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1
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George J, Lu Y, Tsuchishima M, Tsutsumi M. Cellular and molecular mechanisms of hepatic ischemia-reperfusion injury: The role of oxidative stress and therapeutic approaches. Redox Biol 2024; 75:103258. [PMID: 38970988 PMCID: PMC11279328 DOI: 10.1016/j.redox.2024.103258] [Citation(s) in RCA: 19] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2024] [Revised: 06/20/2024] [Accepted: 06/25/2024] [Indexed: 07/08/2024] Open
Abstract
Ischemia-reperfusion (IR) or reoxygenation injury is the paradoxical exacerbation of cellular impairment following restoration of blood flow after a period of ischemia during surgical procedures or other conditions. Acute interruption of blood supply to the liver and subsequent reperfusion can result in hepatocyte injury, apoptosis, and necrosis. Since the liver requires a continuous supply of oxygen for many biochemical reactions, any obstruction of blood flow can rapidly lead to hepatic hypoxia, which could quickly progress to absolute anoxia. Reoxygenation results in the increased generation of reactive oxygen species and oxidative stress, which lead to the enhanced production of proinflammatory cytokines, chemokines, and other signaling molecules. Consequent acute inflammatory cascades lead to significant impairment of hepatocytes and nonparenchymal cells. Furthermore, the expression of several vascular growth factors results in the heterogeneous closure of numerous hepatic sinusoids, which leads to reduced oxygen supply in certain areas of the liver even after reperfusion. Therefore, it is vital to identify appropriate therapeutic modalities to mitigate hepatic IR injury and subsequent tissue damage. This review covers all the major aspects of cellular and molecular mechanisms underlying the pathogenesis of hepatic ischemia-reperfusion injury, with special emphasis on oxidative stress, associated inflammation and complications, and prospective therapeutic approaches.
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Affiliation(s)
- Joseph George
- Department of Cancer Biology, Mayo Clinic, Jacksonville, FL, 32224, USA; Department of Hepatology, Kanazawa Medical University, Uchinada, Ishikawa, 920-0293, Japan; Center for Regenerative Medicine, Kanazawa Medical University Hospital, Uchinada, Ishikawa, 920-0293, Japan.
| | - Yongke Lu
- Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, 25755, USA
| | - Mutsumi Tsuchishima
- Department of Hepatology, Kanazawa Medical University, Uchinada, Ishikawa, 920-0293, Japan
| | - Mikihiro Tsutsumi
- Department of Hepatology, Kanazawa Medical University, Uchinada, Ishikawa, 920-0293, Japan; Center for Regenerative Medicine, Kanazawa Medical University Hospital, Uchinada, Ishikawa, 920-0293, Japan
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2
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Mitrić A, Castellano I. Targeting gamma-glutamyl transpeptidase: A pleiotropic enzyme involved in glutathione metabolism and in the control of redox homeostasis. Free Radic Biol Med 2023; 208:672-683. [PMID: 37739139 DOI: 10.1016/j.freeradbiomed.2023.09.020] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/31/2023] [Revised: 09/07/2023] [Accepted: 09/18/2023] [Indexed: 09/24/2023]
Abstract
Gamma-glutamyl transpeptidase (GGT) is an enzyme located on the outer membrane of the cells where it regulates the metabolism of glutathione (GSH), the most abundant intracellular antioxidant thiol. GGT plays a key role in the control of redox homeostasis, by hydrolyzing extracellular GSH and providing the cell with the recovery of cysteine, which is necessary for de novo intracellular GSH and protein biosynthesis. Therefore, the upregulation of GGT confers to the cell greater resistance to oxidative stress and the advantage of growing fast. Indeed, GGT is upregulated in inflammatory conditions and in the progression of various human tumors and it is involved in many physiological disorders related to oxidative stress, such as cardiovascular disease and diabetes. Currently, increased GGT expression is considered a marker of liver damage, cancer, and low-grade chronic inflammation. This review addresses the current knowledge on the structure-function relationship of GGT, focusing on human GGT, and provides information on the pleiotropic biological role and relevance of the enzyme as a target of drugs aimed at alleviating oxidative stress-related diseases. The development of new GGT inhibitors is critically discussed, as are the advantages and disadvantages of their potential use in clinics. Considering its pleiotropic activities and evolved functions, GGT is a potential "moonlighting protein".
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Affiliation(s)
- Aleksandra Mitrić
- Institute of Clinical and Molecular Virology, Friedrich-Alexander University Erlangen-Nürnberg, 91054, Erlangen, Germany
| | - Immacolata Castellano
- Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, 80131, Naples, Italy; Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Villa Comunale, Naples, Italy.
