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1
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Bist G, Luong NT, Mahabubur Rahman KM, Ruszaj DM, Li C, Hanigan MH, You Y. SAR of L-ABBA analogs for GGT1 inhibitory activity and L-ABBA's effect on plasma cysteine and GSH species. Bioorg Med Chem Lett 2023:129406. [PMID: 37423504 DOI: 10.1016/j.bmcl.2023.129406] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2023] [Revised: 06/20/2023] [Accepted: 07/04/2023] [Indexed: 07/11/2023]
Abstract
Gamma-glutamyl transferase 1 (GGT1) is a critical enzyme involved in the hydrolysis and/or transfer of gamma-glutamyl groups of glutathione, which helps maintain cysteine levels in plasma. In this study, we synthesized L-ABBA analogs to investigate their inhibitory effect on GGT1 hydrolysis and transpeptidase activity, with the goal of defining the pharmacophore of L-ABBA. Our structure-activity relationship (SAR) study revealed that an α-COO- and α-NH3+ group, as well as a two-CH2 unit distance between α-C and boronic acid, are essential for the activity. The addition of an R (alkyl) group at the α-C reduced the activity of GGT1 inhibition, with L-ABBA being the most potent inhibitor among the analogs. Next, we investigated the impact of L-ABBA on plasma levels of cysteine and GSH species, with the expectation of observing reduced cysteine levels and enhanced GSH levels due to its GGT1 inhibition. We administered L-ABBA intraperitoneally and determined the plasma levels of cysteine, cystine, GSH, and GSSG using LCMS. Our results showed time- and L-ABBA dose-dependent changes in total plasma cysteine and GSH levels. This study is the first to demonstrate the regulation of plasma thiol species upon GGT1 inhibition, with plasma cystine levels reduced by up to ∼75% with L-ABBA (0.3 mg/dose). Cancer cells are highly dependent on the uptake of cysteine from plasma for maintaining high levels of intracellular glutathione. Thus, our findings suggest that GGT1 inhibitors, such as L-ABBA, have a potential to be used for GSH reduction thereby inducing oxidative stress in cancer cells and reducing their resistance to many chemotherapeutic agents.
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Affiliation(s)
- Ganesh Bist
- Department of Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, NY 14214, United States
| | - Nguyen T Luong
- Department of Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, NY 14214, United States
| | - Kazi Md Mahabubur Rahman
- Department of Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, NY 14214, United States
| | - Donna M Ruszaj
- Department of Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, NY 14214, United States
| | - Chenglong Li
- Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610, United States
| | - Marie H Hanigan
- Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, United States
| | - Youngjae You
- Department of Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, NY 14214, United States.
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2
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Paties Montagner G, Dominici S, Piaggi S, Pompella A, Corti A. Redox Mechanisms Underlying the Cytostatic Effects of Boric Acid on Cancer Cells-An Issue Still Open. Antioxidants (Basel) 2023; 12:1302. [PMID: 37372032 DOI: 10.3390/antiox12061302] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2023] [Revised: 06/05/2023] [Accepted: 06/13/2023] [Indexed: 06/29/2023] Open
Abstract
Boric acid (BA) is the dominant form of boron in plasma, playing a role in different physiological mechanisms such as cell replication. Toxic effects have been reported, both for high doses of boron and its deficiency. Contrasting results were, however, reported about the cytotoxicity of pharmacological BA concentrations on cancer cells. The aim of this review is to briefly summarize the main findings in the field ranging from the proposed mechanisms of BA uptake and actions to its effects on cancer cells.
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Affiliation(s)
- Giulia Paties Montagner
- Department of Translational Research NTMS, University of Pisa Medical School, 56126 Pisa, Italy
| | - Silvia Dominici
- Department of Translational Research NTMS, University of Pisa Medical School, 56126 Pisa, Italy
| | - Simona Piaggi
- Department of Translational Research NTMS, University of Pisa Medical School, 56126 Pisa, Italy
| | - Alfonso Pompella
- Department of Translational Research NTMS, University of Pisa Medical School, 56126 Pisa, Italy
| | - Alessandro Corti
- Department of Translational Research NTMS, University of Pisa Medical School, 56126 Pisa, Italy
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3
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Reza MH, Sanyal K. Defective in utilizing glutathione 3, DUG3, is required for conidiation and host infection in the rice blast fungus Magnaporthe oryzae. MICROPUBLICATION BIOLOGY 2022; 2022:10.17912/micropub.biology.000550. [PMID: 35622524 PMCID: PMC9012958 DOI: 10.17912/micropub.biology.000550] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/03/2022] [Revised: 04/06/2022] [Accepted: 04/12/2022] [Indexed: 11/19/2022]
Abstract
Glutathione, an important redox buffer of the cell, also functions as a source of sulphur and nitrogen under starvation conditions. The metabolism and maintenance of glutathione homeostasis are vital for the appropriate functioning of the cell. In addition to the γ-glutamyl transpeptidase, the fungus-specific alternative pathway involving DUG1, DUG2 and DUG3 genes also mediate glutathione degradation. Here, we studied the functional significance of DUG3 in the vegetative growth and infection cycle of the cereal blast fungus Magnaporthe oryzae . Cells lacking the DUG3 gene displayed reduced conidiation, delayed appressorium formation, and a decrease in the severity of host infection. Further, we show that the γ-glutamyl transpeptidase inhibitor severely compromises the vegetative growth of the M. oryzae cells lacking the DUG3 gene. Taken together, our results suggest a significant role of glutathione metabolism in the growth and virulence of M. oryzae .
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Affiliation(s)
- Md. Hashim Reza
- Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru 560 064, India
,
Bharat Chattoo Genome Research Centre, Department of Microbiology & Biotechnology Centre, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390 002, Gujarat, India
,
Correspondence to: Md. Hashim Reza (
)
| | - Kaustuv Sanyal
- Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru 560 064, India
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4
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Bovine serum albumin-encapsulated gold nanoclusters-Cu2+ synergize and promote calcein chemiluminescence for glutathione detection in human whole blood. Microchem J 2021. [DOI: 10.1016/j.microc.2021.106749] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
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5
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Saini M, Kashyap A, Bindal S, Saini K, Gupta R. Bacterial Gamma-Glutamyl Transpeptidase, an Emerging Biocatalyst: Insights Into Structure-Function Relationship and Its Biotechnological Applications. Front Microbiol 2021; 12:641251. [PMID: 33897647 PMCID: PMC8062742 DOI: 10.3389/fmicb.2021.641251] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2020] [Accepted: 02/15/2021] [Indexed: 12/16/2022] Open
Abstract
Gamma-glutamyl transpeptidase (GGT) enzyme is ubiquitously present in all life forms and plays a variety of roles in diverse organisms. Higher eukaryotes mainly utilize GGT for glutathione degradation, and mammalian GGTs have implications in many physiological disorders also. GGTs from unicellular prokaryotes serve different physiological functions in Gram-positive and Gram-negative bacteria. In the present review, the physiological significance of bacterial GGTs has been discussed categorizing GGTs from Gram-negative bacteria like Escherichia coli as glutathione degraders and from pathogenic species like Helicobacter pylori as virulence factors. Gram-positive bacilli, however, are considered separately as poly-γ-glutamic acid (PGA) degraders. The structure-function relationship of the GGT is also discussed mainly focusing on the crystallization of bacterial GGTs along with functional characterization of conserved regions by site-directed mutagenesis that unravels molecular aspects of autoprocessing and catalysis. Only a few crystal structures have been deciphered so far. Further, different reports on heterologous expression of bacterial GGTs in E. coli and Bacillus subtilis as hosts have been presented in a table pointing toward the lack of fermentation studies for large-scale production. Physicochemical properties of bacterial GGTs have also been described, followed by a detailed discussion on various applications of bacterial GGTs in different biotechnological sectors. This review emphasizes the potential of bacterial GGTs as an industrial biocatalyst relevant to the current switch toward green chemistry.
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Affiliation(s)
| | | | | | | | - Rani Gupta
- Department of Microbiology, University of Delhi South Campus, New Delhi, India
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6
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A Systematic Review of Serum γ-Glutamyltransferase as a Prognostic Biomarker in Patients with Genitourinary Cancer. Antioxidants (Basel) 2021; 10:antiox10040549. [PMID: 33916150 PMCID: PMC8066142 DOI: 10.3390/antiox10040549] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2021] [Revised: 03/24/2021] [Accepted: 03/29/2021] [Indexed: 12/11/2022] Open
Abstract
γ-Glutamyltransferase (GGT), a membrane-bound enzyme, contributes to the metabolism of glutathione (GSH), which plays a critical physiological role in protecting cells against oxidative stress. GGT has been proposed as a biomarker of carcinogenesis and tumor progression given that GGT activity is important during both the promotion and invasion phases in cancer cells. Moreover, GGT expression is reportedly related to drug-resistance possibly because a wide range of drugs are conjugated with GSH, the availability of which is influenced by GGT activity. While serum GGT activity is commonly used as a quick, inexpensive, yet reliable means of assessing liver function, recent epidemiological studies have shown that it may also be an indicator of an increased risk of prostate cancer development. Moreover, elevated serum GGT is reportedly an adverse prognostic predictor in patients with urologic neoplasms, including renal cell carcinoma, prostate cancer, and urothelial carcinoma, although the background mechanisms have still not been well-characterized. The present review article summarizes the possible role of GGT in cancer cells and focuses on evidence evaluation through a systematic review of the latest literature on the prognostic role of serum GGT in patients with genitourinary cancer.
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Abstract
The mercapturic acid pathway is a major route for the biotransformation of xenobiotic and endobiotic electrophilic compounds and their metabolites. Mercapturic acids (N-acetyl-l-cysteine S-conjugates) are formed by the sequential action of the glutathione transferases, γ-glutamyltransferases, dipeptidases, and cysteine S-conjugate N-acetyltransferase to yield glutathione S-conjugates, l-cysteinylglycine S-conjugates, l-cysteine S-conjugates, and mercapturic acids; these metabolites constitute a "mercapturomic" profile. Aminoacylases catalyze the hydrolysis of mercapturic acids to form cysteine S-conjugates. Several renal transport systems facilitate the urinary elimination of mercapturic acids; urinary mercapturic acids may serve as biomarkers for exposure to chemicals. Although mercapturic acid formation and elimination is a detoxication reaction, l-cysteine S-conjugates may undergo bioactivation by cysteine S-conjugate β-lyase. Moreover, some l-cysteine S-conjugates, particularly l-cysteinyl-leukotrienes, exert significant pathophysiological effects. Finally, some enzymes of the mercapturic acid pathway are described as the so-called "moonlighting proteins," catalytic proteins that exert multiple biochemical or biophysical functions apart from catalysis.
