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Biochemistry Mar 2019Non-typhoidal Salmonella can colonize the gastrointestinal system of cattle and can also cause significant food-borne disease in humans. The use of a library of...
Non-typhoidal Salmonella can colonize the gastrointestinal system of cattle and can also cause significant food-borne disease in humans. The use of a library of single-gene deletions in Salmonella enterica serotype Typhimurium allowed identification of several proteins that are under selection in the intestine of cattle. STM2437 ( yfeJ) encodes one of these proteins, and it is currently annotated as a type I glutamine amidotransferase. STM2437 was purified to homogeneity, and its catalytic properties with a wide range of γ-glutamyl derivatives were determined. The catalytic efficiency toward the hydrolysis of l-glutamine was extremely weak with a k/ K value of 20 M s. γ-l-Glutamyl hydroxamate was identified as the best substrate for STM2437, with a k/ K value of 9.6 × 10 M s. A homology model of STM2437 was constructed on the basis of the known crystal structure of a protein of unknown function (Protein Data Bank entry 3L7N ), and γ-l-glutamyl hydroxamate was docked into the active site based on the binding of l-glutamine in the active site of carbamoyl phosphate synthetase. Acivicin was shown to inactivate the enzyme by reaction with the active site cysteine residue and the subsequent loss of HCl. Mutation of Cys91 to serine completely abolished catalytic activity. Inactivation of STM2437 did not affect the ability of this strain to colonize mice, but it inhibited the growth of S. enterica Typhimurium in bacteriologic media containing γ-l-glutamyl hydroxamate.
Topics: Animals; Bacterial Proteins; Cattle; Cattle Diseases; Colitis; Enzyme Activation; Escherichia coli; Glutamates; Hydroxamic Acids; Hydroxylamine; Isoxazoles; Mice, Inbred C57BL; Mutagenesis, Site-Directed; Nitrogenous Group Transferases; Protein Conformation; Salmonella Infections, Animal; Salmonella typhimurium; Substrate Specificity
PubMed: 30715856
DOI: 10.1021/acs.biochem.8b01283 -
Proceedings of the National Academy of... Feb 2019Glutamate is the most abundant excitatory neurotransmitter, present at the bulk of cortical synapses, and participating in many physiologic and pathologic processes...
Glutamate is the most abundant excitatory neurotransmitter, present at the bulk of cortical synapses, and participating in many physiologic and pathologic processes ranging from learning and memory to stroke. The tripeptide, glutathione, is one-third glutamate and present at up to low millimolar intracellular concentrations in brain, mediating antioxidant defenses and drug detoxification. Because of the substantial amounts of brain glutathione and its rapid turnover under homeostatic control, we hypothesized that glutathione is a relevant reservoir of glutamate and could influence synaptic excitability. We find that drugs that inhibit generation of glutamate by the glutathione cycle elicit decreases in cytosolic glutamate and decreased miniature excitatory postsynaptic potential (mEPSC) frequency. In contrast, pharmacologically decreasing the biosynthesis of glutathione leads to increases in cytosolic glutamate and enhanced mEPSC frequency. The glutathione cycle can compensate for decreased excitatory neurotransmission when the glutamate-glutamine shuttle is inhibited. Glutathione may be a physiologic reservoir of glutamate neurotransmitter.
Topics: Animals; Cells, Cultured; Excitatory Postsynaptic Potentials; Glutamic Acid; Glutathione; Homeostasis; Neurons; Rats, Sprague-Dawley; Synapses; Synaptic Transmission
PubMed: 30692251
DOI: 10.1073/pnas.1817885116 -
Cellular Microbiology Mar 2019Helicobacter saguini is a novel enterohepatic Helicobacter species isolated from captive cotton top tamarins with chronic colitis and colon cancer. Monoassociated...
BACKGROUND
Helicobacter saguini is a novel enterohepatic Helicobacter species isolated from captive cotton top tamarins with chronic colitis and colon cancer. Monoassociated H. saguini infection in gnotobiotic IL-10 mice causes typhlocolitis and dysplasia; however, the virulent mechanisms of this species are unknown. Gamma-glutamyltranspeptidase (GGT) is an enzymatic virulence factor expressed by pathogenic Helicobacter and Campylobacter species that inhibits host cellular proliferation and promotes inflammatory-mediated gastrointestinal pathology. The aim of this study was to determine if H. saguini expresses an enzymatically active GGT homologue with virulence properties.
EXPERIMENTAL PROCEDURES
Two putative GGT paralogs (HSGGT1 and HSGGT2) identified in the H. saguini genome were bioinformatically analysed to predict enzymatic functionality and virulence potential. An isogenic knockout mutant strain and purified recombinant protein of HSGGT1 were created to study enzymatic activity and virulence properties by in vitro biochemical and cell culture experiments.