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3
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Zhang C, Xu H, Netto KG, Sokulsky LA, Miao Y, Mo Z, Meng Y, Du Y, Wu C, Han L, Zhang L, Liu C, Zhang G, Li F, Yang M. Inhibition of γ-glutamyl transferase suppresses airway hyperresponsiveness and airway inflammation in a mouse model of steroid resistant asthma exacerbation. Front Immunol 2023; 14:1132939. [PMID: 37377967 PMCID: PMC10292800 DOI: 10.3389/fimmu.2023.1132939] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2022] [Accepted: 05/30/2023] [Indexed: 06/29/2023] Open
Abstract
Introduction Despite recent advances, there are limited treatments available for acute asthma exacerbations. Here, we investigated the therapeutic potential of GGsTop, a γ-glutamyl transferase inhibitor, on the disease with a murine model of asthma exacerbation. Methods GGsTop was administered to mice that received lipopolysaccharide (LPS) and ovalbumin (OVA) challenges. Airway hyperresponsiveness (AHR), lung histology, mucus hypersecretion, and collagen deposition were analyzed to evaluate the hallmark features of asthma exacerbation. The level of proinflammatory cytokines and glutathione were determined with/without GGsTop. The transcription profiles were also examined. Results GGsTop attenuates hallmark features of the disease with a murine model of LPS and OVA driven asthma exacerbation. Airway hyperresponsiveness (AHR), mucus hypersecretion, collagen deposition, and expression of inflammatory cytokines were dramatically inhibited by GGsTop treatment. Additionally, GGsTop restored the level of glutathione. Using RNA-sequencing and pathway analysis, we demonstrated that the activation of LPS/NFκB signaling pathway in airway was downregulated by GGsTop. Interestingly, further analysis revealed that GGsTop significantly inhibited not only IFNγ responses but also the expression of glucocorticoid-associated molecules, implicating that GGsTop profoundly attenuates inflammatory pathways. Conclusions Our study suggests that GGsTop is a viable treatment for asthma exacerbation by broadly inhibiting the activation of multiple inflammatory pathways.
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Affiliation(s)
- Cancan Zhang
- Academy of Medical Sciences & Department of Immunology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China
| | - Huisha Xu
- Academy of Medical Sciences & Department of Immunology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China
| | - Keilah G. Netto
- Priority Research Centre for Healthy Lungs, School of Biomedical Sciences & Pharmacy, Faculty of Health and Hunter Medical Research Institute, The University of Newcastle, Callaghan, New South Wales, Australia
| | - Leon A. Sokulsky
- Priority Research Centre for Healthy Lungs, School of Biomedical Sciences & Pharmacy, Faculty of Health and Hunter Medical Research Institute, The University of Newcastle, Callaghan, New South Wales, Australia
| | - Yiyan Miao
- Academy of Medical Sciences & Department of Immunology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China
| | - Zhongyuan Mo
- Academy of Medical Sciences & Department of Immunology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China
| | - Yan Meng
- Academy of Medical Sciences & Department of Immunology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China
| | - Yingying Du
- Academy of Medical Sciences & Department of Immunology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China
| | - Chengyong Wu
- Academy of Medical Sciences & Department of Immunology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China
| | - Liyou Han
- Institute for Liberal Arts and Sciences, Kyoto University, Kyoto, Japan
| | - Lirong Zhang
- Department of Pharmacology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China
| | - Chi Liu
- Department of Physiology, School of Basic Medicine Science, Central South University, Changsha, China
| | - Guojun Zhang
- Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Fuguang Li
- Academy of Medical Sciences & Department of Immunology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China
| | - Ming Yang
- Academy of Medical Sciences & Department of Immunology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China
- Priority Research Centre for Healthy Lungs, School of Biomedical Sciences & Pharmacy, Faculty of Health and Hunter Medical Research Institute, The University of Newcastle, Callaghan, New South Wales, Australia
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4
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Rapid imaging of thymoma and thymic carcinoma with a fluorogenic probe targeting γ-glutamyltranspeptidase. Sci Rep 2023; 13:3757. [PMID: 36882498 PMCID: PMC9992351 DOI: 10.1038/s41598-023-30753-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2022] [Accepted: 02/28/2023] [Indexed: 03/09/2023] Open
Abstract
In recent years, thoracoscopic and robotic surgical procedures have increasingly replaced median sternotomy for thymoma and thymic carcinoma. In cases of partial thymectomy, the prognosis is greatly improved by ensuring a sufficient margin from the tumor, and therefore intraoperative fluorescent imaging of the tumor is especially valuable in thoracoscopic and robotic surgery, where tactile information is not available. γ-Glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) has been applied for fluorescence imaging of some types of tumors in the resected tissues, and here we aimed to examine its validity for the imaging of thymoma and thymic carcinoma. 22 patients with thymoma or thymic carcinoma who underwent surgery between February 2013 and January 2021 were included in the study. Ex vivo imaging of specimens was performed, and the sensitivity and specificity of gGlu-HMRG were 77.3% and 100%, respectively. Immunohistochemistry (IHC) staining was performed to confirm expression of gGlu-HMRG's target enzyme, γ-glutamyltranspeptidase (GGT). IHC revealed high GGT expression in thymoma and thymic carcinoma in contrast to absent or low expression in normal thymic parenchyma and fat tissue. These results suggest the utility of gGlu-HMRG as a fluorescence probe for intraoperative visualization of thymomas and thymic carcinomas.