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Affiliation(s)
- Patrick E Hanna
- Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN, USA
| | - M W Anders
- Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY, USA
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8
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Milito A, Brancaccio M, Lisurek M, Masullo M, Palumbo A, Castellano I. Probing the Interactions of Sulfur-Containing Histidine Compounds with Human Gamma-Glutamyl Transpeptidase. Mar Drugs 2019; 17:md17120650. [PMID: 31757046 PMCID: PMC6949936 DOI: 10.3390/md17120650] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2019] [Revised: 11/11/2019] [Accepted: 11/18/2019] [Indexed: 02/07/2023] Open
Abstract
Gamma-glutamyl transpeptidase (GGT) is a cell surface enzyme involved in glutathione metabolism and maintenance of redox homeostasis. High expression of GGT on tumor cells is associated with an increase of cell proliferation and resistance against chemotherapy. GGT inhibitors that have been evaluated in clinical trials are too toxic for human use. We have previously identified ovothiols, 5(Nπ)-methyl-thiohistidines of marine origin, as non-competitive-like inhibitors of GGT that are more potent than the known GGT inhibitor, 6-diazo-5-oxo-l-norleucine (DON), and are not toxic for human embryonic cells. We extended these studies to the desmethylated form of ovothiol, 5-thiohistidine, and confirmed that this ovothiol derivative also acts as a non-competitive-like GGT inhibitor, with a potency comparable to ovothiol. We also found that both 5-thiohistidine derivatives act as reversible GGT inhibitors compared to the irreversible DON. Finally, we probed the interactions of 5-thiohistidines with GGT by docking analysis and compared them with the 2-thiohistidine ergothioneine, the physiological substrate glutathione, and the DON inhibitor. Overall, our results provide new insight for further development of 5-thiohistidine derivatives as therapeutics for GGT-positive tumors.
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Affiliation(s)
- Alfonsina Milito
- Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, 80121 Naples, Italy; (A.M.); (M.B.); (A.P.)
| | - Mariarita Brancaccio
- Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, 80121 Naples, Italy; (A.M.); (M.B.); (A.P.)
| | - Michael Lisurek
- Department of Computational Chemistry and Drug Design, Leibniz-Forschungsinstitut für Molekulare Pharmakologie, 13125 Berlin, Germany;
| | - Mariorosario Masullo
- Department of Human Movement Sciences and Wellbeing, University of Naples “Parthenope”, 80133 Naples, Italy;
| | - Anna Palumbo
- Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, 80121 Naples, Italy; (A.M.); (M.B.); (A.P.)
| | - Immacolata Castellano
- Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, 80121 Naples, Italy; (A.M.); (M.B.); (A.P.)
- Correspondence: ; Tel.: +39-081-5833206
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9
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Philips JG, Dumin W, Winefield C. Functional Characterization of the Grapevine γ-Glutamyl Transferase/Transpeptidase (E.C. 2.3.2.2) Gene Family Reveals a Single Functional Gene Whose Encoded Protein Product Is Not Located in Either the Vacuole or Apoplast. FRONTIERS IN PLANT SCIENCE 2019; 10:1402. [PMID: 31749820 PMCID: PMC6843540 DOI: 10.3389/fpls.2019.01402] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/16/2019] [Accepted: 10/10/2019] [Indexed: 06/08/2023]
Abstract
γ-glutamyl transferases/transpeptidases (E.C. 2.3.2.2, GGTs) are involved in the catabolism of many compounds that are conjugated to glutathione (GSH), which have a variety of roles. GSH can act as storage and transport vehicle for reduced sulfur; it is involved in the detoxification of xenobiotics and also acts as a redox buffer by utilizing its thiol residue to protect against reactive oxygen species, which accumulate in response to biotic and abiotic stress. Furthermore, many distinctive flavor and aroma compounds in Sauvignon blanc wines originate from odorless C5- and C6-GSH conjugates or their GGT catabolized derivatives. These precursors are then processed into their volatile forms by yeast during fermentation. In many plant species, two or more isoforms of GGTs exist that target GSH-conjugates to either the apoplast or the vacuole. A bioinformatics approach identified multiple GGT candidates in grapevine (Vitis vinifera). However, only a single candidate, VvGGT3, has all the conserved residues needed for GGT activity. This is intriguing given the variety of roles of GSH and GGTs in plant cells. Characterization of VvGGT3 from cv. Sauvignon blanc was then undertaken. The VvGGT3 transcript is present in roots, leaves, inflorescences, and tendril and at equal abundance in the skin, pulp, and seed of mature berries and shows steady accumulation over the course of whole berry development. In addition, the VvGGT3 transcript in whole berries is upregulated upon Botrytis cinerea infection as well as mechanical damage to leaf tissue. VvGGT3-GFP fusion proteins transiently over-expressed in onion cells were used to study subcellular localization. To confirm VvGGT3 activity and localization in vivo, the fluorescent γ-glutamyl-7-amido-4-methylcoumarin substrate was added to Nicotiana benthamiana leaves transiently over-expressing VvGGT3. In combination, these results suggest that the functional VvGGT3 is associated with membrane-like structures. This is not consistent with its closely related functionally characterized GGTs from Arabidopsis, radish and garlic.
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Affiliation(s)
| | | | - Christopher Winefield
- Department of Wine Food and Molecular Biosciences, Faculty of Agriculture and Life Sciences, Lincoln University, Christchurch, New Zealand
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10
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Krishnan BB, Selvaraju S, Gowda NKS, Subramanya KB, Pal D, Archana SS, Bhatta R. Dietary boron supplementation enhances sperm quality and immunity through influencing the associated biochemical parameters and modulating the genes expression at testicular tissue. J Trace Elem Med Biol 2019; 55:6-14. [PMID: 31345367 DOI: 10.1016/j.jtemb.2019.05.004] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/16/2019] [Revised: 05/03/2019] [Accepted: 05/08/2019] [Indexed: 12/19/2022]
Abstract
INTRODUCTION Dietary boron improves immune and antioxidant status and calcium metabolism in mammals. However, till date the effects of dietary boron supplementation on male reproduction, especially on sperm production and sperm quality in farm animals are not documented. OBJECTIVE The present study was aimed to investigate the influence of dietary boron on semen production, semen quality, immunity and molecular changes in the testis, blood and seminal plasma and to assess the interrelationship with other minerals in male goats. METHODOLOGY The study was conducted in 21 adult male goats divided into 3 groups (control, boron and selenium supplemented groups, n = 7 each). In boron group, boron was supplemented at 40 ppm and in selenium group, selenium was supplemented at 1 ppm over and above the basal level. In control group, only the basal diet was fed without supplementary boron or selenium. The feeding trial was carried out for 60 days. Selenium was taken as a positive control for the dietary boron supplementation experiment. Following feeding trials, the sperm concentration, kinematics and functional attributes, immunity and molecular level changes in the testis, biomolecular changes in the blood and seminal plasma and also interrelationship with other minerals were studied. RESULTS The average sperm concentration (million/ml) and the total sperm production (million/ejaculate) were significantly (p < 0.05) increased in boron supplemented group when compared to selenium and control groups. The boron levels in blood plasma (r = 0.65) and seminal plasma (r = 0.54) showed a positive correlation with sperm progressive motility. Blood and seminal plasma metabolic biomarker namely, aspartate aminotransferase (AST) (p < 0.01) was significantly lower in the boron and selenium supplemented group than control, while alanine aminotransferase (ALT) (p < 0.05) was significantly lower in the boron supplemented group than selenium and control group. There was a significant increase in the mRNA expression of serine proteinase inhibitor (SERPIN) and interferon γ (IFNγ) in the testis of boron supplemented than the control group. Boron supplementation up-regulated the immune-regulatory gene, interleukin 2 (IL2) and antioxidant gene, catalase (CAT) in the peripheral blood mononuclear cells (PBMC). On contrary, toll-like receptor 2 (TLR2) mRNA expression was significantly (p < 0.05) down-regulated in boron and selenium supplemented groups. CONCLUSION The study revealed that dietary boron supplementation increased the sperm output, sperm motility and enhanced the immune and antioxidant defense capacity in male goats. The improved semen quality can be attributed to enhanced expression of testicular SERPIN, a crucial protein for the regulation of spermatogenesis process.
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Affiliation(s)
- Binsila B Krishnan
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru 560 030, India.
| | - Sellappan Selvaraju
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru 560 030, India
| | - Nisarani Kollurappa Shivakumar Gowda
- Micronutrient Laboratory, Animal Nutrition Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru 560 030, India
| | - Karthik Bhat Subramanya
- Micronutrient Laboratory, Animal Nutrition Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru 560 030, India
| | - Dintaran Pal
- Micronutrient Laboratory, Animal Nutrition Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru 560 030, India
| | - Santhanahalli Siddalingappa Archana
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru 560 030, India
| | - Raghavendra Bhatta
- Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru 560 030, India
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11
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Brancaccio M, Russo M, Masullo M, Palumbo A, Russo GL, Castellano I. Sulfur-containing histidine compounds inhibit γ-glutamyl transpeptidase activity in human cancer cells. J Biol Chem 2019; 294:14603-14614. [PMID: 31375562 DOI: 10.1074/jbc.ra119.009304] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2019] [Revised: 07/04/2019] [Indexed: 12/19/2022] Open
Abstract
γ-Glutamyl transpeptidase (GGT) is an enzyme located on the surface of cellular membranes and involved in GSH metabolism and maintenance of redox homeostasis. High GGT expression on tumor cells is associated with increased cell proliferation and resistance against chemotherapy. GGT inhibitors evaluated so far in clinical trials are too toxic for human use. In this study, using enzyme kinetics analyses, we demonstrate that ovothiols, 5(Nπ)-methyl thiohistidines of marine origin, act as noncompetitive inhibitors of GGT, with an apparent Ki of 21 μm, when we fixed the concentrations of the donor substrate. We found that these compounds are more potent than the known GGT inhibitor 6-diazo-5-oxo-l-norleucine and are not toxic toward human embryonic cells. In particular, cellular process-specific fluorescence-based assays revealed that ovothiols induce a mixed cell-death phenotype of apoptosis and autophagy in GGT-overexpressing cell lines, including human liver cancer and chronic B leukemic cells. The findings of our study provide the basis for further development of 5-thiohistidines as therapeutics for GGT-positive tumors and highlight that GGT inhibition is involved in autophagy.