RESULTS
Bioinformatic analysis predicted that HSGGT1 has enzymatic functionality and is most similar to the virulent homologue expressed by Helicobacter bilis, whereas HSGGT2 contains putatively inactivating mutations. An isogenic knockout mutant strain and recombinant HSGGT1 protein were successfully created and demonstrated that H. saguini has GGT enzymatic activity. Recombinant HSGGT1 protein and sonicate from wild-type but not mutant H. saguini inhibited gastrointestinal epithelial and lymphocyte cell proliferation without evidence of cell death. The antiproliferative effect by H. saguini sonicate or recombinant HSGGT1 protein could be significantly prevented with glutamine supplementation or the GGT-selective inhibitor acivicin. Recombinant HSGGT1 protein also induced proinflammatory gene expression in colon epithelial cells.
CONCLUSIONS
This study shows that H. saguini may express GGT as a potential virulence factor and supports further in vitro and in vitro studies into how GGT expression by enterohepatic Helicobacter species influences the pathogenesis of gastrointestinal inflammatory diseases.
Topics: Animals; Cell Survival; Chronic Disease; Colitis; Computational Biology; Epithelial Cells; Gene Expression; Gene Knockout Techniques; Helicobacter; Interleukin-10; Mice, Knockout; Recombinant Proteins; Saguinus; Virulence Factors; gamma-Glutamyltransferase
PubMed: 30365223
DOI: 10.1111/cmi.12968 -
Journal of Cellular Physiology May 2019Excess reactive oxygen species (ROS) generated in embryos during in vitro culture damage cellular macromolecules and embryo development. Glutathione (GSH) scavenges ROS...
Excess reactive oxygen species (ROS) generated in embryos during in vitro culture damage cellular macromolecules and embryo development. Glutathione (GSH) scavenges ROS and optimizes the culture system. However, how exogenous GSH influences intracellular GSH and improves the embryo developmental rate is poorly understood. In this study, GSH or GSX (a stable GSH isotope) was added to the culture media of bovine in vitro fertilization embryos for 7 days. The cleavage rate, blastocyst rate, and total cell number of blastocysts were calculated. Similarly to GSH, GSX increased the in vitro development rate and embryo quality. We measured intracellular ROS, GSX, and GSH for 0-32-hr postinsemination (hpi) in embryos (including zygotes at G1, S, and G2 phases and cleaved embryos) cultured in medium containing GSX. Intracellular ROS significantly decreased with increasing intracellular GSH in S-stage zygotes (18 hpi) and cleaved embryos (32 hpi). γ-Glutamyltranspeptidase ( GGT) and glutathione synthetase ( GSS) messenger RNA expression increased in zygotes (18 hpi) and cleaved embryos treated with GSH, consistent with the tendency of overall GSH content. GGT activity increased significantly in 18 hpi zygotes. GGT and GCL enzyme inhibition with acivicin and buthionine sulfoximine, respectively, decreased cleavage rate, blastocyst rate, total cell number, and GSH and GSX content. All results indicated that exogenous GSH affects intracellular GSH levels through the γ-glutamyl cycle and improves early embryo development, enhancing our understanding of the redox regulation effects and transport of GSH during embryo culture in vitro.
Topics: Animals; Cattle; Cleavage Stage, Ovum; Embryo Culture Techniques; Enzyme Inhibitors; Female; Fertilization in Vitro; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Glutathione; Glutathione Synthase; Male; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Time Factors; Zygote; gamma-Glutamyltransferase
PubMed: 30362550
DOI: 10.1002/jcp.27497 -
Nucleosides, Nucleotides & Nucleic Acids 2018The pyrimidine de novo nucleotide synthesis consists of 6 sequential steps. Various inhibitors against these enzymes have been developed and evaluated in the clinic for...