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5
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Wei X, Zhang C, He S, Huang J, Huang J, Liew SS, Zeng Z, Pu K. A Dual-Locked Activatable Phototheranostic Probe for Biomarker-Regulated Photodynamic and Photothermal Cancer Therapy. Angew Chem Int Ed Engl 2022; 61:e202202966. [PMID: 35396786 DOI: 10.1002/anie.202202966] [Citation(s) in RCA: 67] [Impact Index Per Article: 22.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2022] [Indexed: 12/29/2022]
Abstract
Activatable phototheranostics holds promise for precision cancer treatment owing to the "turn-on" signals and therapeutic effects. However, most activatable phototheranostic probes only possess photodynamic therapy (PDT) or photothermal therapy (PTT), which suffer from poor therapeutic efficacy due to deficient cellular oxygen and complex tumor microenvironment. We herein report a dual-locked activatable phototheranostic probe that activates near-infrared fluorescence (NIRF) signals in tumor, triggers PDT in response to a tumor-periphery biomarker, and switches from PDT to PTT upon detecting a tumor-core-hypoxia biomarker. This PDT-PTT auto-regulated probe exhibits complete tumor ablation under the photoirradiation of a single laser source by producing cytotoxic singlet oxygen at the tumor periphery and generating hyperthermia at tumor-core where is too hypoxic for PDT. This dual-locked probe represents a promising molecular design approach toward precise cancer phototheranostics.
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Affiliation(s)
- Xin Wei
- School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, 637457, Singapore
| | - Chi Zhang
- School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, 637457, Singapore
| | - Shasha He
- School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, 637457, Singapore
| | - Jiaguo Huang
- School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, 637457, Singapore
| | - Jingsheng Huang
- School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, 637457, Singapore
| | - Si Si Liew
- School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, 637457, Singapore
| | - Ziling Zeng
- School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, 637457, Singapore
| | - Kanyi Pu
- School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, 637457, Singapore.,Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, 636921, Singapore
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6
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Nishijima H, Zunitch MJ, Yoshida M, Kondo K, Yamasoba T, Schwob JE, Holbrook EH. Rapid fluorescent vital imaging of olfactory epithelium. iScience 2022; 25:104222. [PMID: 35494237 PMCID: PMC9046240 DOI: 10.1016/j.isci.2022.104222] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2021] [Revised: 02/28/2022] [Accepted: 04/06/2022] [Indexed: 11/25/2022] Open
Abstract
Olfactory epithelium (OE) undergoes degeneration in disorders such as age-related and post-viral olfactory dysfunction. However, methods for real-time in vivo detection of OE and assessment of total extent within the nasal cavity are currently unavailable. We identified two fluorescence probes for rapidly detecting and evaluating the entire extent of mice OE with topical application. Taking advantage of the differential expression of the enzymes cytochrome p450 (CYP) and γ-glutamyltranspeptidase (GGT) in OE relative to respiratory epithelium, we utilized the conversion of coumarin (a substrate of various CYP subtypes) and gGlu-HRMG (a substrate of GGT) by these enzymes to form metabolites with fluorescent emissions in the duct cells and sustentacular cells of neuron-containing OE. In depleted and regenerated OE model, the emission of these probes remained absent in respiratory metaplasia but appeared in regenerated OE. These substrates could be used to monitor OE degeneration and follow regenerative response to therapeutic interventions.
Enzymes Cyp2a5 and Ggt7 are expressed in olfactory epithelial cells Substrates for Cyp2a5 and Ggt7 can label olfactory epithelium (OE) in situ Lesion recovered, not damaged OE, is labeled with Cyp2a5 and Ggt7 substrates
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Affiliation(s)
- Hironobu Nishijima
- Department of Developmental, Molecular, and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111, USA.,Department of Otolaryngology Head and Neck Surgery, Harvard Medical School, Massachusetts Eye and Ear, 243 Charles Street, Boston, MA 02114, USA.,Department of Otolaryngology-Head and Neck Surgery, The University of Tokyo, Tokyo 113-0033, Japan
| | - Matthew J Zunitch
- Department of Developmental, Molecular, and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111, USA
| | - Masafumi Yoshida
- Department of Otolaryngology-Head and Neck Surgery, The University of Tokyo, Tokyo 113-0033, Japan
| | - Kenji Kondo
- Department of Otolaryngology-Head and Neck Surgery, The University of Tokyo, Tokyo 113-0033, Japan
| | - Tatsuya Yamasoba
- Department of Otolaryngology-Head and Neck Surgery, The University of Tokyo, Tokyo 113-0033, Japan
| | - James E Schwob
- Department of Developmental, Molecular, and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111, USA
| | - Eric H Holbrook
- Department of Developmental, Molecular, and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111, USA.,Department of Otolaryngology Head and Neck Surgery, Harvard Medical School, Massachusetts Eye and Ear, 243 Charles Street, Boston, MA 02114, USA
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7
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Wei X, Zhang C, He S, Huang J, Huang J, Liew SS, Zeng Z, Pu K. A Dual‐locked Activatable Phototheranostic Probe for Biomarkers Regulated Photodynamic and Photothermal Cancer Therapy. Angew Chem Int Ed Engl 2022. [DOI: 10.1002/ange.202202966] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Affiliation(s)
