The lipoxygenase (LOX) cascade is a source of bioactive oxylipins that play a regulatory role in plants, animals, and fungi. Soybean (Glycine max (L.) Merr.) LOXs are the classical models for LOX research. Progress in genomics has uncovered a large diversity of GmLOX isoenzymes. Most of them await biochemical investigations. The catalytic properties of recombinant soybean LOX2 (GmLOX2) are described in the present work. The GmLOX2 gene has been cloned before, but only for nucleotide sequencing, while the recombinant protein was not prepared and studied. In the present work, the recombinant GmLOX2 behavior towards linoleic, α-linolenic, eicosatetraenoic (20:4), eicosapentaenoic (20:5), and hexadecatrienoic (16:3) acids was examined. Linoleic acid was a preferred substrate. Oxidation of linoleic acid afforded 94% optically pure (13S)-hydroperoxide and 6% racemic 9-hydroperoxide. GmLOX2 was less active on other substrates but possessed an even higher degree of regio- and stereospecificity. For example, it converted α-linolenic acid into (13S)-hydroperoxide at about 98% yield. GmLOX2 showed similar specificity towards other substrates, producing (15S)-hydroperoxides (with 20:4 and 20:5) or (11S)-hydroperoxide (with 16:3). Thus, the obtained data demonstrate that soybean GmLOX2 is a specific (13S)-LOX. Overall, the catalytic properties of GmLOX2 are quite similar to those of GmLOX1, but pH is optimum.

Avenanthramides (AVAs) present in oats are amides of anthranilic and cinnamic acids. AVAs are potent antioxidants and have anti-inflammatory properties. There are various potential mechanisms for their anti-inflammatory effects, including inhibition of lipoxygenases (LOX), which catalyse oxygenation of polyunsaturated fatty acids into potent signal molecules involved in inflammatory processes. In this study, AVAs were screened for LOX inhibition in vitro and structure-activity relationships were examined. Twelve different AVAs at 0.6 mM were tested as LOX inhibitors. The corresponding free cinnamic acids, the AVA analogue Tranilast® and the known LOX inhibitor trans-resveratrol were included for comparison. It was found that AVAs comprising caffeic or sinapic acid exhibited significant lipoxygenase inhibition (60-90%) (P < 0.05), whereas low or no inhibition was observed with AVAs containing p-coumaric or ferulic acid. No difference in inhibition was seen on comparing AVAs with their free corresponding cinnamic acids, which implies that the anthranilic acid part of the avenanthramide molecule does not affect inhibition. Trans-resveratrol showed inhibition, whereas no inhibition was seen for Tranilast® at the concentrations used in this study. This study suggests that aventahtramides comprising caffeic acid or sinapic acid partly exert their antioxidant and anti-inflammatory effects via lipoxygenase inhibition.

Post-traumatic lung injury following trauma and hemorrhagic shock (T/HS) is associated with significant morbidity. Leukotriene-induced inflammation has been implicated in the development of post-traumatic lung injury through a mechanism that is only partially understood. Postshock mesenteric lymph returning to the systemic circulation is rich in arachidonic acid, the substrate of 5-lipoxygenase (ALOX5). ALOX5 is the rate-limiting enzyme in leukotriene synthesis and, following T/HS, contributes to the development of lung dysfunction. ALOX5 colocalizes with its cofactor, 5-lipoxygenase-activating protein (ALOX5AP), which is thought to potentiate ALOX5 synthetic activity. We hypothesized that T/HS results in the molecular association and nuclear colocalization of ALOX5 and ALOX5AP, which ultimately increases leukotriene production and potentiates lung injury.

12R-lipoxygenase (12R-LOX) and the epidermal LOX-3 (eLOX-3) constitute a novel LOX pathway involved in terminal differentiation in skin. This view is supported by recent studies showing that inactivating mutations in 12R-LOX and eLOX-3 are linked to the development of autosomal recessive congenital ichthyosis. We show that 12R-LOX deficiency in mice results in a severe impairment of skin barrier function. Loss of barrier function occurs without alterations in proliferation and stratified organization of the keratinocytes, but is associated with ultrastructural anomalies in the upper granular layer, suggesting perturbance of the assembly/extrusion of lamellar bodies. Cornified envelopes from skin of 12R-LOX-deficient mice show increased fragility. Lipid analysis demonstrates a disordered composition of ceramides, in particular a decrease of ester-bound ceramide species. Moreover, processing of profilaggrin to monomeric filaggrin is impaired. This study indicates that the 12R-LOX-eLOX-3 pathway plays a key role in the process of epidermal barrier acquisition by affecting lipid metabolism, as well as protein processing.

