ABSTRACT
Potato is an important crop in the genus Solanum section Petota. Potatoes are susceptible to multiple abiotic and biotic stresses and have undergone constant improvement through breeding programs worldwide. Introgression of wild relatives from section Petota with potato is used as a strategy to enhance the diversity of potato germplasm. The current dataset contributes a phased genome assembly for diploid S. okadae, and short read sequences and de novo assemblies for the genomes of 16 additional wild diploid species in section Petota that were noted for stress resistance and were of interest to potato breeders. Genome sequence data for three additional genomes representing polyploid hybrids with cultivated potato, and an additional genome from non-tuberizing S. etuberosum, which is outside of section Petota, were also included. High quality short reads assemblies were achieved with genome sizes ranging from 575 to 795 Mbp and annotations were performed utilizing transcriptome sequence data. Genomes were compared for presence/absence of genes and phylogenetic analyses were carried out using plastome and nuclear sequences.
Subject(s)
Genome, Plant , Phylogeny , Solanum , Solanum/genetics , Solanum tuberosum/genetics , Hybridization, GeneticABSTRACT
Cold storage (2-4 °C) used in potato production to suppress diseases and sprouting during storage can result in cold-induced sweetening (CIS), where reducing sugars accumulate in tuber tissue leading to undesirable browning, production of bitter flavors, and increased levels of acrylamide with frying. Potato exhibits genetic and environmental variation in resistance to CIS. The current study profiles gene expression in post-harvest tubers before cold storage using transcriptome sequencing and identifies genes whose expression is predictive for CIS. A distance matrix for potato clones based on glucose levels after cold storage was constructed and compared to distance matrices constructed using RNA-seq gene expression data. Congruence between glucose and gene expression distance matrices was tested for each gene. Correlation between glucose and gene expression was also tested. Seventy-three genes were found that had significant p values in the congruence and correlation tests. Twelve genes from the list of 73 genes also had a high correlation between glucose and gene expression as measured by Nanostring nCounter. The gene annotations indicated functions in protein degradation, nematode resistance, auxin transport, and gibberellin response. These 12 genes were used to build models for prediction of CIS using multiple linear regression. Nine linear models were constructed that used different combinations of the 12 genes. An F-box protein, cellulose synthase, and a putative Lax auxin transporter gene were most frequently used. The findings of this study demonstrate the utility of gene expression profiles in predictive diagnostics for severity of CIS.
Subject(s)
Glucose/metabolism , Plant Proteins/genetics , Solanum tuberosum/genetics , Cold-Shock Response , Gene Expression Regulation, Plant , Glucose/genetics , Plant Proteins/metabolism , Solanum tuberosum/growth & development , Solanum tuberosum/metabolismABSTRACT
NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of 15(S)-hydroxyl group of prostaglandins and lipoxins resulting in the formation of 15-keto metabolites which exhibit greatly reduced biological activities. Therefore, this enzyme has been considered the key enzyme responsible for the inactivation of prostaglandins and lipoxins. Both the cDNA and the genomic DNA of the 15-PGDH gene have been cloned. Structural characterization, transcriptional regulation and biological functions of this enzyme have been investigated. Molecular modeling corroborated with site-directed mutagenesis has identified key residues and domains involved in coenzyme and substrate binding. Catalytic mechanism has been proposed. Studies on the regulation of enzyme expression and activity by physiological and pharmacological agents have begun to uncover its significant roles in cancer, inflammation and reproduction. Apparently, 15-PGDH works with cyclooxygenase-2 to control the cellular levels of prostaglandins. Their reciprocal regulation within the same cells appears to determine the fate of the cells. Because of its ability to inactivate both prostaglandins and lipoxins of two opposite biological activities, the roles of 15-PGDH in cancer and inflammation are particularly intriguing and challenging. Future investigations in these areas are warranted.