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Affiliation(s)
- Mariarita Brancaccio
- Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, 80121 Naples, Italy
| | - Maria Russo
- Institute of Food Sciences, National Research Council, 83100 Avellino, Italy
| | - Mariorosario Masullo
- Department of Movement Sciences and Wellbeing, University of Naples "Parthenope," 80133 Naples, Italy
| | - Anna Palumbo
- Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, 80121 Naples, Italy
| | - Gian Luigi Russo
- Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, 80121 Naples, Italy.,Institute of Food Sciences, National Research Council, 83100 Avellino, Italy
| | - Immacolata Castellano
- Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, 80121 Naples, Italy
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12
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Gifford JL, Nguyen WN, de Koning L, Seiden-Long I. Stabilizing specimens for routine ammonia testing in the clinical laboratory. Clin Chim Acta 2018; 478:37-43. [DOI: 10.1016/j.cca.2017.12.022] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2017] [Revised: 11/21/2017] [Accepted: 12/15/2017] [Indexed: 01/09/2023]
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13
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Abstract
Many potentially toxic electrophilic xenobiotics and some endogenous compounds are detoxified by conversion to the corresponding glutathione S-conjugate, which is metabolized to the N-acetylcysteine S-conjugate (mercapturate) and excreted. Some mercapturate pathway components, however, are toxic. Bioactivation (toxification) may occur when the glutathione S-conjugate (or mercapturate) is converted to a cysteine S-conjugate that undergoes a β-lyase reaction. If the sulfhydryl-containing fragment produced in this reaction is reactive, toxicity may ensue. Some drugs and halogenated workplace/environmental contaminants are bioactivated by this mechanism. On the other hand, cysteine S-conjugate β-lyases occur in nature as a means of generating some biologically useful sulfhydryl-containing compounds.
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14
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Hatem E, El Banna N, Huang ME. Multifaceted Roles of Glutathione and Glutathione-Based Systems in Carcinogenesis and Anticancer Drug Resistance. Antioxid Redox Signal 2017; 27:1217-1234. [PMID: 28537430 DOI: 10.1089/ars.2017.7134] [Citation(s) in RCA: 79] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
SIGNIFICANCE Glutathione is the most abundant antioxidant molecule in living organisms and has multiple functions. Intracellular glutathione homeostasis, through its synthesis, consumption, and degradation, is an intricately balanced process. Glutathione levels are often high in tumor cells before treatment, and there is a strong correlation between elevated levels of intracellular glutathione/sustained glutathione-mediated redox activity and resistance to pro-oxidant anticancer therapy. Recent Advances: Ample evidence demonstrates that glutathione and glutathione-based systems are particularly relevant in cancer initiation, progression, and the development of anticancer drug resistance. CRITICAL ISSUES This review highlights the multifaceted roles of glutathione and glutathione-based systems in carcinogenesis, anticancer drug resistance, and clinical applications. FUTURE DIRECTIONS The evidence summarized here underscores the important role played by glutathione and the glutathione-based systems in carcinogenesis and anticancer drug resistance. Future studies should address mechanistic questions regarding the distinct roles of glutathione in different stages of cancer development and cancer cell death. It will be important to study how metabolic alterations in cancer cells can influence glutathione homeostasis. Sensitive approaches to monitor glutathione dynamics in subcellular compartments will be an indispensible step. Therapeutic perspectives should focus on mechanism-based rational drug combinations that are directed against multiple redox targets using effective, specific, and clinically safe inhibitors. This new strategy is expected to produce a synergistic effect, prevent drug resistance, and diminish doses of single drugs. Antioxid. Redox Signal. 27, 1217-1234.
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Affiliation(s)
- Elie Hatem
- 1 CNRS UMR3348, Institut Curie, PSL Research University , Orsay, France .,2 CNRS UMR3348, Université Paris Sud, Université Paris-Saclay , Orsay, France
| | - Nadine El Banna
- 1 CNRS UMR3348, Institut Curie, PSL Research University , Orsay, France .,2 CNRS UMR3348, Université Paris Sud, Université Paris-Saclay , Orsay, France
| | - Meng-Er Huang
- 1 CNRS UMR3348, Institut Curie, PSL Research University , Orsay, France .,2 CNRS UMR3348, Université Paris Sud, Université Paris-Saclay , Orsay, France
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15
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Terzyan SS, Cook PF, Heroux A, Hanigan MH. Structure of 6-diazo-5-oxo-norleucine-bound human gamma-glutamyl transpeptidase 1, a novel mechanism of inactivation. Protein Sci 2017; 26:1196-1205. [PMID: 28378915 PMCID: PMC5441403 DOI: 10.1002/pro.3172] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2017] [Revised: 03/29/2017] [Accepted: 03/30/2017] [Indexed: 01/26/2023]
Abstract
Intense efforts are underway to identify inhibitors of the enzyme gamma-glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma-glutamyl compounds and contributes to the pathology of asthma, reperfusion injury and cancer. The glutamate analog, 6-diazo-5-oxo-norleucine (DON), inhibits GGT1. DON also inhibits many essential glutamine metabolizing enzymes rendering it too toxic for use in the clinic as a GGT1 inhibitor. We investigated the molecular mechanism of human GGT1 (hGGT1) inhibition by DON to determine possible strategies for increasing its specificity for hGGT1. DON is an irreversible inhibitor of hGGT1. The second order rate constant of inactivation was 0.052 mM-1 min-1 and the Ki was 2.7 ± 0.7 mM. The crystal structure of DON-inactivated hGGT1 contained a molecule of DON without the diazo-nitrogen atoms in the active site. The overall structure of the hGGT1-DON complex resembled the structure of the apo-enzyme; however, shifts were detected in the loop forming the oxyanion hole and elements of the main chain that form the entrance to the active site. The structure of hGGT1-DON complex revealed two covalent bonds between the enzyme and inhibitor which were part of a six membered ring. The ring included the OG atom of Thr381, the reactive nucleophile of hGGT1 and the α-amine of Thr381. The structure of DON-bound hGGT1 has led to the discovery of a new mechanism of inactivation by DON that differs from its inactivation of other glutamine metabolizing enzymes, and insight into the activation of the catalytic nucleophile that initiates the hGGT1 reaction.
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Affiliation(s)
- Simon S. Terzyan
- Laboratory of Biomolecular Structure and FunctionUniversity of Oklahoma Health Sciences CenterOklahoma CityOklahoma73104
| | - Paul F. Cook
- Department of Chemistry and BiochemistryUniversity of OklahomaNormanOklahoma73019
| | - Annie Heroux
- Energy Sciences Directorate/Photon Science DivisionBrookhaven National LaboratoryUptonNew York11973
| | - Marie H. Hanigan
- Department of Cell BiologyUniversity of Oklahoma Health Sciences CenterOklahoma CityOklahoma73104
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16
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Kayıhan DS, Kayıhan C, Çiftçi YÖ. Excess boron responsive regulations of antioxidative mechanism at physio-biochemical and molecular levels in Arabidopsis thaliana. PLANT PHYSIOLOGY AND BIOCHEMISTRY : PPB 2016; 109:337-345. [PMID: 27794275 DOI: 10.1016/j.plaphy.2016.10.016] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/06/2016] [Revised: 10/10/2016] [Accepted: 10/16/2016] [Indexed: 05/07/2023]
Abstract
This work was aimed to evaluate the effect of boron (B) toxicity on oxidative damage level, non-enzymatic antioxidant accumulation such as anthocyanin, flavonoid and proline and expression levels of antioxidant enzymes including superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) and their respective activities as well as expression levels of miR398 and miR408 in Arabidopsis thaliana. Plants were germinated and grown on MS medium containing 1 mM B (1B) and 3 mM B (3B) for 14 d. Toxic B led to a decrease of photosynthetic pigments and an increase in accumulation of total soluble and insoluble sugars in accordance with phenotypically viewed chlorosis of seedlings through increasing level of B concentration. Along with these inhibitions, a corresponding increase in contents of flavonoid, anthocyanin and proline occurred that provoked oxidative stress tolerance. 3B caused a remarkable increase in total SOD activity whereas the activities of APX, GR and CAT remained unchanged as verified by expected increase in H2O2 content. In contrast to GR, the coincidence was found between the expressions of SOD and APX genes and their respective activities. 1B induced mir398 expression, whereas 3B did not cause any significant change in expression of mir408 and mir398. Expression levels of GR genes were coordinately regulated with DHAR2 expression. Moreover, the changes in expression level of MDAR2 was in accordance with changes in APX6 expression and total APX activity, indicating fine-tuned regulation of ascorbate-glutathione cycle which might trigger antioxidative responses against B toxicity in Arabidopsis thaliana.
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Affiliation(s)
- Doğa Selin Kayıhan
- Department of Molecular Biology and Genetics, Gebze Technical University, Kocaeli, Turkey
| | - Ceyhun Kayıhan
- Department of Molecular Biology and Genetics, Gebze Technical University, Kocaeli, Turkey
| | - Yelda Özden Çiftçi
- Department of Molecular Biology and Genetics, Gebze Technical University, Kocaeli, Turkey.