- Xin Wei
- Nanyang Technological University School of chemical and biomedical Engineering SINGAPORE
| | - Chi Zhang
- Nanyang Technological University School of Chemical and Biomedical Engineering SINGAPORE
| | - Shasha He
- Nanyang Technological University School of Chemical and Biomedical Engineering SINGAPORE
| | - Jiaguo Huang
- Sun Yat-Sen University School of Pharmaceutical Sciences SINGAPORE
| | - Jingsheng Huang
- Nanyang Technological University School of Chemical and Biomedical Engineering SINGAPORE
| | - Si Si Liew
- Nanyang Technological University School of Chemical and Biomedical Engineering SINGAPORE
| | - Ziling Zeng
- Nanyang Technological University School of Chemical and Biomedical Engineering SINGAPORE
| | - Kanyi Pu
- Nanyang Technological University School of Chemical and Biomedical Engieering 70 Nanyang Drive 637457 Singapore SINGAPORE
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8
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Terzyan SS, Nguyen LT, Burgett AWG, Heroux A, Smith CA, You Y, Hanigan MH. Crystal structures of glutathione- and inhibitor-bound human GGT1: critical interactions within the cysteinylglycine binding site. J Biol Chem 2021; 296:100066. [PMID: 33187988 PMCID: PMC7949050 DOI: 10.1074/jbc.ra120.016265] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2020] [Revised: 11/06/2020] [Accepted: 11/13/2020] [Indexed: 11/06/2022] Open
Abstract
Overexpression of γ-glutamyl transpeptidase (GGT1) has been implicated in an array of human diseases including asthma, reperfusion injury, and cancer. Inhibitors are needed for therapy, but development of potent, specific inhibitors of GGT1 has been hampered by a lack of structural information regarding substrate binding and cleavage. To enhance our understanding of the molecular mechanism of substrate cleavage, we have solved the crystal structures of human GGT1 (hGGT1) with glutathione (a substrate) and a phosphate-glutathione analog (an irreversible inhibitor) bound in the active site. These are the first structures of any eukaryotic GGT with the cysteinylglycine region of the substrate-binding site occupied. These structures and the structure of apo-hGGT reveal movement of amino acid residues within the active site as the substrate binds. Asn-401 and Thr-381 each form hydrogen bonds with two atoms of GSH spanning the γ-glutamyl bond. Three different atoms of hGGT1 interact with the carboxyl oxygen of the cysteine of GSH. Interactions between the enzyme and substrate change as the substrate moves deeper into the active site cleft. The substrate reorients and a new hydrogen bond is formed between the substrate and the oxyanion hole. Thr-381 is locked into a single conformation as an acyl bond forms between the substrate and the enzyme. These data provide insight on a molecular level into the substrate specificity of hGGT1 and provide an explanation for seemingly disparate observations regarding the enzymatic activity of hGGT1 mutants. This knowledge will aid in the design of clinically useful hGGT1 inhibitors.
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Affiliation(s)
- Simon S Terzyan
- Laboratory of Biomolecular Structure and Function, Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA
| | - Luong T Nguyen
- Department of Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, New York, USA
| | - Anthony W G Burgett
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA
| | - Annie Heroux
- Energy Sciences Directorate/Photon Science Division, Brookhaven National Laboratory, Upton, New York, USA
| | - Clyde A Smith
- Stanford Synchrotron Radiation Lightsource, Stanford University, Menlo Park, California, USA
| | - Youngjae You
- Department of Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, New York, USA
| | - Marie H Hanigan
- Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
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9
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Corti A, Belcastro E, Dominici S, Maellaro E, Pompella A. The dark side of gamma-glutamyltransferase (GGT): Pathogenic effects of an 'antioxidant' enzyme. Free Radic Biol Med 2020; 160:807-819. [PMID: 32916278 DOI: 10.1016/j.freeradbiomed.2020.09.005] [Citation(s) in RCA: 64] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/29/2020] [Revised: 08/26/2020] [Accepted: 09/01/2020] [Indexed: 12/16/2022]
Abstract
Having long been regarded as just a member in the cellular antioxidant systems, as well as a clinical biomarker of hepatobiliary diseases and alcohol abuse, gamma-glutamyltransferase (GGT) enzyme activity has been highlighted by more recent research as a critical factor in modulation of redox equilibria within the cell and in its surroundings. Moreover, due to the prooxidant reactions which can originate during its metabolic function in selected conditions, experimental and clinical studies are increasingly involving GGT in the pathogenesis of several important disease conditions, such as atherosclerosis, cardiovascular diseases, cancer, lung inflammation, neuroinflammation and bone disorders. The present article is an overview of the laboratory findings that have prompted an evolution in interpretation of the significance of GGT in human pathophysiology.
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Affiliation(s)
- Alessandro Corti
- Dept. of Translational Research NTMS, University of Pisa Medical School, Italy
| | - Eugenia Belcastro
- Dept. of Translational Research NTMS, University of Pisa Medical School, Italy
| | - Silvia Dominici
- Dept. of Translational Research NTMS, University of Pisa Medical School, Italy
| | - Emilia Maellaro
- Dept. of Molecular and Developmental Medicine, University of Siena, Italy
| | - Alfonso Pompella
- Dept. of Translational Research NTMS, University of Pisa Medical School, Italy.
