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Dynamics of Radical Intermediates in Prostaglandin H Synthase-1 Cyclooxygenase Reactions is Modulated by Multiple Factors.

ABSTRACT: Prostaglandin H synthase (PGHS) catalyzes the biosynthesis of PGG2 and PGH2, the precursor of all prostanoids, from arachidonic acid (AA). PGHS exhibits two enzymatic activities following a branched-chain radical mechanism: 1) a peroxidase activity (POX) that utilizes hydroperoxide through heme redox cycles to generate the critical Tyr385 tyrosyl radical for coupling both enzyme activities; 2) the cyclooxygenase (COX) activity inserting two oxygen molecules into AA to generate endoperoxide/hydroperoxide PGG2 through a series of radical intermediates. Upon the generation of Tyr385 radical, COX catalysis is initiated, with C13 pro-S hydrogen abstraction from AA by Tyr385 radical to generate arachidonyl substrate radical. Oxygen provides a large driving force for the subsequent fast steps leading to the formation of PGG2, including radical redistributions, ring formations, and rearrangements. On the other hand, if the supply of oxygen is severed, equilibrium between arachidonyl radical and tyrosyl radical(s) biases largely towards the latter. In this study, we demonstrate that such equilibrium is shifted by many factors, including temperature, chemical structures of fatty acid substrates and limited supply of oxygen. We also, for the first time, reveal that this equilibrium is significantly affected by co-substrates of POX. The presence of efficient POX co-substrates, which reduces heme to its ferric state, apparently biases the equilibrium towards arachidonyl radical. Therefore a dynamic interplay exists between the two activities of PGHS.

SUBMITTER: Wu G

PROVIDER: S-EPMC8376226 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Cyclooxygenase competitive inhibitors alter tyrosyl radical dynamics in prostaglandin H synthase-2.

Project description:Reaction of prostaglandin H synthase (PGHS) isoforms 1 or 2 with peroxide forms a radical at Tyr385 that is required for cyclooxygenase catalysis and another radical at Tyr504, whose function is unknown. Both tyrosyl radicals are transient and rapidly dissipated by reductants, suggesting that cyclooxygenase catalysis might be vulnerable to suppression by intracellular antioxidants. Our initial hypothesis was that the two radicals are in equilibrium and that their proportions and stability are altered upon binding of fatty acid substrate. As a test, we examined the effects of three competitive inhibitors (nimesulide, flurbiprofen, and diclofenac) on the proportions and stability of the two radicals in PGHS-2 pretreated with peroxide. Adding nimesulide after ethyl peroxide led to some narrowing of the tyrosyl radical signal detected by EPR spectroscopy, consistent with a small increase in the proportion of the Tyr504 radical. Neither flurbiprofen nor diclofenac changed the EPR line width when added after peroxide. In contrast, the effects of cyclooxygenase inhibitors on the stability of the preformed tyrosyl radicals were dramatic. The half-life of total tyrosyl radical was 4.1 min in the control, >10 h with added nimesulide, 48 min with flurbiprofen, and 0.8 min with diclofenac. Stabilization of the tyrosyl radicals was evident even at substoichiometric levels of nimesulide. Thus, the inhibitors had potent, structure-dependent, effects on the stability of both tyrosyl radicals. This dramatic modulation of tyrosyl radical stability by cyclooxygenase site ligands suggests a mechanism for regulating the reactivity of PGHS tyrosyl radicals with cellular antioxidants.

| S-EPMC2805951 | biostudies-literature

Role of Tyr348 in Tyr385 radical dynamics and cyclooxygenase inhibitor interactions in prostaglandin H synthase-2.

Project description:Both prostaglandin H synthase (PGHS) isoforms utilize a radical at Tyr385 to abstract a hydrogen atom from arachidonic acid, initializing prostaglandin synthesis. A Tyr348-Tyr385 hydrogen bond appears to be conserved in both isoforms; this hydrogen bonding has the potential to modulate the positioning and reactivity of the Tyr385 side chain. The EPR signal from the Tyr385 radical undergoes a time-dependent transition from a wide doublet to a wide singlet species in both isoforms. In PGHS-2, this transition results from radical migration from Tyr385 to Tyr504. Localization of the radical to Tyr385 in the recombinant human PGHS-2 Y504F mutant was exploited in examining the effects of blocking Tyr385 hydrogen bonding by introduction of a further Y348F mutation. Cyclooxygenase and peroxidase activities were found to be maintained in the Y348F/Y504F mutant, but the Tyr385 radical was formed more slowly and had greater rotational freedom, as evidenced by observation of a transition from an initial wide doublet species to a narrow singlet species, a transition not seen in the parent Y504F mutant. The effect of disrupting Tyr385 hydrogen bonding on the cyclooxygenase active site structure was probed by examination of cyclooxygenase inhibitor kinetics. Aspirin treatment eliminated all oxygenase activity in the Y348F/Y504F double mutant, with no indication of the lipoxygenase activity observed in aspirin-treated wild-type PGHS-2. Introduction of the Y348F mutation also strengthened the time-dependent inhibitory action of nimesulide. These results suggest that removal of Tyr348-Tyr385 hydrogen bonding in PGHS-2 allows greater conformational flexibility in the cyclooxygenase active site, resulting in altered interactions with inhibitors and altered Tyr385 radical behavior.

