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The Journal of Biological Chemistry Oct 2012Deamidase of Pup (Dop), the prokaryotic ubiquitin-like protein (Pup)-deconjugating enzyme, is critical for the full virulence of Mycobacterium tuberculosis and is unique...
Deamidase of Pup (Dop), the prokaryotic ubiquitin-like protein (Pup)-deconjugating enzyme, is critical for the full virulence of Mycobacterium tuberculosis and is unique to bacteria, providing an ideal target for the development of selective chemotherapies. We used a combination of genetics and chemical biology to characterize the mechanism of depupylation. We identified an aspartate as a potential nucleophile in the active site of Dop, suggesting a novel protease activity to target for inhibitor development.
Topics: Amidohydrolases; Amino Acid Motifs; Amino Acid Sequence; Amino Acid Substitution; Bacterial Proteins; Biocatalysis; Catalytic Domain; Diazooxonorleucine; Enzyme Inhibitors; Hydrolysis; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mycobacterium tuberculosis; Structural Homology, Protein
PubMed: 22942282
DOI: 10.1074/jbc.M112.384784 -
Journal of Genetics and Genomics = Yi... Sep 2011CTP synthase is compartmentalized within a subcellular structure, termed the cytoophidium, in a range of organisms including bacteria, yeast, fruit fly and rat. Here we...
CTP synthase is compartmentalized within a subcellular structure, termed the cytoophidium, in a range of organisms including bacteria, yeast, fruit fly and rat. Here we show that CTP synthase is also compartmentalized into cytoophidia in human cells. Surprisingly, the occurrence of cytoophidia in human cells increases upon treatment with a glutamine analog 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of glutamine-dependent enzymes including CTP synthase. Experiments in flies confirmed that DON globally promotes cytoophidium assembly. Clonal analysis via CTP synthase RNA interference in somatic cells indicates that CTP synthase expression level is critical for the formation of cytoophidia. Moreover, DON facilitates cytoophidium assembly even when CTP synthase level is low. A second glutamine analog azaserine also promotes cytoophidum formation. Our data demonstrate that glutamine analogs serve as useful tools in the study of cytoophidia.
Topics: Animals; Apoptosis; Azaserine; Carbon-Nitrogen Ligases; Cell Compartmentation; Cell Cycle; Diazooxonorleucine; Drosophila melanogaster; Enzyme Inhibitors; Female; Glutamine; HeLa Cells; Humans; Intracellular Space; Male; Oogenesis; RNA Interference
PubMed: 21930098
DOI: 10.1016/j.jgg.2011.08.004 -
The FEBS Journal Oct 2011GMP synthetase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the nucleotide xanthosine 5'-monophosphate (XMP) to form guanosine...
GMP synthetase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the nucleotide xanthosine 5'-monophosphate (XMP) to form guanosine 5'-monophosphate (GMP). Functional coordination of domains in glutamine amidotransferases leads to upregulation of glutamine hydrolysis in the presence of acceptor substrates and is a common feature in this class of enzymes. We have shown earlier that binding of substrates to the acceptor domain of Plasmodium falciparum GMP synthetase (PfGMPS) leads to enhancement in both glutaminase activity and rate of glutaminase inactivation, by the irreversible inhibitors acivicin and diazo-oxonorleucine [Bhat JY et al. (2008) Biochem J409, 263-273], a process that must be driven by conformational alterations. In this paper, through the combined use of biochemical assays, optical spectroscopy and mass spectrometry, we demonstrate that PfGMPS undergoes conformational transitions upon binding of substrates to the acceptor domain. Limited proteolysis and hydrogen-deuterium exchange in conjunction with mass spectrometry unveil region-specific conformational changes in the ATP + XMP bound state of PfGMPS. Decreased accessibility of R294 and K428 residues to trypsin in the ATP pyrophosphatase domain and reduced deuterium incorporation in the 143-155 region, pertaining to the glutaminase domain, suggest that in PfGMPS ligand-induced conformational changes are not only local but also transmitted over a long range across the domains. Overall, these results provide a detailed understanding of the substrate-induced changes in PfGMPS that could be essential for the overall catalytic process.
Topics: Carbon-Nitrogen Ligases; Circular Dichroism; Deuterium Exchange Measurement; Guanosine Monophosphate; Models, Molecular; Molecular Structure; Plasmodium falciparum; Protein Binding; Protein Conformation; Protozoan Proteins; Spectrometry, Fluorescence; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 21827625
DOI: 10.1111/j.1742-4658.2011.08296.x -
American Journal of Physiology. Cell... Oct 2011Here we report and validate a simple method for measuring intracellular activities of glial glutamine synthetase (GS) and glutaminase (GLNase) in intact glial cells....
