Pontus Forsell | Karolinska Institutet (original) (raw)
Papers by Pontus Forsell
Additional file 1: Figure S1. Characterization of differentiated SH-SY5Y cells. Figure S2. Cytoto... more Additional file 1: Figure S1. Characterization of differentiated SH-SY5Y cells. Figure S2. Cytotoxicity of compounds evaluated in the HTS. Figure S3. Effects of luteolin in respiratory capacity and ΔΨm. Figure S4. Effects of luteolin in mitochondrial biogenesis and cristae organization. Figure S5. Effects of luteolin on calcium levels and characterization of isolated mitochondrial fractions.
Additional file 2. Prestwick Chemical Library compounds used in the HTS and the corresponding ATP... more Additional file 2. Prestwick Chemical Library compounds used in the HTS and the corresponding ATP and cytotoxicity z-scores (data related with Fig. 2b and Additional file 1: Figure S2A).
Additional file 3. ATP and cytotoxicity z-scores obtained for the 3-CRC (data related with Fig. 2... more Additional file 3. ATP and cytotoxicity z-scores obtained for the 3-CRC (data related with Fig. 2c and Additional file 1: Figure S2D).
Bioorganic & medicinal chemistry letters, 2015
Investigation of 1N-substituted pyrazole-3-carboxanilides as 15-lipoxygenase-1 (15-LOX-1) inhibit... more Investigation of 1N-substituted pyrazole-3-carboxanilides as 15-lipoxygenase-1 (15-LOX-1) inhibitors demonstrated that the 1N-substituent was not essential for activity or selectivity. Additional halogen substituents on the pyrazole ring, however, increased activity. Further development led to triazole-4-carboxanilides and 2-(3-pyrazolyl) benzoxazoles, which are potent and selective 15-LOX-1 inhibitors.
Bioorganic & Medicinal Chemistry Letters, 2015
High-throughput screening was used to find selective inhibitors of human 15-lipoxygenase-1 (15-LO... more High-throughput screening was used to find selective inhibitors of human 15-lipoxygenase-1 (15-LOX-1). One hit, a 1-benzoyl substituted pyrazole-3-carboxanilide (1a), was used as a starting point in a program to develop potent and selective 15-LOX-1 inhibitors.
Annual Reports in Medicinal Chemistry, 2014
Abstract Neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, a... more Abstract Neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease are currently lacking disease-modifying treatments. A substantial body of evidence links increases in neurotrophin (NT) signaling in neurodegenerative disorders with biological mechanisms that could have a positive effect on disease outcome. NTs like NGF and BDNF signal through Trk receptors. This signaling is of high importance to support neuronal survival, differentiation, neurogenesis, and synaptic plasticity. The poor pharmacokinetics of NTs makes them unsuitable as drugs. In addition, the NTs have pleiotropic effects that may give side effects. Therefore, the identification of small molecules that promote NT signaling is of increasing interest. Such small-molecule activators of NT signaling will support neuronal survival in the CNS, and thus they could possibly function as disease modifiers. Herein, we review the current status of small-molecule compounds that are able to activate NT receptors.
European Journal of Biochemistry, 1999
Recently, we reported the human 88-kDa calcium-independent phospholipase A2 (iPLA2) cDNA sequence... more Recently, we reported the human 88-kDa calcium-independent phospholipase A2 (iPLA2) cDNA sequence, as well as extensive alternative splicing of the iPLA2 mRNA. In this report we identified the gene coding for iPLA2, which was localized on chromosome 22q13.1. The gene consists of at least 17 exons spanning > 69 kb. Based on the iPLA2 gene organization the splice variants can be explained. The putative promotor for the iPLA2 gene lacks a TATA-box and contains a CpG island as well as several potential Sp-1-binding sites. Furthermore, the 5'-flanking region also contains one medium reiteration frequency repeat (MER53) and an Alu repetitive sequence. Northern blot analysis of iPLA2 mRNA in various human tissues demonstrated tissue-specific expression of four distinct iPLA2 transcripts. The native human 3.2-kb iPLA2 transcript was predominantly expressed in heart, brain, skeletal muscle, prostate, testis, thyroid and spinal cord, and to a lesser extent in peripheral blood leucocytes, stomach, trachea and bone marrow. Studies on the subcellular localization of the native iPLA2 protein were performed in COS-7 cells overexpressing this enzyme. The cytosolic fraction of untransfected and cells overexpressing iPLA2 contained equal amounts of calcium-independent PLA2 activity. However, the membrane fraction displayed a 5.5-fold increased activity in iPLA2 overexpressing cells. This increased calcium-independent PLA2 activity correlated with the presence of iPLA2 immunoreactive protein in the membrane fraction, indicating that this form of iPLA2 protein was membrane associated. Studies of iPLA2 in rat vascular smooth muscle cells verified the membrane association of this form of iPLA2. The major difference between this form of iPLA2 enzyme and the soluble forms of iPLA2 studied previously is the presence of 54 additional amino acid residues derived from exon 9. We suggest that the addition of these 54 amino acids leads to a membrane-associated protein. In summary, these results demonstrate that alternative splicing of the human iPLA2 transcript generates multiple iPLA2 isoforms with distinct tissue distribution and cellular localization.