Subject(s)
Hydroxyprostaglandin Dehydrogenases/chemistry , Hydroxyprostaglandin Dehydrogenases/metabolism , Prostaglandins/metabolism , Animals , Anti-Inflammatory Agents/pharmacology , Catalytic Domain/genetics , Cyclooxygenase 2/metabolism , Gene Expression Regulation/drug effects , Humans , Hydroxyprostaglandin Dehydrogenases/genetics , Lipoxins/metabolism , Lung Neoplasms/enzymology , Lung Neoplasms/genetics , Mice , Mice, Knockout , Models, Animal , Mutagenesis, Site-Directed , Protein Conformation , Substrate Specificity , Urinary Bladder Neoplasms/enzymology , Urinary Bladder Neoplasms/geneticsABSTRACT
15-Hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes NAD(+)-dependent oxidation of 15(S)-hydroxyl group of prostaglandins and has been considered a key enzyme involved in biological inactivation of prostaglandins. This enzyme is markedly induced by androgens in hormone-sensitive human prostate cancer cells (Tong M., Tai H. H. Biochem Biophys Res Commun 2000; 276: 77-81) and may be involved in tumorigenesis. Inhibition of this enzyme may be of value in anticancer therapy. Non-steroidal anti-inflammatory drugs (NSAIDs) which inhibit cyclooxygenases (COXs) have been shown to be chemopreventive in epidemiological and animal-model studies. However, chemoprevention by these drugs may not be directly related to their inhibition of COXs. Other targets may be also involved in their chemopreventive activity. We have examined a variety of NSAIDs including COX-2 selective inhibitors, peroxisome proliferator-activated receptor (PPAR) gamma agonists and phytophenolic compounds which have been shown to be chemopreventive for their effect on 15-PGDH. It was found that most of these compounds were potent inhibitors of 15-PGDH. Among these compounds, ciglitazone appeared to be the most powerful inhibitor (IC(50)=2.7 microM). Inhibition by ciglitazone was non-competitive with respect to NAD(+) and uncompetitive with respect to PGE(2).
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cyclooxygenase Inhibitors/pharmacology , Hydroxyprostaglandin Dehydrogenases/antagonists & inhibitors , Hydroxyprostaglandin Dehydrogenases/metabolism , NAD/metabolism , Thiazolidinediones , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Dinoprostone/metabolism , Humans , Inhibitory Concentration 50 , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Kinetics , Membrane Proteins , Placenta/enzymology , Prostaglandin-Endoperoxide Synthases/metabolism , Thiazoles/pharmacologyABSTRACT
NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase family, catalyzes the first step in the catabolic pathway of the prostaglandins. This enzyme oxidizes the 15-hydroxyl group of prostaglandins to produce 15-keto metabolites which are usually biologically inactive. A relatively conserved threonine residue corresponding to threonine 11 of 15-PGDH is proposed to be involved in the interaction with NAD(+). Site-directed mutagenesis was used to examine the important role of this residue. Threonine 11 was changed to alanine (T11A), cysteine (T11C), serine (T11S) or tyrosine (T11Y) and the mutant proteins were expressed in E. coli. Western-blot analysis showed that the expression levels of mutant proteins were comparable to that of the wild-type enzyme. Mutants T11A, T11C and T11Y were found to be inactive. Mutant T11S still retained substantial activity and the K(m) value for prostaglandin E(2) (PGE(2)) was similar to the wild-type enzyme; however, the K(m) value for NAD(+) was increased over 23-fold. These results suggest that threonine 11 may be involved in the interaction with NAD(+) either directly or indirectly and contributes to the full catalytic activity of 15-PGDH.
Subject(s)
Hydroxyprostaglandin Dehydrogenases/metabolism , NAD/metabolism , Threonine/metabolism , Amino Acid Sequence , Blotting, Western , Catalysis , Conserved Sequence , Dinoprostone/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Fluorometry , Humans , Hydroxyprostaglandin Dehydrogenases/genetics , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Sequence Homology, Amino Acid , Threonine/geneticsABSTRACT
Prostaglandins (PGs) play a key role in the onset of labor in many species and regulate uterine contractility and cervical dilatation. Therefore, the regulation of prostaglandin output by PG synthesizing (PGHS-I and PGHS-II) and metabolizing (PGDH) enzymes in the human myometrium may determine uterine activity patterns in human labor both at preterm and at term. We hypothesized that expression of PGHS isozymes and PGDH in myometrium from women at preterm and term labor would change to favor increased uterotonin (PG) production. Myometrial samples were obtained from the lower uterine segment during cesarean section deliveries from women presenting in preterm, no labor; preterm, labor; term, no labor; term, labor. Immunoreactive (ir-) PGHS and PGDH protein was localized using immunohistochemistry, and changes in protein levels were determined by Western blotting. Ir-PGHS-I and PGHS-II proteins were localized only to myocytes. Ir-PGDH was localized to myocytes in all samples of myometrium examined, but using dual immunofluorescence and immunohistochemistry, ir-PGDH was also detected in cells of the connective tissue. Levels of ir-PGHS-I and PGHS-II protein were not significantly different between no labor and labor tissues, either at preterm or at term. There was no significant effect of gestational age on levels of PGDH, PGHS-I, and PGHS-II protein, but there was a significant decrease in ir-PGDH protein levels in myometrium with labor both at preterm and at term. In addition, there was a decrease in PGDH activity in myometrium from women in labor, both at preterm and at term. Therefore, we conclude that PGDH, PGHS-I, and PGHS-II protein localize within the myocytes of the human pregnant myometrium. A decrease in PGDH protein and activity occurs in association with active labor and may contribute to the amount of bioactive PGs available to act within the human pregnant myometrium at that time.