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17
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Lukic A, Ji J, Idborg H, Samuelsson B, Palmberg L, Gabrielsson S, Rådmark O. Pulmonary epithelial cancer cells and their exosomes metabolize myeloid cell-derived leukotriene C4 to leukotriene D4. J Lipid Res 2016; 57:1659-69. [PMID: 27436590 DOI: 10.1194/jlr.m066910] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2016] [Indexed: 01/03/2023] Open
Abstract
Leukotrienes (LTs) play major roles in lung immune responses, and LTD4 is the most potent agonist for cysteinyl LT1, leading to bronchoconstriction and tissue remodeling. Here, we studied LT crosstalk between myeloid cells and pulmonary epithelial cells. Monocytic cells (Mono Mac 6 cell line, primary dendritic cells) and eosinophils produced primarily LTC4 In coincubations of these myeloid cells and epithelial cells, LTD4 became a prominent product. LTC4 released from the myeloid cells was further transformed by the epithelial cells in a transcellular manner. Formation of LTD4 was rapid when catalyzed by γ-glutamyl transpeptidase (GGT)1 in the A549 epithelial lung cancer cell line, but considerably slower when catalyzed by GGT5 in primary bronchial epithelial cells. When A549 cells were cultured in the presence of IL-1β, GGT1 expression increased about 2-fold. Also exosomes from A549 cells contained GGT1 and augmented LTD4 formation. Serine-borate complex (SBC), an inhibitor of GGT, inhibited conversion of LTC4 to LTD4 Unexpectedly, SBC also upregulated translocation of 5-lipoxygenase (LO) to the nucleus in Mono Mac 6 cells, and 5-LO activity. Our results demonstrate an active role for epithelial cells in biosynthesis of LTD4, which may be of particular relevance in the lung.
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Affiliation(s)
- Ana Lukic
- Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, 171 77 Stockholm, Sweden
| | - Jie Ji
- Lung and Airway Research, Institute of Environmental Medicine, Karolinska Institutet, 171 77 Stockholm, Sweden
| | - Helena Idborg
- Rheumatology Unit, Department of Medicine, Karolinska Institutet, 171 77 Stockholm, Sweden
| | - Bengt Samuelsson
- Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, 171 77 Stockholm, Sweden
| | - Lena Palmberg
- Lung and Airway Research, Institute of Environmental Medicine, Karolinska Institutet, 171 77 Stockholm, Sweden
| | - Susanne Gabrielsson
- Department of Medicine Solna, Unit for Immunology and Allergy, Karolinska Institutet, 171 77 Stockholm, Sweden
| | - Olof Rådmark
- Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, 171 77 Stockholm, Sweden
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18
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Li L, Shi W, Wu X, Gong Q, Li X, Ma H. Monitoring γ-glutamyl transpeptidase activity and evaluating its inhibitors by a water-soluble near-infrared fluorescent probe. Biosens Bioelectron 2016; 81:395-400. [DOI: 10.1016/j.bios.2016.03.021] [Citation(s) in RCA: 79] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2016] [Revised: 03/01/2016] [Accepted: 03/11/2016] [Indexed: 11/28/2022]
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Yardimci T, Yaman A, Ulutin O. Characterization of Platelet Gamma Glutamyltransferase and Its Alteration in Cases of Atherosclerosis. Clin Appl Thromb Hemost 2016. [DOI: 10.1177/107602969500100202] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Among the various functional and biochemical alterations in the platelets of cases of atherosclerosis, the membrane alterations occupy an important place. The platelet intrinsic membrane protein gamma glutamyltransferase (GGT), which is involved in glutathione metabolism, has shown decreased activity in cases of atherosclerosis. To add new insights into the pathogenesis of atherosclerosis, GGT is characterized and correlated with other alterations. Triton X-100 solubilized membrane fractions of frozen and thawed platelets of atherosclerotic and normal subjects had 18.66 ± 2.86 mU/109 platelets and 35.67 ± 3.01 mU/109 platelets, respectively. The Km values were the same, 2.08 mmol/L for gamma glutamyl- p-nitroanilide and 5.87 mmol/L for glycylglycine. The Vmax values were reduced from 100 mU/109 platelets to 41.66 mU/109 platelets for gamma glutamyl- p-nitroanilide and from 45.45 mU/109 platelets to 38.46 mU/109 platelets for glycylglycine. Optimum pH of GGT activity was 8.2, and optimum temperature was 37°C. It had thermal stability with a 64% relative activity at 36°C for 30 min. Serine against borate was detected as the competitive inhibitor and bromcresol green as the noncompetitive inhibitor. In vivo administration of the antithrombotic drug defibrotide increased the platelet GGT levels to those of normals, from 14.72 ± 7.27 mU/109 platelets to 31.80 ± 12.21 mU/109 platelets in 2 hs. Cholesterol, high-density lipoprotein cholesterol in the membrane fractions, and platelet glutathione levels were unaltered. The lipid per-oxidation (membrane malondialdehyde) level was increased, and glucose and histidine active transport systems were impaired in atherosclerotics. All of these changes are discussed in relation to GGT. Key Words: Human platelets—Atherosclerosis—Gamma glutamyltransferase—Gamma glutamyl transpeptidase— Defibrotide—Biological membranes.
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Affiliation(s)
- Turay Yardimci
- Department of Biochemistry, Faculty of Pharmacy, Marmara University
| | - Azize Yaman
- Department of Biochemistry, Faculty of Pharmacy, Marmara University
| | - Orhan Ulutin
- Hemostasis and Thrombosis Research Center, Cerrahpala Medical School of Istanbul, Istanbul, Turkey
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20
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Borate-fructose complex: A novel mediator for laccase and its new function for fructose determination. FEBS Lett 2015; 589:3107-12. [DOI: 10.1016/j.febslet.2015.08.032] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2015] [Revised: 08/14/2015] [Accepted: 08/17/2015] [Indexed: 11/19/2022]
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21
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Ling SSM, Khoo LHB, Hwang LA, Yeoh KG, Ho B. Instrumental Role of Helicobacter pylori γ-Glutamyl Transpeptidase in VacA-Dependent Vacuolation in Gastric Epithelial Cells. PLoS One 2015; 10:e0131460. [PMID: 26111186 PMCID: PMC4482420 DOI: 10.1371/journal.pone.0131460] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2015] [Accepted: 06/02/2015] [Indexed: 01/27/2023] Open
Abstract
Helicobacter pylori causes cellular vacuolation in host cells, a cytotoxic event attributed to vacuolating cytotoxin (VacA) and the presence of permeant weak bases such as ammonia. We report here the role of γ-glutamyl transpeptidase (GGT), a constitutively expressed secretory enzyme of H. pylori, in potentiating VacA-dependent vacuolation formation in H. pylori-infected AGS and primary gastric cells. The enhancement is brought about by GGT hydrolysing glutamine present in the extracellular medium, thereby releasing ammonia which accentuates the VacA-induced vacuolation. The events of vacuolation in H. pylori wild type (WT)- and Δggt-infected AGS cells were first captured and visualized by real-time phase-contrast microscopy where WT was observed to induce more vacuoles than Δggt. By using semi-quantitative neutral red uptake assay, we next showed that Δggt induced significantly less vacuolation in AGS and primary gastric epithelial cells as compared to the parental strain (P<0.05) indicating that GGT potentiates the vacuolating effect of VacA. Notably, vacuolation induced by WT was significantly reduced in the absence of GGT substrate, glutamine (P<0.05) or in the presence of a competitive GGT inhibitor, serine-borate complex. Furthermore, the vacuolating ability of Δggt was markedly restored when co-incubated with purified recombinant GGT (rGGT), although rGGT itself did not induce vacuolation independently. Similarly, the addition of exogenous ammonium chloride as a source of ammonia also rescued the ability of Δggt to induce vacuolation. Additionally, we also show that monoclonal antibodies against GGT effectively inhibited GGT activity and successfully suppressed H. pylori-induced vacuolation. Collectively, our results clearly demonstrate that generation of ammonia by GGT through glutamine hydrolysis is responsible for enhancing VacA-dependent vacuolation. Our findings provide a new perspective on GGT as an important virulence factor and a promising target in the management of H. pylori-associated gastric diseases.
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Affiliation(s)
- Samantha Shi Min Ling
- Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
| | | | - Le-Ann Hwang
- Monoclonal Antibody Unit, Institute of Molecular and Cell Biology, Singapore, Singapore
| | - Khay Guan Yeoh
- Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
| | - Bow Ho
- Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
- * E-mail:
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22
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Terzyan SS, Burgett AWG, Heroux A, Smith CA, Mooers BHM, Hanigan MH. Human γ-Glutamyl Transpeptidase 1: STRUCTURES OF THE FREE ENZYME, INHIBITOR-BOUND TETRAHEDRAL TRANSITION STATES, AND GLUTAMATE-BOUND ENZYME REVEAL NOVEL MOVEMENT WITHIN THE ACTIVE SITE DURING CATALYSIS. J Biol Chem 2015; 290:17576-86. [PMID: 26013825 DOI: 10.1074/jbc.m115.659680] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2015] [Indexed: 12/31/2022] Open
Abstract
γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within the active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzyme's tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. These data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use.
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Affiliation(s)
- Simon S Terzyan
- From the Macromolecular Crystallography Laboratory, Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104
| | - Anthony W G Burgett
- the Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019
| | - Annie Heroux
- the Energy Sciences Directorate/Photon Science Division, Brookhaven National Laboratory, Upton, New York 11973
| | - Clyde A Smith
- the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California 94025
| | - Blaine H M Mooers
- the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, and
| | - Marie H Hanigan
- the Department of Cell Biology, University of Oklahoma Health Sciences Center, Stanton L. Young Biomedical Research Center, Oklahoma City, Oklahoma 73104
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23
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Synaptic NMDA receptor activity is coupled to the transcriptional control of the glutathione system. Nat Commun 2015; 6:6761. [PMID: 25854456 PMCID: PMC4403319 DOI: 10.1038/ncomms7761] [Citation(s) in RCA: 113] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2014] [Accepted: 02/25/2015] [Indexed: 02/07/2023] Open
Abstract
How the brain’s antioxidant defenses adapt to changing demand is incompletely understood. Here we show that synaptic activity is coupled, via the NMDA receptor (NMDAR), to control of the glutathione antioxidant system. This tunes antioxidant capacity to reflect the elevated needs of an active neuron, guards against future increased demand and maintains redox balance in the brain. This control is mediated via a programme of gene expression changes that boosts the synthesis, recycling and utilization of glutathione, facilitating ROS detoxification and preventing Puma-dependent neuronal apoptosis. Of particular importance to the developing brain is the direct NMDAR-dependent transcriptional control of glutathione biosynthesis, disruption of which can lead to degeneration. Notably, these activity-dependent cell-autonomous mechanisms were found to cooperate with non-cell-autonomous Nrf2-driven support from astrocytes to maintain neuronal GSH levels in the face of oxidative insults. Thus, developmental NMDAR hypofunction and glutathione system deficits, separately implicated in several neurodevelopmental disorders, are mechanistically linked. How the brain’s antioxidant defenses adapt to changing demand is not well understood. Here the authors demonstrate that synaptic activity is coupled to transcriptional control of the glutathione antioxidant system via NMDA receptors, enabling neurons to tune their antioxidant defenses.