Lipids, 2012
Human 15-lipoxygenase-1 (15-LO-1) can metabolize arachidonic acid (ARA) into pro-inflammatory med... more Human 15-lipoxygenase-1 (15-LO-1) can metabolize arachidonic acid (ARA) into pro-inflammatory mediators such as the eoxins, 15-hydroperoxyeicosatetraenoic acid (HPETE), and 15-hydroxyeicosatetraenoyl-phosphatidylethanolamine. We have in this study investigated the formation of various lipid hydroperoxide by either purified 15-LO-1 or in the Hodgkin lymphoma cell line L1236, which contain abundant amount of 15-LO-1. Both purified 15-LO-1 and L1236 cells produced lipid hydroperoxides more efficiently when anandamide (AEA) or 2-arachidonoyl-glycerol ester was used as substrate than with ARA. Furthermore, L1236 cells converted AEA to a novel class of cysteinyl-containing metabolites. Based on RP-HPLC, mass spectrometry and comparison to synthetic products, these metabolites were identified as the ethanolamide of the eoxin (EX) C(4) and EXD(4). By using the epoxide EXA(4)-ethanol amide, it was also found that platelets have the capacity to produce the ethanolamide of EXC(4) and EXD(4). We suggest that the ethanolamides of the eoxins should be referred to as eoxamides, in analogy to the ethanolamides of prostaglandins which are named prostamides. The metabolism of AEA into eoxamides might engender molecules with novel biological effects. Alternatively, it might represent a new mechanism for the termination of AEA signalling.
Journal of Biomolecular Screening, 2010
15-Lipoxygenase-1 catalyzes the introduction of molecular oxygen into polyunsaturated fatty acids... more 15-Lipoxygenase-1 catalyzes the introduction of molecular oxygen into polyunsaturated fatty acids to form a lipid hydroperoxide. The authors have developed an assay for the detection of lipid hydroperoxides formed by human 15-lipoxygenase (15-LO) in enzyme or cellular assays using either a 96-well or a 384-well format. The assays described take advantage of the ability of lipid hydroperoxides to oxidize nonfluorescent diphenyl-1-pyrenylphosphine (DPPP) to a fluorescent phosphine oxide. Oxidation of DPPP yields a fluorescent compound, which is not sensitive to temperature and is stable for more than 2 h. The assay is sensitive toward inhibition and robust with a Z′ value of 0.79 and 0.4 in a 96- and 384-well format, respectively, and thus amenable for high-throughput screening. The utility of DPPP as a marker for 15-lipoxygenase activity was demonstrated with both enzyme- and cell-based assays for the identification of hits and to determine potency by IC50determinations.