Subject(s)
Hydroxyprostaglandin Dehydrogenases/metabolism , Isoenzymes/metabolism , Myometrium/enzymology , Pregnancy/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Female , Humans , Labor, Obstetric/metabolism , Obstetric Labor, Premature/enzymology , Tissue DistributionABSTRACT
The thromboxane A(2) receptor (TP), which mediates vasoconstriction, mitogenesis, and platelet aggregation, has been shown to undergo rapid agonist-induced desensitization. Two isoforms (alpha and beta) of TP have been recognized. The potential role of the G protein-coupled receptor kinases (GRKs) in the phosphorylation and desensitization of TP alpha was investigated. Human embryonic kidney (HEK) 293 cells stably transfected with the His-tagged TP alpha was used to study the phosphorylation and desensitization of the receptor. Rapid isolation of the (32)P-labeled receptor was achieved by Ni(2+)-nitrilotriacetic acid agarose after agonist stimulation of HEK293 cells prelabeled with (32)P(i). [1S-[1 alpha,2 alpha(Z),3 beta(1E,3S*),4 alpha]]-7-[3-[3-Hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptenoic acid (I-BOP) induced receptor phosphorylation and Ca(2+) release in a time- and dose-dependent manner. Pretreatment of cells with I-BOP abolished subsequent induction of Ca(2+) release through a second dose of I-BOP. Transfection with expression plasmids encoding the cDNA of GRK5 or GRK6 augmented I-BOP-induced phosphorylation and inhibited I-BOP-stimulated Ca(2+) release. Both I-BOP-induced and GRK-mediated phosphorylation and phorbol ester-induced phosphorylation were blocked by the addition of 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) (GF 109203X). This indicates that GF 109203X, a known protein kinase C (PKC) inhibitor, also inhibits GRKs. This finding was further supported by in vitro studies in which preparations of GRK5 and GRK6 were found to be inhibited by GF 109203X. These results suggest that GRK5 and GRK6 may phosphorylate the TP alpha in an agonist-dependent manner. Furthermore, the results obtained with PKC inhibitors in assessing the role of PKC in agonist-induced receptor phosphorylation should be interpreted with caution.
Subject(s)
Protein Serine-Threonine Kinases/metabolism , Receptors, Thromboxane/metabolism , Amino Acid Sequence , Blotting, Western , Calcium/metabolism , Cell Membrane/chemistry , Cell Membrane/metabolism , Chelating Agents/chemistry , DNA, Complementary/isolation & purification , G-Protein-Coupled Receptor Kinase 5 , G-Protein-Coupled Receptor Kinases , Glutathione Transferase/metabolism , Humans , Ligands , Molecular Sequence Data , Phosphorylation , Receptors, Thromboxane/drug effectsABSTRACT
Human embryonic kidney (HEK)293 cells stably transfected with the His-tagged thromboxane receptor alpha (TPalpha) was used to study the phosphorylation and desensitization of the receptor induced by 8-bromo-cyclic GMP (8-Br-cGMP), sodium nitroprusside (SNP), or S-nitroso-glutathione (SNG). These agents are known to activate cGMP-dependent protein kinase (PKG). Pretreatment of cells with these agents attenuated significantly agonist I-BOP induced Ca(2+) release. These agents also induced dose-dependent phosphorylation of the TPalpha as demonstrated by increased (32)P-labeling of the receptor from cells prelabeled with (32)Pi. To facilitate the identification of the intracellular domains involved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used as substrates for the purified PKG. It was found that only the GST-C-terminal tail fusion protein could serve as a substrate for the PKG. To identify the specific serine/threonine residues in the C-terminal tail being phosphorylated, various alanine mutants of these serine/threonine residues were checked for their ability to serve as substrates. It was found that the Ser-331 of the C-terminal tail was primarily involved in the PKG-mediated phosphorylation. That Ser-331 is a predominant site of phosphorylation was supported by in vivo studies in which HEK293 cells expressing the S331A mutant receptor showed little phosphorylation induced by any of the above three agents. Furthermore, HEK293 cells expressing the S331A mutant receptor pretreated with any of the above three agents became responsive to the agonist I-BOP-induced Ca(2+) release. These results indicate that Ser-331 of the TPalpha is the primary site responsible for the phosphorylation and the desensitization of the receptor induced by agents that activate the PKG.
