Small-wedge synchrotron and serial XFEL datasets for Cysteinyl leukotriene GPCRs.

Structural studies of challenging targets such as G protein-coupled receptors (GPCRs) have accelerated during the last several years due to the development of new approaches, including small-wedge and serial crystallography. Here, we describe the deposition of seven datasets consisting of X-ray diffraction images acquired from lipidic cubic phase (LCP) grown microcrystals of two human GPCRs, Cysteinyl leukotriene receptors 1 and 2 (CysLT1R and CysLT2R), in complex with various antagonists. Five datasets were collected using small-wedge synchrotron crystallography (SWSX) at the European Synchrotron Radiation Facility with multiple crystals under cryo-conditions. Two datasets were collected using X-ray free electron laser (XFEL) serial femtosecond crystallography (SFX) at the Linac Coherent Light Source, with microcrystals delivered at room temperature into the beam within LCP matrix by a viscous media microextrusion injector. All seven datasets have been deposited in the open-access databases Zenodo and CXIDB. Here, we describe sample preparation and annotate crystallization conditions for each partial and full datasets. We also document full processing pipelines and provide wrapper scripts for SWSX and SFX data processing.

Development and Characterization of an Allergic Asthma Rat Model for Interventional Studies.

Allergic asthma is one of the most common chronic diseases of the airways, however it still remains underdiagnosed and hence undertreated. Therefore, an allergic asthma rat model would be useful to be applied in future therapeutic strategy studies. The aim of the present study was to develop an objective model of allergic asthma in atopic rats that allows the induction and quantification of anaphylactic shock with quantitative variables. Female Brown Norway rats were intraperitoneally sensitized with ovalbumin (OVA), alum and Bordetella pertussis toxin and boosted a week later with OVA in alum. At day 28, all rats received an intranasal challenge with OVA. Anaphylactic response was accurately assessed by changes in motor activity and body temperature. Leukotriene concentration was determined in the bronchoalveolar lavage fluid (BALF), and total and IgE anti-OVA antibodies were quantified in blood and BALF samples. The asthmatic animals' motility and body temperature were reduced after the shock for at least 20 h. The asthmatic animals developed anti-OVA IgE antibodies both in BALF and in serum. These results show an effective and relatively rapid model of allergic asthma in female Brown Norway rats that allows the quantification of the anaphylactic response.

Roles of cysteinyl leukotrienes and their receptors in immune cell-related functions.

The cysteinyl leukotrienes (cys-LTs), leukotriene C4, (LTC4), LTD4, and LTE4, are lipid mediators of inflammation. LTC4 is the only intracellularly synthesized cys-LT through the 5-lipoxygenase and LTC4 synthase pathway and after transport is metabolized to LTD4 and LTE4 by specific extracellular peptidases. Each cys-LT has a preferred functional receptor in vivo; LTD4 to the type 1 cys-LT receptor (CysLT1R), LTC4 to CysLT2R, and LTE4 to CysLT3R (OXGR1 or GPR99). Recent studies in mouse models revealed that there are multiple regulatory mechanisms for these receptor functions and each receptor plays a distinct role as observed in different mouse models of inflammation and immune responses. This review focuses on the integrated host responses to the cys-LT/CysLTR pathway composed of sequential ligands with preferred receptors as seen from mouse models. It also discusses potential therapeutic targets for LTC4 synthase, CysLT2R, and CysLT3R.

Structural and functional insights into the reaction specificity of catalase-related hydroperoxide lyase: A shift from lyase activity to allene oxide synthase by site-directed mutagenesis.