| S-EPMC2851202 | biostudies-literature

Hypoxia activates the cyclooxygenase-2-prostaglandin E synthase axis.

Project description:Hypoxia-inducible factors (HIFs), in particular HIF-1alpha, have been implicated in tumor biology. However, HIF target genes in the esophageal tumor microenvironment remain elusive. Gene expression profiling was performed upon hypoxia-exposed non-transformed immortalized human esophageal epithelial cells, EPC2-hTERT, and comparing with a gene signature of esophageal squamous cell carcinoma (ESCC). In addition to known HIF-1alpha target genes such as carbonic anhydrase 9, insulin-like growth factor binding protein-3 (IGFBP3) and cyclooxygenase (COX)-2, prostaglandin E synthase (PTGES) was identified as a novel target gene among the commonly upregulated genes in ESCC as well as the cells exposed to hypoxia. The PTGES induction was augmented upon stabilization of HIF-1alpha by hypoxia or cobalt chloride under normoxic conditions and suppressed by dominant-negative HIF-1alpha. Whereas PTGES messenger RNA (mRNA) was negatively regulated by normoxia, PTGES protein remained stable upon reoxygenation. Prostaglandin E(2) (PGE(2)) biosynthesis was documented in transformed human esophageal cells by ectopic expression of PTGES as well as RNA interference directed against PTGES. Moreover, hypoxia stimulated PGE(2) production in a HIF-1alpha-dependent manner. In ESCC, PTGES was overexpressed frequently at the mRNA and protein levels. Finally, COX-2 and PTGES were colocalized in primary tumors along with HIF-1alpha and IGFBP3. Activation of the COX-2-PTGES axis in primary tumors was further corroborated by concomitant upregulation of interleukin-1beta and downregulation of hydroxylprostaglandin dehydrogenase. Thus, PTGES is a novel HIF-1alpha target gene, involved in prostaglandin E biosynthesis in the esophageal tumor hypoxic microenvironment, and this has implications in diverse tumors types, especially of squamous origin.

| S-EPMC2832548 | biostudies-literature

Genetic-deletion of Cyclooxygenase-2 Downstream Prostacyclin Synthase Suppresses Inflammatory Reactions but Facilitates Carcinogenesis, unlike Deletion of Microsomal Prostaglandin E Synthase-1.

Project description:Prostacyclin synthase (PGIS) and microsomal prostaglandin E synthase-1 (mPGES-1) are prostaglandin (PG) terminal synthases that function downstream of inducible cyclooxygenase (COX)-2 in the PGI2 and PGE2 biosynthetic pathways, respectively. mPGES-1 has been shown to be involved in various COX-2-related diseases such as inflammatory diseases and cancers, but it is not yet known how PGIS is involved in these COX-2-related diseases. Here, to clarify the pathophysiological role of PGIS, we investigated the phenotypes of PGIS and mPGES-1 individual knockout (KO) or double KO (DKO) mice. The results indicate that a thioglycollate-induced exudation of leukocytes into the peritoneal cavity was suppressed by the genetic-deletion of PGIS. In the PGIS KO mice, lipopolysaccharide-primed pain nociception (as assessed by the acetic acid-induced writhing reaction) was also reduced. Both of these reactions were suppressed more effectively in the PGIS/mPGES-1 DKO mice than in the PGIS KO mice. On the other hand, unlike mPGES-1 deficiency (which suppressed azoxymethane-induced colon carcinogenesis), PGIS deficiency up-regulated both aberrant crypt foci formation at the early stage of carcinogenesis and polyp formation at the late stage. These results indicate that PGIS and mPGES-1 cooperatively exacerbate inflammatory reactions but have opposing effects on carcinogenesis, and that PGIS-derived PGI2 has anti-carcinogenic effects.

| S-EPMC4661703 | biostudies-literature

Cyclooxygenase reaction mechanism of prostaglandin H synthase from deuterium kinetic isotope effects.