The lipoxygenases comprise a family of fatty acid dioxygenases that are involved in a variety of inflammatory conditions. Various approaches have been taken in order to understand the different regiospecificities of the different lipoxygenases. Here we have reviewed the current knowledge of the structural features of the substrate and of the enzyme that form the basis of the regiospecificity of 15-lipoxygenase. Earlier experiments on the structural features of the substrate were reviewed, as well as more recent results of site-directed mutagenesis studies. The structure of the soybean lipoxygenase isoform-1 was also briefly reviewed.

Early healing after myocardial infarction (MI) is characterized by a strong inflammatory reaction. Most leukotrienes are pro-inflammatory and are therefore potential mediators of healing and remodeling after myocardial ischemia. The enzyme 5-lipoxygenase (5-LOX) has a key role in the transformation of arachidonic acid in leukotrienes. Thus, we tested the effect of 5-LOX on healing after MI. After chronic coronary artery ligation, early mortality was significantly increased in 5-LOX(-/-) when compared to matching wildtype (WT) mice due to left ventricular rupture. This effect could be reproduced in mice treated with the 5-LOX inhibitor Zileuton. A perfusion mismatch due to the vasoactive potential of leukotrienes is not responsible for left ventricular rupture since local blood flow assessed by magnetic resonance perfusion measurements was not different. However, after MI, there was an accentuation of the inflammatory reaction with an increase of pro-inflammatory macrophages. Yet, mortality was not changed in chimeric mice (WT vs. 5-LOX(-/-) bone marrow in 5-LOX(-/-) animals), indicating that an altered function of 5-LOX(-/-) inflammatory cells is not responsible for the phenotype. Collagen production and accumulation of fibroblasts were significantly reduced in 5-LOX(-/-) mice in vivo after MI. This might be due to an impaired migration of 5-LOX(-/-) fibroblasts, as shown in vitro to serum. In conclusion, a lack or inhibition of 5-LOX increases mortality after MI because of healing defects. This is not mediated by a change in local blood flow, but through an altered inflammation and/or fibroblast function.

Endocannabinoids, such as anandamide (ANA) and 2-arachidonoylglycerol (2AG), are lipid-signaling molecules that can be oxidized by lipid-peroxidizing enzymes, and this oxidation alters the bioactivity of these lipid mediators. Here, under strictly comparable experimental conditions, we explored whether ANA and 2AG function as substrates for four human (ALOX15, ALOX15B, ALOX12, ALOX5) and three mice Alox isoforms (Alox15, Alox12, Alox5) and compared the rates of product formation with those of arachidonic acid oxygenation. Except for ALOX5, the two endocannabinoids were more efficiently oxygenated than arachidonic acid by human ALOX isoforms. Mice Alox15 oxygenated ANA more efficiently than arachidonic acid, but the other mice Alox isoforms exhibited reduced reaction rates for endocannabinoid conversion. Like its human ortholog, mice Alox5 did not oxygenate ANA, but the formation of 5-HETE-containing 2AG derivatives was observed for this enzyme. 1AG and 2AG were similarly effective substrates for human ALOX isoforms. Molecular docking studies, the pattern of oxygenation products, and site-directed mutagenesis experiments suggested a similar substrate alignment of arachidonic acid and endocannabinoids at the active site of ALOX15 orthologs. The product specificity of arachidonic acid oxygenation was conserved for endocannabinoid metabolization, and the triad concept describing the molecular basis for the reaction specificity of ALOX15 orthologs is applicable for endocannabinoid oxygenation. Taken together, these data indicate that, except for ALOX5 orthologs, endocannabinoids are suitable substrates for most mammalian ALOX isoforms.