Two highly identical fusion proteins, an allene oxide synthase-lipoxygenase (AOS-LOX) and a hydroperoxide lyase-lipoxygenase (HPL-LOX), were identified in the soft coral Capnella imbricata. Both enzymes initially catalyze the formation of 8R-hydroperoxy-eicosatetraenoic acid (8R-HpETE) from arachidonic acid by the C-terminal lipoxygenase (LOX) domain. Despite the fact that the defined catalytically important residues of N-terminal catalase-related allene oxide synthase (cAOS) domain are also conserved in C. imbricata hydroperoxide lyase (cHPL), their reaction specificities differ. In the present study, we tested which of the amino acid substitutions around the active site of cHPL are responsible for a control in the reaction specificity. The possible candidates were determined via comparative sequence and structural analysis of the substrate channel and the heme region of coral cAOSs and C. imbricata cHPL. The amino acid replacements in cHPL-R56G, ME59-60LK, P65A, F150L, YS176-177NL, I357V, and SSSAGE155-160PVKEGD-with the corresponding residues of cAOS were conducted by site-directed mutagenesis. Although all these mutations influenced the catalytic efficiency of cHPL, only F150L and YS176-177NL substitutions caused a shift in the reaction specificity from HPL to AOS. The docking analysis of P. homomalla cAOS with 8R-HpETE substrate revealed that the Leu150 of cAOS interacts with the C5-C6 double bond and the Leu177 with the hydrophobic tail of 8R-HpETE. We propose that the corresponding residues in cHPL, Phe150 and Ser177, are involved in a proper coordination of the epoxy allylic radical intermediate necessary for aldehyde formation in the hydroperoxide lyase reaction.

Polypharmacology of Small-Molecule Modulators of the 5-Lipoxygenase Activating Protein (FLAP) Observed via a High-throughput Lipidomics Platform.

Leukotrienes (LTs) and related species are proinflammatory lipid mediators derived from arachidonic acid (AA) that have pathological roles in autoimmune and inflammatory conditions, cardiovascular diseases, and cancer. 5-Lipoxygenase activating protein (FLAP) plays a critical accessory role in the conversion of AA to LTA4, and its subsequent conversion to LTC4 by LTC4 synthase. Pharmacological inhibition of FLAP results in a loss of LT production by preventing the biosynthesis of both LTB4 and LTC4, making it an attractive target for the treatment of inflammatory diseases in which LTs likely play a role. Small-molecule (SM) drugs often exhibit polypharmacology through various pathways, which may explain the differential therapeutic efficacies of compounds sharing structural similarity. We have profiled a series of SM FLAP modulators for their selectivity across enzymes of AA cascade in human whole blood (HWB), using a recently developed LC/MS (liquid chromatography-mass spectrometry)-based high-throughput lipidomics platform that monitors 122 eicosanoids in multiplex. Highly efficient data acquisition coupled with fast and accurate data analysis allowed facile compound profiling from ex vivo study samples. This platform allowed us to quantitatively map the effects of those SMs on the entire AA cascade, demonstrating its potential to discriminate structurally related compounds.

Differential mobility separation of leukotrienes and protectins.

Differential mobility spectrometry (DMS) is capable of separating stereoisomeric molecular ions based on their mobility in an oscillating electrical field with an asymmetric waveform. Thus, it is an "orthogonal" technique to chromatography and (tandem) mass spectrometry. Bioactive lipids, particularly of the eicosanoid and docosanoid class feature numerous stereoisomers, which exhibit a highly specific structure-activity relationship. Moreover, the geometry of these compounds also reflects their biochemical origin. Therefore, the unambiguous characterization of related isomers of the eicosanoid and docosanoid classes is of fundamental importance to the understanding of their origin and function in many biological processes. Here we show, that SelexION DMS technology coupled to µLC-MS/MS is capable of differentiating at least five closely related leukotrienes partially coeluting and (almost) unresolvable using LC-MS/MS only. We applied the developed method to the separation of LTB4 and its coeluting isomer 5S,12S-diHETE in murine peritoneal exudate cells, showing that LTB4 is present only after zymosan A injection while its isomer 5S,12S-diHETE is produced after saline (PBS) administration. Additionally, we show that the SelexION technology can also be applied to the separation of PD1 and PDX (10S,17S-diHDHA), two isomeric protectins.

Pathophysiology of the hepoxilins.