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10
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Kubota R, Hayashi N, Kinoshita K, Saito T, Ozaki K, Ueda Y, Tsuchishima M, Tsutsumi M, George J. Inhibition of γ-glutamyltransferase ameliorates ischaemia-reoxygenation tissue damage in rats with hepatic steatosis. Br J Pharmacol 2020; 177:5195-5207. [PMID: 32910829 DOI: 10.1111/bph.15258] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2020] [Revised: 08/20/2020] [Accepted: 09/07/2020] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND AND PURPOSE Hepatic steatosis may be associated with an increased γ-glutamyltransferase (γ-GT) levels. Ischaemia-reoxygenation (IR) injury causes several deleterious effects. We evaluated the protective effects of a selective inhibitor of γ-GT in experimentally induced IR injury in rats with obesity and steatosis. EXPERIMENTAL APPROACH Otsuka Long-Evans Tokushima Fatty (OLETF) rats with hepatic steatosis were used in the current study. The portal vein and hepatic artery of left lateral and median lobes were clamped to induce ischaemia. Before clamping, 1 ml of saline (IR group) or 1-ml saline containing 1 mg·kg-1 body weight of GGsTop (γ-GT inhibitor; IR-GGsTop group) was injected into the liver via the inferior vena cava. Blood flow was restored after at 30 min of the start of ischaemia. Blood was collected before, at 30 min after ischaemia and at 2 h and 6 h after reoxygenation. All the animals were killed at 6 h and the livers were collected. KEY RESULTS Treatment with GGsTop resulted in significant reduction of serum ALT, AST and γ-GT levels and hepatic γ-GT, malondialdehyde, 4-hydroxy-2-nonenal and HMGB1 at 6 h after reoxygenation. Inhibition of γ-GT retained normal hepatic glutathione levels. There was prominent hepatic necrosis in IR group, which is significantly reduced in IR-GGsTop group. CONCLUSION AND IMPLICATIONS Treatment with GGsTop significantly increased hepatic glutathione content, reduced hepatic MDA, 4-HNE and HMGB1 levels and, remarkably, ameliorated hepatic necrosis after ischaemia-reoxygenation. The results indicated that GGsTop could be an appropriate therapeutic agent to reduce IR-induced liver injury in obesity and steatosis.
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Affiliation(s)
- Ryuichi Kubota
- Department of Hepatology, Kanazawa Medical University, Uchinada, Japan
| | - Nobuhiko Hayashi
- Department of Hepatology, Kanazawa Medical University, Uchinada, Japan.,Center for Regenerative Medicine, Kanazawa Medical University Hospital, Uchinada, Japan
| | - Kaori Kinoshita
- Department of Hepatology, Kanazawa Medical University, Uchinada, Japan.,Center for Regenerative Medicine, Kanazawa Medical University Hospital, Uchinada, Japan
| | - Takashi Saito
- Department of Hepatology, Kanazawa Medical University, Uchinada, Japan
| | - Kazuaki Ozaki
- Department of Hepatology, Kanazawa Medical University, Uchinada, Japan
| | - Yoshimichi Ueda
- Department of Pathology II, Kanazawa Medical University, Uchinada, Japan
| | | | - Mikihiro Tsutsumi
- Department of Hepatology, Kanazawa Medical University, Uchinada, Japan.,Center for Regenerative Medicine, Kanazawa Medical University Hospital, Uchinada, Japan
| | - Joseph George
- Department of Hepatology, Kanazawa Medical University, Uchinada, Japan.,Center for Regenerative Medicine, Kanazawa Medical University Hospital, Uchinada, Japan
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11
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Mendiola AS, Ryu JK, Bardehle S, Meyer-Franke A, Ang KKH, Wilson C, Baeten KM, Hanspers K, Merlini M, Thomas S, Petersen MA, Williams A, Thomas R, Rafalski VA, Meza-Acevedo R, Tognatta R, Yan Z, Pfaff SJ, Machado MR, Bedard C, Rios Coronado PE, Jiang X, Wang J, Pleiss MA, Green AJ, Zamvil SS, Pico AR, Bruneau BG, Arkin MR, Akassoglou K. Transcriptional profiling and therapeutic targeting of oxidative stress in neuroinflammation. Nat Immunol 2020; 21:513-524. [PMID: 32284594 PMCID: PMC7523413 DOI: 10.1038/s41590-020-0654-0] [Citation(s) in RCA: 138] [Impact Index Per Article: 27.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2019] [Accepted: 03/04/2020] [Indexed: 12/11/2022]
Abstract
Oxidative stress is a central part of innate-immune induced neurodegeneration. However, the transcriptomic landscape of the central nervous system (CNS) innate immune cells contributing to oxidative stress is unknown, and therapies to target their neurotoxic functions are not widely available. Here, we provide the oxidative stress innate immune cell atlas in neuroinflammatory disease, and report the discovery of new druggable pathways. Transcriptional profiling of oxidative stress-producing CNS innate immune cells (Tox-seq) identified a core oxidative stress gene signature coupled to coagulation and glutathione pathway genes shared between a microglia cluster and infiltrating macrophages. Tox-seq followed by a microglia high-throughput screen (HTS) and oxidative stress gene network analysis, identified the glutathione regulating compound acivicin with potent therapeutic effects decreasing oxidative stress and axonal damage in chronic and relapsing multiple sclerosis (MS) models. Thus, oxidative stress transcriptomics identified neurotoxic CNS innate immune populations and may enable the discovery of selective neuroprotective strategies.