Here we report and validate a simple method for measuring intracellular activities of glial glutamine synthetase (GS) and glutaminase (GLNase) in intact glial cells. These enzymes are responsible for glutamate and glutamine recycling in the brain, where glutamate and glutamine transport from the blood stream is strongly limited by the blood-brain barrier. The intracellular levels of glutamate and glutamine are dependent on activities of numerous enzymatic processes, including 1) cytosolic production of glutamine from glutamate by GS, 2) production of glutamate from glutamine by GLNase that is primarily localized between mitochondrial membranes, and 3) mitochondrial conversion of glutamate to the tricarboxylic cycle intermediate α-ketoglutarate in the reactions of oxidative deamination and transamination. We measured intracellular activities of GS and GLNase by quantifying enzymatic interconversions of L-[(3)H]glutamate and L-[(3)H]glutamine in cultured rat astrocytes. The intracellular substrate and the products of enzymatic reactions were separated in one step using commercially available anion exchange columns and quantified using a scintillation counter. The involvement of GS and GLNase in the conversion of (3)H-labeled substrates was verified using irreversible pharmacological inhibitors for each of the enzymes and additionally validated by measuring intracellular amino acid levels using an HPLC. Overall, this paper describes optimized conditions and pharmacological controls for measuring GS and GLNase activities in intact glial cells.
Topics: Animals; Astrocytes; Diazooxonorleucine; Enzyme Assays; Glutamate-Ammonia Ligase; Glutaminase; Methionine Sulfoximine; Neuroglia; Rats; Rats, Sprague-Dawley
PubMed: 21734190
DOI: 10.1152/ajpcell.00035.2011 -
Arteriosclerosis, Thrombosis, and... May 2011Trivalent chromium (Cr3+) is an essential micronutrient. Findings since the 1950s suggest that Cr3+ might benefit cholesterol homeostasis. Here we present mechanistic...
OBJECTIVE
Trivalent chromium (Cr3+) is an essential micronutrient. Findings since the 1950s suggest that Cr3+ might benefit cholesterol homeostasis. Here we present mechanistic evidence in support of this role of Cr3+.
METHODS AND RESULTS
High-density lipoprotein cholesterol generation in 3T3-L1 adipocytes, which are rendered ineffective by the hyperinsulinemia that is known to accompany disorders of lipid metabolism, was corrected by Cr3+. Mechanistically, Cr3+ reversed hyperinsulinemia-induced cellular cholesterol accrual and associated defects in cholesterol transporter ATP-binding cassette transporter-A1 trafficking and apolipoprotein A1-mediated cholesterol efflux. Moreover, direct activation of AMP-activated protein kinase, which is known to be activated by Cr3+, or inhibition of hexosamine biosynthesis pathway activity, which is known to be elevated by hyperinsulinemia, mimics Cr3+ action.
CONCLUSIONS
These findings suggest a mechanism of Cr3+ action that fits with long-standing claims of its role in cholesterol homeostasis. Furthermore, these data imply a mechanistic basis for the coexistence of dyslipidemia with hyperinsulinemia.
Topics: AMP-Activated Protein Kinases; ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; Adipocytes; Aminoimidazole Carboxamide; Animals; Apolipoprotein A-I; Cholesterol; Cholesterol, HDL; Diazooxonorleucine; Enzyme Activation; Enzyme Activators; Enzyme Inhibitors; Hexosamines; Homeostasis; Hyperinsulinism; Insulin; Mice; NIH 3T3 Cells; Picolinic Acids; Protein Transport; Ribonucleotides; Time Factors; rab GTP-Binding Proteins
PubMed: 21311039
DOI: 10.1161/ATVBAHA.110.222158 -
Glia Feb 2011High blood ammonia, elevated glutamine, and hyponatremia are pathogenic factors contributing to astrocytic swelling and brain edema in liver failure. We investigated the...
