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Endocrine Journal Apr 1993We have previously shown that methotrexate (MTX) and hydroxyurea (HU) stimulate expression of the human chorionic gonadotropin alpha and placental alkaline phosphatase...
We have previously shown that methotrexate (MTX) and hydroxyurea (HU) stimulate expression of the human chorionic gonadotropin alpha and placental alkaline phosphatase genes and repress the expression of the c-myc oncogene in BeWo choriocarcinoma cells. In order to determine whether c-myc downregulation played a role in the induction of these placental genes, we treated BeWo choriocarcinoma cells with aphidicolin (APH), and 6-diazo-5-oxo-L-norleucine (DON), and compared the effects of these drugs with that of MTX. All of these drugs downregulate c-myc gene expression. At the doses used, both APH and DON repress c-myc expression to approximately the same extent, as well as inducing morphologic changes in BeWo similar to that of MTX, although neither stimulated hCG alpha expression. Both DON and APH stimulate placental alkaline phosphatase gene expression, but only MTX and DON stimulate cholesterol side chain cleavage enzyme gene expression. This indicates that the downregulation of c-myc gene expression is insufficient to stimulate the expression of all the placental genes stimulated by MTX.
Topics: Alkaline Phosphatase; Aphidicolin; Cholesterol; Choriocarcinoma; Chorionic Gonadotropin; DNA, Neoplasm; Diazooxonorleucine; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Methotrexate; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Uterine Neoplasms
PubMed: 7951513
DOI: 10.1507/endocrj.40.263 -
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 -
Virology Aug 2017Infection of weanling C57BL/6 mice with the TE strain of Sindbis virus (SINV) causes nonfatal encephalomyelitis associated with hippocampal-based memory impairment that...
Infection of weanling C57BL/6 mice with the TE strain of Sindbis virus (SINV) causes nonfatal encephalomyelitis associated with hippocampal-based memory impairment that is partially prevented by treatment with 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist (Potter et al., J Neurovirol 21:159, 2015). To determine the mechanism(s) of protection, lymph node and central nervous system (CNS) tissues from SINV-infected mice treated daily for 1 week with low (0.3mg/kg) or high (0.6mg/kg) dose DON were examined. DON treatment suppressed lymphocyte proliferation in cervical lymph nodes resulting in reduced CNS immune cell infiltration, inflammation, and cell death compared to untreated SINV-infected mice. Production of SINV-specific antibody and interferon-gamma were also impaired by DON treatment with a delay in virus clearance. Cessation of treatment allowed activation of the antiviral immune response and viral clearance, but revived CNS pathology, demonstrating the ability of the immune response to mediate both CNS damage and virus clearance.
Topics: Alphavirus Infections; Animals; Antiviral Agents; Diazooxonorleucine; Encephalomyelitis; Glutamine; Humans; Interferon-gamma; Male; Mice; Mice, Inbred C57BL; Sindbis Virus
PubMed: 28531865
DOI: 10.1016/j.virol.2017.05.013 -
Oncotarget Sep 2015Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We...
Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention.
Topics: Adenocarcinoma; Aged; Amidophosphoribosyltransferase; Aneuploidy; Animals; Biomarkers, Tumor; Carboxy-Lyases; Cell Line, Tumor; Cell Proliferation; Chickens; Diazooxonorleucine; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glutamine; Humans; Lung Neoplasms; Male; Mice; Middle Aged; Neoplasm Invasiveness; Neoplasm Transplantation; Oligonucleotide Array Sequence Analysis; Peptide Synthases; Prognosis; Purines
PubMed: 26140362
DOI: 10.18632/oncotarget.4352 -
Frontiers in Immunology 2020Transplant tolerance in the absence of long-term immunosuppression has been an elusive goal for solid organ transplantation. Recently, it has become clear that metabolic...
Transplant tolerance in the absence of long-term immunosuppression has been an elusive goal for solid organ transplantation. Recently, it has become clear that metabolic reprogramming plays a critical role in promoting T cell activation, differentiation, and function. Targeting metabolism can preferentially inhibit T cell effector generation while simultaneously promoting the generation of T regulatory cells. We hypothesized that costimulatory blockade with CTLA4Ig in combination with targeting T cell metabolism might provide a novel platform to promote the induction of transplant tolerance.
Topics: Abatacept; Allografts; Animals; Deoxyglucose; Diazooxonorleucine; Glycolysis; Immunosuppression Therapy; Immunosuppressive Agents; Lymphocyte Activation; Metformin; Mice; T-Lymphocytes; Transplantation Tolerance
PubMed: 32328063
DOI: 10.3389/fimmu.2020.00572 -
The Biochemical Journal Jul 1999The growth arrest and DNA damage-inducible (gadd) genes are co-ordinately activated by a variety of genotoxic agents and/or growth-cessation signals. The regulation of...
The growth arrest and DNA damage-inducible (gadd) genes are co-ordinately activated by a variety of genotoxic agents and/or growth-cessation signals. The regulation of gadd153 mRNA was investigated in renal proximal tubular epithelial cells (LLC-PK1) cultured in a nutrient- and serum-deprived medium. The addition of glutamine alone to LLC-PK1 cells cultured in Earl's balanced salt solution (EBSS) is sufficient to suppress gadd153 mRNA expression, and the removal of only glutamine from Dulbecco's modified Eagle's medium (DMEM) is also sufficient to induce gadd153 mRNA expression. Consistent with these findings, the inhibition of glutamine utilization with acivicin and 6-diazo-5-oxo-l-norleucine (DON) in cells grown in a glutamine-containing medium effectively induces gadd153 expression. Glutamine can be used as an energy source in cultured mammalian cells. However, it is unlikely that deficits in cellular energy stores (ATP) are coupled to gadd153 mRNA expression, because concentrations of ATP, UTP and GTP are all elevated in EBSS-exposed cells, and the addition of alpha-oxoglutarate to cells grown in EBSS has no effect on gadd153 mRNA expression. In contrast, concentrations of CTP decline substantially in EBSS and glutamine-deprived DMEM-cultured cells. Glutamine also serves as a precursor for the synthesis of protein and DNA. The addition of glutamine to cells grown in EBSS partly restores CTP concentrations. The addition of pyrimidine ribonucleosides (cytidine and uridine) to LLC-PK1 cells also restores CTP concentrations, in a manner commensurate with their relative abilities to overcome gadd153 expression. Finally, glutamine does not completely suppress DNA damage-induced gadd153 expression, suggesting that multiple signalling pathways lead to the expression of gadd153 mRNA under conditions of nutrient deprivation and DNA damage.
Topics: Animals; Aspartic Acid; CCAAT-Enhancer-Binding Proteins; Cells, Cultured; Culture Media; Cytidine; Cytidine Triphosphate; DNA Damage; DNA-Binding Proteins; Diazooxonorleucine; Epithelial Cells; Gene Expression Regulation; Glutamine; Isoxazoles; Ketoglutaric Acids; Kidney Tubules, Proximal; RNA, Messenger; Salts; Swine; Transcription Factor CHOP; Transcription Factors; Uridine
PubMed: 10377266
DOI: No ID Found -
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 -
The Tohoku Journal of Experimental... Feb 2009Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system. Despite a variety of anti-inflammatory or...
Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system. Despite a variety of anti-inflammatory or immunomodulation drugs including interferon-beta are effective to reduce relapse risk, most patients have progressive neurological deterioration due to axonal degeneration. Accumulation of activated microglia is a pathological hallmark of active MS lesion. Microglia can act as not only antigen-presenting cells but also effector cells to damage other cells in the central nervous system. Especially, glutamate released by activated microglia induces excito-neurotoxicity and may contribute to neurodegeneration in MS. Gap junction is a major cell-to-cell channel and is composed of paired hemichannels on coupled cells. Recent studies showed that cells release various small molecules (including ions, ATP, and amino acids) from unpaired hemichannel of gap junction that is openly exposed to the extracellular space. We have previously revealed that activated microglia produce glutamate via glutaminase and release it through hemichannels of gap junctions. Thus, in this study, we examined whether the glutaminase inhibitor and the gap junction blocker relieved experimental autoimmune encephalomyelitis (EAE) that is an animal model of MS. Here we show that the gap junction blocker carbenoxolone (CBX) and the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON) decreased glutamate release from activated microglia and rescued neuronal death in a dose-dependent manner in vitro. In EAE mice, treatment with CBX or DON also attenuated EAE clinical symptoms. Thus, blockade of glutamate release from activated microglia with CBX or DON may be an effective therapeutic strategy against neurodegeneration in MS.
Topics: Animals; Carbenoxolone; Cell Death; Diazooxonorleucine; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Enzyme Inhibitors; Gap Junctions; Glutamic Acid; Glutaminase; Mice; Mice, Inbred C57BL; Microglia; Neurons; Spinal Cord
PubMed: 19212100
DOI: 10.1620/tjem.217.87 -
Scientific Reports Jan 2014Glutaminase is a metabolic enzyme responsible for glutaminolysis, a process harnessed by cancer cells to feed their accelerated growth and proliferation. Among the...
Glutaminase is a metabolic enzyme responsible for glutaminolysis, a process harnessed by cancer cells to feed their accelerated growth and proliferation. Among the glutaminase isoforms, human kidney-type glutaminase (KGA) is often upregulated in cancer and is thus touted as an attractive drug target. Here we report the active site inhibition mechanism of KGA through the crystal structure of the catalytic domain of KGA (cKGA) in complex with 6-diazo-5-oxo-L-norleucine (DON), a substrate analogue of glutamine. DON covalently binds with the active site Ser286 and interacts with residues such as Tyr249, Asn335, Glu381, Asn388, Tyr414, Tyr466 and Val484. The nucleophilic attack of Ser286 sidechain on DON releases the diazo group (N2) from the inhibitor and results in the formation of an enzyme-inhibitor complex. Mutational studies confirmed the key role of these residues in the activity of KGA. This study will be important in the development of KGA active site inhibitors for therapeutic interventions.
Topics: Binding Sites; Catalytic Domain; Crystallography, X-Ray; Diazooxonorleucine; Glutaminase; Glutamine; Humans; Kidney; Kinetics; Models, Molecular; Mutation; Protein Binding; Protein Conformation; Substrate Specificity
PubMed: 24451979
DOI: 10.1038/srep03827 -
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