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Affiliation(s)
| | - Jae Kyu Ryu
- Gladstone Institutes, San Francisco, CA, USA.,Department of Neurology and Weill Institute for Neurosciences, University of California San Francisco, San Francisco, CA, USA
| | | | | | - Kenny Kean-Hooi Ang
- Small Molecule Discovery Center, University of California San Francisco, San Francisco, CA, USA.,Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, CA, USA
| | - Chris Wilson
- Small Molecule Discovery Center, University of California San Francisco, San Francisco, CA, USA.,Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, CA, USA
| | | | | | | | - Sean Thomas
- Gladstone Institutes, San Francisco, CA, USA
| | - Mark A Petersen
- Gladstone Institutes, San Francisco, CA, USA.,Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA
| | | | | | | | | | | | - Zhaoqi Yan
- Gladstone Institutes, San Francisco, CA, USA
| | - Samuel J Pfaff
- Small Molecule Discovery Center, University of California San Francisco, San Francisco, CA, USA.,Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, CA, USA
| | | | | | | | - Xiqian Jiang
- Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, TX, USA
| | - Jin Wang
- Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, TX, USA
| | | | - Ari J Green
- Department of Neurology and Weill Institute for Neurosciences, University of California San Francisco, San Francisco, CA, USA.,Department of Ophthalmology, University of California San Francisco, San Francisco, CA, USA
| | - Scott S Zamvil
- Department of Neurology and Weill Institute for Neurosciences, University of California San Francisco, San Francisco, CA, USA
| | | | - Benoit G Bruneau
- Gladstone Institutes, San Francisco, CA, USA.,Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA.,Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA
| | - Michelle R Arkin
- Small Molecule Discovery Center, University of California San Francisco, San Francisco, CA, USA.,Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, CA, USA
| | - Katerina Akassoglou
- Gladstone Institutes, San Francisco, CA, USA. .,Department of Neurology and Weill Institute for Neurosciences, University of California San Francisco, San Francisco, CA, USA.
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12
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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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13
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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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14
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Koyama T, Tsubota A, Sawano T, Tawa M, Watanabe B, Hiratake J, Nakagawa K, Matsumura Y, Ohkita M. Involvement of γ-Glutamyl Transpeptidase in Ischemia/Reperfusion-Induced Cardiac Dysfunction in Isolated Rat Hearts. Biol Pharm Bull 2019; 42:1947-1952. [DOI: 10.1248/bpb.b19-00434] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Affiliation(s)
- Takeshi Koyama
- Laboratory of Pathological and Molecular Pharmacology, Osaka University of Pharmaceutical Sciences
| | - Akari Tsubota
- Laboratory of Pathological and Molecular Pharmacology, Osaka University of Pharmaceutical Sciences
| | - Tatsuya Sawano
- Laboratory of Pathological and Molecular Pharmacology, Osaka University of Pharmaceutical Sciences
- Division of Pharmacology, Faculty of Medicine, Tottori University
| | - Masashi Tawa
- Laboratory of Pathological and Molecular Pharmacology, Osaka University of Pharmaceutical Sciences
- Department of Pharmacology, Kanazawa Medical University
| | | | - Jun Hiratake
- Institute for Chemical Research, Kyoto University
| | - Keisuke Nakagawa
- Laboratory of Pathological and Molecular Pharmacology, Osaka University of Pharmaceutical Sciences
| | - Yasuo Matsumura
- Laboratory of Pathological and Molecular Pharmacology, Osaka University of Pharmaceutical Sciences
| | - Mamoru Ohkita
- Laboratory of Pathological and Molecular Pharmacology, Osaka University of Pharmaceutical Sciences
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15
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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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16
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Altınkaynak Y, Kural B, Akcan BA, Bodur A, Özer S, Yuluğ E, Munğan S, Kaya C, Örem A. Protective effects of L-theanine against doxorubicin-induced nephrotoxicity in rats. Biomed Pharmacother 2018; 108:1524-1534. [DOI: 10.1016/j.biopha.2018.09.171] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2018] [Revised: 09/28/2018] [Accepted: 09/28/2018] [Indexed: 12/20/2022] Open
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17
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Plant-Derived Anticancer Agents: Lessons from the Pharmacology of Geniposide and Its Aglycone, Genipin. Biomedicines 2018; 6:biomedicines6020039. [PMID: 29587429 PMCID: PMC6027249 DOI: 10.3390/biomedicines6020039] [Citation(s) in RCA: 55] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2018] [Revised: 03/21/2018] [Accepted: 03/22/2018] [Indexed: 12/24/2022] Open
Abstract
For centuries, plants have been exploited by mankind as sources of numerous cancer chemotherapeutic agents. Good examples of anticancer compounds of clinical significance today include the taxanes (e.g., taxol), vincristine, vinblastine, and the podophyllotoxin analogues that all trace their origin to higher plants. While all these drugs, along with the various other available therapeutic options, brought some relief in cancer management, a real breakthrough or cure has not yet been achieved. This critical review is a reflection on the lessons learnt from decades of research on the iridoid glycoside geniposide and its aglycone, genipin, which are currently used as gold standard reference compounds in cancer studies. Their effects on tumour development (carcinogenesis), cancer cell survival, and death, with particular emphasis on their mechanisms of actions, are discussed. Particular attention is also given to mechanisms related to the dual pro-oxidant and antioxidant effects of these compounds, the mitochondrial mechanism of cancer cell killing through reactive oxygen species (ROS), including that generated through the uncoupling protein-2 (UCP-2), the inflammatory mechanism, and cell cycle regulation. The implications of various studies for the evaluation of glycosidic and aglycone forms of natural products in vitro and in vivo through pharmacokinetic scrutiny are also addressed.