Journal of Biological Chemistry, 2009
The nomenclature of lipoxygenases (LOXs) is partly based on the positional specificity of arachid... more The nomenclature of lipoxygenases (LOXs) is partly based on the positional specificity of arachidonic acid oxygenation, but there is no unifying concept explaining the mechanistic basis of this enzyme property. According to the triad model, Phe-353, Ile-418, and Ile-593 of the rabbit 12/15-LOX form the bottom of the substrate-binding pocket, and introduction of less spacefilling residues at either of these positions favors arachidonic acid 12-lipoxygenation. The present study was aimed at exploring the validity of the triad concept for two novel primate 12/15-LOX (Macaca mulatta and Pongo pygmaeus) and for five known members of the mammalian LOX family (human 12/15-LOX, mouse 12/15-LOX, human 15-LOX2, human platelet type 12-LOX, and mouse (12R)-LOX). The enzymes were expressed as N-terminal His tag fusion proteins in E. coli, the potential sequence determinants were mutated, and the specificity of arachidonic acid oxygenation was quantified. Taken together, our data indicate that the triad concept explains the positional specificity of all 12/15-LOXs tested (rabbit, human, M. mulatta, P. pygmaeus, and mouse). For the new enzymes of M. mulatta and P. pygmaeus, the concept had predictive value because the positional specificity predicted on the basis of the amino acid sequence was confirmed experimentally. The specificity of the platelet 12-LOX was partly explained by the triad hypothesis, but the concept was not applicable for 15-LOX2 and (12R)-LOX.
The Journal of biological chemistry, 2007
Although protein-tyrosine phosphatase 1B (PTP-1B) is a negative regulator of insulin action, adip... more Although protein-tyrosine phosphatase 1B (PTP-1B) is a negative regulator of insulin action, adipose tissue from PTP-1B-/- mice does not show enhanced insulin-stimulated insulin receptor phosphorylation. Investigation of glucose uptake in isolated adipocytes revealed that the adipocytes from PTP-1B-/- mice have a significantly attenuated insulin response as compared with PTP-1B+/+ adipocytes. This insulin resistance manifests in PTP-1B-/- animals older than 16 weeks of age and could be partially rescued by adenoviral expression of PTP-1B in null adipocytes. Examination of adipose signaling pathways found that the basal p70S6K activity was at least 50% higher in adipose from PTP-1B-/- mice compared with wild type animals. The increased basal activity of p70S6K in PTP-1B-/- adipose correlated with decreases in IR substrate-1 protein levels and insulin-stimulated Akt/protein kinase B activity, explaining the decrease in insulin sensitivity even as insulin receptor phosphorylation was u...
Experimental Cell Research, 2012
Hodgkin lymphoma (HL) is a unique entity among the lymphomas, with a minority of malignant Hodgki... more Hodgkin lymphoma (HL) is a unique entity among the lymphomas, with a minority of malignant Hodgkin and Reed-Sternberg (H-RS) cells surrounded by a broad range of infiltrating cells. The infiltration of certain inflammatory cells has been reported to predict prognosis of the disease. In HL tumor microenvironment, the primary H-RS cells and those inflammatory cells interact interdependently. The aberrant cytokine production of H-RS cells has been suggested to contribute to this interdependency. However, little is known in terms of the mechanisms involved in the abnormal cytokine secretion by H-RS cells. Previous studies suggested that several pro-inflammatory molecules likely contribute to the aberrant cytokine secretion of HL, including cysteinyl-leukotrienes receptor type I (CysLT 1 R) and 15-lipoxygenase-1(15-LOX-1) that are highly expressed in primary H-RS cells and cultured HL-derived L1236 cells. Previous and present studies in cultured HL cells demonstrate that CysLT 1 R mediates transcription and secretion of cytokines, including interleukin (IL)-6, IL-8 and tumor necrosis factor-α, upon stimulation by leukotriene D 4 (LTD 4). This lipid mediator is formed from arachidonic acid through the 5-lipoxygenase (5-LOX) pathway and several types of inflammatory cells surrounding H-RS cells can produce cysteinylleukotrienes. To depict the intracellular signaling pathways that bridge the LTD 4-CysLT 1 R ligation to cytokine induction, a mechanistic study was carried out in L1236 cells. The results demonstrated that the transcription factor early growth response (EGR)-1 is involved in the LTD 4-triggered cytokine transcriptional induction. The regulatory mechanisms implicated in 15-LOX-1 trans-activation in HL have been obscure. This study has also assessed the epigenetic modulation of 15-LOX-1 in different aspects. The results revealed that signal transducer and activator of transcription (STAT)-6 positively regulates 15-LOX-1 transcription by binding to its promoter, in which three putative STAT-6 binding motifs are identified to be required for full activation. The accessibility of STAT-6 to the 15-LOX-1 promoter is controlled by DNA methylation and histone modification. The histone H3 lysine (K)-4 specific methyltransferase SMYD3 was found to exhibit an important role in this multi-step regulation. Although the H3K27me3 demethylase UTX mediates 15-LOX-1 transactivation by H3K27 demethylation upon IL-4 stimulation in lung carcinoma A549 cells, a crucial histone H3K27-demethylase-independent role of UTX in 15-LOX-1 transcriptional regulation in L1236 cells was demonstrated. In conclusion, this study has evaluated the biology of HL by using in vitro models, focusing on lipoxygenases regulation and function. The results not only demonstrated a signaling pathway that hypothetically bridges 5-LOX activity to the striking inflammatory microenvironment in HL, but also uncovered epigenetic regulation mechanisms involved in 15-LOX-1 expression in HL-derived cells. Our findings suggest that lipid signaling pathways might play critical roles in HL pathogenesis, thus warranting further HL research.