Project description:Cyclooxygenase catalysis by prostaglandin H synthase (PGHS) is thought to involve a multistep mechanism with several radical intermediates. The proposed mechanism begins with transfer of the C13 pro-(S) hydrogen atom from the substrate arachidonic acid (AA) to the Tyr385 radical in PGHS, followed by oxygen insertion and several bond rearrangements. The importance of the hydrogen-transfer step to controlling the overall kinetics of cyclooxygenase catalysis has not been directly examined. We quantified the non-competitive primary kinetic isotope effect (KIE) for both PGHS-1 and -2 using unlabeled AA and several deuterated AAs, including 13-pro-(S) d-AA, 13,13-d(2)-AA and 10, 10, 13,13-d(4)-AA. The primary KIE for steady-state cyclooxygenase catalysis, (D)k(cat), ranged between 1.8 and 2.3 in oxygen electrode measurements. The intrinsic KIE of AA radical formation by C13 pro-(S) hydrogen abstraction in PGHS-1 was estimated to be 1.9-2.3 using rapid freeze-quench EPR kinetic analysis of anaerobic reactions and computer modeling to a mechanism that includes slow formation of a pentadienyl AA radical and rapid equilibration of the AA radical with a tyrosyl radical, NS1c. The observation of similar values for steady-state and pre-steady state KIEs suggests that hydrogen abstraction is a rate-limiting step in cyclooxygenase catalysis. The large difference of the observed KIE from that of lipoxygenase indicates very different mechanism of hydrogen transfer.

| S-EPMC3049311 | biostudies-literature

Polymorphisms in prostaglandin synthase 2/cyclooxygenase 2 (PTGS2/COX2) and risk of colorectal cancer.

Project description:Inflammation plays a key role in the development of colorectal cancers. We have investigated the relationship between PTGS2 (COX2) polymorphisms and colorectal cancer risk in a hospital based case-control study. We recruited 292 patients with colorectal cancer and 274 controls from new patients admitted to Bellvitge Hospital, Barcelona, Spain, from 1996 to 1998. Subjects responded to a questionnaire on risk factors. Genotypes of the eight more frequent polymorphisms of PTGS2 were determined. Two polymorphisms are located in the promoter sequence, one in the untranslated region of exon 1, one in exon 3, one in intron 5, two in the untranslated region of exon 10, and one downstream of the last polyadenylation (poly-A) signal. Associations were analysed with logistic regression models assuming a dominant effect for rare variants to increase statistical power. An association was detected between colorectal cancer and a polymorphism in the untranslated region of exon 10 of PTGS2, with an odds ratio (OR) of 2.49, 95% confidence interval (CI) of 1.17-5.32, P=0.01. A nearby polymorphism downstream of the last poly-A signal also showed a nonsignificant increase in risk (OR 2.17, 95% CI 0.99-4.78, P=0.05). Analysis of haplotypes confirmed that individuals with these variants were at increased risk of colorectal cancer (OR compared to the most frequent haplotype: 2.17, 95% CI 0.97-4.84, P=0.06) Interactions between PTGS2 genotype and use of nonsteroidal anti-inflammatory drugs and risk of colorectal cancer were also explored.

| S-EPMC2409800 | biostudies-literature

Radical intermediates in photoinduced reactions on TiO2 (an EPR spin trapping study).

Project description:The radical intermediates formed upon UVA irradiation of titanium dioxide suspensions in aqueous and non-aqueous environments were investigated applying the EPR spin trapping technique. The results showed that the generation of reactive species and their consecutive reactions are influenced by the solvent properties (e.g., polarity, solubility of molecular oxygen, rate constant for the reaction of hydroxyl radicals with the solvent). The formation of hydroxyl radicals, evidenced as the corresponding spin-adducts, dominated in the irradiated TiO2 aqueous suspensions. The addition of 17O-enriched water caused changes in the EPR spectra reflecting the interaction of an unpaired electron with the 17O nucleus. The photoexcitation of TiO2 in non-aqueous solvents (dimethylsulfoxide, acetonitrile, methanol and ethanol) in the presence of 5,5-dimethyl-1-pyrroline N-oxide spin trap displayed a stabilization of the superoxide radical anions generated via electron transfer reaction to molecular oxygen, and various oxygen- and carbon-centered radicals from the solvents were generated. The character and origin of the carbon-centered spin-adducts was confirmed using nitroso spin trapping agents.

| S-EPMC6271711 | biostudies-literature

Polymorphic human prostaglandin H synthase-2 proteins and their interactions with cyclooxygenase substrates and inhibitors.