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18
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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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19
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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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20
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Zhou X, Wang L, Wang G, Cheng X, Hu S, Ke W, Li M, Zhang Y, Song Z, Zheng Q. A new plasma biomarker enhance the clinical prediction of postoperative acute kidney injury in patients with hepatocellular carcinoma. Clin Chim Acta 2017; 475:128-136. [PMID: 29031454 DOI: 10.1016/j.cca.2017.10.008] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2017] [Revised: 09/07/2017] [Accepted: 10/10/2017] [Indexed: 01/09/2023]
Abstract
BACKGROUND The ratio of serum γ-glutamyl transferase (GGT) to alanine aminotransferase (ALT) (GGT/ALT) is a marker for evaluating effects to antivirotic treatment and a helpful predictive factor for the prognosis of Child-Pugh A hepatocellular carcinoma (HCC) patients after surgery. The relationship between the incidence of postoperative acute kidney injury (AKI) and preoperative GGT/ALT is studied in hepatectomized hepatitis B- or C- associated HCC patients. METHODS A total of 253 hepatitis B or C virus-related HCC patients undergoing hepatectomy between September 2012 and August 2016 at our hospital were included in the retrospective study. Serum ALT and GGT value were recorded, and the GGT/ALT was computed. AKI was defined that based on the "Kidney Disease Improving Global Outcomes (KDIGO) criteria". RESULTS AKI was observed in 22 (8.7%) patients. Mean GGT/ALT of patients with AKI was significantly higher than in those without it (6.0 vs 2.1, P<0.001). Multivariate analysis revealed an increase in GGT/ALT as an independent risk factor for AKI in hepatitis B- or C- associated HCC patients, particularly in patients with Barcelona Clinic Liver Cancer (BCLC) stage 0 or A staged HCC (odds ratio (OR) 1.400, P<0.001). Multivariate analysis showed that ALT (OR 0.966, P=0.044) was somewhat inversely associated with the incidence of AKI in hepatitis B- or C- associated HCC patients. The best cutoff point of GGT/ALT was 2.92. Multivariate analysis showed that preoperative GGT/ALT ≥2.92 predicted poor prognosis of postoperative AKI in patients with HCC after hepatectomy (odds ratio 17.697, P<0.001). After propensity score matching, preoperative GGT/ALT ≥2.92 remained an independent risk factor for AKI in HCC patients (OR 13.947, P=0.003). CONCLUSIONS The GGT/ALT of patients with AKI was significantly higher than those without it. Evaluation of GGT/ALT before surgery can be a helpful predictive tool for postoperative AKI in hepatitis B- or C- associated HCC patients undergoing hepatectomy, particularly in patients with BCLC stage 0 or A staged HCC. Hepatitis B- or C- associated HCC patients with low ALT especially within the normal range may have a high risk of AKI. However, the reason remains to be elucidated.
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Affiliation(s)
- Xing Zhou
- Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Liyu Wang
- Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Guoliang Wang
- Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Xiang Cheng
- Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Shaobo Hu
- Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Wenbo Ke
- Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Min Li
- Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Yong Zhang
- Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Zifang Song
- Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Qichang Zheng
- Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
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21
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Watanabe B, Tabuchi Y, Wada K, Hiratake J. Synthesis and evaluation of the inhibitory activity of the four stereoisomers of the potent and selective human γ-glutamyl transpeptidase inhibitor GGsTop. Bioorg Med Chem Lett 2017; 27:4920-4924. [PMID: 28985998 DOI: 10.1016/j.bmcl.2017.09.017] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2017] [Revised: 09/06/2017] [Accepted: 09/07/2017] [Indexed: 12/30/2022]
Abstract
2-Amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop) is a potent, highly selective, nontoxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT). GGsTop has been widely used in academic and medicinal research, and also as an active ingredient (Nahlsgen) in commercial anti-aging cosmetics. GGsTop consists of four stereoisomers due to the presence of two stereogenic centers, i.e., the α-carbon atom of the glutamate mimic (l/d) and the phosphorus atom (RP/SP). In this study, each stereoisomer of GGsTop was synthesized stereoselectively and their inhibitory activity against human GGT was evaluated. The l- and d-configurations of each stereoisomer were determined by a combination of a chiral pool synthesis and chiral HPLC analysis. The synthesis of the four stereoisomers of GGsTop used chiral synthetic precursors that were separated by chiral HPLC on a preparative scale. With respect to the configuration of the α-carbon atom of the glutamate mimic, the l-isomer (kon=174M-1s-1) was ca. 8-fold more potent than the d-isomer (kon=21.5M-1s-1). In contrast, the configuration of the phosphorus atom is critical for GGT inhibitory activity. Based on a molecular modeling approach, the absolute configuration of the phosphorus atom of the active GGsTop isomers was postulated to be SP. The SP-isomers inhibited human GGT (kon=21.5-174M-1s-1), while the RP-isomers were inactive even at concentrations of 0.1mM.