Experimental Cell Research, 2004
15-Lipoxygenase type 1 (15-LO), a lipid-peroxidating enzyme implicated in physiological membrane ... more 15-Lipoxygenase type 1 (15-LO), a lipid-peroxidating enzyme implicated in physiological membrane remodeling and the pathogenesis of atherosclerosis, inflammation, and carcinogenesis, is highly regulated and expressed in a tissue-and cell-type-specific fashion. It is known that interleukins (IL) 4 and 13 play important roles in transactivating the 15-LO gene. However, the fact that they only exert such effects on a few types of cells suggests additional mechanism(s) for the profile control of 15-LO expression. In the present study, we demonstrate that hyper-and hypomethylation of CpG islands in the 15-LO promoter region is intimately associated with the transcriptional repression and activation of the 15-LO gene, respectively. The 15-LO promoter was exclusively methylated in all examined cells incapable of expressing 15-LO (certain solid tumor and human lymphoma cell lines and human T lymphocytes) while unmethylated in 15-LO-competent cells (the human airway epithelial cell line A549 and human monocytes) where 15-LO expression is IL4-inducible. Inhibition of DNA methylation in L428 lymphoma cells restores IL4 inducibility to 15-LO expression. Consistent with this, the unmethylated 15-LO promoter reporter construct exhibited threefold higher activity in A549 cells compared to its methylated counterpart. Taken together, demethylation of the 15-LO promoter is a prerequisite for the gene transactivation, which contributes to tissue-and cell-type-specific regulation of 15-LO expression.
Biochemical Pharmacology, 2005
![Research paper thumbnail of Corrigendum to “Interaction of human 15-lipoxygenase-1 with phosphatidylinositol bisphosphates results in increased enzyme activity” [Biochim. Biophys. Acta 1761 (2006) 1498–1505]](https://attachments.academia-assets.com/83715424/thumbnails/1.jpg)
](Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2009
Corrigendum to "Interaction of human 15-lipoxygenase-1 with phosphatidylinositol bisphosphates re... more Corrigendum to "Interaction of human 15-lipoxygenase-1 with phosphatidylinositol bisphosphates results in increased enzyme activity" [Biochim. Biophys.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2006
15-lipoxygenase-1 (15-LO-1) can oxygenate both free fatty acids and fatty acids bound to membrane... more 15-lipoxygenase-1 (15-LO-1) can oxygenate both free fatty acids and fatty acids bound to membrane phospholipids. The regulation of the activity of membrane associated 15-LO-1 is poorly understood. Here we demonstrate that calcium ionophore stimulates the translocation of 15-LO-1 to the plasma membrane in human dendritic cells. In a protein-lipid overlay assay, 15-LO-1 was capable of interacting with several phosphoinositides. In the presence of calcium, addition of phosphatidylinositol-4.5-bisphosphate (PI(4.5)P 2) or PI(3.4)P 2 to the vesicles containing arachidonic acid, led to the formation of approximately three times more 15-HETE than vesicles without phosphoinositides and up to seven times more 15-HETE than vesicles without both calcium and phosphoinositides. The Vmax was unchanged but the apparent Km of 15-LO-1 towards arachidonic acid was significantly lower in the presence of PI(4.5)P 2 or PI(3.4)P 2 in the vesicles in comparison to vesicles with PC only. Taken together, this report demonstrates that human 15-LO-1 binds to PI(4.5)P 2 and PI(3.4)P 2 and that these phospholipids stimulate enzyme activity in the presence of calcium in a vesicle based assay.