Human 5-lipoxygenase (5-LOX) is a well-validated target for anti-inflammatory therapy. Development of novel 5-LOX inhibitors with higher activities is highly demanded. In previous study, we have built a model for the active conformation of human 5-LOX, and identified naphthalen-1-yl 3,5-dinitrobenzoate (JMC-4) as a 5-LOX inhibitor by virtual screening. In the present work, 3,5-dinitrobenzoate-based 5-lipoxygenase inhibitors were developed. Twenty aryl 3,5-dinitrobenzoates, N-aryl 3,5-dinitrobenzamides and analogues were designed and synthesized. Several of them were found with significantly increased activities according to cell-free assay and human whole blood assay. The structure-activity relationship study may provide useful insights for designing effective 5-LOX inhibitors.

Among the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectrometry. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LO in vitro.

The relationship between glioblastoma (GBM) and fatty acid metabolism could be the key to elucidate more effective therapeutic targets. 15-lipoxygenase-1 (15-LOX), a linolenic acid and arachidonic acid metabolizing enzyme, induces both pro- and antitumorigenic effects in different cancer types. Its role in glioma activity has not yet been clearly described. The objective of this study was to identify the influence of 15-LOX and its metabolites on glioblastoma cell activity.

Oxylipins generated by the lipoxygenase (LOX) pathway play an important role in plant defense against biotic and abiotic stress. In chitosan-treated Adelostemma gracillimum seedlings, obvious accumulation of 9-LOX-derived oxylipins, namely 9,10,11-trihydroxy-12-octadecenoic acid, was detected. Using degenerate primers, a LOX-specific fragment putatively encoding LOX was obtained by RT-PCR, and a 2.9-kb full-length cDNA named AgLOX1 was isolated by RACE from chitosan-induced A. gracillimum seedlings. Genomic Southern analysis implied that there was only one copy of AgLOX1 in the A. gracillimum genome. AgLOX1 was expressed in Escherichia coli and the recombinant protein was partially purified. The enzyme converted linoleic and linolenic acids almost exclusively to their 9-hydroperoxides. AgLOX1 encoded a 9-lipoxygenase. Northern blot analysis indicated that chitosan-induced AgLOX1 transcript accumulation peaked at 8 h after initiation of treatment, whereas trihydroxy derivatives accumulation was highest at 24 h after elicitation. Results showed that chitosan-induced AgLOX1 encoded a 9-lipoxygenase potentially involved in the defense response through 9-LOX pathway leading to biosynthesis of antimicrobial compounds in A. gracillimum seedlings.

Infections and vaccines can induce enhanced long-term responses in innate immune cells, establishing an innate immunological memory termed trained immunity. Here, we show that monocytes with a trained immunity phenotype, due to exposure to the Bacillus Calmette-Guérin (BCG) vaccine, are characterized by an increased biosynthesis of different lipid mediators (LM) derived from long-chain polyunsaturated fatty acids (PUFA). Pharmacological and genetic approaches show that long-chain PUFA synthesis and lipoxygenase-derived LM are essential for the BCG-induced trained immunity responses of human monocytes. Furthermore, products of 12-lipoxygenase activity increase in monocytes of healthy individuals after BCG vaccination. Grasping the underscoring lipid metabolic pathways contributes to our understanding of trained immunity and may help to identify therapeutic tools and targets for the modulation of innate immune responses.

The opportunistic human pathogen Aspergillus fumigatus initiates invasive growth through a programmed germination process that progresses from dormant spore to swollen spore (SS) to germling (GL) and ultimately invasive hyphal growth. We find a lipoxygenase with considerable homology to human Alox5 and Alox15, LoxB, that impacts the transitions of programmed spore germination. Overexpression of loxB (OE::loxB) increases germination with rapid advance to the GL stage. However, deletion of loxB (ΔloxB) or its signal peptide only delays progression to the SS stage in the presence of arachidonic acid (AA); no delay is observed in minimal media. This delay is remediated by the addition of the oxygenated AA oxylipin 5-hydroxyeicosatetraenoic acid (5-HETE) that is a product of human Alox5. We propose that A. fumigatus acquisition of LoxB (found in few fungi) enhances germination rates in polyunsaturated fatty acid-rich environments.

5-Lipoxygenase (5-LO) converts arachidonic acid into leukotrienes (LTs) and is involved in inflammation. At present, the participation of 5-LO in acetaminophen (APAP)-induced hepatotoxicity and liver damage has not been addressed. 5-LO deficient (5-LO⁻/⁻) mice and background wild type mice were challenged with APAP (0.3-6 g/kg) or saline. The lethality, liver damage, neutrophil and macrophage recruitment, LTB₄, cytokine production, and oxidative stress were assessed. APAP induced a dose-dependent mortality, and the dose of 3 g/kg was selected for next experiments. APAP induced LTB4 production in the liver, the primary target organ in APAP toxicity. Histopathological analysis revealed that 5-LO⁻/⁻ mice presented reduced APAP-induced liver necrosis and inflammation compared with WT mice. APAP-induced lethality, increase of plasma levels of aspartate aminotransferase and alanine aminotransferase, liver cytokine (IL-1β, TNF-α , IFN- γ, and IL-10), superoxide anion, and thiobarbituric acid reactive substances production, myeloperoxidase and N-acetyl-β-D-glucosaminidase activity, Nrf2 and gp91(phox) mRNA expression, and decrease of reduced glutathione and antioxidant capacity measured by 2,2'-azinobis(3-ethylbenzothiazoline 6-sulfonate) assay were prevented in 5-LO⁻/⁻ mice compared to WT mice. Therefore, 5-LO deficiency resulted in reduced mortality due to reduced liver inflammatory and oxidative damage, suggesting 5-LO is a promising target to reduce APAP-induced lethality and liver inflammatory/oxidative damage.

Leukotriene B4 (LTB4), a proinflammatory mediator produced by the enzyme 5-lipoxygenase (5-LO), is associated with the development of many inflammatory diseases. In this study, we evaluated the participation of the 5-LO/LTB4 axis in graft-versus-host disease (GVHD) pathogenesis by transplanting 5-LO-deficient leukocytes and investigated the effect of pharmacologic 5-LO inhibition by zileuton and LTB4 inhibition by CP-105,696. Mice that received allogeneic transplant showed an increase in nuclear 5-LO expression in splenocytes, indicating enzyme activation after GVHD. Mice receiving 5-LO-deficient cell transplant or zileuton treatment had prolonged survival, reduced GVHD clinical scores, reduced intestinal and liver injury, and decreased levels of serum and hepatic LTB4 These results were associated with inhibition of leukocyte recruitment and decreased production of cytokines and chemokines. Treatment with CP-105,696 achieved similar effects. The chimerism or the beneficial graft-versus-leukemia response remained unaffected. Our data provide evidence that the 5-LO/LTB4 axis orchestrates GVHD development and suggest it could be a target for the development of novel therapeutic strategies for GVHD treatment.

Radiation therapy is a commonly used treatment modality for cancer. Although effective in providing local tumor control, radiation causes oxidative stress, inflammation, immunomodulatory and mitogenic cytokine production, extracellular matrix production, and premature senescence in lung parenchyma. The senescence associated secretory phenotype (SASP) can promote inflammation and stimulate alterations in the surrounding tissue. Therefore, we hypothesized that radiation-induced senescent parenchymal cells in irradiated lung would enhance tumor growth. Using a murine syngeneic tumor model of melanoma and non-small cell lung cancer lung metastasis, we demonstrate that radiation causes a significant increase in markers of premature senescence in lung parenchyma within 4 to 8 weeks. Further, injection of B16F0 (melanoma) or Lewis Lung carcinoma (epidermoid lung cancer) cells at these time points after radiation results in an increase in the number and size of pulmonary tumor nodules relative to unirradiated mice. Treatment of irradiated mice with a senolytic agent (ABT-737) or agents that prevent senescence (rapamycin, INK-128) was sufficient to reduce radiation-induced lung parenchymal senescence and to mitigate radiation-enhanced tumor growth. These agents abrogated radiation-induced expression of 12-Lipoxygenase (12-LOX), a molecule implicated in several deleterious effects of senescence. Deficiency of 12-LOX prevented radiation-enhanced tumor growth. Together, these data demonstrate the pro-tumorigenic role of radiation-induced senescence, introduces the dual TORC inhibitor INK-128 as an effective agent for prevention of radiation-induced normal tissue senescence, and identifies senescence-associated 12-LOX activity as an important component of the pro-tumorigenic irradiated tissue microenvironment. These studies suggest that combining senotherapeutic agents with radiotherapy may decrease post-therapy tumor growth.

