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... 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 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. 2c and Additional file 1: Figure S2D).
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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,... 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.
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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... 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.
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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... 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.
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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... 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.
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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... 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.
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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... 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.
Research Interests: Fluorescence, Screening, Antioxidants, Humans, High throughput screening, and 12 moreOrganophosphorus Compounds, High Performance Liquid Chromatography, Enzyme, High Pressure Liquid Chromatography, Molecular cloning, Reproducibility of Results, Biological Assay, Oxidation-Reduction, Arachidonic Acid, Biochemistry and cell biology, Minimum inhibitory concentration, and Lipid peroxides
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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... 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...
Research Interests: Biology, Biological Chemistry, Medicine, Biological Sciences, Insulin Resistance, and 14 moreAdipose tissue, Humans, Protein Kinases, Glucose, Mice, Animals, Biological, Phosphorylation, Protein tyrosine phosphatase, CHEMICAL SCIENCES, Adipocytes, Sirolimus, Receptor Insulin, and Medical and Health Sciences
Research Interests: Transcription Regulation, Cell line, Humans, Chromatin remodeling, Clinical Sciences, and 11 moreSpectrum, Chromatin Immunoprecipitation, Transfection, Biological activity, Protein Binding, Binding Site, Histone Acetylation, Hodgkin disease, Response Elements, Biochemistry and cell biology, and Oxidative Metabolism
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Research Interests: Chemistry, Calcium, Medicine, Humans, Platelet aggregation, and 13 morePolychlorinated Biphenyls, Phosphorylation, Biochemical, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Polychlorinated Biphenyl, Tamoxifen, BIOCHEMICAL PHARMACOLOGY, Protein Transport, Arachidonic Acid, Intracellular Calcium, Blood platelets, Pharmacology and pharmaceutical sciences, and In Vitro Techniques
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Research Interests: Chemistry, Kinetics, Medicine, Dendritic Cells, Biological Sciences, and 13 moreHumans, Physical sciences, Dendritic cell, Fatty Acid, Enzyme activity, Free Fatty Acid, Subcellular Fractions, Recombinant Proteins, Plasma Membrane, Arachidonic Acid, Linoleic Acid, Medical and Health Sciences, and In Vitro Techniques
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It has been demonstrated that equine neutro- phils, but not eosinophils, require exogenous arachidonic acid for calcium ionophore A23187-induced leukotriene synthesis. Because cytosolic phospholipase A 2 (cPLA 2 ) plays an essential role... more
It has been demonstrated that equine neutro- phils, but not eosinophils, require exogenous arachidonic acid for calcium ionophore A23187-induced leukotriene synthesis. Because cytosolic phospholipase A 2 (cPLA 2 ) plays an essential role in leukotriene formation in leukocytes, we investigated the presence of a functional cPLA 2 in equine neutrophils. To determine whether cPLA 2 from neutrophils was catalytically active, we purified the enzyme . 6,500 fold with 3% recovery from equine neutrophils. The full-length cDNA sequence encoded a 749-amino acid protein. The de- duced amino acid sequence demonstrated 95% identity with human and mouse cPLA 2 , as well as 83 and 73% identity with chicken and zebra fish cPLA 2 protein, respectively. The equine cPLA 2 possessed some properties that distinguished the equine enzyme from the human enzyme. First, the en- zyme activity of the equine cPLA 2 was differently influenced by cations as compared with the human cPLA 2 . Second, the equine neutr...
Research Interests: Gene expression, Western blotting, Humans, Sequence alignment, Animals, and 15 morePolymerase Chain Reaction, Horses, Phosphorylation, Enzyme, Neutrophils, Eosinophils, Substrate Specificity, Lipid, Amino Acid Sequence, Catalytic Activity, Arachidonic Acid, Full Length Movies, Biochemistry and cell biology, Molecular Sequence Data, and Medical biochemistry and metabolomics
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Alzheimer’s disease (AD) is the most common neurodegenerative disorder and results in severe neurodegeneration and progressive cognitive decline. Neurotrophins are growth factors involved in the development and survival of neurons, but... more
Alzheimer’s disease (AD) is the most common neurodegenerative disorder and results in severe neurodegeneration and progressive cognitive decline. Neurotrophins are growth factors involved in the development and survival of neurons, but also in underlying mechanisms for memory formation such as hippocampal long-term potentiation. Our aim was to identify small molecules with stimulatory effects on the signaling of two neurotrophins, the nerve growth factor (NGF) and the brain derived neurotrophic factor (BDNF). To identify molecules that could potentiate neurotrophin signaling, 25,000 molecules were screened, which led to the identification of the triazinetrione derivatives ACD855 (Ponazuril) and later on ACD856, as positive allosteric modulators of tropomyosin related kinase (Trk) receptors. ACD855 or ACD856 potentiated the cellular signaling of the neurotrophin receptors with EC50 values of 1.9 and 3.2 or 0.38 and 0.30 µM, respectively, for TrkA or TrkB. ACD855 increased acetylcholi...
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Background Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a... more
Background Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. Results Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitocho...
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Research Interests:
PTP-1B is a ubiquitously expressed intracellular protein tyrosine phosphatase (PTP) that has been implicated in the negative regulation of insulin signaling. Mice deficient in PTP-1B were found to have an enhanced insulin sensitivity and... more
PTP-1B is a ubiquitously expressed intracellular protein tyrosine phosphatase (PTP) that has been implicated in the negative regulation of insulin signaling. Mice deficient in PTP-1B were found to have an enhanced insulin sensitivity and a resistance to diet-induced obesity. Interestingly, the human PTP-1B gene maps to chromosome 20q13.1 in a region that has been associated with diabetes and obesity. Although there has been a partial characterization of the 3' end of the human PTP-1B gene, the complete gene organization has not been described. In order to further characterize the PTP-1B gene, we have cloned and determined the genomic organization for both the human and mouse PTP-1B genes including the promoter. The human gene spans >74 kb and features a large first intron of >54 kb; the mouse gene likewise contains a large first intron, although the exact size has not been determined. The organization of the human and mouse PTP-1B genes is identical except for an additional exon at the 3' end of the human that is absent in the mouse. The mouse PTP-1B gene maps to the distal arm of mouse chromosome 2 in the region H2-H3. This region is associated with a mouse obesity quantitative trait locus (QTL) and is syntenic with human chromosome 20. The promoter region of both the human and mouse genes contain no TATA box but multiple GC-rich sequences that contain a number of consensus SP-1 binding sites. The basal activity of the human PTP-1B promoter was characterized in Hep G2 cells using up to 8 kb of 5' flanking sequence. A 432 bp promoter construct immediately upstream of the ATG was able to confer maximal promoter activity. Within this sequence, there are at least three GC-rich sequences and one CCAAT box, and deletion of any of these elements results in decreased promoter activity. In addition, the promoter in a number of mouse strains contains, 3.5 kb upstream of the start codon, an insertion of an intracisternal a particle (IAP) element that possibly could alter the expression of PTP-1B mRNA in these strains.
Research Interests: Genetics, Gene expression, DNA, Gene Mapping, Humans, and 15 moreMice, Animals, Male, Insulin signaling, Gene, Alkaline phosphatase, Genes, Introns, Molecular cloning, Genome Organization, Fluorescent in situ hybridization, Amino Acid Sequence, Base Sequence, Binding Site, and Gene Expression Regulation
Leukotrienes (LT) exert stimulatory effects on myelopoiesis, beside their inflammatory and immunomodulating effects. Here, we have studied the expression and activity of the enzymes involved in the synthesis of leukotriene B4 (LTB4) in... more
Leukotrienes (LT) exert stimulatory effects on myelopoiesis, beside their inflammatory and immunomodulating effects. Here, we have studied the expression and activity of the enzymes involved in the synthesis of leukotriene B4 (LTB4) in acute myeloid leukemia (AML) cells (16 clones) and G-CSF mobilized peripheral blood CD34+ cells. CD34+ cells from patients with non-myeloid malignancies expressed cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase activating protein (FLAP), and leukotriene A4 (LTA4) hydrolase but not 5-lipoxygenase (5-LO). The enzyme cPLA2 was abundantly expressed in AML cells and the activity of the enzyme was high in certain AML clones. The expression of 5-LO, FLAP, and LTA4 hydrolase in AML clones was in general lower than in healthy donor polymorphonuclear leukocytes (PMNL). The calcium ionophore A23187-induced release of [14C] arachidonic acid (AA) in AML cells was low, compared with PMNL, and did not correlate with the expression of cPLA2 protein. Biosynthesis of LTB4, upon calcium ionophore A23187 activation, was only observed in five of the investigated AML clones and only three of the most differentiated clones produced similar amounts of LTB4 as PMNL. The capacity of various cell clones to produce LTs could neither be explained by the difference in [1 − 14C] AA release nor 5-LO expression. Taken together, these results indicate that LT synthesis is under development during early myelopoiesis and the capacity to produce LTs is gained upon maturation. High expression of cPLA2 in AML suggests a putative role of this enzyme in the pathophysiology of this disease.
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Alzheimer's Disease (AD) is the most common form of dementia, affecting approximately 36 million people worldwide. To date there is no preventive or curative treatment available for AD, and in absence of major progress in therapeutic... more
Alzheimer's Disease (AD) is the most common form of dementia, affecting approximately 36 million people worldwide. To date there is no preventive or curative treatment available for AD, and in absence of major progress in therapeutic development, AD manifests a concrete socioeconomic threat. The awareness of the growing problem of AD is increasing, exemplified by the recent G8 Dementia Summit, a meeting held in order to set the stage and steer the compass for the future. Simultaneously, and paradoxically, we have seen key players in the pharmaceutical industry that have recently closed or significantly decreased their R&D spending on AD and other CNS disorders. Given the pressing need for new treatments in this area, other actors need to step-in and enter this drug discovery arena complementing the industrial efforts, in order to turn biological and technological progress into novel therapeutics. In this article, we present an example of a novel drug discovery initiative that in...
The TrkA-PathHunter cell-based assay was used in high-throughput screening (HTS) to identify compounds that inhibit nerve growth factor (NGF)/TrkA signaling. The assay was conducted in a 384-well format, and typical... more
The TrkA-PathHunter cell-based assay was used in high-throughput screening (HTS) to identify compounds that inhibit nerve growth factor (NGF)/TrkA signaling. The assay was conducted in a 384-well format, and typical Z' values during HTS ranged from 0.3 to 0.8. The reproducibility of IC50 values was good, and the use of cryopreserved cells was well tolerated, as judged by assay parameters such as Z' and S/B and by comparison of IC50 values obtained with cells in culture. During hit deconvolution, TrkA-kinase inhibitors were identified with ATP-competitive as well as non-ATP-competitive mechanisms of action. Furthermore, other mechanisms of action such as NGF and TrkA antagonists were also identified. Because of the different molecular mechanisms identified, it is possible that subsequent optimization work to increase affinity and selectivity might lead to compounds that could have a better chance to evoke clinical efficacy without the adverse effects observed for nonselective TrkA inhibitors.
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Research Interests: Gene expression, Western blotting, Humans, Sequence alignment, Animals, and 15 morePolymerase Chain Reaction, Horses, Phosphorylation, Enzyme, Neutrophils, Eosinophils, Substrate Specificity, Amino Acid Profile, Lipid, Amino Acid Sequence, Catalytic Activity, Arachidonic Acid, Full Length Movies, Biochemistry and cell biology, and Molecular Sequence Data
Research Interests: Gene regulation, Transcription Regulation, Enzyme Inhibitors, Cell line, Humans, and 18 moreDNA methylation, CpG islands, Enzyme, Clinical Sciences, IL, T lymphocytes, Histones, Chip, Epithelial cells, STAT, Monocytes, Acetylation, Neoplasms, MSP, CpG island, Biochemistry and cell biology, Interleukin, and Cell Membrane
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Research Interests:
Research Interests:
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... 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 unaffected. The insulin resistance of the of the PTP-1B-/- adipocytes could also be rescued by treatment with rapamycin, suggesting that in adipose the loss of PTP-1B results in basal activation of mTOR (mammalian target of rapamycin) complex 1 leading to a tissue-specific insulin resistance.
Research Interests:
Research Interests:
Research Interests:
PTP-1B is a ubiquitously expressed intracellular protein tyrosine phosphatase (PTP) that has been implicated in the negative regulation of insulin signaling. Mice deficient in PTP-1B were found to have an enhanced insulin sensitivity and... more
PTP-1B is a ubiquitously expressed intracellular protein tyrosine phosphatase (PTP) that has been implicated in the negative regulation of insulin signaling. Mice deficient in PTP-1B were found to have an enhanced insulin sensitivity and a resistance to diet-induced obesity. Interestingly, the human PTP-1B gene maps to chromosome 20q13.1 in a region that has been associated with diabetes and obesity. Although there has been a partial characterization of the 3' end of the human PTP-1B gene, the complete gene organization has not been described. In order to further characterize the PTP-1B gene, we have cloned and determined the genomic organization for both the human and mouse PTP-1B genes including the promoter. The human gene spans >74 kb and features a large first intron of >54 kb; the mouse gene likewise contains a large first intron, although the exact size has not been determined. The organization of the human and mouse PTP-1B genes is identical except for an additional exon at the 3' end of the human that is absent in the mouse. The mouse PTP-1B gene maps to the distal arm of mouse chromosome 2 in the region H2-H3. This region is associated with a mouse obesity quantitative trait locus (QTL) and is syntenic with human chromosome 20. The promoter region of both the human and mouse genes contain no TATA box but multiple GC-rich sequences that contain a number of consensus SP-1 binding sites. The basal activity of the human PTP-1B promoter was characterized in Hep G2 cells using up to 8 kb of 5' flanking sequence. A 432 bp promoter construct immediately upstream of the ATG was able to confer maximal promoter activity. Within this sequence, there are at least three GC-rich sequences and one CCAAT box, and deletion of any of these elements results in decreased promoter activity. In addition, the promoter in a number of mouse strains contains, 3.5 kb upstream of the start codon, an insertion of an intracisternal a particle (IAP) element that possibly could alter the expression of PTP-1B mRNA in these strains.