Project description:The cyclooxygenase (COX) activity of prostaglandin H synthase-2 (PGHS-2) is implicated in colorectal cancer and is targeted by nonsteroidal anti-inflammatory drugs (NSAIDs) and dietary n-3 fatty acids. We used purified, recombinant proteins to evaluate the functional impacts of the R228H, E488G, V511A and G587R PGHS-2 polymorphisms on COX activity, fatty acid selectivity and NSAID actions. Compared to wild-type PGHS-2, COX activity with arachidonate was ?20% lower in 488G and ?20% higher in 511A. All variants showed time-dependent inhibition by the COX-2-specific inhibitor (coxib) nimesulide, but 488G and 511A had 30-60% higher residual COX activity; 511A also showed up to 70% higher residual activity with other time-dependent inhibitors. In addition, 488G and 511A differed significantly from wild type in Vmax values with the two fatty acids: 488G showed ?20% less and 511A showed ?20% more discrimination against eicosapentaenoic acid. The Vmax value for eicosapentaenoate was not affected in 228H or 587R, nor were the Km values or the COX activation efficiency (with arachidonate) significantly altered in any variant. Thus, the E488G and V511A PGHS-2 polymorphisms may predict who will most likely benefit from interventions with some NSAIDs or n-3 fatty acids.

| S-EPMC4465550 | biostudies-literature

Haplotype of prostaglandin synthase 2/cyclooxygenase 2 is involved in the susceptibility to inflammatory bowel disease.

Project description:AimProstaglandin G/H synthase 2 (PTGS2 or COX2) is one of the key factors in the cellular response to inflammation. PTGS2 is expressed in the affected intestinal segments of patients with inflammatory bowel diseases (IBD). In IBD patients, non-steroidal anti-inflammatory drugs, which have been shown to reduce both the production and activity of PTGS2, may activate IBD and aggravate the symptoms. We aimed at examining genetic variants of PTGS2 that may be risk factors for IBD.MethodsWe have genotyped 291 individuals diagnosed with IBD and 367 controls from the Dutch population for the five most frequent polymorphisms of the PTGS2 gene. Clinical data were collected on all patients. DNA was extracted via normal laboratory methods. Genotyping was carried out using multiplex PCR followed by the Invader Assay and the 5' exonuclease assay (TaqMan). New polymorphism screening was performed by pre-screening with denaturing high-performance liquid chromatography, followed by fluorescent sequencing.ResultsAllele 5209G was weakly associated with Crohn's disease (odds ratio (OR) 1.63, 95% confidence interval (CI) 1.03-2.57), and allele 8473T with ulcerative colitis (OR 1.50, 95%CI 1.00-2.27). The haplotype including both alleles showed a strong association with IBD (OR 13.15, 95%CI 3.17-116.15). This haplotype, while rare (-0.3%) in the general population, is found more frequently in the patients (3.5%).ConclusionOur data suggest that this haplotype of PTGS2 contributes to the susceptibility of IBD.

| S-EPMC4436724 | biostudies-literature

Prognostic Value of Prostaglandin-endoperoxide Synthase 2 Polymorphisms in Prostate Cancer Recurrence after Radical Prostatectomy.

Project description:UnlabelledBackgroud: Increasing evidence suggests the involvement of chronic inflammation in the progression of prostate cancer, and prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase-2, catalyzes the rate-limiting steps of the pathway. We hypothesized that genetic variants of PTGS2 can influence the outcome of prostate cancer patients.MethodsWe genotyped five haplotype-tagging single-nucleotide polymorphisms (SNPs) to detect common genetic variations across the PTGS2 region in 458 prostate cancer patients treated with radical prostatectomy.ResultsOne SNP, rs4648302, was associated with disease recurrence. Five-year recurrence-free survival rate increased according to the number of variant alleles inherited (55.6%, 70.7%, and 100.0% for patients with different genotypes; P = 0.037), and the effect was maintained in multivariable analysis. Public dataset analyses also suggested that PTGS2 expression was correlated with prostate cancer prognosis.ConclusionOur results indicated that PTGS2 could be a potential prognostic marker to improve the prediction of disease recurrence in prostate cancer patients.

| S-EPMC5027188 | biostudies-literature

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