There is increasing evidence from various scientific groups that hepoxilins represent novel inflammatory mediators. In vitro studies have shown that the hepoxilins cause mobilization of intracellular calcium in human neutrophils, cause plasma leakage, and potently stimulate chemotaxis of human neutrophils. In vivo, the hepoxilin pathway is activated in conditions of inflammation, e.g. after pathogen infection, in inflamed conditions (psoriasis, arthritis), and hepoxilins promote inflammatory hyperalgesia and allodynia. Although much work has demonstrated an effect of hepoxilins on neutrophils, the hepoxilin pathway has been demonstrated in a variety of tissues, including the lung, brain, pituitary, pancreatic islets, skin, etc. A genetic defect linked to a deficiency in hepoxilin formation has been described and believed to be responsible for the scaly skin observed in ichthyosis. Despite their biological and chemical instability, the involvement of the hepoxilin pathway in pathology has been demonstrated in vitro and in vivo through either isolation of the hepoxilins themselves (or their metabolites) or implied through the use of stable hepoxilin analogs. These analogs have additionally shown efficacy in animal models of lung fibrosis, cancer, thrombosis and diabetes. Research on these compounds has merely scratched the surface, but results published to date have suggested that the hepoxilin pathway is a distinct and novel pathway leading to inflammation and hepoxilin antagonists may provide the means of controlling early aspects of the acute inflammatory phase. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

The hydroperoxide moiety of aliphatic lipid hydroperoxides is not affected by hypochlorous acid.

The oxidation of polyunsaturated fatty acids to the corresponding hydroperoxide by plant and animal lipoxygenases is an important step for the generation of bioactive lipid mediators. Thereby fatty acid hydroperoxide represent a common intermediate, also in human innate immune cells, like neutrophil granulocytes. In these cells a further key component is the heme protein myeloperoxidase producing HOCl as a reactive oxidant. On the basis of different investigation a reaction of the fatty acid hydroperoxide and hypochlorous acid (HOCl) could be assumed. Here, chromatographic and spectrometric analysis revealed that the hydroperoxide moiety of 15S-​hydroperoxy-​5Z,​8Z,​11Z,​13E-​eicosatetraenoic acid (15-HpETE) and 13S-​hydroperoxy-​9Z,​11E-​octadecadienoic acid (13-HpODE) is not affected by HOCl. No reduction of the hydroperoxide group due to a reaction with HOCl could be measured. It could be demonstrated that the double bonds of the fatty acid hydroperoxides are the major target of HOCl, present either as reagent or formed by the myeloperoxidase-hydrogen peroxide-chloride system.

ATP allosterically activates the human 5-lipoxygenase molecular mechanism of arachidonic acid and 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid.

5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid. This work investigates the kinetic mechanism of these two processes and the role of ATP in their activation. Specifically, it was determined that epoxidation of 5(S)-HpETE (dehydration of the hydroperoxide) has a rate of substrate capture (Vmax/Km) significantly lower than that of AA hydroperoxidation (oxidation of AA to form the hydroperoxide); however, hyperbolic kinetic parameters for ATP activation indicate a similar activation for AA and 5(S)-HpETE. Solvent isotope effect results for both hydroperoxidation and epoxidation indicate that a specific step in its molecular mechanism is changed, possibly because of a lowering of the dependence of the rate-limiting step on hydrogen atom abstraction and an increase in the dependency on hydrogen bond rearrangement. Therefore, changes in ATP concentration in the cell could affect the production of 5-LOX products, such as leukotrienes and lipoxins, and thus have wide implications for the regulation of cellular inflammation.

Anti-inflammatory potential of alpha-linolenic acid mediated through selective COX inhibition: computational and experimental data.

The present work investigates the anti-inflammatory activity of alpha-linolenic acid (ALA) and linoleic acid (LA) using computational and experimental analysis. The binding affinity of ALA and LA was appraised for cyclooxygenase 1 (COX-1), cyclooxygenase 2 (COX-2), and 5-lipoxygenase (5-LOX) using AutoDock 4.2 and AutoDock Vina 1.1.2. Anti-inflammatory activity of ALA (2 and 4 ml/kg, i.p.) (55.65 % v/v) and LA (2 and 4 ml/kg, i.p.) (55 % v/v) was further assayed using the rat paw edema test against a variety of phlogistic agents including carrageenan, arachidonic acid, prostaglandin, and leukotriene, respectively. ALA (2 and 4 ml/kg, i.p.) and LA (2 and 4 ml/kg, i.p.) were further tested for their efficacy against complete Freund's adjuvant (CFA)-induced (0.05 ml) arthritis in albino rats. Following CFA-induced arthritis, ALA and LA were tested for their inhibitory proficiency against COX-1, COX-2, and 5-LOX in vitro. The present study commends that the anti-inflammatory potential of ALA could be attributed to COX inhibition, in particular, COX-2.