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24
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Abstract
The expression of gamma-glutamyl transpeptidase (GGT) is essential to maintaining cysteine levels in the body. GGT is a cell surface enzyme that hydrolyzes the gamma-glutamyl bond of extracellular reduced and oxidized glutathione, initiating their cleavage into glutamate, cysteine (cystine), and glycine. GGT is normally expressed on the apical surface of ducts and glands, salvaging the amino acids from glutathione in the ductal fluids. GGT in tumors is expressed over the entire cell membrane and provides tumors with access to additional cysteine and cystine from reduced and oxidized glutathione in the blood and interstitial fluid. Cysteine is rate-limiting for glutathione synthesis in cells under oxidative stress. The induction of GGT is observed in tumors with elevated levels of intracellular glutathione. Studies in models of hepatocarcinogenesis show that GGT expression in foci of preneoplastic hepatocytes provides a selective advantage to the cells during tumor promotion with agents that deplete intracellular glutathione. Similarly, expression of GGT in tumors enables cells to maintain elevated levels of intracellular glutathione and to rapidly replenish glutathione during treatment with prooxidant anticancer therapy. In the clinic, the expression of GGT in tumors is correlated with drug resistance. The inhibitors of GGT block GGT-positive tumors from accessing the cysteine in extracellular glutathione. They also inhibit GGT activity in the kidney, which results in the excretion of GSH in the urine and a rapid decrease in blood cysteine levels, leading to depletion of intracellular GSH in both GGT-positive and GGT-negative tumors. GGT inhibitors are being developed for clinical use to sensitize tumors to chemotherapy.
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Affiliation(s)
- Marie H Hanigan
- Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
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25
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Landi M, Guidi L, Pardossi A, Tattini M, Gould KS. Photoprotection by foliar anthocyanins mitigates effects of boron toxicity in sweet basil (Ocimum basilicum). PLANTA 2014; 240:941-53. [PMID: 24903358 DOI: 10.1007/s00425-014-2087-1] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/02/2013] [Accepted: 04/22/2014] [Indexed: 05/08/2023]
Abstract
Boron (B) toxicity is an important agricultural problem in arid environments. Excess edaphic B compromises photosynthetic efficiency, limits growth and reduces crop yield. However, some purple-leafed cultivars of sweet basil (Ocimum basilicum) exhibit greater tolerance to high B concentrations than do green-leafed cultivars. We hypothesised that foliar anthocyanins protect basil leaf mesophyll from photo-oxidative stress when chloroplast function is compromised by B toxicity. Purple-leafed 'Red Rubin' and green-leafed 'Tigullio' cultivars, grown with high or negligible edaphic B, were given a photoinhibitory light treatment. Possible effects of photoabatement by anthocyanins were simulated by superimposing a purple polycarbonate filter on the green leaves. An ameliorative effect of light filtering on photosynthetic quantum yield and on photo-oxidative load was observed in B-stressed plants. In addition, when green protoplasts from both cultivars were treated with B and illuminated through a screen of anthocyanic protoplasts or a polycarbonate film which approximated cyanidin-3-O-glucoside optical properties, the degree of photoinhibition, hydrogen peroxide production, and malondialdehyde content were reduced. The data provide evidence that anthocyanins exert a photoprotective role in purple-leafed basil mesophyll cells, thereby contributing to improved tolerance to high B concentrations.
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Affiliation(s)
- Marco Landi
- Department of Agriculture, Food and Environment, Via del Borghetto 80, 56124, Pisa, Italy
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26
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Balakrishna S, Prabhune AA. Gamma-glutamyl transferases: A structural, mechanistic and physiological perspective. ACTA ACUST UNITED AC 2014. [DOI: 10.1007/s11515-014-1288-0] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
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27
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Morelli CF, Calvio C, Biagiotti M, Speranza G. pH-Dependent hydrolase, glutaminase, transpeptidase and autotranspeptidase activities ofBacillus subtilisγ-glutamyltransferase. FEBS J 2013; 281:232-45. [DOI: 10.1111/febs.12591] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2013] [Revised: 10/25/2013] [Accepted: 10/28/2013] [Indexed: 01/01/2023]
Affiliation(s)
- Carlo F. Morelli
- Department of Chemistry; University of Milan; Italy
- Italian Biocatalysis Center (IBC); c/o Dipartimento di Scienza del Farmaco; University of Pavia; Italy
| | - Cinzia Calvio
- Department of Biology and Biotechnology; University of Pavia; Italy
| | | | - Giovanna Speranza
- Department of Chemistry; University of Milan; Italy
- Italian Biocatalysis Center (IBC); c/o Dipartimento di Scienza del Farmaco; University of Pavia; Italy
- Istituto di Scienze e Tecnologie Molecolari (INSTM); CNR; c/o Department of Chemistry; University of Milan; Italy
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28
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Tolin S, Arrigoni G, Trentin AR, Veljovic-Jovanovic S, Pivato M, Zechman B, Masi A. Biochemical and quantitative proteomics investigations in Arabidopsisggt1mutant leaves reveal a role for the gamma-glutamyl cycle in plant's adaptation to environment. Proteomics 2013; 13:2031-45. [DOI: 10.1002/pmic.201200479] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2012] [Revised: 04/13/2013] [Accepted: 04/24/2013] [Indexed: 11/07/2022]
Affiliation(s)
- Serena Tolin
- DAFNAE, University of Padova; Legnaro Italy
- Proteomics Center of Padova University; VIMM, Padova University Hospital; Padova Italy
| | - Giorgio Arrigoni
- Proteomics Center of Padova University; VIMM, Padova University Hospital; Padova Italy
- Department of Biomedical Sciences; University of Padova; Padova Italy
| | | | | | | | - Bernd Zechman
- Karl-Franzens-University of Graz; Institute of Plant Sciences; Graz Austria
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29
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Role of glutathione in cancer progression and chemoresistance. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2013; 2013:972913. [PMID: 23766865 PMCID: PMC3673338 DOI: 10.1155/2013/972913] [Citation(s) in RCA: 802] [Impact Index Per Article: 66.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/22/2013] [Revised: 04/26/2013] [Accepted: 04/29/2013] [Indexed: 01/19/2023]
Abstract
Glutathione (GSH) plays an important role in a multitude of cellular processes, including cell differentiation, proliferation, and apoptosis, and disturbances in GSH homeostasis are involved in the etiology and progression of many human diseases including cancer. While GSH deficiency, or a decrease in the GSH/glutathione disulphide (GSSG) ratio, leads to an increased susceptibility to oxidative stress implicated in the progression of cancer, elevated GSH levels increase the antioxidant capacity and the resistance to oxidative stress as observed in many cancer cells. The present review highlights the role of GSH and related cytoprotective effects in the susceptibility to carcinogenesis and in the sensitivity of tumors to the cytotoxic effects of anticancer agents.
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30
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Inhibition of human γ-glutamyl transpeptidase: development of more potent, physiologically relevant, uncompetitive inhibitors. Biochem J 2013; 450:547-57. [PMID: 23301618 DOI: 10.1042/bj20121435] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
GGT (γ-glutamyl transpeptidase) is an essential enzyme for maintaining cysteine homoeostasis, leukotriene synthesis, metabolism of glutathione conjugates and catabolism of extracellular glutathione. Overexpression of GGT has been implicated in many pathologies, and clinical inhibitors of GGT are under development for use in the treatment of asthma, cancer and other diseases. Inhibitors are generally characterized using synthetic GGT substrates. The present study of uncompetitive inhibitors of GGT, has revealed that the potency with which compounds inhibit GGT activity in the standard biochemical assay does not correlate with the potency with which they inhibit the physiological reaction catalysed by GGT. Kinetic studies provided insight into the mechanism of inhibition. Modifications to the sulfobenzene or distal benzene ring of the uncompetitive inhibitor OU749 affected activity. One of the most potent inhibitors was identified among a novel group of analogues with an amine group para on the benzosulfonamide ring. New more potent uncompetitive inhibitors of the physiological GGT reaction were found to be less toxic than the glutamine analogues that have been tested clinically. Development of non-toxic inhibitors is essential for exploiting GGT as a therapeutic target.
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31
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Castellano I, Merlino A. Gamma-Glutamyl Transpeptidases: Structure and Function. GAMMA-GLUTAMYL TRANSPEPTIDASES 2013. [DOI: 10.1007/978-3-0348-0682-4_1] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
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A fluorometric method to quantify protein glutathionylation using glutathione derivatization with 2,3-naphthalenedicarboxaldehyde. Anal Biochem 2012; 433:132-6. [PMID: 23072983 DOI: 10.1016/j.ab.2012.10.009] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2012] [Revised: 10/03/2012] [Accepted: 10/05/2012] [Indexed: 01/13/2023]
Abstract
This study reports the development of a new assay for the rapid determination of protein glutathionylation in tissues and cell lines using commercially available reagents and standard instrumentation. In this method cells are homogenized in the presence of N-ethylmaleimide to eliminate free thiols and the proteins are precipitated with acetone. Subsequently, the disulfide-bound glutathione is eluted from the protein by the addition of tris(2-carboxyethyl)phosphine and reacted with 2,3-napthalenedicarboxaldehyde to generate a highly fluorescent product. Lymphoblastoid cell lines were found to have glutathionylation levels in the range of 0.3-3 nmol/mg protein, which were significantly elevated after treatment of the cells with S-nitrosoglutathione. Mouse tissues including liver, kidney, lung, heart, brain, spleen, and testes were found to have glutathionylation levels between 1 and 2.5 nmol/mg protein and the levels tended to increase after treatment of mice with doxorubicin. In contrast, mouse skeletal muscle glutathionylation was significantly higher (4.2 ± 0.33 nmol/mg, p < 0.001) than in other tissues in untreated mice and decreased to 1.9 ± 0.15 nmol/mg after doxorubicin treatment. This new method allows rapid measurement of cellular glutathionylation in a high-throughput 96-well plate format.