Subject(s)
Cyclic GMP-Dependent Protein Kinases/metabolism , Receptors, Thromboxane/chemistry , Receptors, Thromboxane/metabolism , Base Sequence , Binding Sites , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line , Cyclic GMP/analogs & derivatives , Cyclic GMP/pharmacology , DNA Primers/genetics , Enzyme Activation/drug effects , Fatty Acids, Unsaturated/pharmacology , Glutathione/analogs & derivatives , Glutathione/pharmacology , Humans , In Vitro Techniques , Mutagenesis, Site-Directed , Nitroprusside/pharmacology , Nitroso Compounds/pharmacology , Phosphorylation , Receptors, Thromboxane/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , S-Nitrosoglutathione , Serine/chemistryABSTRACT
Prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs) are generated by the action of cyclooxygenase-2 on the endocannabinoids 2-arachidonylglycerol (2-AG) and arachidonylethanolamide, respectively. These novel eicosanoids may have unique pharmacological properties and/or serve as latent sources of prostaglandins at sites remote from their tissue of origin. Therefore, we investigated the metabolism of PG-Gs and PG-EAs in vitro and in vivo. PGE(2)-G was rapidly hydrolyzed in rat plasma to generate PGE(2) (t(1/2) = 14 s) but was only slowly metabolized in human plasma (t(1/2) > 10 min). An intermediate extent of metabolism of PGE(2)-G was observed in human whole blood (t(1/2) approximately 7 min). The parent arachidonylglycerol, 2-AG, and the more stable regioisomer, 1-AG, also were much more rapidly metabolized in rat plasma compared with human plasma. PGE(2)-EA was not significantly hydrolyzed in plasma, undergoing slow dehydration/isomerization to PGB(2)-EA. Both PGE(2)-G and PGE(2)-EA were stable in canine, bovine, and human cerebrospinal fluid. Human 15-hydroxyprostaglandin dehydrogenase, the enzyme responsible for the initial step in PG inactivation in vivo, oxidized both PGE(2)-G and PGE(2)-EA less efficiently than the free acid. The sterically hindered glyceryl prostaglandin was the poorest substrate examined in the E series. Minimal 15-hydroxyprostaglandin dehydrogenase oxidation of PGF(2 alpha)-G was observed. PGE(2)-G and PGE(2)-EA pharmacokinetics were assessed in rats. PGE(2)-G was not detected in plasma 5 min following an intravenous dose of 2 mg/kg. However, PGE(2)-EA was detectable up to 2 h following an identical dose, displaying a large apparent volume of distribution and a half-life of over 6 min. The results suggest that endocannabinoid-derived PG-like compounds may be sufficiently stable in humans to exert actions systemically. Furthermore, these results suggest that the rat is not an adequate model for investigating the biological activities of 2-arachidonylglycerol or glyceryl prostaglandins in humans.