Arachidonate 15-lipoxygenase enzyme products increase platelet aggregation and thrombin generation.

Atherosclerotic cardiovascular diseases are the leading causes of morbidity and mortality worldwide. We have previously shown that arachidonate 15-lipoxygenase B (ALOX15B) is highly expressed in atherosclerotic carotid plaques, and elucidation of mechanisms downstream of activated lipoxygenases may be relevant to our understanding of the genesis of atherosclerotic diseases. We examined 120 carotid plaques from patients with symptomatic carotid artery stenosis and showed that the extent of ALOX15B staining was significantly increased in carotid plaques with thrombosis. Impedance aggregometry analyses showed that the ALOX15B enzyme products 15-HETE and 15-HPETE increased platelet aggregation. By using a calibrated automatic thrombin assay, we showed that the ALOX15B products also increased both peak levels of thrombin and the total endogenous thrombin potential. Moreover, platelet aggregation was increased by addition of cell lysates from ischemic human macrophages, whereas platelet aggregation was reduced after knockdown of ALOX15B in human macrophages. Our data show that ALOX15B expression in human carotid plaques is associated with thrombus formation and that enzyme products of ALOX15B increase platelet aggregation and thrombin generation. We therefore propose that activated ALOX15B in macrophages may play a role in the induction of atherothrombotic events by increasing platelet aggregation and thrombin generation.

Increased cysteinyl leukotriene concentration and receptor expression in tonsillar tissues of Chinese children with sleep-disordered breathing.

BACKGROUND: Evidence suggests that increased leukotriene level and receptor protein expression emerged in the adenotonsillar tissue of children with sleep-disordered breathing (SDB). Nevertheless, few studies have investigated the contribution of disease severity in the cysteinyl leukotriene (CysLT) production and cysteinyl leukotriene receptor (CysLTR) expression. Furthermore, the relationship between local upper-airway and systemic inflammatory responses remains undefined. METHODS: A prospective, observational study that included standard questionnaires, physical examinations and overnight polysomnography. CysLTs were determined from serum, urine and tonsillar homogenate supernatant by means of enzyme-linked immunosorbent assays. The protein expressions of CysLTR were measured using Western blot analysis. RESULTS: Children with SDB had increased intratonsillar CysLT levels as well as CysLT subtype 1 receptor (CysLT1-R) and CysLT subtype 2 receptor (CysLT2-R) protein expression than the control group. CysLT concentration was positively correlated with body mass index z-score and apnea-hypopnea index (r=0.454 and 0.487, p<0.001 respectively), and negatively correlated with pulse oximetric saturation nadir (r=-0.518, p<0.001). Upper-airway intratonsillar CysLT production was positively correlated with systemic production (vs. urinary LTE4: r=0.456, p<0.001; vs. serum CysLTs: r=0.440, p<0.001). Immunoblots showed that CysLT1-R protein expressions were modestly higher in the severe group when compared to the mild group. In contrast, there were no differences in CysLT2-R protein appearing among the SDB subgroups. CONCLUSIONS: Increased CysLT level and receptor expression in upper-airway tonsillar tissues are related to disease severity in SDB children. The local and systemic CysLT production were positively correlated.

Quantitative measurement of cysteinyl leukotrienes and leukotriene B4 in human sputum using ultra high pressure liquid chromatography-tandem mass spectrometry.

