited the PG-LXR/LTB(4)DH eicosanoid oxidoreductase (EOR) by 80% while other COX-2 inhibitors such as nimesulide and NS-398 did not inhibit this enzyme. These results indicate that certain clinically useful NSAIDs such as diclofenac and indomethacin, in addition to inhibiting cyclooxygenases (1 and 2), also interfere with eicosanoid degradation by blocking PG-LXR/LTB(4)DH (EOR) and are members of a new class of dual cyclooxygenase (COX)-EOR inhibitors. Moreover, they suggest that the impact of NSAIDs on PG-LXR/LTB(4)DH activities as targets in the local tissue regulation of eicosanoid-mediated processes should be taken into account.

Antioxidative function and substrate specificity of NAD(P)H-dependent alkenal/one oxidoreductase. A new role for leukotriene B4 12-hydroxydehydrogenase/15-oxoprostaglandin 13-reductase.

There are several known routes for the metabolic detoxication of alpha,beta-unsaturated aldehydes and ketones, including conjugation to glutathione and reduction and oxidation of the aldehyde to an alcohol and a carboxylic acid, respectively. In this study, we describe a fourth class of detoxication that involves the reduction of the alpha,beta-carbon=carbon double bond to a single bond. This reaction is catalyzed by NAD(P)H-dependent alkenal/one oxidoreductase (AO), an enzyme heretofore known as leukotriene B4 12-hydroxydehydrogenase, 15-oxoprostaglandin 13-reductase, and dithiolethione-inducible gene-1. AO is shown to effectively reduce cytotoxic lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE) (k(cat) = 4.0 x 10(3) min(-1); k(cat)/K(m) = 3.3 x 10(7) min(-1) M(-1)) and acrolein (k(cat) = 2.2 x 10(2) min(-1); k(cat)/K(m) = 1.5 x 10(6) min(-1) M(-1)) and common industrial compounds such as ethyl vinyl ketone (k(cat) = 9.6 x 10(3) min(-1); k(cat)/K(m) = 8.8 x 10(7) min(-1) M(-1)) and 15-oxoprostaglandin E1 (k(cat) = 2.4 x 10(3) min(-1); k(cat)/K(m) = 2.4 x 10(9) min(-1) M(-1)). Furthermore, transfection of human embryonic kidney cells with a rat liver AO expression vector protected these cells from challenge with HNE. The concentration of HNE at which 50% of the cells were killed after 24 h increased from approximately 15 microM in control cells to approximately 70 microM in AO-transfected cells. Overexpression of AO also completely abolished protein alkylation by HNE at all concentrations tested (up to 30 microM). Thus, we describe a novel antioxidative activity of a previously characterized bioactive lipid-metabolizing enzyme that could prove to be therapeutically or prophylactically useful due to its high catalytic rate and inducibility.

Oxidoreductases in lipoxin A4 metabolic inactivation: a novel role for 15-onoprostaglandin 13-reductase/leukotriene B4 12-hydroxydehydrogenase in inflammation.

The lipoxins (LX) are autacoids that act within a local inflammatory milieu to dampen neutrophil recruitment and promote resolution. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) and 15-oxoprostaglandin 13-reductase, also termed leukotriene B(4) 12-hydroxydehydrogenase (PGR/LTB(4)DH), are two enzymatic activities appreciated for their roles in the metabolism of prostaglandins and LTB(4). Here, we determined whether these oxidoreductases also catalyze the conversion of lipoxin A(4) (LXA(4)) and assessed the activities of these LXA(4) metabolites. 15-Oxo-LXA(4) was generated by incubating LXA(4) with 15-PGDH and NAD(+) for studies of its further conversion. PGR/LTB(4)DH catalyzed the NADH-dependent reduction of 15-oxo-LXA(4) to yield 13,14-dihydro-15-oxo-LXA(4). With NADH as a cofactor, 15-PGDH acted as a 15-carbonyl reductase and catalyzed the conversion of 13,14-dihydro-15-oxo-LXA(4) to 13, 14-dihydro-LXA(4). Human polymorphonuclear leukocytes (PMN) exposed to native LXA(4), 15-oxo-LXA(4), or 13,14-dihydro-LXA(4) did not produce superoxide anions. At concentrations where LXA(4) and a metabolically stable LXA(4) analog potently inhibited leukotriene B(4)-induced superoxide anion generation, the further metabolites were devoid of activity. Neither 15-oxo-LXA(4) nor 13, 14-dihydro-LXA(4) effectively competed with (3)H-labeled LXA(4) for specific binding to recombinant LXA(4) receptor (ALXR). In addition, introducing recombinant PGR/LTB(4)DH into a murine exudative model of inflammation increased PMN number by approximately 2-fold, suggesting that this enzyme participates in the regulation of PMN trafficking. These results establish the structures of LXA(4) further metabolites and indicate that conversion of LXA(4) to oxo- and dihydro- products represents a mode of LXA(4) inactivation in inflammation. Moreover, they suggest that these eicosanoid oxidoreductases have multifaceted roles controlling the levels of specific eicosanoids involved in the regulation of inflammation.

Purification, cDNA cloning and expression of 15-oxoprostaglandin 13-reductase from pig lung.

15-Oxoprostaglandin 13-reductase (PGR) has been purified to apparent homogeneity from pig lung. The enzyme was estimated to have a molecular mass of 36 kDa by both SDS/PAGE and non-denaturing PAGE, indicating that the enzyme is a monomer. 15-Oxo-PGE1, 15-oxo-PGE2 and 15-oxo-PGF2alpha were found to be substrates for the enzyme, whereas the corresponding 15-hydroxyprostaglandins were not. The reverse reaction, the oxidation of 13,14-dihydro-15-oxo-PGE1 to 15-oxo-PGE1, was not observed. Either NADH or NADPH could serve as a coenzyme. However, the Vmax with NADH was approx. 3-fold that with NADPH, while the Km for NADPH was approx. one-tenth that for NADH. Cloning of the cDNA was achieved by PCR and library screening. A 600 bp PCR product containing the sequences of three different tryptic peptides derived from purified PGR was used for cDNA library screening by plaque hybridization. A cDNA clone that contained the entire PGR coding sequence of 987 bp was obtained. The sequence codes for a protein of 329 amino acid residues with a calculated molecular mass of 35791 Da. Homology analysis indicated that the sequence is virtually identical with that of leukotriene B4 (LTB4) 12-hydroxydehydrogenase [Yokomizo, Ogawa, Uozumi, Kume, Izumi and Shimizu (1996) J. Biol. Chem. 271, 2844-2850]. Expression of this cDNA in Escherichia coli resulted in a protein exhibiting both PGR and LTB4 12-hydroxydehydrogenase activities. However, the specific activity of PGR with 15-oxo-PGE1 as a substrate was approx. 300-fold that of LTB4 12-hydroxydehydrogenase. These results indicate that the cloned cDNA codes for a protein with two different enzyme activities, with 15-oxoprostaglandins as the preferred substrates.

Multiplicity of rat liver 15-ketoprostaglandin delta 13-reductases.

Five kinds of 15-keto-PG delta 13-reductases (enzymes I, II, III, IV and V) were separated and purified from rat liver cytosol. Four (enzymes I,II, III and IV) out of these enzymes were homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of enzymes I, II, III and IV were estimated to be 40,000, 25,000, 64,000 and 70,000 by the electrophoresis, and 42,000, 23,000, 66,000 and 72,000 by gel filtration on a Sephadex G-200 column, respectively. All of these enzymes exhibited the NADPH-dependent activities. In the cases of enzymes I, III and V, NADH was also effective as an electron donor, but to a lesser extent in enzymes I and III. The apparent K(m) values of enzymes I, II, III, IV and V for 15-keto-PGF2 alpha with NADPH were 276, 875, 842, 948 and 2767 nM. The enzymes had isoelectric points at 4.5, 4.9, 6.2, 6.4 and 5.4, respectively. Enzyme I exhibited the double bond reductase activities toward alpha, beta-ketoalkenes such as trans-benzylidene-acetone and trans-phenyl-1-propenylketone. Enzymes III and IV also catalyzed the double bond reduction of trans-phenyl-1-propenyl-ketone. All of these enzymes were markedly inhibited by various chemicals such as dicumarol, quercitrin, p-chloromercuri-benzoic acid, 5,5'-dithio-bis(2-nitrobenzoic acid) and so on.