Subject(s)
Dinoprostone/analogs & derivatives , Ethanolamines/metabolism , Glycerides/metabolism , Prostaglandins/metabolism , Animals , Cannabinoid Receptor Modulators , Dinoprostone/metabolism , Dinoprostone/pharmacokinetics , Drug Stability , Esters/metabolism , Ethanolamines/pharmacokinetics , Glycerides/pharmacokinetics , Humans , Hydroxyprostaglandin Dehydrogenases/metabolism , Male , Plasma/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of the 15(S) hydroxyl group of prostaglandins to a 15-keto group resulting in a significant reduction of the biological activities of prostaglandins. Although the key residues involved in NAD+ binding and in catalytic activity have been partially identified, the sites of interaction of the enzyme with the prostaglandin substrates are yet to be determined. Homology analysis of the primary structures of 15-PGDH from human, mouse and rat indicates that the sequences are almost homologous except for two regions near the C-terminus. The involvement of the C-terminal region in catalytic activity was examined by studies on C-terminally truncated enzymes and on human/rat chimeric enzymes. When three to four amino acids were removed successively from the C-terminal end of human 15-PGDH, the truncated enzymes exhibited decreasing Vmax/Km ratios and increasing Km values for PGE2 as the chain was shortened. Similarly, when the C-terminal 14 amino acids of human 15-PGDH were replaced by the C-terminal 14 amino acids of rat 15-PGDH or vice versa, the Vmax/Km ratios and the Km values for prostaglandin E2 of the chimeric enzymes were in between those of the two wild-type enzymes. This indicates that the catalytic effectiveness of human 15-PGDH decreases as the C-terminal region is gradually removed or replaced by rat sequences. The C-terminal region appears to be more important for the interaction of the enzyme with the prostaglandin substrates than with the coenzyme.
Subject(s)
Hydroxyprostaglandin Dehydrogenases/metabolism , NAD/metabolism , Prostaglandins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Catalysis , DNA Primers , DNA, Complementary , Electrophoresis, Polyacrylamide Gel , Humans , Hydroxyprostaglandin Dehydrogenases/chemistry , Hydroxyprostaglandin Dehydrogenases/genetics , Hydroxyprostaglandin Dehydrogenases/isolation & purification , Molecular Sequence Data , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Species Specificity , Substrate SpecificityABSTRACT
We have previously reported a macrophage-mediated gender difference in postburn immunosuppression, which was dependent upon elevated levels of circulating 17beta-estradiol (E(2)) and, in part, interleukin-6. Herein we examined the role of prostaglandin E(2) (PGE(2)), a potent suppressor of cell-mediated immunity. Circulating levels of PGE(2) were significantly elevated in females but not males at 10 days postburn (P < 0.01), and indomethacin treatment fully restored the delayed-type hypersensitivity and splenocyte proliferative responses of thermally injured females. While there was no difference in cyclooxygenase-2 protein expression in the lungs and liver of thermally injured male and female mice, there was a marked decrease in the protein expression of 15-hydroxyprostaglandin dehydrogenase in females. These data demonstrate that PGE(2) is a critical mediator of immunosuppression in thermally injured female mice and that the increase in circulating PGE(2) is derived, in part, from decreased degradation and clearance of PGE(2).
Subject(s)
Burns/immunology , Dinoprostone/immunology , Hydroxyprostaglandin Dehydrogenases/biosynthesis , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Burns/metabolism , Cell Division/drug effects , Cells, Cultured , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/pharmacology , Dinitrofluorobenzene/administration & dosage , Dinitrofluorobenzene/immunology , Dinoprostone/biosynthesis , Dinoprostone/pharmacology , Female , Hypersensitivity, Delayed/immunology , Indomethacin/pharmacology , Irritants/administration & dosage , Irritants/immunology , Isoenzymes/metabolism , Lung/metabolism , Macrophages/drug effects , Macrophages/metabolism , Male , Mice , Mice, Inbred BALB C , Prostaglandin-Endoperoxide Synthases/metabolism , Sex Characteristics , Spleen/cytologyABSTRACT
Prostate cancer cells are known to express cyclooxygenases (COXs) and synthesize prostaglandins. Catabolism of prostaglandins in these cells remains to be determined. Induction of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key metabolic inactivation enzyme, was investigated in androgen-sensitive LNCaP cells and in hormone-independent PC3 cells. 15-PGDH was found to be induced by dihydrotestosterone or testosterone in a time- and dose-dependent manner in LNCaP but not in PC3 cells as shown by activity assay and immunoblot analysis. However, prostaglandin synthetic enzymes, COX-1 and COX-2, were not found to be induced by androgens. Induction was also achieved by 17beta-estradiol and progesterone, although to a lesser extent. Induction of 15-PGDH was not blocked by steroid receptor antagonist, RU 486, nor by antiandrogen, flutamide. However, induction was inhibited by tyrosine kinase inhibitor, genistein, and by ERK kinase inhibitor, PD 98059, but not by protein kinase C inhibitor, GF109203X. These results suggest that androgens induce 15-PGDH gene expression through an unconventional nongenomic pathway.
Subject(s)
Dihydrotestosterone/pharmacology , Hydroxyprostaglandin Dehydrogenases/biosynthesis , Prostatic Neoplasms/enzymology , Testosterone/pharmacology , Dose-Response Relationship, Drug , Humans , Male , Prostaglandins/biosynthesis , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
15-Oxoprostaglandin 13-Reductase/physiology , Alcohol Oxidoreductases/physiology , Hydroxyeicosatetraenoic Acids/metabolism , Inflammation/enzymology , Lipoxins , Oxidoreductases/physiology , Animals , Anions/metabolism , Chromatography, High Pressure Liquid , Chromatography, Liquid , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Escherichia coli/metabolism , Horses , Humans , Male , Mass Spectrometry , Mice , Mice, Inbred BALB C , Models, Chemical , Neutrophils/enzymology , Recombinant Proteins/metabolism , Superoxides , Swine , Time FactorsABSTRACT
Pro-inflammatory prostaglandins are known to be first catabolized by NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) to inactive metabolites. This enzyme is under regulatory control by various inflammation-related agents. Regulation of this enzyme was investigated in human promonocytic U937 cells. 15-PGDH activity was found to be optimally induced by phorbol 12-myristate 13-acetate (PMA) at 10 nM after 24 h of treatment. The induction was blocked by staurosporine or GF 109203X indicating that the induction was mediated by protein kinase C. The induction by PMA was inhibited by the concurrent addition of dexamethasone. Nearly complete inhibition was observed at 50 nM. Other glucocorticoids, such as hydrocortisone and corticosterone, but not sex hormones, were also inhibitory. Inhibition by dexamethasone could be reversed by the concurrent addition of antagonist mifepristone (RU-486) indicating that the inhibition was a receptor-mediated event. Either induction by PMA or inhibition by dexamethasone the 15-PGDH activity correlated well with the enzyme protein expression as shown by the Western blot analysis. These results provide the first evidence that prostaglandin catabolism is regulated by glucocorticoids at the therapeutic level.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Dexamethasone/pharmacology , Hydroxyprostaglandin Dehydrogenases/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Dose-Response Relationship, Drug , Enzyme Induction/drug effects , Humans , Indoles/pharmacology , Maleimides/pharmacology , Mifepristone/pharmacology , Phorbol Esters/pharmacology , Staurosporine/pharmacology , Time Factors , U937 CellsABSTRACT
Human CXCR4 was expressed in Sf9 insect cells using the Bac-to-Bac baculovirus expression system. The recombinant receptor exhibited ligand binding activities with a K(d) value (3.3 nM) comparable to that of the native receptor. The role of four conserved cysteinyl residues was explored by site-directed mutagenesis. Each cysteine was individually changed to an alanine residue. All of the four mutants showed decreased ligand binding activity with increased K(d) values although comparable levels of receptor expression were observed. These results suggest that each of these four cysteinyl residues may be important for the ligand binding of the receptor. Evidence suggests that the ionic interaction may be involved in ligand binding. Point mutation of several relatively conserved acidic residues (Asp-10, Asp-262, Glu-275, and Glu-277) to an alanine residue greatly decreased the ligand binding activity and affinity. Since SDF-1alpha is a highly basic protein, these acidic residues may interact with the basic residues of SDF-1alpha by ionic pairing in addition to other molecular interactions and play an important role in ligand binding.