The pyrimidine de novo nucleotide synthesis consists of 6 sequential steps. Various inhibitors against these enzymes have been developed and evaluated in the clinic for their potential anticancer activity: acivicin inhibits carbamoyl-phosphate-synthase-II, N-(phosphonacetyl)-L- aspartate (PALA) inhibits aspartate-transcarbamylase, Brequinar sodium and dichloroallyl-lawsone (DCL) inhibit dihydroorotate-dehydrogenase, and pyrazofurin (PF) inhibits orotate-phosphoribosyltransferase. We compared their growth inhibition against 3 cell lines from head-and-neck-cancer (HEP-2, UMSCC-14B and UMSCC-14C) and related the sensitivity to their effects on nucleotide pools. In all cell lines Brequinar and PF were the most active compounds with IC50 (50% growth inhibition) values between 0.06-0.37 µM, Acivicin was as potent (IC50s 0.26-1 µM), but DCL was 20-31-fold less active. PALA was most inactive (24-128 µM). At equitoxic concentrations, all pure antipyrimidine de novo inhibitors depleted UTP and CTP after 24 hr exposure, which was most pronounced for Brequinar (between 6-10% of UTP left, and 12-36% CTP), followed by DCL and PF, which were almost similar (6-16% UTP and 12-27% CTP), while PALA was the least active compound (10-70% UTP and 13-68% CTP). Acivicin is a multi-target inhibitor of more glutamine requiring enzymes (including GMP synthetase) and no decrease of UTP was found, but a pronounced decrease in GTP (31-72% left). In conclusion, these 5 inhibitors of the pyrimidine de novo nucleotide synthesis varied considerably in their efficacy and effect on pyrimidine nucleotide pools. Inhibitors of DHO-DH were most effective suggesting a primary role of this enzyme in controlling pyrimidine nucleotide pools.
Topics: Amides; Antineoplastic Agents; Aspartate Carbamoyltransferase; Aspartic Acid; Biphenyl Compounds; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); Carcinoma, Squamous Cell; Cell Line, Tumor; Dihydroorotate Dehydrogenase; Head and Neck Neoplasms; Humans; Isoxazoles; Naphthoquinones; Orotate Phosphoribosyltransferase; Oxidoreductases Acting on CH-CH Group Donors; Phosphonoacetic Acid; Purine Nucleotides; Pyrazoles; Pyrimidine Nucleotides; Ribonucleosides; Ribose
PubMed: 29723133
DOI: 10.1080/15257770.2018.1460479 -
Experimental Eye Research Jan 2018In this study, we have investigated whether the lens was capable of exporting the antioxidant glutathione. Pairs of rat lenses were cultured in isosmotic artificial...
In this study, we have investigated whether the lens was capable of exporting the antioxidant glutathione. Pairs of rat lenses were cultured in isosmotic artificial aqueous humour for one, two, three, or six hours in low oxygen conditions (90% N, 5% CO, 5% O), and reduced glutathione (GSH) and oxidised glutathione (GSSG) levels measured in the media and lenses. We show that the rat lens is capable of releasing ∼5 nmol GSH for each time point suggesting that GSH release is regulated since it does not appreciably increase over time. We also demonstrated that the predominant form of glutathione released was the reduced form. We next cultured lenses in the absence or presence of acivicin, a γ-glutamyl transpeptidase (GGT) inhibitor, and found that GSH levels were significantly increased (p < 0.001) in the presence of this inhibitor, which indicated that GSH released by the lens undergoes degradation into its constituent amino acids. GSH release was significantly decreased (p < 0.001) in the presence of 100 μM MK571, a multidrug resistance-associated protein (Mrp) inhibitor, suggesting that Mrp transporters mediate GSH efflux from the lens. Culturing lenses in low (10 μM) or high (70 μM) concentrations of HO for one hour significantly increased total glutathione levels (p < 0.05) relative to controls, due to the increased release of GSSG. Our results show that in response to oxidative stress, the rat lens is able to release GSH or GSSG, thereby serving to maintain lens redox state or potentially influence the redox state of nearby tissues.
Topics: Animals; Aqueous Humor; Glutathione; Hydrogen Peroxide; L-Lactate Dehydrogenase; Lens, Crystalline; Models, Animal; Oxidative Stress; Rats; Rats, Wistar
PubMed: 29032155
DOI: 10.1016/j.exer.2017.10.010 -
ChemMedChem Apr 2017The natural product acivicin inhibits the glutaminase activity of cytidine triphosphate (CTP) synthetase and is a potent lead compound for drug discovery in the area of...
The natural product acivicin inhibits the glutaminase activity of cytidine triphosphate (CTP) synthetase and is a potent lead compound for drug discovery in the area of neglected tropical diseases, specifically trypanosomaisis. A 2.1-Å-resolution crystal structure of the acivicin adduct with the glutaminase domain from Trypanosoma brucei CTP synthetase has been deposited in the RCSB Protein Data Bank (PDB) and provides a template for structure-based approaches to design new inhibitors. However, our assessment of that data identified deficiencies in the model. We now report an improved and corrected inhibitor structure with changes to the chirality at one position, the orientation and covalent structure of the isoxazoline moiety, and the location of a chloride ion in an oxyanion binding site that is exploited during catalysis. The model is now in agreement with established chemical principles and allows an accurate description of molecular recognition of the ligand and the mode of binding in a potentially valuable drug target.
Topics: Bacillus subtilis; Carbon-Nitrogen Ligases; Catalytic Domain; Glutaminase; Helicobacter pylori; Hydrogen Bonding; Isoxazoles; Ligands; Trypanocidal Agents; Trypanosoma brucei brucei; gamma-Glutamyltransferase
PubMed: 28333400
DOI: 10.1002/cmdc.201700118 -
The Protein Journal Feb 2017Gamma glutamyl transpeptidase, (GGT) is a ubiquitous protein which plays a central role in glutathione metabolism and has myriad clinical implications. It has been shown...
Gamma glutamyl transpeptidase, (GGT) is a ubiquitous protein which plays a central role in glutathione metabolism and has myriad clinical implications. It has been shown to be a virulence factor for pathogenic bacteria, inhibition of which results in reduced colonization potential. However, existing inhibitors are effective but toxic and therefore search is on for novel inhibitors, which makes it imperative to understand the interactions of various inhibitors with the protein in substantial detail. High resolution structures of protein bound to different inhibitors can serve this purpose. Gamma glutamyl transpeptidase from Bacillus licheniformis is one of the model systems that have been used to understand the structure-function correlation of the protein. The structures of the native protein (PDB code 4OTT), of its complex with glutamate (PDB code 4OTU) and that of its precursor mimic (PDB code 4Y23) are available, although at moderate/low resolution. In the present study, we are reporting the preliminary analysis of, high resolution X-ray diffraction data collected for the co-crystals of B. licheniformis, Gamma glutamyl transpeptidase, with its inhibitor, Acivicin. Crystals belong to the orthorhombic space group P222 and diffract X-ray to 1.45 Å resolution. This is the highest resolution data reported for all GGT structures available till now. The use of SUMO fused expression system enhanced yield of the target protein in the soluble fraction, facilitating recovery of protein with high purity. The preliminary analysis of this data set shows clear density for the inhibitor, acivicin, in the protein active site.
Topics: Bacillus licheniformis; Gene Expression; Isoxazoles; Recombinant Fusion Proteins; SUMO-1 Protein; X-Ray Diffraction; gamma-Glutamyltransferase
PubMed: 28120227
DOI: 10.1007/s10930-017-9693-2 -
Thrombosis Journal 2016Besides maintaining intracellular glutathione stores, gamma-glutamyltransferase(GGT) generates reactive oxygen species and activates NFkB, a redox-sensitive...
BACKGROUND
Besides maintaining intracellular glutathione stores, gamma-glutamyltransferase(GGT) generates reactive oxygen species and activates NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the principal initiator of the clotting cascade. Thus, GGT might be involved in TF-mediated coagulation processes, an assumption untested insofar.
METHODS
Experiments were run with either equine, enzymatically active GGT or human recombinant (hr) GGT, a wheat germ-derived protein enzymatically inert because of missing post-translational glycosylation. TF Procoagulant Activity (PCA, one-stage clotting assay), TF antigen(ELISA) and TFmRNA(real-time PCR) were assessed in unpooled human peripheral blood mononuclear cell(PBMC) suspensions obtained from healthy donors through discontinuous Ficoll/Hystopaque density gradient.
RESULTS
Equine GGT increased PCA, an effect insensitive to GGT inhibition by acivicin suggesting mechanisms independent of its enzymatic activity, a possibility confirmed by the maintained stimulation in response to hrGGT, an enzymatically inactive molecule. Endotoxin(LPS) contamination of GGT preparations was excluded by heat inactivation studies and direct determination(LAL method) of LPS concentrations <0.1 ng/mL practically devoid of procoagulant effect. Inhibition by anti-GGT antibodies corroborated that conclusion. Upregulation by hrGGT of TF antigen and mRNA and its downregulation by BAY-11-7082, a NFkB inhibitor, and N-acetyl-L-cysteine, an antioxidant, was consistent with a NFkB-driven, redox-sensitive transcriptional site of action.
CONCLUSIONS
GGT upregulates TF expression independent of its enzymatic activity, a cytokine-like behaviour mediated by NFκB activation, a mechanism contributing to promote acute thrombotic events, a possibility in need, however, of further evaluation.
PubMed: 27822142
DOI: 10.1186/s12959-016-0119-8 -
The Journal of General and Applied... Nov 2016
Topics: Antifungal Agents; Culture Media; Fusarium; Genes, Fungal; Isoxazoles; Mycelium; Trichothecenes
PubMed: 27600357
DOI: 10.2323/jgam.2016.04.002