The role of leukotrienes (LTs) in airway inflammatory diseases, such as asthma, has been extensively reported. The measurement of LTs in sputum supernatants, which is commonly done via enzyme immunoassays (EIAs), may prove to be useful for assessing airway inflammation. Despite the many advantages of EIA, these methods suffer from a lack of selectivity. Therefore, a selective and reliable method for the analysis of LTs in human sputum is needed. In this study we developed and validated a sensitive and specific method using ultra high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), to measure simultaneously cysteinyl leukotrienes (CysLTs) and leukotriene B4 (LTB4) in human sputum. Sputum supernatants obtained by ultracentrifugation were stabilized by protease inhibitors, spiked with stable isotopic internal standards, and subjected to solid phase extraction (SPE) and UHPLC separation. Multiple reaction monitoring (MRM) transitions were optimized and measured on a mass spectrometer. The limit of detection (LOD) for LTE4 and LTB4 was 9.8 and 19.5 pg/mL, respectively. The lower limit of quantitation (LLOQ) for LTE4 and LTB4 was 19.5 and 39.0 pg/mL, respectively. The dynamic range of the LTE4 assay was from 9.8 to 5000 pg/mL, whereas for the LTB4 assay was from 19.5 to 10,000 pg/mL. The intra- and inter-day % coefficient of variation (%CV) was <6.5% and <10%, for both LTE4 and LTB4, respectively. Spike recovery ranged from 105% to 111% for both analytes. In addition, twenty-two sputum samples were analyzed for cysLTs and LTB4. Fourteen of these samples were purchased commercially and eight were collected during the course of a clinical trial. LTB4 was detectable in all samples tested and it ranged from 79 to 7220 pg/mL. LTE4 was detectable in most of the sputum samples (12.3-891 pg/mL), whereas LTC4 and LTD4 were below limit of detection for majority of sputum samples. The in vitro conversion of LTC4 and LTD4 into LTE4 was observed. The measurement of LTB4 was sensitive to low pH and high temperature. The use of UHPLC-MS/MS method will allow a more accurate and reliable quantitation of LTs in human sputum, which in turn, may lead to a better understanding of the role of LTs in airway disease pathways and the application in associated clinical treatments.

Cysteinyl-leukotrienes induce vascular endothelial growth factor production in human monocytes and bronchial smooth muscle cells.

BACKGROUND: Cysteinyl leukotrienes (cysLTs) are suggested to be implicated in the process of airway remodelling in asthma. OBJECTIVE: We investigated the potential for cysLTs to modulate vascular endothelial growth factor (VEGF) expression, a growth factor involved in the angiogenesis of airway remodelling. METHODS: VEGF mRNA and protein were quantified by real-time PCR and ELISA, respectively. VEGF promoter activation was assessed using luciferase gene-tagged promoter constructs. RESULTS: We found that LTD(4) induction of VEGF in human monocytes and bronchial smooth muscle cells is cysLT1 dependent. Stimulation of HEK293 cells stably expressing cysLT1 or cysLT2 with cysLTs showed a concentration-dependent activation of the VEGF promoter and a time-dependent increase in VEGF mRNA and protein. For the cysLT1-mediated response, mutations of hypoxia-induced factor-1 (HIF-1) sites failed to reduce cysLT-induced VEGF promoter activation and 5' deletions showed that the proximal region containing one AP-1 and four specificity protein 1 (Sp1) sites was necessary. Pretreatment with inhibitors of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), but not p38, and an overexpression of dominant negative forms of c-Jun, c-Fos or Ras suggested the implication of mitogen-activated protein kinases and AP-1. Mutation of the AP-1-binding element failed to prevent VEGF transactivation suggesting that AP-1 might not act directly on the promoter. Moreover, inhibition of Sp1-dependent transcription by mithramycin completely inhibited VEGF promoter transactivation and VEGF mRNA expression by LTD(4) . Finally, mutations of Sp1 binding elements prevented VEGF promoter transactivation. CONCLUSION AND CLINICAL RELEVANCE: Our data indicate for the first time that cysLTs can transcriptionally activate VEGF production via cysLT1 receptors, with the involvement of JNK, ERK, the AP-1 complex and Sp1. These findings suggest that cysLTs may be important in the angiogenic process of airway remodelling and potentially provide a previously unknown benefit of using cysLT1 receptor antagonists in the prevention or treatment of airway remodelling in asthma.

Eosinophil cysteinyl leukotriene synthesis mediated by exogenous secreted phospholipase A2 group X.

Secreted phospholipase A(2) group X (sPLA(2)-X) has recently been identified in the airways of patients with asthma and may participate in cysteinyl leukotriene (CysLT; C(4), D(4), and E(4)) synthesis. We examined CysLT synthesis and arachidonic acid (AA) and lysophospholipid release by eosinophils mediated by recombinant human sPLA(2)-X. We found that recombinant sPLA(2)-X caused marked AA release and a rapid onset of CysLT synthesis in human eosinophils that was blocked by a selective sPLA(2)-X inhibitor. Exogenous sPLA(2)-X released lysophospholipid species that arise from phospholipids enriched in AA in eosinophils, including phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine as well as plasmenyl phosphatidylcholine and phosphatidylethanolamine. CysLT synthesis mediated by sPLA(2)-X but not AA release could be suppressed by inhibition of cPLA(2)α. Exogenous sPLA(2)-X initiated Ser(505) phosphorylation of cPLA(2)α, an intracellular Ca(2+) flux, and translocation of cPLA(2)α and 5-lipoxygenase in eosinophils. Synthesis of CysLTs in response to sPLA(2)-X or lysophosphatidylcholine was inhibited by p38 or JNK inhibitors but not by a MEK 1/2 inhibitor. A further increase in CysLT synthesis was induced by the addition of sPLA(2)-X to eosinophils under conditions of N-formyl-methionyl-leucyl-phenylalanine-mediated cPLA(2)α activation. These results indicate that sPLA(2)-X participates in AA and lysophospholipid release, resulting in CysLT synthesis in eosinophils through a mechanism involving p38 and JNK MAPK, cPLA(2)α, and 5-lipoxygenase activation and resulting in the amplification of CysLT synthesis during cPLA(2)α activation. Transactivation of eosinophils by sPLA(2)-X may be an important mechanism leading to CysLT formation in the airways of patients with asthma.

Leukotrienes produced in allergic lung inflammation activate alveolar macrophages.

It has been well-documented that leukotrienes (LTs) are released in allergic lung inflammation and that they participate in the physiopathology of asthma. A role for LTs in innate immunity has recently emerged: Cys-LTs were shown to enhance FcgammaR-mediated phagocytosis by alveolar macrophages (AMs). Thus, using a rat model of asthma, we evaluated FcgammaR-mediated phagocytosis and killing of Klebsiella pneumoniae by AMs. The effect of treatment with a cys-LT antagonist (montelukast) on macrophage function was also investigated. Male Wistar rats were immunized twice with OVA/alumen intraperitoneally and challenged with OVA aerosol. After 24 h, the animals were killed, and the AMs were obtained by bronchoalveolar lavage. Macrophages were cultured with IgG-opsonized red blood cells (50:1) or IgG-opsonized K. pneumoniae (30:1), and phagocytosis or killing was evaluated. Leukotriene C(4) and nitric oxide were quantified by the EIA and Griess methods, respectively. The results showed that AMs from sensitized and challenged rats presented a markedly increased phagocytic capacity via FcgammaR (10X compared to controls) and enhanced killing of K. pneumoniae (4X higher than controls). The increased phagocytosis was inhibited 15X and killing 3X by treatment of the rats with montelukast, as compared to the non-treated group. cys-LT addition increased phagocytosis in control AMs but had no effect on macrophages from allergic lungs. Montelukast reduced nitric oxide (39%) and LTC(4) (73%). These results suggest that LTs produced during allergic lung inflammation potentiate the capacity of AMs to phagocytose and kill K. pneumonia via FcgammaR.

Algal pheromone biosynthesis: stereochemical analysis and mechanistic implications in gametes of Ectocarpus siliculosus.

During sexual reproduction, female gametes or eggs of brown algae release pheromones to attract their male mating partners. The biologically active compounds comprise linear or alicyclic unsaturated hydrocarbons derived from the aliphatic terminus of C(20) polyunsaturated fatty acids (PUFAs) by oxidative cleavage. The current study addresses the stereochemical course of the pheromone biosynthesis using female gametes of the marine brown alga E. siliculosus and chiral deuterium-labeled arachidonic acids. The biosynthetic sequence is likely to proceed via an intermediary 9-hydroperoxyarachidonic acid, which is cleaved with loss of the C(16)-H(R) into the C(11)-hydrocarbon dictyopterene C and 9-oxonona-(5Z,7E)-dienoic acid.

Mechanistic investigations of human reticulocyte 15- and platelet 12-lipoxygenases with arachidonic acid.

Human reticulocyte 15-lipoxygenase-1 (15-hLO-1) and human platelet 12-lipoxygenase (12-hLO) have been implicated in a number of diseases, with differences in their relative activity potentially playing a central role. In this work, we characterize the catalytic mechanism of these two enzymes with arachidonic acid (AA) as the substrate. Using variable-temperature kinetic isotope effects (KIE) and solvent isotope effects (SIE), we demonstrate that both k(cat)/K(M) and k(cat) for 15-hLO-1 and 12-hLO involve multiple rate-limiting steps that include a solvent-dependent step and hydrogen atom abstraction. A relatively low k(cat)/K(M) KIE of 8 was determined for 15-hLO-1, which increases to 18 upon the addition of the allosteric effector molecule, 12-hydroxyeicosatetraenoic acid (12-HETE), indicating a tunneling mechanism. Furthermore, the addition of 12-HETE lowers the observed k(cat)/K(M) SIE from 2.2 to 1.4, indicating that the rate-limiting contribution from a solvent sensitive step in the reaction mechanism of 15-hLO-1 has decreased, with a concomitant increase in the C-H bond abstraction contribution. Finally, the allosteric binding of 12-HETE to 15-hLO-1 decreases the K(M)[O(2)] for AA to 15 microM but increases the K(M)[O(2)] for linoleic acid (LA) to 22 microM, such that the k(cat)/K(M)[O(2)] values become similar for both substrates (approximately 0.3 s(-1) microM(-1)). Considering that the oxygen concentration in cancerous tissue can be less than 5 microM, this result may have cellular implications with respect to the substrate specificity of 15-hLO-1.

Sialic acid attenuates the cytotoxicity of the lipid hydroperoxides HpODE and HpETE.

Reduction of peroxide molecular species is an essential function in living organisms. In previous studies, we proposed a new function for the sialic acid N-acetylneuraminic acid (Neu5Ac)--that of antioxidant/hydrogen peroxide scavenging agent. On the basis of the reaction scheme, Neu5Ac is thought to act as a general antioxidant of all hydroperoxide-type species (R-OOHs). The concentration of tert-butyl hydroperoxide (t-BuOOH) decreased after co-incubation with N-acetylneuraminic acid. Neu5Ac also decreased the R-OOH concentration in solutions of peroxylinolenic acid (13(S)-hydroperoxy-(9Z,11E)-octadecadienoic acid, HpODE) and peroxyarachidonic acid (15(S)-hydroperoxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid, HpETE)--two lipid hydroperoxides that participate in many physiological events. Moreover, the cytotoxicity of both these lipid hydroperoxides was attenuated by reaction with Neu5Ac acid. Our results suggest that N-acetylneuraminic acid is a potential antioxidant of most hydroperoxides that accumulate in organisms.

Effects of (15S)-hydroperoxyeicosatetraenoic acid and (15S)-hydroxyeicosatetraenoic acid on the acute- lymphoblastic-leukaemia cell line Jurkat: activation of the Fas-mediated death pathway.

The antiproliferative effects of 15-LOX (15-lipoxygenase) metabolites of arachidonic acid {(15S)-HPETE [(15S)-hydroperoxyeicosatetraenoic acid] and (15S)-HETE [(15S)-hydroxyeicosatetraenoic acid]} and the mechanism(s) involved were studied in the human T-cell leukaemia cell line Jurkat. (15S)-HPETE, the hydroperoxy metabolite of 15-LOX, inhibited the growth of Jurkat cells 3 h after exposure and with an IC(50) value of 10 microM. The hydroxy metabolite of 15-LOX, (15S)-HETE, on the other hand, inhibited the growth of Jurkat cells after 6 h of exposure and with an IC(50) value of 40 microM. The cells exposed to 10 microM (15S)-HPETE for 3 h or to 40 microM (15S)-HETE for 6 h showed increased expression of Fas ligand and FADD (Fas-associated death domain), caspase 8 activation, Bid (BH3-interacting domain death agonist) cleavage, decrease in mitochondrial membrane potential, cytochrome c release, caspase 3 activation, PARP-1 [poly(ADP-ribose) polymerase-1] cleavage and DNA fragmentation, suggesting the involvement of both extrinsic and intrinsic death pathways. Further studies on ROS (reactive oxygen species) generation revealed the involvement of NADPH oxidase. In conclusion, the present study indicates that NADPH oxidase-induced ROS generation activates the Fas-mediated death pathway.