Subject(s)
Point Mutation , Receptors, CXCR4/genetics , Receptors, CXCR4/metabolism , Animals , Baculoviridae/genetics , Base Sequence , Binding Sites/genetics , Cell Line , Cysteine/chemistry , DNA Primers/genetics , Electrochemistry , Humans , In Vitro Techniques , Kinetics , Ligands , Mutagenesis, Site-Directed , Receptors, CXCR4/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SpodopteraABSTRACT
OBJECTIVE: The aim of this study was to examine the relationship between prostaglandin synthesis and prostaglandin degradation in a model of burn injury with infection. METHODS: Male B2D6F1 mice were assigned to control, burn (16% dorsal scald burn), or burn with infection (burn with topical application of 1,000 colony forming units of Pseudomonas aeruginosa) groups. Lung tissue was harvested at 1, 2, and 3 days after burn injury and subsequently processed for total RNA and protein. Northern and Western blot analyses were used to examine differences in cyclooxygenase 2 (COX-2) and prostaglandin 15-OH dehydrogenase (PGDH) protein and mRNA expression. Total RNA was probed with the riboprobe for murine PGDH and COX-2 and the 100,000g protein fraction was immunoblotted by using an rabbit anti-murine PGDH and anti-murine COX-2 antibody. RESULTS: COX-2 expression was elevated in the burn with infection animals on day 1 and day 2 after burn injury. At these time points in the burn + infection group, PGDH was significantly depressed. Burn injury increased COX-2 expression on day 1, but by day 2, COX-2 expression had decreased to control values. A corresponding increase in PGDH expression was observed on day 2 in the burned mice. The mRNA expression of COX-2 was followed by a similar increase in COX-2 protein expression at all time points in the injured animals. This was not the case with PGDH expression. On day 1, PGDH mRNA expression was depressed in the burn with infection mice with no change in PGDH protein expression. This finding indicates that PGDH is subject to regulation at both the transcriptional and posttranscriptional levels. CONCLUSION: Burn wound infection depressed both PGDH mRNA and protein expression and increased COX-2 mRNA and protein expression. Therefore, increases in circulating prostaglandin E2 levels during septic injury are derived from alterations in synthesis and degradation of prostaglandin E2.
Subject(s)
Burns/complications , Dinoprostone/biosynthesis , Dinoprostone/metabolism , Gene Expression Regulation, Enzymologic/genetics , Hydroxyprostaglandin Dehydrogenases/analysis , Hydroxyprostaglandin Dehydrogenases/genetics , Isoenzymes/analysis , Isoenzymes/genetics , Lung/chemistry , Prostaglandin-Endoperoxide Synthases/analysis , Prostaglandin-Endoperoxide Synthases/genetics , Pseudomonas Infections/etiology , Pseudomonas Infections/metabolism , Wound Infection/etiology , Wound Infection/metabolism , Animals , Blotting, Northern , Blotting, Western , Cyclooxygenase 2 , Disease Models, Animal , Male , Mice , Mice, Inbred Strains , Pseudomonas Infections/pathology , RNA, Messenger/analysis , Rabbits , Random Allocation , Transcription, Genetic/genetics , Wound Infection/pathologyABSTRACT
Eicosanoid receptors exhibit a highly conserved ERY(C)XXV(I)XXPL sequence in the second intracellular loop. The carboxyl end of this motif contains a bulky hydrophobic amino acid (L,I,V, or F). In human thromboxane A2 receptor (TXA(2)R), phenylalanine 138 is located at the carboxyl end of this highly conserved motif. This study examined the function of the F138 in G protein coupling. F138 was mutated to aspartic acid (D) and tyrosine (Y), respectively. Both mutants F138D and F138Y showed similar ligand binding activity to that of the wild type TXA(2)R. The Kd and Bmax values of either mutant were comparable to those of the wild type receptor. However, both mutants showed significant impairment of agonist induced Ca(2+) signaling and phospholipase C activation. These results suggest that the F138 plays a key role in G protein coupling.
Subject(s)
GTP-Binding Proteins/metabolism , Phenylalanine/metabolism , Receptors, Cell Surface/metabolism , Receptors, Thromboxane/metabolism , Amino Acid Sequence , Cells, Cultured , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Phenylalanine/chemistry , Protein Binding , Protein Conformation , Receptors, Thromboxane/chemistry , Sequence Homology, Amino AcidABSTRACT
The cDNA of the human CXCR4/fusin was isolated from a human HeLa cell cDNA library by PCR and functionally expressed in Sf9 insect cells. The recombinant receptor was found to bind its natural ligand SDF-1alpha with an affinity comparable to that of the native receptor. Sequence-specific antibodies against each of the four extracellular domains were generated and used to investigate the interactions between the different domains of the receptor and the ligand. Each of the four antibodies was found to be able to inhibit ligand binding. CXCR4 was shown to be a glycoprotein. The role of N-glycosylation of CXCR4 in ligand binding was investigated in the insect cells overexpressed with recombinant CXCR4. Two potential N-glycosylation sites (Asn-11 and Asn-176) were either singly or doubly mutated to a leucine residue. Both single mutant receptors exhibited a significant decrease in ligand binding activity and affinity. The double mutant receptor showed little binding activity. Our data suggest that all of the extracellular domains are involved in ligand-receptor interactions and that N-glycosylation is required to maintain high-affinity ligand binding.
Subject(s)
Chemokines, CXC/metabolism , Peptide Fragments/metabolism , Receptors, CXCR4/metabolism , Animals , Antibody Specificity , Baculoviridae , Chemokine CXCL12 , DNA, Complementary , Gene Library , Glycosylation , HeLa Cells , Humans , Ligands , Peptide Fragments/genetics , Peptide Fragments/immunology , Peptide Fragments/isolation & purification , Protein Processing, Post-Translational , Receptors, CXCR4/genetics , Receptors, CXCR4/immunology , Receptors, CXCR4/isolation & purification , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , SpodopteraABSTRACT
A human PGHS-2 promoter fragment (300 BP) linked to the luciferase reporter was used to study the regulation of PGHS-2 gene expression in human amnion-derived WISH cells. A cyclic AMP (cAMP) response element (CRE) was found to be important in the induction of PGHS-2 gene expression. This was demonstrated by showing that coexpression of CREB stimulated native but not CRE mutant promoter and that IL-1beta and PMA induced less activity with the mutant promoter as compared to the native promoter. The effect of dexamethasone on IL-1beta and PMA induced promoter activities was further examined. IL-1beta or PMA induced activity was blocked by dexamethasone, whereas IL-1beta or PMA induced mutant activity was not responsive to dexamethasone. Direct activation of CRE by a cAMP elevating agent, isoproterenol, was found to be inhibited significantly dexamethasone. These results suggest that CRE may mediate the induction of PGHS-2 by IL-1beta and PMA as well as the suppression of expression by dexamethasone in amnion-derived cells.
Subject(s)
Amnion/metabolism , Cyclic AMP/genetics , Dexamethasone/pharmacology , Isoenzymes/genetics , Prostaglandin-Endoperoxide Synthases/genetics , Cell Line , Cyclooxygenase 2 , DNA-Binding Proteins/genetics , Down-Regulation , Gene Expression Regulation/drug effects , Genes, Reporter , Humans , Interleukin-1/pharmacology , Isoproterenol/pharmacology , Membrane Proteins , Mutagenesis, Site-Directed , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , Tetradecanoylphorbol Acetate/pharmacology , Transcription Factors/genetics , TransfectionABSTRACT
Previous studies have shown that the reduced nicotinamide adenine dinucleotide phosphate (NADPH)- binding domain of rat liver microsomal steroid 5alpha-reductase isozyme-1 (r5alphaR-1) is in a highly conserved region of the polypeptide sequence (residues 160-190). In this study, we investigated, by site-directed mutagenesis, the role of hydroxylated and aromatic amino acids within the NADPH-binding domain. The r5alphaR-1 cDNA was cloned into a pCMV vector, and the double strand site-directed mutagenesis method was used to create mutants Y179F, Y179S, Y189F, Y189S, S164A, S164T, and Y187F, which were subsequently expressed in COS-1 cells. Kinetic studies of the expressed enzymes showed that the mutation Y179F resulted in an approximately 40-fold increase in the Km for NADPH versus wild-type, with only a 2-fold increase in the Km for testosterone. The mutants Y189F and S164A showed smaller increases (4 and 6-fold) in Kms for NADPH and no significant change in the Km for testosterone, whereas Y189S had kinetic properties similar to the wild-type r5alphaR-1. Mutants Y179S and S164T both resulted in inactive enzymes, whereas F187Y showed an approximately 5-fold decrease in Km for NADPH and a significant increase (approximately 18-fold) in the Km for testosterone. The results suggest that the -OH functionality of Y179 is involved in cofactor binding, but is not essential for the activity of the enzyme, whereas the -OH functionalities of Y189 and S164 play lesser roles in cofactor binding to r5alphaR-1 and may not be required for enzyme activity. On the other hand, the residue F187 may be important for the binding of both NADPH and testosterone.