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Dahboul F, Leroy P, Maguin Gate K, Boudier A, Gaucher C, Liminana P, Lartaud I, Pompella A, Perrin-Sarrado C. Endothelial γ-glutamyltransferase contributes to the vasorelaxant effect of S-nitrosoglutathione in rat aorta. PLoS One 2012; 7:e43190. [PMID: 22984412 PMCID: PMC3439434 DOI: 10.1371/journal.pone.0043190] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2012] [Accepted: 07/20/2012] [Indexed: 11/28/2022] Open
Abstract
S-nitrosoglutathione (GSNO) involved in storage and transport of nitric oxide (•NO) plays an important role in vascular homeostasis. Breakdown of GSNO can be catalyzed by γ-glutamyltransferase (GGT). We investigated whether vascular GGT influences the vasorelaxant effect of GSNO in isolated rat aorta. Histochemical localization of GGT and measurement of its activity were performed by using chromogenic substrates in sections and in aorta homogenates, respectively. The role of GGT in GSNO metabolism was evaluated by measuring GSNO consumption rate (absorbance decay at 334 nm), •NO release was visualized and quantified with the fluorescent probe 4,5-diaminofluorescein diacetate. The vasorelaxant effect of GSNO was assayed using isolated rat aortic rings (in the presence or absence of endothelium). The role of GGT was assessed by stimulating enzyme activity with cosubstrate glycylglycine, as well as using two independent inhibitors, competitive serine borate complex and non-competitive acivicin. Specific GGT activity was histochemically localized in the endothelium. Consumption of GSNO and release of free •NO decreased and increased in presence of serine borate complex and glycylglycine, respectively. In vasorelaxation experiments with endothelium-intact aorta, the half maximal effective concentration of GSNO (EC50 = 3.2±0.5.10−7 M) increased in the presence of the two distinct GGT inhibitors, serine borate complex (1.6±0.2.10−6 M) and acivicin (8.3±0.6.10−7 M), while it decreased with glycylglycine (4.7±0.9.10−8 M). In endothelium-denuded aorta, EC50 for GSNO alone increased to 2.3±0.3.10−6 M, with no change in the presence of serine borate complex. These data demonstrate the important role of endothelial GGT activity in mediating the vasorelaxant effect of GSNO in rat aorta under physiological conditions. Because therapeutic treatments based on GSNO are presently under development, this endothelium-dependent mechanism involved in the vascular effects of GSNO should be taken into account in a pharmacological perspective.
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Affiliation(s)
- Fatima Dahboul
- EA3452 CITHEFOR “Drug targets, formulation and preclinical assessment”, Faculté de Pharmacie, Université de Lorraine, Nancy, France
| | - Pierre Leroy
- EA3452 CITHEFOR “Drug targets, formulation and preclinical assessment”, Faculté de Pharmacie, Université de Lorraine, Nancy, France
| | - Katy Maguin Gate
- EA3452 CITHEFOR “Drug targets, formulation and preclinical assessment”, Faculté de Pharmacie, Université de Lorraine, Nancy, France
| | - Ariane Boudier
- EA3452 CITHEFOR “Drug targets, formulation and preclinical assessment”, Faculté de Pharmacie, Université de Lorraine, Nancy, France
| | - Caroline Gaucher
- EA3452 CITHEFOR “Drug targets, formulation and preclinical assessment”, Faculté de Pharmacie, Université de Lorraine, Nancy, France
| | - Patrick Liminana
- EA3452 CITHEFOR “Drug targets, formulation and preclinical assessment”, Faculté de Pharmacie, Université de Lorraine, Nancy, France
| | - Isabelle Lartaud
- EA3452 CITHEFOR “Drug targets, formulation and preclinical assessment”, Faculté de Pharmacie, Université de Lorraine, Nancy, France
| | - Alfonso Pompella
- EA3452 CITHEFOR “Drug targets, formulation and preclinical assessment”, Faculté de Pharmacie, Université de Lorraine, Nancy, France
| | - Caroline Perrin-Sarrado
- EA3452 CITHEFOR “Drug targets, formulation and preclinical assessment”, Faculté de Pharmacie, Université de Lorraine, Nancy, France
- * E-mail:
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Smoum R, Rubinstein A, Dembitsky VM, Srebnik M. Boron containing compounds as protease inhibitors. Chem Rev 2012; 112:4156-220. [PMID: 22519511 DOI: 10.1021/cr608202m] [Citation(s) in RCA: 320] [Impact Index Per Article: 24.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Affiliation(s)
- Reem Smoum
- The School of Pharmacy, Institute for Drug Research, The Hebrew University of Jerusalem, Faculty of Medicine, Jerusalem, Israel.
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Scorei RI, Rotaru P. Calcium fructoborate--potential anti-inflammatory agent. Biol Trace Elem Res 2011; 143:1223-38. [PMID: 21274653 DOI: 10.1007/s12011-011-8972-6] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/06/2010] [Accepted: 01/13/2011] [Indexed: 12/28/2022]
Abstract
Calcium fructoborate is a boron-based nutritional supplement. Its chemical structure is similar to one of the natural forms of boron such as bis-manitol, bis-sorbitol, bis-fructose, and bis-sucrose borate complexes found in edible plants. In vitro studies revealed that calcium fructoborate is a superoxide ion scavenger and anti-inflammatory agent. It may influence macrophage production of inflammatory mediators, can be beneficial for the suppression of cytokine production, and inhibits progression of endotoxin-associated diseases, as well as the boric acid and other boron sources. The mechanisms by which calcium fructoborate exerts its beneficial anti-inflammatory effects are not entirely clear, but some of its molecular biological in vitro activities are understood: inhibition of the superoxide within the cell; inhibition of the interleukin-1β, interleukin-6, and nitric oxide release in the culture media; and increase of the tumor necrosis factor-α production. Also, calcium fructoborate has no effects on lipopolysaccharide-induced cyclooxygenase-2 protein express. The studies on animals and humans with a dose range of 1-7 mg calcium fructoborate (0.025-0.175 mg elemental boron)/kg body weight/day exhibited a good anti-inflammatory activity, and it also seemed to have negligible adverse effect on humans.
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Affiliation(s)
- Romulus Ion Scorei
- Department of Biochemistry, University of Craiova, A.I. Cuza Str., Nr. 13, Craiova, Romania.
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Wickham S, Regan N, West MB, Kumar VP, Thai J, Li PK, Cook PF, Hanigan MH. Divergent effects of compounds on the hydrolysis and transpeptidation reactions of γ-glutamyl transpeptidase. J Enzyme Inhib Med Chem 2011; 27:476-89. [PMID: 21864033 DOI: 10.3109/14756366.2011.597748] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
A novel class of inhibitors of the enzyme γ-glutamyl transpeptidase (GGT) were evaluated. The analog OU749 was shown previously to be an uncompetitive inhibitor of the GGT transpeptidation reaction. The data in this study show that it is an equally potent uncompetitive inhibitor of the hydrolysis reaction, the primary reaction catalyzed by GGT in vivo. A series of structural analogs of OU749 were evaluated. For many of the analogs, the potency of the inhibition differed between the hydrolysis and transpeptidation reactions, providing insight into the malleability of the active site of the enzyme. Analogs with electron withdrawing groups on the benzosulfonamide ring, accelerated the hydrolysis reaction, but inhibited the transpeptidation reaction by competing with a dipeptide acceptor. Several of the OU749 analogs inhibited the transpeptidation reaction by slow onset kinetics, similar to acivicin. Further development of inhibitors of the GGT hydrolysis reaction is necessary to provide new therapeutic compounds.
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Affiliation(s)
- Stephanie Wickham
- Department of Cell Biology, University of Oklahoma Health Sciences Centre, Oklahoma City, OK 73104, USA
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Joyce-Brady M, Hiratake J. Inhibiting Glutathione Metabolism in Lung Lining Fluid as a Strategy to Augment Antioxidant Defense. ACTA ACUST UNITED AC 2011; 7:71-78. [PMID: 22485086 PMCID: PMC3319921 DOI: 10.2174/157340811796575308] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2011] [Revised: 03/25/2011] [Accepted: 04/08/2011] [Indexed: 12/22/2022]
Abstract
Glutathione is abundant in the lining fluid that bathes the gas exchange surface of the lung. On the one hand glutathione in this extracellular pool functions in antioxidant defense to protect cells and proteins in the alveolar space from oxidant injury; on the other hand, it functions as a source of cysteine to maintain cellular glutathione and protein synthesis. These seemingly opposing functions are regulated through metabolism by gamma-glutamyl transferase (GGT, EC 2.3.2.2). Even under normal physiologic conditions, lung lining fluid (LLF) contains a concentrated pool of GGT activity exceeding that of whole lung by about 7-fold and indicating increased turnover of glutathione at the epithelial surface of the lung. With oxidant stress LLF GGT activity is amplified even further as glutathione turnover is accelerated to meet the increased demands of cells for cysteine. Mouse models of GGT deficiency confirmed this biological role of LLF GGT activity and revealed the robust expansiveness and antioxidant capacity of the LLF glutathione pool in the absence of metabolism. Acivicin, an irreversible inhibitor of GGT, can be utilized to augment LLF fluid glutathione content in normal mice and novel GGT inhibitors have now been defined that provide advantages over acivicin. Inhibiting LLF GGT activity is a novel strategy to selectively augment the extracellular LLF glutathione pool. The enhanced antioxidant capacity can maintain lung epithelial cell integrity and barrier function under oxidant stress.
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Affiliation(s)
- Martin Joyce-Brady
- The Pulmonary Center, Boston University School of Medicine, Boston, MA 02118, USA
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Gamma-glutamyl compounds: substrate specificity of gamma-glutamyl transpeptidase enzymes. Anal Biochem 2011; 414:208-14. [PMID: 21447318 DOI: 10.1016/j.ab.2011.03.026] [Citation(s) in RCA: 100] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2011] [Revised: 03/19/2011] [Accepted: 03/23/2011] [Indexed: 11/23/2022]
Abstract
Gamma-glutamyl compounds include antioxidants, inflammatory molecules, drug metabolites, and neuroactive compounds. Two cell surface enzymes that metabolize gamma-glutamyl compounds have been identified: gamma-glutamyl transpeptidase (GGT1) and gamma-glutamyl leukotrienase (GGT5). There is controversy in the literature regarding the substrate specificity of these enzymes. To address this issue, we have developed a method for comprehensive kinetic analysis of compounds as substrates for GGT enzymes. Our assay is sensitive, quantitative, and conducted at physiological pH. We evaluated a series of gamma-glutamyl compounds as substrates for human GGT1 and human GGT5. The K(m) value for reduced glutathione was 11μM for both GGT1 and GGT5. However, the K(m) values for oxidized glutathione were 9μM for GGT1 and 43μM for GGT5. Our data show that the K(m) values for leukotriene C(4) are equivalent for GGT1 and GGT5 at 10.8 and 10.2μM, respectively. This assay was also used to evaluate serine-borate, a well-known inhibitor of GGT1, which was 8-fold more potent in inhibiting GGT1 than in inhibiting GGT5. These data provide essential information regarding the target enzymes for developing treatments for inflammatory diseases such as asthma and cardiovascular disease in humans. This assay is invaluable for studies of oxidative stress, drug metabolism, and other pathways that involve gamma-glutamyl compounds.
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Accurate measurement of reduced glutathione in gamma-glutamyltransferase-rich brain microvessel fractions. Brain Res 2011; 1369:95-102. [DOI: 10.1016/j.brainres.2010.10.100] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2010] [Revised: 10/26/2010] [Accepted: 10/27/2010] [Indexed: 11/18/2022]
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Destro T, Prasad D, Martignago D, Lliso Bernet I, Trentin AR, Renu IK, Ferretti M, Masi A. Compensatory expression and substrate inducibility of gamma-glutamyl transferase GGT2 isoform in Arabidopsis thaliana. JOURNAL OF EXPERIMENTAL BOTANY 2011; 62:805-14. [PMID: 20959624 PMCID: PMC3003821 DOI: 10.1093/jxb/erq316] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/01/2010] [Revised: 09/10/2010] [Accepted: 09/20/2010] [Indexed: 05/19/2023]
Abstract
γ-Glutamyl transferases (GGT; EC 2.3.2.2) are glutathione-degrading enzymes that are represented in Arabidopsis thaliana by a small gene family of four members. Two isoforms, GGT1 and GGT2, are apoplastic, sharing broad similarities in their amino acid sequences, but they are differently expressed in the tissues: GGT1 is expressed in roots, leaves, and siliques, while GGT2 was thought to be expressed only in siliques. It is demonstrated here that GGT2 is also expressed in wild-type roots, albeit in very small amounts. GGT2 expression is enhanced in ggt1 knockout mutants, suggesting a compensatory effect to restore GGT activity in the root apoplast. Supplementation with 100 μM glutathione (GSH) resulted in the up-regulation of GGT2 gene expression in wild-type and ggt1 knockout roots, and of GGT1 gene expression in wild-type roots. Glutathione recovery was hampered by the GGT inhibitor serine/borate, suggesting a major role for apoplastic GGTs in this process. These findings can explain the ability of ggt1 knockout mutants to retrieve exogenously added glutathione from the growth medium.
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Affiliation(s)
| | | | | | | | | | | | | | - Antonio Masi
- To whom correspondence should be addressed. E-mail:
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Suenaga H, Nakashima K, Mikami M, Yamamoto H, James TD, Sandanayake KRAS, Shinkai S. Screening of arylboronic acids to search for a strong inhibitor for γ-glutamyl transpeptidase (γ-GTP). ACTA ACUST UNITED AC 2010. [DOI: 10.1002/recl.19961150109] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
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Gong M, Ling SSM, Lui SY, Yeoh KG, Ho B. Helicobacter pylori gamma-glutamyl transpeptidase is a pathogenic factor in the development of peptic ulcer disease. Gastroenterology 2010; 139:564-73. [PMID: 20347814 DOI: 10.1053/j.gastro.2010.03.050] [Citation(s) in RCA: 93] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/18/2008] [Revised: 02/03/2010] [Accepted: 03/11/2010] [Indexed: 12/14/2022]
Abstract
BACKGROUND & AIMS gamma-Glutamyl transpeptidase (GGT) has been reported to be a virulence factor of Helicobacter pylori associated with bacterial colonization and cell apoptosis. But its mechanism of pathogenesis is not firmly established. This study aims to examine its role in H pylori-mediated infection. METHODS Various H pylori isogenic mutants were constructed by a polymerase chain reaction (PCR) approach. H pylori native GGT protein (HP-nGGT) was purified with ion-exchange and gel-filtration chromatography. Generation of H2O2 was measured with fluorimetric analysis, whereas nuclear factor-kappaB (NF-kappaB) activation was determined by luciferase assay and Western blot. Cytokine production was examined by enzyme-linked immunoabsorbent assay and real-time PCR. DNA damage was assessed with comet assay and flow cytometry. The GGT activity of 98 H pylori isolates was analyzed by an enzymatic assay. RESULTS Purified HP-nGGT generated H2O2 in primary gastric epithelial cells and AGS gastric cancer cells, resulting in the activation of NF-kappaB and up-regulation of interleukin-8 (IL-8) production. In addition, HP-nGGT caused an increase in the level of 8-OH-dG, indicative of oxidative DNA damage. In contrast, Deltaggt showed significantly reduced levels of H2O2 generation, IL-8 production, and DNA damage in cells compared with the wild type (P<.05). The clinical importance of GGT was indicated by significantly higher (P<.001) activity in H pylori isolates obtained from patients with peptic ulcer disease (n=54) than isolates from patients with nonulcer dyspepsia (n=44). CONCLUSION Our findings provide evidence that GGT is a pathogenic factor associated with H pylori-induced peptic ulcer disease.
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Affiliation(s)
- Min Gong
- Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
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De Donatis GM, Moschini R, Cappiello M, Del Corso A, Mura U. Cysteinyl-glycine in the control of glutathione homeostasis in bovine lenses. Mol Vis 2010; 16:1025-33. [PMID: 20577593 PMCID: PMC2890556] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2010] [Accepted: 06/01/2010] [Indexed: 11/29/2022] Open
Abstract
PURPOSE To define a possible metabolic and/or signaling role for Cys-Gly in glutathione homeostasis in bovine eye lenses. METHODS Bovine lenses were cultured up to 24 h in a medium containing 0.5 mM reduced glutathione (GSH) under different conditions. The intracellular and the extracellular contents of thiol compounds were evaluated using a free zone capillary electrophoresis method. RESULTS Culture of lenses in the presence of GSH and the gamma-glutamyl transferase inhibitor serine-borate demonstrated a 1.5 fold increase in the level of extra-lenticular glutathione with respect to the initial value. Cys-Gly exogenously added impaired the extra-lenticular accumulation of glutathione. Both cysteine and gamma-Glu-Cys were ineffective in reducing extra-lenticular glutathione accumulation. In all conditions no differences in reduced and total intra-lenticular glutathione levels were observed. CONCLUSIONS The impairment of Cys-Gly generation correlated with inhibition of gamma-glutamyl transferase by serine/borate, resulting in high extra-lenticular concentration of glutathione effluxed from the bovine lens. The possibility that Cys-Gly may intervene either in the replenishment processes for cysteine in the GSH biosynthetic step or in the function of the efflux GSH-transporters is considered.
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Stangoulis J, Tate M, Graham R, Bucknall M, Palmer L, Boughton B, Reid R. The mechanism of boron mobility in wheat and canola phloem. PLANT PHYSIOLOGY 2010; 153:876-81. [PMID: 20413647 PMCID: PMC2879810 DOI: 10.1104/pp.110.155655] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/02/2023]
Abstract
Low-molecular-weight borate complexes were isolated from canola (Brassica napus) and wheat (Triticum aestivum) phloem exudates, as well as the cytoplasm of the fresh-water alga Chara corallina, and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Phloem exudate was collected from field-grown canola inflorescence stalks by shallow incision, while wheat phloem exudate was collected by aphid stylectomy. Chara cytoplasm was collected by careful manual separation of the cell wall, vacuole, and cytosolic compartments. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry showed the presence of isotopic borate complexes, at mass-to-charge ratio of 690.22/691.22 in the canola and wheat phloem and at 300.11/301.11 in canola phloem and Chara cytoplasm. Using reference compounds, the borate complexes with mass-to-charge ratio 690.22/691.22 was identified as a bis-sucrose (Suc) borate complex in which the 4,6-hydroxyl pairs from the two alpha-glucopyranoside moieties formed an [L(2)B](-1) complex. Further investigation using liquid chromatography electrospray ionization triple quadrupole mass spectrometry analysis confirmed the presence of the bis-Suc borate complex in wheat phloem with a concentration up to 220 microm. The 300.11/301.11 complex was putatively identified as a bis-N-acetyl-serine borate complex but its concentration was below the detection limits of the liquid chromatography electrospray ionization triple quadrupole mass spectrometer so could not be quantified. The presence of borate complexes in the phloem provides a mechanistic explanation for the observed phloem boron mobility in canola and wheat and other species that transport Suc as their primary photoassimilate.
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Affiliation(s)
- James Stangoulis
- School of Biological Science, Flinders University, Bedford Park, South Australia 5042, Australia.
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Abstract
Many potentially toxic electrophiles react with glutathione to form glutathione S-conjugates in reactions catalyzed or enhanced by glutathione S-transferases. The glutathione S-conjugate is sequentially converted to the cysteinylglycine-, cysteine- and N-acetyl-cysteine S-conjugate (mercapturate). The mercapturate is generally more polar and water soluble than the parent electrophile and is readily excreted. Excretion of the mercapturate represents a detoxication mechanism. Some endogenous compounds, such as leukotrienes, prostaglandin (PG) A2, 15-deoxy-Δ12,14-PGJ2, and hydroxynonenal can also be metabolized to mercapturates and excreted. On occasion, however, formation of glutathione S- and cysteine S-conjugates are bioactivation events as the metabolites are mutagenic and/or cytotoxic. When the cysteine S-conjugate contains a strong electron-withdrawing group attached at the sulfur, it may be converted by cysteine S-conjugate β-lyases to pyruvate, ammonium and the original electrophile modified to contain an –SH group. If this modified electrophile is highly reactive then the enzymes of the mercapturate pathway together with the cysteine S-conjugate β-lyases constitute a bioactivation pathway. Some endogenous halogenated environmental contaminants and drugs are bioactivated by this mechanism. Recent studies suggest that coupling of enzymes of the mercapturate pathway to cysteine S-conjugate β-lyases may be more common in nature and more widespread in the metabolism of electrophilic xenobiotics than previously realized.
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Gürdöl F, Nwose OM, Mikhailidis DP. Gamma-glutamyl Transferase Activity in Human Platelets: Quantification of Activity, Isoenzyme Characterization and Potential Clinical Relevance. Platelets 2009; 6:200-3. [DOI: 10.3109/09537109509078455] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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King JB, West MB, Cook PF, Hanigan MH. A novel, species-specific class of uncompetitive inhibitors of gamma-glutamyl transpeptidase. J Biol Chem 2009; 284:9059-65. [PMID: 19203993 DOI: 10.1074/jbc.m809608200] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Expression of gamma-glutamyl transpeptidase (GGT) in tumors contributes to resistance to radiation and chemotherapy. GGT is inhibited by glutamine analogues that compete with the substrate for the gamma-glutamyl binding site. However, the glutamine analogues that have been evaluated in clinical trials are too toxic for use in humans. We have used high throughput screening to evaluate small molecules for their ability to inhibit GGT and have identified a novel class of inhibitors that are not glutamine analogues. These compounds are uncompetitive inhibitors, binding the gamma-glutamyl enzyme complex. OU749, the lead compound, has an intrinsic K(i) of 17.6 microm. It is a competitive inhibitor of the acceptor glycyl-glycine, which indicates that OU749 occupies the acceptor site while binding to the gamma-glutamyl substrate complex. OU749 is more than 150-fold less toxic than the GGT inhibitor acivicin toward dividing cells. Inhibition of GGT by OU749 is species-specific, inhibiting GGT isolated from human kidney with 7-10-fold greater potency than GGT isolated from rat or mouse kidney. OU749 does not inhibit GGT from pig cells. Human GGT expressed in mouse fibroblasts is inhibited by OU749 similarly to GGT from human cells, which indicates that the species specificity is determined by differences in the primary structure of the protein rather than species-specific, post-translational modifications. These studies have identified a novel class of inhibitors of GGT, providing the basis for further development of a new group of therapeutics that inhibit GGT by a mechanism distinct from the toxic glutamine analogues.
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Affiliation(s)
- Jarrod B King
- Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA
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Ferretti M, Destro T, Tosatto SCE, La Rocca N, Rascio N, Masi A. Gamma-glutamyl transferase in the cell wall participates in extracellular glutathione salvage from the root apoplast. THE NEW PHYTOLOGIST 2009; 181:115-126. [PMID: 19076720 DOI: 10.1111/j.1469-8137.2008.02653.x] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/03/2023]
Abstract
The molecular properties and subcellular location of bound gamma-glutamyl transferase (GGT) were studied, and an experimental setup devised to assess its functions in barley roots. Enzyme histochemistry was used to detect GGT activity at tissue level; immunocytochemistry to localize the protein at subcellular level; and modelling studies to investigate its surface charge properties. GGT activity in vivo was measured for the first time. Functions were explored by applying chemical treatments with inhibitors and the thiol-oxidizing drug diamide, performing time-course chromatographic and spectrophotometric analyses on low-molecular-weight thiols. Gamma-glutamyl transferase activity was found to be high in the root apical region and the protein was anchored to root cell wall components, probably by basic amino acid residues. The results show that GGT is essential to the recovery of apoplastic glutathione provided exogenously or extruded by oxidative treatment. It is demonstrated that GGT activity helps to salvage extracellular glutathione and may contribute to redox control of the extracellular environment, thus providing evidence of a functional role for gamma-glutamyl cycle in roots.
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Affiliation(s)
- M Ferretti
- Department of Agricultural Biotechnology, University of Padova, Viale dell'Universita' 16, I-35020 Legnaro (PD), Italy;Department of Biology, University of Padova, Via Trieste 75, I-35100 Padova, Italy;CRIBI Biotech Centre, University of Padova, Via Trieste 75, I-35100 Padova, Italy
| | - T Destro
- Department of Agricultural Biotechnology, University of Padova, Viale dell'Universita' 16, I-35020 Legnaro (PD), Italy;Department of Biology, University of Padova, Via Trieste 75, I-35100 Padova, Italy;CRIBI Biotech Centre, University of Padova, Via Trieste 75, I-35100 Padova, Italy
| | - S C E Tosatto
- Department of Agricultural Biotechnology, University of Padova, Viale dell'Universita' 16, I-35020 Legnaro (PD), Italy;Department of Biology, University of Padova, Via Trieste 75, I-35100 Padova, Italy;CRIBI Biotech Centre, University of Padova, Via Trieste 75, I-35100 Padova, Italy
| | - N La Rocca
- Department of Agricultural Biotechnology, University of Padova, Viale dell'Universita' 16, I-35020 Legnaro (PD), Italy;Department of Biology, University of Padova, Via Trieste 75, I-35100 Padova, Italy;CRIBI Biotech Centre, University of Padova, Via Trieste 75, I-35100 Padova, Italy
| | - N Rascio
- Department of Agricultural Biotechnology, University of Padova, Viale dell'Universita' 16, I-35020 Legnaro (PD), Italy;Department of Biology, University of Padova, Via Trieste 75, I-35100 Padova, Italy;CRIBI Biotech Centre, University of Padova, Via Trieste 75, I-35100 Padova, Italy
| | - A Masi
- Department of Agricultural Biotechnology, University of Padova, Viale dell'Universita' 16, I-35020 Legnaro (PD), Italy;Department of Biology, University of Padova, Via Trieste 75, I-35100 Padova, Italy;CRIBI Biotech Centre, University of Padova, Via Trieste 75, I-35100 Padova, Italy
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Zarnowski R, Cooper KG, Brunold LS, Calaycay J, Woods JP. Histoplasma capsulatum secreted gamma-glutamyltransferase reduces iron by generating an efficient ferric reductant. Mol Microbiol 2008; 70:352-68. [PMID: 18761625 DOI: 10.1111/j.1365-2958.2008.06410.x] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The intracellular fungal pathogen Histoplasma capsulatum (Hc) resides in mammalian macrophages and causes respiratory and systemic disease. Iron limitation is an important host antimicrobial defence, and iron acquisition is critical for microbial pathogenesis. Hc displays several iron acquisition mechanisms, including secreted glutathione-dependent ferric reductase activity (GSH-FeR). We purified this enzyme from culture supernatant and identified a novel extracellular iron reduction strategy involving gamma-glutamyltransferase (Ggt1) activity. The 320 kDa complex was composed of glycosylated protein subunits of about 50 and 37 kDa. The purified enzyme exhibited gamma-glutamyl transfer activity as well as iron reduction activity in the presence of glutathione. We cloned and manipulated expression of the encoding gene. Overexpression or RNAi silencing affected both GGT and GSH-FeR activities concurrently. Enzyme inhibition experiments showed that the activity is complex and involves two reactions. First, Ggt1 initiates enzymatic breakdown of GSH by cleavage of the gamma-glutamyl bond and release of cysteinylglycine. Second, the thiol group of the released dipeptide reduces ferric to ferrous iron. A combination of kinetic properties of both reactions resulted in efficient iron reduction over a broad pH range. Our findings provide novel insight into Hc iron acquisition strategies and reveal a unique aspect of Ggt1 function in this dimorphic mycopathogen.
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Affiliation(s)
- Robert Zarnowski
- Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI, USA.
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Meister A, Griffith OW, Novogrodsky A, Tate SS. New aspects of glutathione metabolism and translocation in mammals. CIBA FOUNDATION SYMPOSIUM 2008:135-61. [PMID: 45011 DOI: 10.1002/9780470720554.ch9] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
An appreciable fraction of the sulphur present in the mammal occurs in the form of glutathione, whose concentration in various tissues ranges from about 0.8 to about 8 mM; the extracellular concentration of glutatione (largely present as the disulphide) is in the micromolecular range. The synthesis of glutathione and its utilization take place by the reactions of the gamma-glutamyl cycle, which include those catalysed by gamma-glutamylcysteine and glutathione synthetases, gamma-glutamyl transpeptidase, cysteinylglycinase, gamma-glutamyl cyclotransferease, and 5-oxoprolinase. gamma-Glutamyl transpeptidase catalyses transpeptidation (with amino acids and dipeptides) and hydrolysis reactions with both blutathione and its disulphide. The transpeptidase is membrane-boudn, apparently to the outer surface of the cell, and is found in certain epithelial cells in anatomical sites that are involved in transport and secretory activities (e.g., renal tubule, jejunal villi, choroid plexus, ciliary body). Evidence that the reactions of the gamma-glutamyl cycle take place in vivo has come from studies with labelled metabolites and selective enzyme inhibitors, and on inborn errors of metabolism associated with specific enzyme deficiencies. Inhibition in vivo of gamma-glutamyl cyclotransferase and 5-oxoprolinase leads, respectively, to decreased and increased renal levels of 5-oxoproline. Administration of a specific inhibitor of gamma-glutamylcysteine synthetase, such as buthionine sulphoximine, leads to a rapid decline in the glutamylcysteine synthetase, such as buthionine sulphoximine, leads to a rapid decline in the glutathione level of the kidney and other tissues, reflecting the appreciable rate of glutathione utilization. When gamma-glutamyl transpeptidase is inhibited in vivo by injection of L- or D-gamma-glutamyl-(o-carboxy)phenylhydrazide, there is extensive glutathionuria and the blood plasma level of glutathione increases. Studies in which inhibitors of glutathione synthesis and transpeptidation were given to mice showed that transport of intracellular glutathione to membrane-bound transpeptidase is a discrete step in the gamma-glutamyl cycle, and that the level of plasma glutatione reflects (a) synthesis of glutathione and its export by liver, muscle, and other tissues and (b) utilization of glutatione by kidney and other tissues. Studies on several lymphoid cell lines show that these cells also actively translocate glutathione out of the cell. A summary scheme is given for the metabolism of glutathione in which glutathione is translocated to the cell membrane where it may be utilized as such or oxidized to glutathione disulphide. Oxidation is inhibited, and transpeptidation is promoted by the presence of amino acids that are substrates of the transpeptidase. Glutathione exported from cells that have membrane-bound transpeptidase may be recovered by the cell transport of gamma-glutamyl amino acids and free amino acids...
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