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Affiliation(s)
- Bunta Watanabe
- Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
| | - Yukiko Tabuchi
- Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
| | - Kei Wada
- Department of Medical Sciences, University of Miyazaki, Miyazaki 889-1692, Japan
| | - Jun Hiratake
- Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
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22
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Watanabe B, Morikita T, Tabuchi Y, Kobayashi R, Li C, Yamamoto M, Koeduka T, Hiratake J. An improved synthesis of the potent and selective γ-glutamyl transpeptidase inhibitor GGsTop together with an inhibitory activity evaluation of its potential hydrolysis products. Tetrahedron Lett 2017. [DOI: 10.1016/j.tetlet.2017.08.019] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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23
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Miyata Y, Ishizawa T, Kamiya M, Yamashita S, Hasegawa K, Ushiku A, Shibahara J, Fukayama M, Urano Y, Kokudo N. Intraoperative imaging of hepatic cancers using γ-glutamyltranspeptidase-specific fluorophore enabling real-time identification and estimation of recurrence. Sci Rep 2017; 7:3542. [PMID: 28615696 PMCID: PMC5471246 DOI: 10.1038/s41598-017-03760-3] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2017] [Accepted: 05/03/2017] [Indexed: 01/10/2023] Open
Abstract
γ-Glutamyltranspeptidase (GGT) is upregulated in a variety of human cancers including primary and secondary hepatic tumors. This motivated us to use γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), a novel fluorophore emitting light at around 520 nm following enzymatic reaction with GGT, as a tool for the intraoperative identification of hepatic tumors. gGlu-HMRG was topically applied to 103 freshly resected hepatic specimens. Fluorescence imaging using gGlu-HMRG identified hepatic tumors with the sensitivity/specificity of 48%/96% for hepatocellular carcinoma, 100%/100% for intrahepatic cholangiocarcinoma, and 87%/100% for colorectal liver metastasis. High gGlu-HMRG fluorescence intensity was positively associated with the incidence of microscopic vascular invasion in HCC and the risk of early postoperative recurrence in CRLM. These results suggest that gGlu-HMRG imaging could not only be a useful intraoperative navigation tool but also provide information related to postoperative disease recurrence.
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Affiliation(s)
- Yoichi Miyata
- Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Takeaki Ishizawa
- Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. .,Department of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan.
| | - Mako Kamiya
- Laboratory of Chemical Biology and Molecular Imaging, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.,PRESTO, Japan Science and Technology Agency, Saitama, Japan
| | - Suguru Yamashita
- Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Kiyoshi Hasegawa
- Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Aya Ushiku
- Department of Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Junji Shibahara
- Department of Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Masashi Fukayama
- Department of Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Yasuteru Urano
- Laboratory of Chemical Biology and Molecular Imaging, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. .,Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan. .,CREST, Japan Agency for Medical Research and Development, Tokyo, Japan.
| | - Norihiro Kokudo
- Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
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24
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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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25
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Phosphonate-based irreversible inhibitors of human γ-glutamyl transpeptidase (GGT). GGsTop is a non-toxic and highly selective inhibitor with critical electrostatic interaction with an active-site residue Lys562 for enhanced inhibitory activity. Bioorg Med Chem 2016; 24:5340-5352. [PMID: 27622749 DOI: 10.1016/j.bmc.2016.08.050] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2016] [Revised: 08/25/2016] [Accepted: 08/27/2016] [Indexed: 11/22/2022]
Abstract
γ-Glutamyl transpeptidase (GGT, EC 2.3.2.2) that catalyzes the hydrolysis and transpeptidation of glutathione and its S-conjugates is involved in a number of physiological and pathological processes through glutathione metabolism and is an attractive pharmaceutical target. We report here the evaluation of a phosphonate-based irreversible inhibitor, 2-amino-4-{[3-(carboxymethyl)phenoxy](methoyl)phosphoryl}butanoic acid (GGsTop) and its analogues as a mechanism-based inhibitor of human GGT. GGsTop is a stable compound, but inactivated the human enzyme significantly faster than the other phosphonates, and importantly did not inhibit a glutamine amidotransferase. The structure-activity relationships, X-ray crystallography with Escherichia coli GGT, sequence alignment and site-directed mutagenesis of human GGT revealed a critical electrostatic interaction between the terminal carboxylate of GGsTop and the active-site residue Lys562 of human GGT for potent inhibition. GGsTop showed no cytotoxicity toward human fibroblasts and hepatic stellate cells up to 1mM. GGsTop serves as a non-toxic, selective and highly potent irreversible GGT inhibitor that could be used for various in vivo as well as in vitro biochemical studies.
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Tamura K, Hayashi N, George J, Toshikuni N, Arisawa T, Hiratake J, Tsuchishima M, Tsutsumi M. GGsTop, a novel and specific γ-glutamyl transpeptidase inhibitor, protects hepatic ischemia-reperfusion injury in rats. Am J Physiol Gastrointest Liver Physiol 2016; 311:G305-12. [PMID: 27365338 DOI: 10.1152/ajpgi.00439.2015] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/11/2015] [Accepted: 06/27/2016] [Indexed: 01/31/2023]
Abstract
Ischemia-reperfusion (IR) injury is a major clinical problem and is associated with numerous adverse effects. GGsTop [2-amino-4{[3-(carboxymethyl)phenyl](methyl)phosphono}butanoic acid] is a highly specific and irreversible γ-glutamyl transpeptidase (γ-GT) inhibitor. We studied the protective effects of GGsTop on IR-induced hepatic injury in rats. Ischemia was induced by clamping the portal vein and hepatic artery of left lateral and median lobes of the liver. Before clamping, saline (IR group) or saline containing 1 mg/kg body wt of GGsTop (IR-GGsTop group) was injected into the liver through the inferior vena cava. At 90 min of ischemia, blood flow was restored. Blood was collected before induction of ischemia and prior to restoration of blood flow and at 12, 24, and 48 h after reperfusion. All the animals were euthanized at 48 h after reperfusion and the livers were harvested. Serum levels of alanine transaminase, aspartate transaminase, and γ-GT were significantly lower after reperfusion in the IR-GGsTop group compared with the IR group. