Search

Polyunsaturated fatty acids (PUFAs) have been extensively studied for their health benefits because they can be oxidized by lipoxygenases to form bioactive oxylipins. In this study, we investigated the impact of double bond placement on the kinetic properties and product profiles of human platelet 12-lipoxygenase (h12-LOX), human reticulocyte 15-lipoxygenase-1 (h15-LOX-1), and human endothelial 15-lipoxygenase-2 (h15-LOX-2) by using 22-carbon (C22) fatty acid substrates with differing double bond content. With respect to kcat/KM values, the loss of Δ4 and Δ19 led to an 18-fold loss of kinetic activity for h12-LOX, no change in kinetic capability for h15-LOX-1, but a 24-fold loss for h15-LOX-2 for both C22-FAs. With respect to the product profiles, h12-LOX produced mainly 14-oxylipins. For h15-LOX-1, the 14-oxylipin production increased with the loss of either Δ4 and Δ19, however, the 17-oxylipin became the major species upon loss of both Δ4 and Δ19. h15-LOX-2 produced mostly the 17-oxylipin products throughout the fatty acid series. This study also investigated the effects of various 17-oxylipins on platelet activation. The results revealed that both 17(S)-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-DHA (17-HDHA) and 17-hydroxy-4Z,7Z,10Z,13Z,15E-DPAn6 (17-HDPAn6) demonstrated anti-aggregation properties with thrombin or collagen stimulation. 17-hydroxy-7Z,10Z,13Z,15E,19Z-DPAn3 (17-HDPAn3) exhibited agonistic properties, and 17-hydroxy-7Z,10Z,13Z,15E-DTA (17-HDTA) showed biphasic effects, inhibiting collagen-induced aggregation at lower concentrationsbut promoting aggregation at higher concentrations. Both 17-hydroxy-13Z,15E,19Z-DTrA (17-HDTrA), and 17-hydroxy-13Z,15E-DDiA (17-HDDiA) induced platelet aggregation. In summary, the number and placement of the double bonds affect platelet activation, with the general trend being that more double bonds generally inhibit aggregation, while less double bonds promote aggregation. These findings provide insights into the potential role of specific fatty acids and their metabolizing LOX isozymes with respect to cardiovascular health.

The antiplatelet effect of polyunsaturated fatty acids is primarily attributed to its metabolism to bioactive metabolites by oxygenases, such as lipoxygenases (LOX). Platelets have demonstrated the ability to generate 15-LOX-derived metabolites (15-oxylipins); however, whether 15-LOX is in the platelet or is required for the formation of 15-oxylipins remains unclear. This study seeks to elucidate whether 15-LOX is required for the formation of 15-oxylipins in the platelet and determine their mechanistic effects on platelet reactivity. In this study, 15-HETrE, 15-HETE, and 15-HEPE attenuated collagen-induced platelet aggregation, and 15-HETrE inhibited platelet aggregation induced by different agonists. The observed anti-aggregatory effect was due to the inhibition of intracellular signaling including αIIbβ3 and protein kinase C activities, calcium mobilization, and granule secretion. While 15-HETrE inhibited platelets partially through activation of peroxisome proliferator-activated receptor β (PPARβ), 15-HETE also inhibited platelets partially through activation of PPARα. 15-HETrE, 15-HETE, or 15-HEPE inhibited 12-LOX in vitro, with arachidonic acid as the substrate. Additionally, a 15-oxylipin-dependent attenuation of 12-HETE level was observed in platelets following ex vivo treatment with 15-HETrE, 15-HETE, or 15-HEPE. Platelets treated with DGLA formed 15-HETrE and collagen-induced platelet aggregation was attenuated only in the presence of ML355 or aspirin, but not in the presence of 15-LOX-1 or 15-LOX-2 inhibitors. Expression of 15-LOX-1, but not 15-LOX-2, was decreased in leukocyte-depleted platelets compared to non-depleted platelets. Taken together, these findings suggest that 15-oxylipins regulate platelet reactivity; however, platelet expression of 15-LOX-1 is low, suggesting that 15-oxylipins may be formed in the platelet through a 15-LOX-independent pathway.

Human 15-lipoxygenases (LOX) are critical enzymes in the inflammatory process, producing various pro-resolution molecules, such as lipoxins and resolvins, but the exact role each of the two 15-LOXs in these biosynthetic pathways remains elusive. Previously, it was observed that h15-LOX-1 reacted with 5S-HETE in a non-canonical manner, producing primarily the 5S,12S-diHETE product. To determine the active site constraints of h15-LOX-1 in achieving this reactivity, amino acids involved in the fatty acid binding were investigated. It was observed that R402L did not have a large effect on 5S-HETE catalysis, but F414 appeared to π-π stack with 5S-HETE, as seen with AA binding, indicating an aromatic interaction between a double bond of 5S-HETE and F414. Decreasing the size of F352 and I417 shifted oxygenation of 5S-HETE to C12, while increasing the size of these residues reversed the positional specificity of 5S-HETE to C15. Mutants at these locations demonstrated a similar effect with 7S-HDHA as the substrate, indicating that the depth of the active site regulates product specificity for both substrates. Together, these data indicate that of the three regions proposed to control positional specificity, π-π stacking and active site cavity depth are the primary determinants of positional specificity with 5S-HETE and h15-LOX-1. Finally, the altered reactivity of h15-LOX-1 was also observed with 5S-HEPE, producing 5S,12S-diHEPE instead of 5S,15S-diHEPE (aka resolvin E4 (RvE4). However, h15-LOX-2 efficiently produces 5S,15S-diHEPE from 5S-HEPE. This result is important with respect to the biosynthesis of the RvE4 since it obscures which LOX isozyme is involved in its biosynthesis. Future work detailing the expression levels of the lipoxygenase isoforms in immune cells and selective inhibition during the inflammatory response will be required for a comprehensive understanding of RvE4 biosynthesis.

Altered redox biology challenges all cells, with compensatory responses often determining a cell's fate. When 15 lipoxygenase 1 (15LO1), a lipid-peroxidizing enzyme abundant in asthmatic human airway epithelial cells (HAECs), binds phosphatidylethanolamine-binding protein 1 (PEBP1), hydroperoxy-phospholipids, which drive ferroptotic cell death, are generated. Peroxidases, including glutathione peroxidase 4 (GPX4), metabolize hydroperoxy-phospholipids to hydroxy derivatives to prevent ferroptotic death, but consume reduced glutathione (GSH). The cystine transporter SLC7A11 critically restores/maintains intracellular GSH. We hypothesized that high 15LO1, PEBP1, and GPX4 activity drives abnormal asthmatic redox biology, evidenced by lower bronchoalveolar lavage (BAL) fluid and intraepithelial cell GSH:oxidized GSH (GSSG) ratios, to enhance type 2 (T2) inflammatory responses. GSH, GSSG (enzymatic assays), 15LO1, GPX4, SLC7A11, and T2 biomarkers (Western blot and RNA-Seq) were measured in asthmatic and healthy control (HC) cells and fluids, with siRNA knockdown as appropriate. GSSG was higher and GSH:GSSG lower in asthmatic compared with HC BAL fluid, while intracellular GSH was lower in asthma. In vitro, a T2 cytokine (IL-13) induced 15LO1 generation of hydroperoxy-phospholipids, which lowered intracellular GSH and increased extracellular GSSG. Lowering GSH further by inhibiting SLC7A11 enhanced T2 inflammatory protein expression and ferroptosis. Ex vivo, redox imbalances corresponded to 15LO1 and SLC7A11 expression, T2 biomarkers, and worsened clinical outcomes. Thus, 15LO1 pathway-induced redox biology perturbations worsen T2 inflammation and asthma control, supporting 15LO1 as a therapeutic target.

It is essential to further characterize liver injury aimed at developing novel therapeutic approaches. This study investigated the mechanistic basis of genipin against carbon tetrachloride (CCl4)-triggered acute liver injury concerning ferroptosis, a novel discovered modality of regulated cell death. All experiments were performed using hepatotoxic models upon CCl4 exposure in mice and human hepatocytes in vitro. Immunohistochemistry, immunoblotting, molecular docking, RNA-sequencing and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were conducted. CCl4 intoxication was manifested with lipid peroxidation-dictated ferroptotic cell death, together with changes in a cascade of ferroptosis-associated events and several regulatory pathways. Both the administration of genipin and ferrostatin-1 (Fer-1) significantly prevented this hepatotoxicity in response to CCl4 intoxication via upregulating GPX4 and xCT (i.e., critical regulators of ferroptosis). RNA-sequencing unraveled that arachidonic acid metabolism was considerably influenced upon genipin treatment. Accordingly, genipin treatment attenuated arachidonate 15-lipoxygenase (ALOX15)-launched lipid peroxidation in terms of UHPLC-MS/MS analysis and inflammation. In vitro, genipin supplementation rescued erastin-induced hepatocellular inviability and lipid ROS accumulation. The siRNA knockdown of GPX4 partially abrogated the protective effects of genipin on erastin-induced cytotoxicity, whereas the cytotoxicity was less severe in the presence of diminished ALOX15 expression in L-O2 cells. In conclusion, our findings uncovered that genipin treatment protects against CCl4-triggered acute liver injury by abrogating hepatocyte ferroptosis, wherein the pharmacological modification of dysregulated GPX4 and ALOX15-launched lipid peroxidation was responsible for underlying medicinal effects as molecular basis. [ABSTRACT FROM AUTHOR]

Human epithelial 15-lipoxygenase-2 (h15-LOX-2, ALOX15B) is expressed in many tissues and has been implicated in atherosclerosis, cystic fibrosis and ferroptosis. However, there are few reported potent/selective inhibitors that are active ex vivo. In the current work, we report newly discovered molecules that are more potent and structurally distinct from our previous inhibitors, MLS000545091 and MLS000536924 (Jameson et al, PLoS One, 2014, 9, e104094), in that they contain a central imidazole ring, which is substituted at the 1-position with a phenyl moiety and with a benzylthio moiety at the 2-position. The initial three molecules were mixed-type, non-reductive inhibitors, with IC50 values of 0.34 ± 0.05 μM for MLS000327069, 0.53 ± 0.04 μM for MLS000327186 and 0.87 ± 0.06 μM for MLS000327206 and greater than 50-fold selectivity versus h5-LOX, h12-LOX, h15-LOX-1, COX-1 and COX-2. A small set of focused analogs was synthesized to demonstrate the validity of the hits. In addition, a binding model was developed for the three imidazole inhibitors based on computational docking and a co-structure of h15-LOX-2 with MLS000536924. Hydrogen/deuterium exchange (HDX) results indicate a similar binding mode between MLS000536924 and MLS000327069, however, the latter restricts protein motion of helix-α2 more, consistent with its greater potency. Given these results, we designed, docked, and synthesized novel inhibitors of the imidazole scaffold and confirmed our binding mode hypothesis. Importantly, four of the five inhibitors mentioned above are active in an h15-LOX-2/HEK293 cell assay and thus they could be important tool compounds in gaining a better understanding of h15-LOX-2's role in human biology. As such, a suite of similar pharmacophores that target h15-LOX-2 both in vitro and ex vivo are presented in the hope of developing them as therapeutic agents.

In this paper, human platelet 12-lipoxygenase [h12-LOX (ALOX12)], human reticulocyte 15-lipoxygenase-1 [h15-LOX-1 (ALOX15)], and human epithelial 15-lipoxygenase-2 [h15-LOX-2 (ALOX15B)] were observed to react with docosahexaenoic acid (DHA) and produce 17S-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid (17S-HpDHA). The kcat/KM values with DHA for h12-LOX, h15-LOX-1, and h15-LOX-2 were 12, 0.35, and 0.43 s-1 μM-1, respectively, which demonstrate h12-LOX as the most efficient of the three. These values are comparable to their counterpart kcat/KM values with arachidonic acid (AA), 14, 0.98, and 0.24 s-1 μM-1, respectively. Comparison of their product profiles with DHA demonstrates that the three LOX isozymes produce 11S-HpDHA, 14S-HpDHA, and 17S-HpDHA, to varying degrees, with 17S-HpDHA being the majority product only for the 15-LOX isozymes. The effective kcat/KM values (kcat/KM × percent product formation) for 17S-HpDHA of the three isozymes indicate that the in vitro value of h12-LOX was 2.8-fold greater than that of h15-LOX-1 and 1.3-fold greater than that of h15-LOX-2. 17S-HpDHA was an effective substrate for h12-LOX and h15-LOX-1, with four products being observed under reducing conditions: protectin DX (PDX), 16S,17S-epoxy-4Z,7Z,10Z,12E,14E,19Z-docosahexaenoic acid (16S,17S-epoxyDHA), the key intermediate in neuroprotection D1 biosynthesis [NPD1, also known as protectin D1 (PD1)], 11,17S-diHDHA, and 16,17S-diHDHA. However, h15-LOX-2 did not react with 17-HpDHA. With respect to their effective kcat/KM values, h12-LOX was markedly less effective than h15-LOX-1 in reacting with 17S-HpDHA, with a 55-fold lower effective kcat/KM in producing 16S,17S-epoxyDHA and a 27-fold lower effective kcat/KM in generating PDX. This is the first direct demonstration of h15-LOX-1 catalyzing this reaction and reveals an in vitro pathway for PDX and NPD1 intermediate biosynthesis. In addition, epoxide formation from 17S-HpDHA and h15-LOX-1 was negatively affected via allosteric regulation by 17S-HpDHA (Kd = 5.9 μM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (Kd = 2.5 μM), and 17S-hydroxy-13Z,15E,19Z-docosatrienoic acid (17S-HDTA) (Kd = 1.4 μM), suggesting a possible regulatory pathway in reducing epoxide formation. Finally, 17S-HpDHA and PDX inhibited platelet aggregation, with EC50 values of approximately 1 and 3 μM, respectively. The in vitro results presented here may help advise in vivo PDX and NPD1 intermediate (i.e., 16S,17S-epoxyDHA) biosynthetic investigations and support the benefits of DHA rich diets.

Macrophages acquire anti-inflammatory and proresolving functions to facilitate resolution of inflammation and promote tissue repair. While alternatively activated macrophages (AAMs), also referred to as M2 macrophages, polarized by type 2 (Th2) cytokines IL-4 or IL-13 contribute to the suppression of inflammatory responses and play a pivotal role in wound healing, contemporaneous exposure to apoptotic cells (ACs) potentiates the expression of anti-inflammatory and tissue repair genes. Given that liver X receptors (LXRs), which coordinate sterol metabolism and immune cell function, play an essential role in the clearance of ACs, we investigated whether LXR activation following engulfment of ACs selectively potentiates the expression of Th2 cytokine-dependent genes in primary human AAMs. We show that AC uptake simultaneously upregulates LXR-dependent, but suppresses SREBP-2-dependent gene expression in macrophages, which are both prevented by inhibiting Niemann-Pick C1 (NPC1)-mediated sterol transport from lysosomes. Concurrently, macrophages accumulate sterol biosynthetic intermediates desmosterol, lathosterol, lanosterol, and dihydrolanosterol but not cholesterol-derived oxysterols. Using global transcriptome analysis, we identify anti-inflammatory and proresolving genes including interleukin-1 receptor antagonist (IL1RN) and arachidonate 15-lipoxygenase (ALOX15) whose expression are selectively potentiated in macrophages upon concomitant exposure to ACs or LXR agonist T0901317 (T09) and Th2 cytokines. We show priming macrophages via LXR activation enhances the cellular capacity to synthesize inflammation-suppressing specialized proresolving mediator (SPM) precursors 15-HETE and 17-HDHA as well as resolvin D5. Silencing LXRα and LXRβ in macrophages attenuates the potentiation of ALOX15 expression by concomitant stimulation of ACs or T09 and IL-13. Collectively, we identify a previously unrecognized mechanism of regulation whereby LXR integrates AC uptake to selectively shape Th2-dependent gene expression in AAMs.

Background: Allergic rhinitis (AR) is an inflammatory disorder of the nasal mucosa, caused by exposure to environmental allergens. It is known that 15-lipoxygenase (15-LOX) is involved in the biosynthetic pathways of anti-inflammatory lipid mediators, including resolvins and protectins. Methods: In this study, which was performed on 130 AR patients and 130 healthy controls, we aimed to investigate the association of susceptibility to AR with two selected single-nucleotide polymorphisms (SNPs), that is, rs2619112:A>G and rs7217186:C>T, in the intron regions of arachidonic acid 15-LOX (ALOX15) gene, using SNPinfo and Regulome DB tools. Results: The results showed that the CT genotype of rs7217186: C>T was significantly associated with the increased risk of AR compared to the CC genotype (P= 0.037, OR=1.943, CI: 1.038-0.638). However, there was no strong evidence of the association of rs2619112: A>G with susceptibility to AR (P> 0.05). Conclusions: The present results indicated that rs7217186 polymorphism of ALOX15 gene might be a potential biomarker for susceptibility to AR. [ABSTRACT FROM AUTHOR]

The oxylipins, 5S,12S-dihydroxy-6E,8Z,10E,14Z-eicosatetraenoic acid (5S,12S-diHETE) and 5S,15S-dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid (5S,15S-diHETE), have been identified in cell exudates and have chemotactic activity toward eosinophils and neutrophils. Their biosynthesis has been proposed to occur by sequential oxidations of arachidonic acid (AA) by lipoxygenase enzymes, specifically through oxidation of AA by h5-LOX followed by h12-LOX, h15-LOX-1, or h15-LOX-2. In this work, h15-LOX-1 demonstrates altered positional specificity when reacting with 5S-HETE, producing 90% 5S,12S-diHETE, instead of 5S,15S-diHETE, with kinetics 5-fold greater than that of h12-LOX. This is consistent with previous work in which h15-LOX-1 reacts with 7S-HDHA, producing the noncanonical, DHA-derived, specialized pro-resolving mediator, 7S,14S-diHDHA. It is also determined that oxygenation of 5S-HETE by h15-LOX-2 produces 5S,15S-diHETE and its biosynthetic kcat/KM flux is 2-fold greater than that of h15-LOX-1, suggesting that h15-LOX-2 may have a greater role in lipoxin biosynthesis than previously thought. In addition, it is shown that oxygenation of 12S-HETE and 15S-HETE by h5-LOX is kinetically slow, suggesting that the first step in the in vitro biosynthesis of both 5S,12S-diHETE and 5S,15S-diHETE is the production of 5S-HETE.

The two oxylipins 7S,14S-dihydroxydocosahexaenoic acid (diHDHA) and 7S,17S-diHDHA [resolvin D5 (RvD5)] have been found in macrophages and infectious inflammatory exudates and are believed to function as specialized pro-resolving mediators (SPMs). Their biosynthesis is thought to proceed through sequential oxidations of DHA by lipoxygenase (LOX) enzymes, specifically, by human 5-LOX (h5-LOX) first to 7(S)-hydroxy-4Z,8E,10Z,13Z,16Z,19Z-DHA (7S-HDHA), followed by human platelet 12-LOX (h12-LOX) to form 7(S),14(S)-dihydroxy-4Z,8E,10Z,12E,16Z,19Z-DHA (7S,14S-diHDHA) or human reticulocyte 15-LOX-1 (h15-LOX-1) to form RvD5. In this work, we determined that oxidation of 7(S)-hydroperoxy-4Z,8E,10Z,13Z,16Z,19Z-DHA to 7S,14S-diHDHA is performed with similar kinetics by either h12-LOX or h15-LOX-1. The oxidation at C14 of DHA by h12-LOX was expected, but the noncanonical reaction of h15-LOX-1 to make over 80% 7S,14S-diHDHA was larger than expected. Results of computer modeling suggested that the alcohol on C7 of 7S-HDHA hydrogen bonds with the backbone carbonyl of Ile399, forcing the hydrogen abstraction from C12 to oxygenate on C14 but not C17. This result raised questions regarding the synthesis of RvD5. Strikingly, we found that h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17, forming RvD5 with faster kinetics than does h15-LOX-1. The presence of h15-LOX-2 in neutrophils and macrophages suggests that it may have a greater role in biosynthesizing SPMs than previously thought. We also determined that the reactions of h5-LOX with 14(S)-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-DHA and 17(S)-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-DHA are kinetically slow compared with DHA, suggesting that these reactions may be minor biosynthetic routes in vivo. Additionally, we show that 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micromolar potencies, which could directly regulate clot resolution.

Human reticulocyte 15-lipoxygenase-1 (h15-LOX-1 or ALOX15) and platelet 12-lipoxygenase (h12-LOX or ALOX12) catalysis of docosahexaenoic acid (DHA) and the maresin precursor, 14S-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (14S-HpDHA), were investigated to determine their product profiles and relative rates in the biosynthesis of the key maresin intermediate, 13S,14S-epoxy-4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid (13S,14S-epoxy-DHA). Both enzymes converted DHA to 14S-HpDHA, with h12-LOX having a 39-fold greater kcat/KM value (14.0 ± 0.8 s-1 μM-1) than that of h15-LOX-1 (0.36 ± 0.08 s-1 μM-1) and a 1.8-fold greater 14S-HpDHA product selectivity, 81 and 46%, respectively. However, h12-LOX was markedly less effective at producing 13S,14S-epoxy-DHA from 14S-HpDHA than h15-LOX-1, with a 4.6-fold smaller kcat/KM value, 0.0024 ± 0.0002 and 0.11 ± 0.006 s-1 μM-1, respectively. This is the first evidence of h15-LOX-1 to catalyze this reaction and reveals a novel in vitro pathway for maresin biosynthesis. In addition, epoxidation of 14S-HpDHA is negatively regulated through allosteric oxylipin binding to h15-LOX-1 and h12-LOX. For h15-LOX-1, 14S-HpDHA (Kd = 6.0 μM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (Kd = 3.5 μM), and 14S-hydroxy-7Z,10Z,12E,16Z,19Z-docosapentaenoic acid (14S-HDPAω-3) (Kd = 4.0 μM) were shown to decrease 13S,14S-epoxy-DHA production. h12-LOX was also shown to be allosterically regulated by 14S-HpDHA (Kd = 3.5 μM) and 14S-HDPAω-3 (Kd = 4.0 μM); however, 12S-HETE showed no effect, indicating for the first time an allosteric response by h12-LOX. Finally, 14S-HpDHA inhibited platelet aggregation at a submicrololar concentration, which may have implications in the benefits of diets rich in DHA. These in vitro biosynthetic pathways may help guide in vivo maresin biosynthetic investigations and possibly direct therapeutic interventions.

Alcoholism is one of the leading and increasingly prevalent reasons of liver associated morbidity and mortality worldwide. Alcoholic hepatitis (AH) constitutes a severe disease with currently no satisfying treatment options. Lipoxin A4 (LXA4), a 15-lipoxygenase (ALOX15)-dependent lipid mediator involved in resolution of inflammation, showed promising pre-clinical results in the therapy of several inflammatory diseases. Since inflammation is a main driver of disease progression in alcoholic hepatitis, we investigated the impact of endogenous ALOX15-dependent lipid mediators and exogenously applied LXA4 on AH development. A mouse model for alcoholic steatohepatitis (NIAAA model) was tested in Alox12/15+/+ and Alox12/15-/- mice, with or without supplementation of LXA4. Absence of Alox12/15 aggravated parameters of liver disease, increased hepatic immune cell infiltration in AH, and elevated systemic neutrophils as a marker for systemic inflammation. Interestingly, i.p. injections of LXA4 significantly lowered transaminase levels only in Alox12/15-/- mice and reduced hepatic immune cell infiltration as well as systemic inflammatory cytokine expression in both genotypes, even though steatosis progressed. Thus, while LXA4 injection attenuated selected parameters of disease progression in Alox12/15-/- mice, its beneficial impact on immunity was also apparent in Alox12/15+/+ mice. In conclusion, pro-resolving lipid mediators may be beneficial to reduce inflammation in alcoholic hepatitis.

Global gene-expression analysis has shown remarkable difference between males and females in response to exposure to many diseases. Nevertheless, gene expression studies in asthmatics have so far focused on sex-combined analysis, ignoring inherent variabilities between the sexes, which potentially drive disparities in asthma prevalence. The objectives of this study were to identify (1) sex-specific differentially expressed genes (DEGs), (2) genes that show sex-interaction effects and (3) sex-specific pathways and networks enriched in asthma risk. We analyzed 711 males and 689 females and more than 2.8 million transcripts covering 20 000 genes leveraged from five different tissues and cell types (i.e. epithelial, blood, induced sputum, T cells and lymphoblastoids). Using tissue-specific meta-analysis, we identified 439 male- and 297 female-specific DEGs in all cell types, with 32 genes in common. By linking DEGs to the genome-wide association study (GWAS) catalog and the lung and blood eQTL annotation data from GTEx, we identified four male-specific genes (FBXL7, ITPR3 and RAD51B from epithelial tissue and ALOX15 from blood) and one female-specific gene (HLA-DQA1 from epithelial tissue) that are disregulated during asthma. The hypoxia-inducible factor 1 signaling pathway was enriched only in males, and IL-17 and chemokine signaling pathways were enriched in females. The cytokine-cytokine signaling pathway was enriched in both sexes. The presence of sex-specific genes and pathways demonstrates that sex-combined analysis does not identify genes preferentially expressed in each sex in response to diseases. Linking DEG and molecular eQTLs to GWAS catalog represents an important avenue for identifying biologically and clinically relevant genes.

Many species have experienced dramatic changes in their abundance and distribution during recent climate change, but it is often unclear whether such ecological responses are accompanied by evolutionary change. We used targeted exon sequencing of 294 museum specimens (160 historic, 134 modern) to generate independent temporal genomic contrasts spanning a century of climate change (1911-2012) for two co-distributed chipmunk species: an endemic alpine specialist (Tamias alpinus) undergoing severe range contraction and a stable mid-elevation species (T. speciosus). Using a novel analytical approach, we reconstructed the demographic histories of these populations and tested for evidence of recent positive directional selection. Only the retracting species showed substantial population genetic fragmentation through time and this was coupled with positive selection and substantial shifts in allele frequencies at a gene, Alox15, involved in regulation of inflammation and response to hypoxia. However, these rapid population and gene-level responses were not detected in an analogous temporal contrast from another area where T. alpinus has also undergone severe range contraction. Collectively, these results highlight that evolutionary responses may be variable and context dependent across populations, even when they show seemingly synchronous ecological shifts. Our results demonstrate that temporal genomic contrasts can be used to detect very recent evolutionary responses within and among contemporary populations, even in the face of complex demographic changes. Given the wealth of specimens archived in natural history museums, comparative analyses of temporal population genomic data have the potential to improve our understanding of recent and ongoing evolutionary responses to rapidly changing environments.

12-lipoxygenase (12-LOX) is one of several enzyme isoforms responsible for the metabolism of arachidonic acid and other poly-unsaturated fatty acids to both pro- and anti-inflammatory lipid mediators. Mounting evidence has shown that 12-LOX plays a critical role in the modulation of inflammation at multiple checkpoints during diabetes development. Due to this, interventions to limit pro-inflammatory 12-LOX metabolites either by isoform-specific 12-LOX inhibition, or by providing specific fatty acid substrates via dietary intervention, has the potential to significantly and positively impact health outcomes of patients living with both type 1 and type 2 diabetes. To date, the development of truly specific and efficacious inhibitors has been hampered by homology of LOX family members; however, improvements in high throughput screening have improved the inhibitor landscape. Here, we describe the function and role of human 12-LOX, and mouse 12-LOX and 12/15-LOX, in the development of diabetes and diabetes-related complications, and describe promise in the development of strategies to limit pro-inflammatory metabolites, primarily via new small molecule 12-LOX inhibitors.

12-lipoxygenase (12-LOX) is one of several enzyme isoforms responsible for the metabolism of arachidonic acid and other poly-unsaturated fatty acids to both pro- and anti-inflammatory lipid mediators. Mounting evidence has shown that 12-LOX plays a critical role in the modulation of inflammation at multiple checkpoints during diabetes development. Due to this, interventions to limit pro-inflammatory 12-LOX metabolites either by isoform-specific 12-LOX inhibition, or by providing specific fatty acid substrates via dietary intervention, has the potential to significantly and positively impact health outcomes of patients living with both type 1 and type 2 diabetes. To date, the development of truly specific and efficacious inhibitors has been hampered by homology of LOX family members; however, improvements in high throughput screening have improved the inhibitor landscape. Here, we describe the function and role of human 12-LOX, and mouse 12-LOX and 12/15-LOX, in the development of diabetes and diabetes-related complications, and describe promise in the development of strategies to limit pro-inflammatory metabolites, primarily via new small molecule 12-LOX inhibitors.

Ischemic strokes are caused by one or more blood clots that typically obstruct one of the major arteries in the brain, but frequently also result in leakage of the blood-brain barrier and subsequent hemorrhage. While it has long been known that the enzyme 12/15-lipoxygenase (12/15-LOX) is up-regulated following ischemic strokes and contributes to neuronal cell death, recent research has shown an additional major role for 12/15-LOX in causing this hemorrhagic transformation. These findings have important implications for the use of 12/15-LOX inhibitors in the treatment of stroke.

Prostate carcinoma is one of the most commonly diagnosed visceral malignancies and the fifth leading cause of cancer-related mortality in males. Reportedly, a series of dietary lipids are identified as 1-cis-4-cis-pentadiene polyunsaturated fatty acids (PUFAs), which play a dominant role in prostate carcinogenesis. Four species of human lipoxygenases (LOXs), a family of nonheme iron-containing enzymes, mediate the deoxygenation of the aforementioned PUFAs. 15-LOX-1 in particular metabolizes the ω -6 lipids and generates certain metabolites (e.g., 13-(S)-hydroxyoctadecaenoic acid) which results in vascular homeostasis, cell proliferation and tissue differentiation in the prostate. Furthermore, in prostate cancer (PCa), the expression of 15-LOX-1 is elevated and positively correlated with the Gleason score of the tumor (an indicator of the disease severity). As membrane receptors, kinases and transcriptional factors are all affected by carcinogenic signals of 15-LOX-1, therapeutic agents that directly inhibit this enzyme can be advantageous in the treatment of PCa. To our knowledge, there are limited effective treatments for PCa, and there is no therapy for its metastatic condition. In this respect, 15-LOX-1, as an appropriate candidate for drug development, was subjected to homology modeling, phylogenic assessment, cross-docking analysis and molecular dynamics (MD) simulation to identify an eligible inhibiting agent amongst a library of 30 potential targeting compounds for PCa management. Prostate carcinoma is one of the most commonly diagnosed visceral malignancies and the fifth leading cause of cancer-related mortality in males. To our knowledge, there are limited effective treatments for prostate cancer. In this respect, human 15-Lipoxygenase-1 (15-LOX-1), as an appropriate candidate, was subjected to in-silico explorations. We report the potential for using 15-LOX-1 inhibitors, such as CHEMBL1270113, as a therapeutic approach to restore the balance between the function of lipoxygenase family members and inflammatory mediators. [ABSTRACT FROM AUTHOR]

The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB4. For 12-LOX and 5,15-diHpETE, the kinetic parameters are kcat = 0.17 s-1 and kcat/ KM = 0.011 μM-1 s-1 [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are kcat = 4.6 s-1 and kcat/ KM = 0.21 μM-1 s-1 (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB4 from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 μM; however, LXB4 did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB4 is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA4 is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA4 and LXB4 are produced by 12-LOX but only if LTA4 is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234].

The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB4. For 12-LOX and 5,15-diHpETE, the kinetic parameters are kcat = 0.17 s-1 and kcat/ KM = 0.011 μM-1 s-1 [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are kcat = 4.6 s-1 and kcat/ KM = 0.21 μM-1 s-1 (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB4 from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 μM; however, LXB4 did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB4 is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA4 is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA4 and LXB4 are produced by 12-LOX but only if LTA4 is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234].

Arachidonate 15-lipoxygenase (ALOX15) and arachidonate 15-lipoxygenase, type B (ALOX15B) catalyze the dioxygenation of polyunsaturated fatty acids and are upregulated in human alternatively activated macrophages (AAMs) induced by Th2 cytokine interleukin-4 (IL-4) and/or interleukin-13. Known primarily for roles in bioactive lipid mediator synthesis, 15-lipoxygenases (15-LOXs) have been implicated in various macrophage functions including efferocytosis and ferroptosis. Using a combination of inhibitors and siRNAs to suppress 15-LOX isoforms, we studied the role of 15-LOXs in cellular cholesterol homeostasis and immune function in naïve and AAMs. Silencing or inhibiting the 15-LOX isoforms impaired sterol regulatory element binding protein (SREBP)-2 signaling by inhibiting SREBP-2 processing into mature transcription factor and reduced SREBP-2 binding to sterol regulatory elements and subsequent target gene expression. Silencing ALOX15B reduced cellular cholesterol and the cholesterol intermediates desmosterol, lanosterol, 24,25-dihydrolanosterol, and lathosterol as well as oxysterols in IL-4-stimulated macrophages. In addition, attenuating both 15-LOX isoforms did not generally affect IL-4 gene expression but rather uniquely impacted IL-4-induced CCL17 production in an SREBP-2-dependent manner resulting in reduced T cell migration to macrophage conditioned media. In conclusion, we identified a novel role for ALOX15B, and to a lesser extent ALOX15, in cholesterol homeostasis and CCL17 production in human macrophages.

Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction. This study utilizes site-directed mutagenesis, guided in part by molecular modeling, to gain a better structural understanding of inhibitor interactions within the active site. We have generated eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 to determine whether these active site residues interact with two h15-LOX-1 inhibitors, ML351 and an ML094 derivative, compound 18. IC50 values and steady-state inhibition kinetics were determined for the eight mutants, with four of the mutants affecting inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that ML351 and compound 18, bind in a similar manner in the active site to an aromatic pocket close to F414 but have subtle differences in their specific binding modes. This information establishes the binding mode for ML094 and ML351 and will be leveraged to develop next-generation inhibitors.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause nosocomial and chronic infections in immunocompromised patients. P. aeruginosa secretes a lipoxygenase, LoxA, but the biological role of this enzyme is currently unknown. LoxA is poorly similar in sequence to both soybean LOX-1 (s15-LOX-1) and human 15-LOX-1 (37 and 39%, respectively) yet has kinetics comparably fast versus those of s15-LOX-1 (at pH 6.5, Kcat = 181 ± 6 s(-1) and Kcat/KM = 16 ± 2 μM(-1) s(-1)). LoxA is capable of efficiently catalyzing the peroxidation of a broad range of free fatty acid (FA) substrates (e.g., AA and LA) with high positional specificity, indicating a 15-LOX. Its mechanism includes hydrogen atom abstraction [a kinetic isotope effect (KIE) of >30], yet LoxA is a poor catalyst against phosphoester FAs, suggesting that LoxA is not involved in membrane decomposition. LoxA also does not react with 5- or 15-HETEs, indicating poor involvement in lipoxin production. A LOX high-throughput screen of the LOPAC library yielded a variety of low-micromolar inhibitors; however, none selectively targeted LoxA over the human LOX isozymes. With respect to cellular activity, the level of LoxA expression is increased when P. aeruginosa undergoes the transition to a biofilm mode of growth, but LoxA is not required for biofilm growth on abiotic surfaces. However, LoxA does appear to be required for biofilm growth in association with the host airway epithelium, suggesting a role for LoxA in mediating bacterium-host interactions during colonization.

Lipoxins are an important class of lipid mediators that induce the resolution of inflammation and arise from transcellular exchange of arachidonic acid (AA)-derived lipoxygenase products. Human epithelial 15-lipoxygenase-2 (h15-LOX-2), the major lipoxygenase in macrophages, has exhibited strict regiospecificity, catalyzing only the hydroperoxidation of carbon 15 of AA. To determine the catalytic potential of h15-LOX-2 in transcellular synthesis events, we reacted it with the three lipoxygenase-derived monohydroperoxy-eicosatetraenoic acids (HPETE) in humans: 5-HPETE, 12-HPETE, and 15-HPETE. Only 5-HPETE was a substrate for h15-LOX-2, and the steady-state catalytic efficiency (kcat/Km) of this reaction was 31% of the kcat/Km of AA. The only major product of h15-LOX-2's reaction with 5-HPETE was the proposed lipoxin intermediate, 5,15-dihydroperoxy-eicosatetraenoic acid (5,15-diHPETE). However, h15-LOX-2 did not react further with 5,15-diHPETE to produce lipoxins. This result is consistent with the specificity of h15-LOX-2 despite the increased reactivity of 5,15-diHPETE. Density functional theory calculations determined that the radical, after abstracting the C10 hydrogen atom from 5,15-diHPETE, had an energy 5.4 kJ/mol lower than that of the same radical generated from AA, demonstrating the facility of 5,15-diHPETE to form lipoxins. Interestingly, h15-LOX-2 does react with 5S,6R-diHETE, forming LipoxinA4, indicating the gemdiol does not prohibit h15-LOX-2 reactivity. Taken together, these results demonstrate the strict regiospecificity of h15-LOX-2 that circumscribes its role in transcellular synthesis.

Human reticulocyte 12/15-lipoxygenase (h12/15-LOX) is a lipid-oxidizing enzyme that can directly oxidize lipid membranes in the absence of a phospholipase, leading to a direct attack on organelles, such as the mitochondria. This cytotoxic activity of h12/15-LOX is up-regulated in neurons and endothelial cells after a stroke and thought to contribute to both neuronal cell death and blood-brain barrier leakage. The discovery of inhibitors that selectively target recombinant h12/15-LOX in vitro, as well as possessing activity against the murine ortholog ex vivo, could potentially support a novel therapeutic strategy for the treatment of stroke. Herein, we report a new family of inhibitors discovered in a High Throughput Screen (HTS) that are selective and potent against recombinant h12/15-LOX and cellular mouse 12/15-LOX (m12/15-LOX). MLS000099089 (compound 99089), the parent molecule, exhibits an IC50 potency of 3.4±0.5 μM against h12/15-LOX in vitro and an ex vivo IC50 potency of approximately 10 μM in a mouse neuronal cell line, HT-22. Compound 99089 displays greater than 30-fold selectivity versus h5-LOX and COX-2, 15-fold versus h15-LOX-2 and 10-fold versus h12-LOX, when tested at 20 μM inhibitor concentration. Steady-state inhibition kinetics reveals that the mode of inhibition of 99089 against h12/15-LOX is that of a mixed inhibitor with a Kic of 1.0±0.08 μM and a Kiu of 6.0±3.3 μM. These data indicate that 99089 and related derivatives may serve as a starting point for the development of anti-stroke therapeutics due to their ability to selectively target h12/15-LOX in vitro and m12/15-LOX ex vivo.

• Expression of SSAT1 was upregulated in ischemic penumbra of the mouse cortex. • Knockdown of SSAT1 alleviate cerebral I/R injury in mice. • SSAT1 positively regulated ALOX15 expression in cortical neuron. • ALOX15 inhibition reduced SSAT1 upregulation-induced neuronal loss and ferroptosis. Ferroptosis, an iron-dependent form of non-apoptotic cell death, is reportedly responsible for cerebral ischemia/reperfusion (I/R) injury. Evidence has shown that spermidine/spermine N1-acetyltransferase 1 (SSAT1) activation-induced ferroptosis is associated with upregulation of arachidonate 15-Lipoxygenase (ALOX15). Our previous study has revealed that upregulation of ALOX15 contributes to cerebral I/R injury via inducing microglial activation. The current study aimed to investigate the role of SSAT1/ALOX15 axis in neuronal ferroptosis after I/R. We found that the expression of SSAT1 was upregulated in the cortical penumbra of mice subjected to transient middle cerebral artery occlusion and reperfusion (tMCAO/R). Knockdown of SSAT1 mitigated I/R-induced cerebral infarction and neurological impairments, as well as decreased cortical iron contents, reactive oxygen species (ROS) generation and 4-Hydroxynonenal (4-HNE) level. Further in vitro evidence revealed that knockdown of SSAT1 downregulated the expression of ALOX15 in the primary cortical neurons exposed to tertbutyl-hydroksyperoxide (TBH). In addition, loss of neuronal viability and production of lipid hydroperoxides were inhibited in TBH-treated neurons when SSAT1 was knocked down. Mechanistically, SSAT1 overexpression decreased the expression levels of two key ferroptotic repressors, glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) in TBH-stimulated neurons. Treatment with the ALOX15 inhibitor PD146176 or ferroptosis inhibitor ferrostatin-1 partially reversed SSAT1 upregulation-induced ferroptosis and viability loss in TBH-treated neurons. These results together indicate that the activation of SSAT1/ALOX15 axis may aggravate cerebral I/R injury via triggering neuronal ferroptosis, providing novel insights into cerebral injury associated with lipid peroxidation. [ABSTRACT FROM AUTHOR]

Abstract Background Eosinophilic chronic rhinosinusitis with nasal polyps (ECRSwNP) exhibits a poorer outcome compared with non-eosinophilic chronic rhinosinusitis with nasal polyps (nonECRSwNP), so it is significant to identify effective markers to differentiate ECRSwNP in guiding the treatment strategies of these patients. Although arachidonate 15-lipoxygenase (ALOX15) is positioned as a marker of eosinophilic inflammation, its study in differentiating ECRSwNP has not been reported. The aim of this study is to assess the potential of ALOX15 in distinguishing and predicting ECRSwNP. Methods Forty-eight patients with chronic rhinosinusitis with nasal polyps (CRSwNP), including 30 ECRSwNP and 18 nonECRSwNP patients, were enrolled. ALOX15 mRNA level was determined in polyps by real-time polymerase chain reaction (RT-PCR). The patients’ baseline characteristics were evaluated and analyzed for correlations with ALOX15. Receiver operating characteristic (ROC) curve was used to assess the predictive significance of the potential predictors for ECRSwNP. Results ALOX15 mRNA level was significantly higher in ECRSwNP patients than in nonECRSwNP patients (P

A key challenge facing drug discovery today is variability of the drug target between species, such as with 12/15-lipoxygenase (12/15-LOX), which contributes to ischemic brain injury, but its human and rodent isozymes have different inhibitor specificities. In the current work, we have utilized a quantitative high-throughput (qHTS) screen to identify compound 1 (ML351), a novel chemotype for 12/15-LOX inhibition that has nanomolar potency (IC50 = 200 nM) against human 12/15-LOX and is protective against oxidative glutamate toxicity in mouse neuronal HT22 cells. In addition, it exhibited greater than 250-fold selectivity versus related LOX isozymes, was a mixed inhibitor, and did not reduce the active-site ferric ion. Lastly, 1 significantly reduced infarct size following permanent focal ischemia in a mouse model of ischemic stroke. As such, this represents the first report of a selective inhibitor of human 12/15-LOX with demonstrated in vivo activity in proof-of-concept mouse models of stroke.

Obesity is associated with metabolic perturbations including liver and adipose tissue inflammation, insulin resistance, and type 2 diabetes. Omega-6 fatty acids (ω6) promote and omega-3 fatty acids (ω3) reduce inflammation as they can be metabolized to pro- and anti-inflammatory eicosanoids, respectively. 12/15-lipoxygenase (12/15-LO) enzymatically produces some of these metabolites and is induced by high fat (HF) diet. We investigated the effects of altering dietary ω6/ω3 ratio and 12/15-LO deficiency on HF diet-induced tissue inflammation and insulin resistance. We examined how these conditions affect circulating concentrations of oxidized metabolites of ω6 arachidonic and linoleic acids and innate and adaptive immune system activity in the liver. For 15 weeks, wild-type (WT) mice were fed either a soybean oil-enriched HF diet with high dietary ω6/ω3 ratio (11∶1, HFH), similar to Western-style diet, or a fat Kcal-matched, fish oil-enriched HF diet with a low dietary ω6/ω3 ratio of 2.7∶1 (HFL). Importantly, the total saturated, monounsaturated and polyunsaturated fat content was matched in the two HF diets, which is unlike most published fish oil studies in mice. Despite modestly increased food intake, WT mice fed HFL were protected from HFH-diet induced steatohepatitis, evidenced by decreased hepatic mRNA expression of pro-inflammatory genes and genes involved in lymphocyte homing, and reduced deposition of hepatic triglyceride. Furthermore, oxidized metabolites of ω6 arachidonic acid were decreased in the plasma of WT HFL compared to WT HFH-fed mice. 12/15-LO knockout (KO) mice were also protected from HFH-induced fatty liver and elevated mRNA markers of inflammation and lymphocyte homing. 12/15-LOKO mice were protected from HFH-induced insulin resistance but reducing dietary ω6/ω3 ratio in WT mice did not ameliorate insulin resistance or adipose tissue inflammation. In conclusion, lowering dietary ω6/ω3 ratio in HF diet significantly reduces steatohepatitis.

Lipoxygenases, important enzymes in inflammation, can regulate their substrate specificity by allosteric interactions with their own hydroperoxide products. In this work, addition of both 13-(S)-hydroxy-(9Z,11E)-octadecadienoic acid [13-(S)-HODE] and 13-(S)-hydroperoxy-(6Z,9Z,11E)-octadecatrienoic acid to human epithelial 15-lipoxygenase-2 (15-LOX-2) increases the kcat/KM substrate specificity ratio of arachidonic acid (AA) and γ-linolenic acid (GLA) by 4-fold. 13-(S)-HODE achieves this change by activating kcat/KM(AA) but inhibiting kcat/KM(GLA), which indicates that the allosteric structural changes at the active site discriminate between the length and unsaturation differences of AA and GLA to achieve opposite kinetic effects. The substrate specificity ratio is further increased, 11-fold in total, with an increase in pH, suggesting mechanistic differences between the pH and allosteric effects. Interestingly, the loss of the PLAT domain affects substrate specificity but does not eliminate the allosteric properties of 15-LOX-2, indicating that the allosteric site is located in the catalytic domain. However, the removal of the PLAT domain does change the magnitude of the allosteric effect. These data suggest that the PLAT domain moderates the communication pathway between the allosteric and catalytic sites, thus affecting substrate specificity. These results are discussed in the context of protein dimerization and other structural changes.

Previously, we observed significant increases in spinal 12-lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation-induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15-LOX enzymes in HEK-293T cells and measured by LC-MS/MS the formation of HXB3, 12-HETE, 8-HETE, and 15-HETE from arachidonic acid (AA) at baseline and in the presence of LOX inhibitors (NDGA, AA-861, CDC, baicalein, and PD146176) vs. vehicle-treated and mock-transfected controls. We detected the following primary intrinsic activities: 12-LOX (Alox12, Alox15), 15-LOX (Alox15b), and HXS (Alox12, Alox15). Similar to human and mouse orthologs, proteins encoded by rat Alox12b and Alox12e possessed minimal 12-LOX activity with AA as substrate, while eLOX3 (encoded by Aloxe3) exhibited HXS without 12-LOX activity when coexpressed with Alox12b or supplemented with 12-HpETE. CDC potently inhibited HXS and 12-LOX activity in vitro (relative IC50s: CDC, ~0.5 and 0.8 μM, respectively) and carrageenan-evoked tactile allodynia in vivo. Notably, peripheral inflammation significantly increased spinal eLOX3; intrathecal pretreatment with either siRNA targeting Aloxe3 or an eLOX3-selective antibody attenuated the associated allodynia. These findings implicate spinal eLOX3-mediated hepoxilin synthesis in inflammatory hyperesthesia and underscore the importance of developing more selective 12-LOX/HXS inhibitors.

The 5- and 12/15-lipoxygenase (LOX) isozymes have been implicated to contribute to disease development in CNS disorders such as Alzheimer's disease. These LOX isozymes are distinct in function, with differential effects on neuroinflammation, and the impact of the distinct isozymes in the pathogenesis of Parkinson's disease has not as yet been evaluated. To determine whether the isozymes contribute differently to nigrostriatal vulnerability, the effects of 5- and 12/15-LOX deficiency on dopaminergic tone under naïve and toxicant-challenged conditions were tested. In naïve mice deficient in 5-LOX expression, a modest but significant reduction (18.0% reduction vs. wildtype (WT)) in striatal dopamine (DA) was detected (n=6-8 per genotype). A concomitant decline in striatal tyrosine hydroxylase (TH) enzyme was also revealed in null 5-LOX vs. WT mice (26.2%); however, no changes in levels of DA or TH immunoreactivity were observed in null 12/15-LOX vs. WT mice. When challenged with the selective dopaminergic toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), WT mice showed a marked reduction in DA (31.9%) and robust astrocytic and microglial activation as compared to saline-treated animals. In contrast, null 5-LOX littermates demonstrated no significant striatal DA depletion or astrogliosis (as noted by Western blot analyses for glial acidic fibrillary protein (GFAP) immunoreactivity). In naïve null 12/15-LOX mice, no significant change in striatal DA values was observed compared to WT, and following MPTP treatment, the transgenics revealed striatal DA reduction similar to the challenged WT mice. Taken together, these data provide the first evidence that: (i) LOX isozymes are involved in the maintenance of normal dopaminergic function in the striatum and (ii) the 5- and 12/15-LOX isozymes contribute differentially to striatal vulnerability in response to neurotoxicant challenge.

Targeting newly identified damage pathways in the ischemic brain can help to circumvent the currently severe limitations of acute stroke therapy. Here we show that the activity of 12/15-lipoxygenase was increased in the ischemic mouse brain, and 12/15-lipoxygenase colocalized with a marker for oxidized lipids, MDA2. This colocalization was also detected in the brain of 2 human stroke patients, where it also coincided with increased apoptosis-inducing factor. A novel inhibitor of 12/15-lipoxygenase, LOXBlock-1, protected neuronal HT22 cells against oxidative stress. In a mouse model of transient focal ischemia, the inhibitor reduced infarct sizes both 24 hours and 14 days poststroke, with improved behavioral parameters. Even when treatment was delayed until at least 4 hours after onset of ischemia, LOXBlock-1 was protective. Furthermore, it reduced tissue plasminogen activator-associated hemorrhage in a clot model of ischemia/reperfusion. This study establishes inhibition of 12/15-lipoxygenase as a viable strategy for first-line stroke treatment.

The involvement of lipoxygenases in various pathologies, combined with the unavailability of safe and effective inhibitors of the biosynthesis of their products, is a source of inspiration for the development of new inhibitors. Based on a structural analysis of known inhibitors of lipoxygenase products biosynthesis, a comprehensive structure-activity study was carried out, which led to the discovery of several novel compounds (16a-c, 17a) demonstrating promising potency to inhibit the biosynthesis of products of 5-, 12- and 15-LO. Compounds 16b and 16c outperformed zileuton (1), the only FDA-approved 5-LO inhibitor, as well as known inhibitors such as caffeic acid phenethyl ester (CAPE (2)) and cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC (4)). However, the introduction of a cyano group at the α-position of the carbonyl abolished the activity. Compounds 16a and 17a also inhibited the biosynthesis of 12- and 15-LO products. Compounds 16a, 17a far surpassed baicalein, a known 12-LO inhibitor, as inhibitors of 12-LO products biosynthesis. Compound 17a and CDC (4) showed equivalent inhibition of LO products, proposing that the double bond in the ester moiety is not necessary for the inhibitory activity. The introduction of the cyano group, as in compound 17a, at the α-position of the carbonyl in compound 16a significantly reduced the inhibitory activity against the biosynthesis of 15-LO products. In addition to the interactions with residues His372 and Phe421 also found with zileuton and CAPE, compounds 16a and 16c each interact with residue His367 as shown by molecular docking. This new interaction may explain their high affinity with the 5-LO active site., (© 2024 Wiley Periodicals LLC.)

The neural cell death in cerebral infarction is suggested to be ferroptosis-like cell death, involving the participation of 15-lipoxygenase (15-LOx). Ferroptosis is induced by lipid radical species generated through the one-electron reduction of lipid hydroperoxides, and it has been shown to propagate intracellularly and intercellularly. At lower oxygen concentration, it appeared that both regiospecificity and stereospecificity of conjugated diene moiety in lipoxygenase-catalysed lipid hydroperoxidation are drastically lost. As a result, in the reaction with linoleic acid, the linoleate 9-peroxyl radical-ferrous lipoxygenase complex dissolves into the linoleate 9-peroxyl radical and ferrous 15-lipoxygenase. Subsequently, the ferrous 15-lipoxygenase then undergoes one-electron reduction of 13-hydroperoxy octadecadienoic acid, generating an alkoxyl radical (pseudoperoxidase reaction). A part of the produced lipid alkoxyl radicals undergoes cleavage of C-C bonds, liberating small molecular hydrocarbon radicals. Particularly, in ω-3 polyunsaturated fatty acids, which are abundant in the vascular and nervous systems, the liberation of small molecular hydrocarbon radicals was more pronounced compared to ω-6 polyunsaturated fatty acids. The involvement of these small molecular hydrocarbon radicals in the propagation of membrane lipid damage is suggested.

Urban particulate matter (PM; uPM) poses significant health risks, particularly to the respiratory system. Fine particles, such as PM2.5, can penetrate deep into the lungs and exacerbate a range of health problems, including emphysema, asthma, and lung cancer. PM exposure is also linked to extrapulmonary disorders such as heart and neurodegenerative diseases. Moreover, prolonged exposure to elevated PM levels can reduce overall life expectancy. Senescence is a dysfunctional cell state typically associated with age but can also be precipitated by environmental stressors. This study aimed to determine whether uPM could drive senescence in macrophages, an essential cell type involved in particulate phagocytosis-mediated clearance. Although it is known that uPM exposure impairs immune function, this deficit is multifaceted and incompletely understood, partly because of the use of particulates such as diesel exhaust particles as a surrogate for true uPM. uPM was collected from several locations in the United States, including Baltimore, Houston, and Phoenix. Bone marrow-derived macrophages were stimulated with uPM or reference particulates (e.g., diesel exhaust particles) to assess senescence-related parameters. We report that uPM-exposed bone marrow-derived macrophages adopt a senescent phenotype characterized by increased IL-1α secretion, senescence-associated β-galactosidase activity, and diminished proliferation. Exposure to allergens failed to elicit such a response, supporting a distinction between different types of environmental exposure. uPM-induced senescence was independent of key macrophage activation pathways, specifically inflammasome and scavenger receptors. However, inhibition of the phagolysosome pathway abrogated senescence markers, supporting this phenotype's attribution to uPM phagocytosis. These data suggest that uPM exposure leads to macrophage senescence, which may contribute to immunopathology., (Copyright © 2024 The Authors.)

There are a variety of lipoxygenases in the human body (hLO), each having a distinct role in cellular biology. Human reticulocyte 15-lipoxygenase-1 (15-hLO-1), which catalyzes the dioxygenation of 1,4-cis,cis-pentadiene-containing polyunsaturated fatty acids, is implicated in a number of diseases including cancer, atherosclerosis, and neurodegenerative conditions. Despite the potential therapeutic relevance of this target, few inhibitors have been reported that are both potent and selective. To this end, we have employed a quantitative high-throughput (qHTS) screen against ∼74000 small molecules in search of reticulocyte 15-hLO-1 selective inhibitors. This screen led to the discovery of a novel chemotype for 15-hLO-1 inhibition, which displays nM potency and is >7500-fold selective against the related isozymes, 5-hLO, platelet 12-hLO, epithelial 15-hLO-2, ovine cyclooxygenase-1, and human cyclooxygenase-2. In addition, kinetic experiments were performed which indicate that this class of inhibitor is tight binding, reversible, and appears not to reduce the active-site ferric ion.

Background: Coronary artery disease (CAD) is major cause of death and morbidity worldwide. Arachidonate 12/15-lipoxygenase (ALOX) is a member of the lipid peroxidizing enzyme family and implicated in the pathogenesis of atherosclerosis, but with contradicting results. Aim: The present study aimed to investigate the association of two polymorphisms in ALOX15 (rs2619112 and rs7217186) and the risk of CAD in North Indian population. Methods: A total of 500 angiographically confirmed CAD patients and 500 control subjects of North Indian population were recruited in the case-control study and genotyped by PCR-RFLP. Results: The data showed a significant association between the two polymorphisms and CAD. Multiple logistic regression revealed heterozygous genotype of both the polymorphisms viz. GA of rs2619112 and CT of rs7217186 to be associated with significant high risk of CAD after adjustment for confounders (p = 0.034, OR = 2.274, 95% CI (1.062–4.870) and p = 0.000, OR = 3.407, 95% CI (2.092–5.548) respectively). Stratified analysis based on gender showed GA and AA of rs2619112 each significantly increased the risk of CAD in females (p = 0.001, OR = 13.120, CI = 2.780–61.928; p = 0.028, OR = 5.393, CI = 1.196–24.316 respectively) whereas only GA increased CAD risk in males (p = 0.005, OR = 2.277, CI = 1.290–4.020). In case of rs7217186, CT and TT showed a significant high risk of CAD in males (p = 0.000, OR = 4.048, CI = 2.678–6.119; p = 0.000, OR = 2.861, CI = 1.928–4.245). Conclusion: The present study shows that rs7217186:C > T and rs2619112:G > A of ALOX15 are associated with increased risk of CAD in the North Indian population.

Allosteric regulation of human lipoxygenase (hLO) activity has recently been implicated in the cellular biology of prostate cancer. In the current work, we present isotope effect, pH, and substrate inhibitor data of epithelial 15-hLO-2, which probe the allosteric effects on its mechanistic behavior. The Dk(cat)/KM for 15-hLO-2, with AA and LA as substrate, is large indicating hydrogen atom abstraction is the principle rate-determining step, involving a tunneling mechanism for both substrates. For AA, there are multiple rate determining steps (RDS) at both high and low temperatures, with both diffusion and hydrogen bonding rearrangements contributing at high temperature, but only hydrogen bonding rearrangements contributing at low temperature. The observed kinetic dependency on the hydrogen bonding rearrangement is eliminated upon addition of the allosteric effector, 13-(S)-hydroxyoctadecadienoic acid (13-HODE), and no allosteric effects were seen on diffusion or hydrogen atom abstraction. The (k(cat)/KM)AA/(k(cat)/KM)LA ratio was observed to have a pH dependence, which was fit with a titration curve (pKa = 7.7), suggesting the protonation of a histidine residue, which could hydrogen bond with the carboxylate of 13-HODE. Assuming this interaction, 13-HODE was docked to the solvent exposed histidines of a 15-hLO-2 homology model and found to bind well with H627, suggesting a potential location for the allosteric site. Utilizing d31-LA as an inhibitor, it was demonstrated that the binding of d31-LA to the allosteric site changes the conformation of 15-hLO-2 such that the affinity for substrate increases. This result suggests that allosteric binding locks the enzyme into a catalytically competent state, which facilitates binding of LA and decreases the (k(cat)/KM)AA/(k(cat)/KM)LA ratio. Finally, the magnitude of the 13-HODE KD for 15-hLO-2 is over 200-fold lower than that of 13-HODE for 15-hLO-1, changing the substrate specificity of 15-hLO-2 to 1.9. This would alter the LO product distribution and increase the production of the pro-tumorigenic, 13-HODE, possibly representing a pro-tumorigenic feedback loop for 13-HODE and 15-hLO-2.

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.

Human reticulocyte 15-lipoxygenase (15-hLO-1) and epithelial 15-lipoxygenase (15-hLO-2) have been implicated in a number of human diseases, with differences in their substrate specificity potentially playing a central role. In this paper, we present a novel method for accurately measuring the substrate specificity of the two 15-hLO isozymes and demonstrate that both cholate and specific LO products affect substrate specificity. The linoleic acid (LA) product, 13-hydroperoxyoctadienoic acid (13-HPODE), changes the ( k cat/ K m) (AA)/( k cat/ K m) (LA) ratio more than 5-fold for 15-hLO-1 and 3-fold for 15-hLO-2, while the arachidonic acid (AA) product, 12-( S)-hydroperoxyeicosatetraenoic acid (12-HPETE), affects only the ratio of 15-hLO-1 (more than 5-fold). In addition, the reduced products, 13-( S)-hydroxyoctadecadienoic acid (13-HODE) and 12-( S)-hydroxyeicosatetraenoic acid (12-HETE), also affect substrate specificity, indicating that iron oxidation is not responsible for the change in the ( k cat/ K m) (AA)/( k cat/ K m) (LA) ratio. These results, coupled with the dependence of the 15-hLO-1 k cat/ K m kinetic isotope effect ( (D) k cat/ K m) on the presence of 12-HPETE and 12-HETE, indicate that the allosteric site, previously identified in 15-hLO-1 [Mogul, R., Johansen, E., and Holman, T. R. (1999) Biochemistry 39, 4801-4807], is responsible for the change in substrate specificity. The ability of LO products to regulate substrate specificity may be relevant with respect to cancer progression and warrants further investigation into the role of this product-feedback loop in the cell.

Lipoxygenases (LOs) catalyze lipid peroxidation and have been implicated in a number of human diseases connected to oxidative stress and inflammation. These enzymes have also attracted considerable attention due to large kinetic isotope effects (30-80) for the rate-limiting hydrogen abstraction step with linoleic acid (LA) as substrate. Herein, we report kinetic isotope effects (KIEs) in the reactions of three human LOs (platelet 12-hLO, reticulocyte 15-hLO-1, and epithelial 15-hLO-2) with arachidonic acid (AA). Surprisingly, the observed KIEs with AA were much smaller than the previously reported values with LA. Investigation into the origins for the smaller KIEs led to the discovery of isotope sensitive branching of the reaction pathways. Product distribution analysis demonstrated an inversion in the regioselectivity of 15-hLO-1, with hydrogen abstraction from C13 being the major pathway with unlabeled AA but abstraction from C10 predominating when the methylene group at position 13 was deuterated. Smaller but clear changes in regioselectivity were also observed for 12-hLO and 15-hLO-2.

Oxidation of low density lipoprotein (LDL) occurs in vivo and significantly contributes to the development of atherosclerosis. An important mechanism of LDL oxidation in vivo is its modification with 12/15-lipoxygenase (LO). We have developed a model of minimally oxidized LDL (mmLDL) in which native LDL is modified by cells expressing 12/15LO. This mmLDL activates macrophages inducing membrane ruffling and cell spreading, activation of ERK1/2 and Akt signaling, and secretion of proinflammatory cytokines. In this study, we found that many of the biological activities of mmLDL were associated with cholesteryl ester (CE) hydroperoxides and were diminished by ebselen, a reducing agent. Liquid chromatography coupled with mass spectroscopy demonstrated the presence of many mono- and polyoxygenated CE species in mmLDL but not in native LDL. Nonpolar lipid extracts of mmLDL activated macrophages, although to a lesser degree than intact mmLDL. The macrophage responses were also induced by LDL directly modified with immobilized 12/15LO, and the nonpolar lipids extracted from 12/15LO-modified LDL contained a similar set of oxidized CE. Cholesteryl arachidonate modified with 12/15LO also activated macrophages and contained a similar collection of oxidized CE molecules. Remarkably, many of these oxidized CE were found in the extracts of atherosclerotic lesions isolated from hyperlipidemic apoE(-/-) mice. These results suggest that CE hydroperoxides constitute a class of biologically active components of mmLDL that may be relevant to proinflammatory activation of macrophages in atherosclerotic lesions.

The lipid-metabolizing enzyme 12/15-lipoxygenase (12/15-LOX) mediates cell death resulting from oxidative stress in both neurons and oligodendrocytes. Specifically, it may contribute to the pathophysiology of stroke and Alzheimer's and Parkinson's diseases. We report here that two of three specific 12/15-LOX inhibitors, derived from a virtual screen by computer modeling and validated by inhibition of recombinant human 15-LOX in vitro, are able to rescue both neuronal as well as oligodendroglial cells from cell death induced by oxidative stress. Thus, in a fairly streamlined process, an initial virtual screen of 50,000 compounds in a library of drug-like molecules has led to the identification of two novel drug candidates for targeting LOX. Future studies of these novel neuroprotective inhibitors of 12/15-LOX may provide new therapeutic opportunities to combat stroke and other neurodegenerative diseases.

Human lipoxygenase (hLO) isozymes have been implicated in a number of disease states and have attracted much attention with respect to their inhibition. One class of inhibitors, the flavonoids, have been shown to be potent lipoxygenase inhibitors but their study has been restricted to those compounds found in nature, which have limited structural variability. We have therefore carried out a comprehensive study to determine the structural requirements for flavonoid potency and selectivity against platelet 12-hLO, reticulocyte 15-hLO-1, and prostate epithelial 15-hLO-2. We conclude from this study that catechols are essential for high potency, that isoflavones and isoflavonones tend to select against 12-hLO, that isoflavons tend to select against 15-hLO-1, but few flavonoids target 15-hLO-2.

We report the discovery of new, low micromolar, small molecule inhibitors of human platelet-type 12- and reticulocyte 15-lipoxygenase-1 (12-hLO and 15-hLO) using structure-based methods. Specifically, we created homology models of 12-hLO and 15-hLO, based on the structure of rabbit 15-lipoxygenase, for in silico screening of a large compound library followed by in vitro screening of 20 top scoring molecules. Eight of these compounds inhibited either 12- or 15-human lipoxygenase with lower than 100 microM affinity. Of these, we obtained IC50 values for the three best inhibitors, all of which displayed low micromolar inhibition. One compound showed specificity for 15-hLO versus 12-hLO; however, a selective inhibitor for 12-hLO was not identified. As a control we screened 20 randomly selected compounds, of which none showed low micromolar inhibition. The new low-micromolar inhibitors appear to be suitable as leads for further inhibitor development efforts against 12-hLO and 15-hLO, based on the fact their size and chemical properties are appropriate to classify them as drug-like compounds. The models of these protein-inhibitor complexes suggest strategies for future development of selective lipoxygenase inhibitors.

The selective inhibition of human 15-lipoxygenase (15-hLO) could serve as a promising therapeutic target for the prevention of atherosclerosis. A screening of marine sponges revealed that crude extracts of Psammocinia sp. exhibited potent 15-hLO inhibitory activity. Bioassay-guided fractionation led to the isolation of chromarols A-E (8-12) as potent and selective inhibitors of 15-hLO. An additional 22 structurally related compounds, including meroditerpenes from the same Psammocinia sp. (3, 4, 13-16) and our pure compound repository (17, 18), commercially available tocopherols (19-24), and synthetic chromanes (25-32), were evaluated for their ability to inhibit human lipoxygenases. The 6-hydroxychromane moiety found in chromarols A-D was identified as essential for the selective redox inhibition of 15-hLO. Furthermore, the oxidized form of the 6-hydroxychromane could be reduced by ascorbate, suggesting a potential regeneration pathway for these inhibitors in the body. This pharmacophore represents a promising paradigm for the development of a unique class of recyclable 15-hLO redox inhibitors for the treatment of atherosclerosis.