High blood ammonia, elevated glutamine, and hyponatremia are pathogenic factors contributing to astrocytic swelling and brain edema in liver failure. We investigated the effects of hypoosmolarity, ammonia, and glutamine on the induction of glial cell swelling in freshly isolated slices of the rat retina. Glutamine, but not ammonia or hypoosmolarity per se, evoked a rapid (within one minute) swelling of retinal glial (Müller) cell bodies under hypoosmotic conditions. Under isoosmotic conditions, glutamine evoked a delayed swelling after 10 min of exposure. The effect of glutamine was concentration-dependent, with half-maximal and maximal effects at ∼ 0.1 and 0.5 mM. Glutamine in hypoosmotic solution induced a dissipation of the mitochondrial membrane potential. The effects on the mitochondrial membrane potential and the glial soma size were reduced by (i) agents which inhibit the transfer of glutamine into mitochondria and its hydrolysis there, (ii) inhibition of the mitochondrial permeability transition, (iii) inhibitors of oxidative-nitrosative stress, and (iv) inhibitors of phospholipase A(2) and cyclooxygenase. Glutamine-induced glial swelling was also prevented by ATP and adenosine, acting at adenosine A(1) receptors. The data suggest that hypoosmolarity accelerates the swelling-inducing effect of glutamine on retinal glial cells, and that swelling induction by glutamine is mediated by inducing oxidative-nitrosative stress, inflammatory lipid mediators, and mitochondrial dysfunction.
Topics: Adenosine; Adenosine A1 Receptor Antagonists; Adenosine Triphosphate; Animals; Arachidonic Acid; Cell Size; Diazooxonorleucine; Dinoprostone; Drug Interactions; Enzyme Inhibitors; Glutamine; Hydrogen Peroxide; In Vitro Techniques; Membrane Potential, Mitochondrial; Neuroglia; Osmosis; Rats; Rats, Long-Evans; Retina; Retinal Ganglion Cells; Xanthines
PubMed: 21154559
DOI: 10.1002/glia.21095 -
International Journal of Cancer Nov 2010Metastatic cancer is a major cause of morbidity and mortality. Current therapeutic options consist of chemotherapy, radiation or targeted therapies. However, these...
Metastatic cancer is a major cause of morbidity and mortality. Current therapeutic options consist of chemotherapy, radiation or targeted therapies. However, these therapies are often toxic, effective over a small range of cancer types or result in drug resistance. Therefore, a more global, less toxic strategy for the management of metastatic cancer is required. Although most cancers display increased glucose metabolism, glutamine is also a major energy substrate for many cancers. We evaluated the antimetastatic potential of 6-diazo-5-oxo-L-norleucine (DON), a glutamine analog, using the new VM mouse model of systemic metastasis. We found that primary tumor growth was ∼20-fold less in DON-treated mice than in untreated control mice. We also found that DON treatment inhibited metastasis to liver, lung and kidney as detected by bioluminescence imaging and histology. Our findings provide proof of concept that metabolic therapies targeting glutamine metabolism can manage systemic metastatic cancer.
Topics: Animals; Antimetabolites, Antineoplastic; Blood Glucose; Body Weight; Brain Neoplasms; Caloric Restriction; Cell Growth Processes; Cell Line, Tumor; Cerebrum; Diazooxonorleucine; Drug Delivery Systems; Female; Glucose; Glutamine; Male; Mice; Neoplasm Metastasis
PubMed: 20473919
DOI: 10.1002/ijc.25431 -
Neuroscience Letters Mar 2010In inflamed tissue, the levels of the excitatory amino acid glutamate are increased. Glutamate sensitizes peripheral axons of primary afferent neurons during...
In inflamed tissue, the levels of the excitatory amino acid glutamate are increased. Glutamate sensitizes peripheral axons of primary afferent neurons during inflammation leading to decreased firing threshold and hyperexcitability. One proposed source of glutamate is the primary afferent. Antagonizing glutamate receptors on peripheral axons of primary afferents during inflammation provides analgesia in animals and humans. The enzyme glutaminase is used by primary sensory neurons to convert glutamine to glutamate, and peripheral inhibition of glutaminase with 6-diazo-5-oxo-l-norleucine (DON) provides long-lasting analgesia during inflammation. In this study, we measured the effects of glutaminase inhibition on carrageenan-induced spinal Fos expression. Rats were given intraplantar injections of carrageenan and treated locally with either vehicle or DON. After 3h of inflammation, hind paw swelling and spinal expression of Fos were examined. CellProfiler was used to automate Fos nuclei counting in five laminar groupings in the spinal cord (I-II, III-IV, V-VI, VII-IX, X). Carrageenan increased hind paw thickness by approximately 70% and spinal Fos expression in superficial (I-II) and deep (V-VI) laminae by 10-fold and 5-fold, respectively. Treatment with DON reduced hind paw swelling by approximately 13% and suppressed Fos expression in the laminae I-II by approximately 54%, but not the deep laminae. Our results further support the notion of glutamate as a peripheral inflammatory mediator and indicate that glutaminase should be considered as a novel therapeutic target for treatment of inflammatory pain.
Topics: Analgesics; Animals; Carrageenan; Diazooxonorleucine; Edema; Glutamic Acid; Glutaminase; Inflammation; Nerve Endings; Posterior Horn Cells; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Spinal Cord
PubMed: 20132864
DOI: 10.1016/j.neulet.2010.01.066 -
Neoplasia (New York, N.Y.) Jan 2010Ovarian carcinoma is the leading cause of death among gynecologic cancers. Although transformation of the outer ovarian epithelium was linked with ovulation, the disease...
Ovarian carcinoma is the leading cause of death among gynecologic cancers. Although transformation of the outer ovarian epithelium was linked with ovulation, the disease is significantly more prevalent and severe in postmenopausal women. We postulated that menopause could augment ovarian cancer progression through the effects of gonadotropins on multifocal seeding to the mesothelial layer lining the peritoneum. This seeding is mediated by integrins as well as by CD44 interaction with hyaluronan (HA). Here, we report the effect of gonadotropins on HA synthesis and degradation and on peritoneal adhesion. A significant concentration- and time-dependent induction in expression levels of HA synthases (HASs) and hyaluronidases (Hyals) was observed in vitro on stimulation of human epithelial ovarian carcinoma cells by gonadotropins. Hormonal regulation of HA-mediated adhesion was manifested in vivo as well, by fluorescence microscopy of stained MLS multicellular tumor spheroids. The number of spheroids adhered to the mesothelium of ovariectomized CD-1 nude mice 9.5 hours after intraperitoneal insertion was significantly higher than in nonovariectomized mice. Inhibition of HA synthesis by 6-diazo-5-oxo-1-norleucine (DON) both in spheroids and ovariectomized mice significantly reduced the number of adhered spheroids. Thus, the change in the hormonal environment during menopause assists in HA-dependent adherence of ovarian cancer spheroids onto the peritoneum. However, HA is antiangiogenic and it can significantly suppress tumor progression. Accordingly, angiogenesis of the adhered spheroids was significantly elevated in DON-treated tumors. These results can explain the selective pressure that can lead to simultaneously increased tumor expression of both HASs and Hyals.
Topics: Animals; Antimetabolites, Antineoplastic; Cell Adhesion; Cell Line, Tumor; Diazooxonorleucine; Dose-Response Relationship, Drug; Female; Follicle Stimulating Hormone; Glucuronosyltransferase; Gonadotropins; Humans; Hyaluronan Synthases; Hyaluronic Acid; Hyaluronoglucosaminidase; In Situ Hybridization; Luteinizing Hormone; Mice; Mice, Nude; Microscopy, Fluorescence; Neovascularization, Pathologic; Ovarian Neoplasms; Ovariectomy; Peritoneum; Reverse Transcriptase Polymerase Chain Reaction; Spheroids, Cellular; Xenograft Model Antitumor Assays
PubMed: 20072653
DOI: 10.1593/neo.91272 -
Neurobiology of Disease Jul 2009Brain L-glutamine (Gln) accumulation and increased activity of the NO/cGMP pathway are immediate consequences of acute exposure to ammonia. This study tested whether...
Brain L-glutamine (Gln) accumulation and increased activity of the NO/cGMP pathway are immediate consequences of acute exposure to ammonia. This study tested whether excess Gln may influence NO and/or cGMP synthesis. Intrastriatal administration of the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine or the system A-specific Gln uptake inhibitor methylaminoisobutyrate increased microdialysate Gln concentration and reduced basal and ammonia-induced NO and cGMP accumulation. Gln applied in vivo (via microdialysis) or in vitro (to rat brain cortical slices) reduced NO and cGMP accumulation in the presence and/or absence of ammonia, but not cGMP synthesis induced by the NO donor sodium nitroprusside. Attenuation of cGMP synthesis by Gln was prevented by administration of L-arginine (Arg). The L-arginine co-substrates of y(+)LAT2 transport system, L-leucine and cyclo-leucine, mimicked the effect of exogenous Gln, suggesting that Gln limits Arg supply for NO synthesis by interfering with y+LAT2-mediated Arg uptake across the cell membrane.
Topics: Ammonia; Animals; Arginine; Butyrates; Chromatography, High Pressure Liquid; Corpus Striatum; Cyclic GMP; Diazooxonorleucine; Dose-Response Relationship, Drug; Drug Interactions; Electrochemical Techniques; Glutamine; In Vitro Techniques; Isobutyrates; Male; Microdialysis; Nitric Oxide; Rats; Rats, Sprague-Dawley; Signal Transduction; Time Factors
PubMed: 19379813
DOI: 10.1016/j.nbd.2009.04.004