Additional file 1: Figure S1. Characterization of differentiated SH-SY5Y cells. Figure S2. Cytoto... more Additional file 1: Figure S1. Characterization of differentiated SH-SY5Y cells. Figure S2. Cytotoxicity of compounds evaluated in the HTS. Figure S3. Effects of luteolin in respiratory capacity and ΔΨm. Figure S4. Effects of luteolin in mitochondrial biogenesis and cristae organization. Figure S5. Effects of luteolin on calcium levels and characterization of isolated mitochondrial fractions.
Additional file 2. Prestwick Chemical Library compounds used in the HTS and the corresponding ATP... more Additional file 2. Prestwick Chemical Library compounds used in the HTS and the corresponding ATP and cytotoxicity z-scores (data related with Fig. 2b and Additional file 1: Figure S2A).
Additional file 3. ATP and cytotoxicity z-scores obtained for the 3-CRC (data related with Fig. 2... more Additional file 3. ATP and cytotoxicity z-scores obtained for the 3-CRC (data related with Fig. 2c and Additional file 1: Figure S2D).
Bioorganic & medicinal chemistry letters, 2015
Investigation of 1N-substituted pyrazole-3-carboxanilides as 15-lipoxygenase-1 (15-LOX-1) inhibit... more Investigation of 1N-substituted pyrazole-3-carboxanilides as 15-lipoxygenase-1 (15-LOX-1) inhibitors demonstrated that the 1N-substituent was not essential for activity or selectivity. Additional halogen substituents on the pyrazole ring, however, increased activity. Further development led to triazole-4-carboxanilides and 2-(3-pyrazolyl) benzoxazoles, which are potent and selective 15-LOX-1 inhibitors.
Bioorganic & Medicinal Chemistry Letters, 2015
High-throughput screening was used to find selective inhibitors of human 15-lipoxygenase-1 (15-LO... more High-throughput screening was used to find selective inhibitors of human 15-lipoxygenase-1 (15-LOX-1). One hit, a 1-benzoyl substituted pyrazole-3-carboxanilide (1a), was used as a starting point in a program to develop potent and selective 15-LOX-1 inhibitors.
Annual Reports in Medicinal Chemistry, 2014
Abstract Neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, a... more Abstract Neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease are currently lacking disease-modifying treatments. A substantial body of evidence links increases in neurotrophin (NT) signaling in neurodegenerative disorders with biological mechanisms that could have a positive effect on disease outcome. NTs like NGF and BDNF signal through Trk receptors. This signaling is of high importance to support neuronal survival, differentiation, neurogenesis, and synaptic plasticity. The poor pharmacokinetics of NTs makes them unsuitable as drugs. In addition, the NTs have pleiotropic effects that may give side effects. Therefore, the identification of small molecules that promote NT signaling is of increasing interest. Such small-molecule activators of NT signaling will support neuronal survival in the CNS, and thus they could possibly function as disease modifiers. Herein, we review the current status of small-molecule compounds that are able to activate NT receptors.
European Journal of Biochemistry, 1999
Recently, we reported the human 88-kDa calcium-independent phospholipase A2 (iPLA2) cDNA sequence... more Recently, we reported the human 88-kDa calcium-independent phospholipase A2 (iPLA2) cDNA sequence, as well as extensive alternative splicing of the iPLA2 mRNA. In this report we identified the gene coding for iPLA2, which was localized on chromosome 22q13.1. The gene consists of at least 17 exons spanning > 69 kb. Based on the iPLA2 gene organization the splice variants can be explained. The putative promotor for the iPLA2 gene lacks a TATA-box and contains a CpG island as well as several potential Sp-1-binding sites. Furthermore, the 5'-flanking region also contains one medium reiteration frequency repeat (MER53) and an Alu repetitive sequence. Northern blot analysis of iPLA2 mRNA in various human tissues demonstrated tissue-specific expression of four distinct iPLA2 transcripts. The native human 3.2-kb iPLA2 transcript was predominantly expressed in heart, brain, skeletal muscle, prostate, testis, thyroid and spinal cord, and to a lesser extent in peripheral blood leucocytes, stomach, trachea and bone marrow. Studies on the subcellular localization of the native iPLA2 protein were performed in COS-7 cells overexpressing this enzyme. The cytosolic fraction of untransfected and cells overexpressing iPLA2 contained equal amounts of calcium-independent PLA2 activity. However, the membrane fraction displayed a 5.5-fold increased activity in iPLA2 overexpressing cells. This increased calcium-independent PLA2 activity correlated with the presence of iPLA2 immunoreactive protein in the membrane fraction, indicating that this form of iPLA2 protein was membrane associated. Studies of iPLA2 in rat vascular smooth muscle cells verified the membrane association of this form of iPLA2. The major difference between this form of iPLA2 enzyme and the soluble forms of iPLA2 studied previously is the presence of 54 additional amino acid residues derived from exon 9. We suggest that the addition of these 54 amino acids leads to a membrane-associated protein. In summary, these results demonstrate that alternative splicing of the human iPLA2 transcript generates multiple iPLA2 isoforms with distinct tissue distribution and cellular localization.
Lipids, 2012
Human 15-lipoxygenase-1 (15-LO-1) can metabolize arachidonic acid (ARA) into pro-inflammatory med... more Human 15-lipoxygenase-1 (15-LO-1) can metabolize arachidonic acid (ARA) into pro-inflammatory mediators such as the eoxins, 15-hydroperoxyeicosatetraenoic acid (HPETE), and 15-hydroxyeicosatetraenoyl-phosphatidylethanolamine. We have in this study investigated the formation of various lipid hydroperoxide by either purified 15-LO-1 or in the Hodgkin lymphoma cell line L1236, which contain abundant amount of 15-LO-1. Both purified 15-LO-1 and L1236 cells produced lipid hydroperoxides more efficiently when anandamide (AEA) or 2-arachidonoyl-glycerol ester was used as substrate than with ARA. Furthermore, L1236 cells converted AEA to a novel class of cysteinyl-containing metabolites. Based on RP-HPLC, mass spectrometry and comparison to synthetic products, these metabolites were identified as the ethanolamide of the eoxin (EX) C(4) and EXD(4). By using the epoxide EXA(4)-ethanol amide, it was also found that platelets have the capacity to produce the ethanolamide of EXC(4) and EXD(4). We suggest that the ethanolamides of the eoxins should be referred to as eoxamides, in analogy to the ethanolamides of prostaglandins which are named prostamides. The metabolism of AEA into eoxamides might engender molecules with novel biological effects. Alternatively, it might represent a new mechanism for the termination of AEA signalling.
Journal of Biomolecular Screening, 2010
15-Lipoxygenase-1 catalyzes the introduction of molecular oxygen into polyunsaturated fatty acids... more 15-Lipoxygenase-1 catalyzes the introduction of molecular oxygen into polyunsaturated fatty acids to form a lipid hydroperoxide. The authors have developed an assay for the detection of lipid hydroperoxides formed by human 15-lipoxygenase (15-LO) in enzyme or cellular assays using either a 96-well or a 384-well format. The assays described take advantage of the ability of lipid hydroperoxides to oxidize nonfluorescent diphenyl-1-pyrenylphosphine (DPPP) to a fluorescent phosphine oxide. Oxidation of DPPP yields a fluorescent compound, which is not sensitive to temperature and is stable for more than 2 h. The assay is sensitive toward inhibition and robust with a Z′ value of 0.79 and 0.4 in a 96- and 384-well format, respectively, and thus amenable for high-throughput screening. The utility of DPPP as a marker for 15-lipoxygenase activity was demonstrated with both enzyme- and cell-based assays for the identification of hits and to determine potency by IC50determinations.
Journal of Biological Chemistry, 2009
The nomenclature of lipoxygenases (LOXs) is partly based on the positional specificity of arachid... more The nomenclature of lipoxygenases (LOXs) is partly based on the positional specificity of arachidonic acid oxygenation, but there is no unifying concept explaining the mechanistic basis of this enzyme property. According to the triad model, Phe-353, Ile-418, and Ile-593 of the rabbit 12/15-LOX form the bottom of the substrate-binding pocket, and introduction of less spacefilling residues at either of these positions favors arachidonic acid 12-lipoxygenation. The present study was aimed at exploring the validity of the triad concept for two novel primate 12/15-LOX (Macaca mulatta and Pongo pygmaeus) and for five known members of the mammalian LOX family (human 12/15-LOX, mouse 12/15-LOX, human 15-LOX2, human platelet type 12-LOX, and mouse (12R)-LOX). The enzymes were expressed as N-terminal His tag fusion proteins in E. coli, the potential sequence determinants were mutated, and the specificity of arachidonic acid oxygenation was quantified. Taken together, our data indicate that the triad concept explains the positional specificity of all 12/15-LOXs tested (rabbit, human, M. mulatta, P. pygmaeus, and mouse). For the new enzymes of M. mulatta and P. pygmaeus, the concept had predictive value because the positional specificity predicted on the basis of the amino acid sequence was confirmed experimentally. The specificity of the platelet 12-LOX was partly explained by the triad hypothesis, but the concept was not applicable for 15-LOX2 and (12R)-LOX.
The Journal of biological chemistry, 2007
Although protein-tyrosine phosphatase 1B (PTP-1B) is a negative regulator of insulin action, adip... more Although protein-tyrosine phosphatase 1B (PTP-1B) is a negative regulator of insulin action, adipose tissue from PTP-1B-/- mice does not show enhanced insulin-stimulated insulin receptor phosphorylation. Investigation of glucose uptake in isolated adipocytes revealed that the adipocytes from PTP-1B-/- mice have a significantly attenuated insulin response as compared with PTP-1B+/+ adipocytes. This insulin resistance manifests in PTP-1B-/- animals older than 16 weeks of age and could be partially rescued by adenoviral expression of PTP-1B in null adipocytes. Examination of adipose signaling pathways found that the basal p70S6K activity was at least 50% higher in adipose from PTP-1B-/- mice compared with wild type animals. The increased basal activity of p70S6K in PTP-1B-/- adipose correlated with decreases in IR substrate-1 protein levels and insulin-stimulated Akt/protein kinase B activity, explaining the decrease in insulin sensitivity even as insulin receptor phosphorylation was u...
Experimental Cell Research, 2012
Hodgkin lymphoma (HL) is a unique entity among the lymphomas, with a minority of malignant Hodgki... more Hodgkin lymphoma (HL) is a unique entity among the lymphomas, with a minority of malignant Hodgkin and Reed-Sternberg (H-RS) cells surrounded by a broad range of infiltrating cells. The infiltration of certain inflammatory cells has been reported to predict prognosis of the disease. In HL tumor microenvironment, the primary H-RS cells and those inflammatory cells interact interdependently. The aberrant cytokine production of H-RS cells has been suggested to contribute to this interdependency. However, little is known in terms of the mechanisms involved in the abnormal cytokine secretion by H-RS cells. Previous studies suggested that several pro-inflammatory molecules likely contribute to the aberrant cytokine secretion of HL, including cysteinyl-leukotrienes receptor type I (CysLT 1 R) and 15-lipoxygenase-1(15-LOX-1) that are highly expressed in primary H-RS cells and cultured HL-derived L1236 cells. Previous and present studies in cultured HL cells demonstrate that CysLT 1 R mediates transcription and secretion of cytokines, including interleukin (IL)-6, IL-8 and tumor necrosis factor-α, upon stimulation by leukotriene D 4 (LTD 4). This lipid mediator is formed from arachidonic acid through the 5-lipoxygenase (5-LOX) pathway and several types of inflammatory cells surrounding H-RS cells can produce cysteinylleukotrienes. To depict the intracellular signaling pathways that bridge the LTD 4-CysLT 1 R ligation to cytokine induction, a mechanistic study was carried out in L1236 cells. The results demonstrated that the transcription factor early growth response (EGR)-1 is involved in the LTD 4-triggered cytokine transcriptional induction. The regulatory mechanisms implicated in 15-LOX-1 trans-activation in HL have been obscure. This study has also assessed the epigenetic modulation of 15-LOX-1 in different aspects. The results revealed that signal transducer and activator of transcription (STAT)-6 positively regulates 15-LOX-1 transcription by binding to its promoter, in which three putative STAT-6 binding motifs are identified to be required for full activation. The accessibility of STAT-6 to the 15-LOX-1 promoter is controlled by DNA methylation and histone modification. The histone H3 lysine (K)-4 specific methyltransferase SMYD3 was found to exhibit an important role in this multi-step regulation. Although the H3K27me3 demethylase UTX mediates 15-LOX-1 transactivation by H3K27 demethylation upon IL-4 stimulation in lung carcinoma A549 cells, a crucial histone H3K27-demethylase-independent role of UTX in 15-LOX-1 transcriptional regulation in L1236 cells was demonstrated. In conclusion, this study has evaluated the biology of HL by using in vitro models, focusing on lipoxygenases regulation and function. The results not only demonstrated a signaling pathway that hypothetically bridges 5-LOX activity to the striking inflammatory microenvironment in HL, but also uncovered epigenetic regulation mechanisms involved in 15-LOX-1 expression in HL-derived cells. Our findings suggest that lipid signaling pathways might play critical roles in HL pathogenesis, thus warranting further HL research.
Experimental Cell Research, 2004
15-Lipoxygenase type 1 (15-LO), a lipid-peroxidating enzyme implicated in physiological membrane ... more 15-Lipoxygenase type 1 (15-LO), a lipid-peroxidating enzyme implicated in physiological membrane remodeling and the pathogenesis of atherosclerosis, inflammation, and carcinogenesis, is highly regulated and expressed in a tissue-and cell-type-specific fashion. It is known that interleukins (IL) 4 and 13 play important roles in transactivating the 15-LO gene. However, the fact that they only exert such effects on a few types of cells suggests additional mechanism(s) for the profile control of 15-LO expression. In the present study, we demonstrate that hyper-and hypomethylation of CpG islands in the 15-LO promoter region is intimately associated with the transcriptional repression and activation of the 15-LO gene, respectively. The 15-LO promoter was exclusively methylated in all examined cells incapable of expressing 15-LO (certain solid tumor and human lymphoma cell lines and human T lymphocytes) while unmethylated in 15-LO-competent cells (the human airway epithelial cell line A549 and human monocytes) where 15-LO expression is IL4-inducible. Inhibition of DNA methylation in L428 lymphoma cells restores IL4 inducibility to 15-LO expression. Consistent with this, the unmethylated 15-LO promoter reporter construct exhibited threefold higher activity in A549 cells compared to its methylated counterpart. Taken together, demethylation of the 15-LO promoter is a prerequisite for the gene transactivation, which contributes to tissue-and cell-type-specific regulation of 15-LO expression.
Biochemical Pharmacology, 2005
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2009
Corrigendum to "Interaction of human 15-lipoxygenase-1 with phosphatidylinositol bisphosphates re... more Corrigendum to "Interaction of human 15-lipoxygenase-1 with phosphatidylinositol bisphosphates results in increased enzyme activity" [Biochim. Biophys.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2006
15-lipoxygenase-1 (15-LO-1) can oxygenate both free fatty acids and fatty acids bound to membrane... more 15-lipoxygenase-1 (15-LO-1) can oxygenate both free fatty acids and fatty acids bound to membrane phospholipids. The regulation of the activity of membrane associated 15-LO-1 is poorly understood. Here we demonstrate that calcium ionophore stimulates the translocation of 15-LO-1 to the plasma membrane in human dendritic cells. In a protein-lipid overlay assay, 15-LO-1 was capable of interacting with several phosphoinositides. In the presence of calcium, addition of phosphatidylinositol-4.5-bisphosphate (PI(4.5)P 2) or PI(3.4)P 2 to the vesicles containing arachidonic acid, led to the formation of approximately three times more 15-HETE than vesicles without phosphoinositides and up to seven times more 15-HETE than vesicles without both calcium and phosphoinositides. The Vmax was unchanged but the apparent Km of 15-LO-1 towards arachidonic acid was significantly lower in the presence of PI(4.5)P 2 or PI(3.4)P 2 in the vesicles in comparison to vesicles with PC only. Taken together, this report demonstrates that human 15-LO-1 binds to PI(4.5)P 2 and PI(3.4)P 2 and that these phospholipids stimulate enzyme activity in the presence of calcium in a vesicle based assay.