Bovine and guinea pig heart homogenates, porcine leukocyte homogenate, and human hemolysate were found to vigorously oxidize linoleic acid, with lipoxygenase-like activity, to its hydroperoxy, epoxy, hydroxy-epoxy, and keto compounds in the presence of calcium chloride. In the absence of calcium, the reaction was significantly reduced. Attempts to characterize this quasi-lipoxygenase activity revealed that calcium potentiated the quasi-lipoxygenase activities of hemoproteins (hemoglobin, myoglobin, myeloperoxidase, catalase, cytochrome c) and hemin at the physiological pH of 7.5. Lipid peroxidation by hemoproteins was inhibited by albumin and erythrocyte membranes in blood, as well as by a low concentration of calcium in cells. However, it seems possible that in extracellular fluid, which contains a high concentration of calcium and a low concentration of albumin, hemoprotein released from damaged cells could oxidize unsaturated fatty acids derived by phospholipase-A2 from phospholipids of damaged cellular membranes. In a model of quasi-lipoxygenase activation under such conditions, lipids of erythrocyte membranes were oxidized by hemoglobin in the presence of phospholipase-A2 and calcium. The effect of nitrogen oxide, paraquat, and bleomycin on oxidation by hemoproteins and hemin was also discussed.

5-Lipoxygenase (5-LO) initiates the biosynthesis of pro-inflammatory leukotrienes from arachidonic acid, which requires the nuclear membrane-bound 5-LO-activating protein (FLAP) for substrate transfer. Here, we identified human 5-LO as a molecular target of melleolides from honey mushroom (Armillaria mellea). Melleolides inhibit 5-LO via an α,β-unsaturated aldehyde serving as Michael acceptor for surface cysteines at the substrate entrance that are revealed as molecular determinants for 5-LO activity. Experiments with 5-LO mutants, where select cysteines had been replaced by serine, indicated that the investigated melleolides suppress 5-LO product formation via two distinct modes of action: (1) by direct interference with 5-LO activity involving two or more of the cysteines 159, 300, 416, and 418, and (2) by preventing 5-LO/FLAP assemblies involving selectively Cys159 in 5-LO. Interestingly, replacement of Cys159 by serine prevented 5-LO/FLAP assemblies as well, implying Cys159 as determinant for 5-LO/FLAP complex formation at the nuclear membrane required for leukotriene biosynthesis.

Lipoxygenases (LOXs) are enzymes that catalyze the addition of an oxygen molecule to unsaturated fatty acids, thus forming hydroperoxides. In plants, these enzymes are encoded by a multigene family found in several organs with varying activity patterns, by which they are classified as LOX9 or LOX13. They are involved in several physiological functions, such as growth, fruit development, and plant defense. Despite several studies on genes of the LOX family in plants, most studies are restricted to a single species or a few closely related species. This study aimed to analyze the diversity, evolution, and expression of LOX genes in angiosperm species. We identified 247 LOX genes among 23 species of angiosperms and basal plants. Phylogenetic analyses identified clades supporting LOX13 and two main clades for LOX9: LOX9_A and LOX9_B. Eudicot species such as Tarenaya hassleriana, Capsella rubella, and Arabidopsis thaliana did not present LOX9_B genes; however, LOX9_B was present in all monocots used in this study. We identified that there were potential new subcellular localization patterns and conserved residues of oxidation for LOX9 and LOX13 yet unexplored. In summary, our study provides a basis for the further functional and evolutionary study of lipoxygenases in angiosperms.

Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesized to be involved in quorum-sensing abilities and invading plant tissue. In this study we used A. nidulans WG505 as an expression host to heterologously express Gaeumannomyces graminis lipoxygenase. The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants. Also, a proteomic analysis of an A. nidulans LOX producing strain indicated that the heterologous protein was degraded before its glycosylation in the secretory pathway. We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly. Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans.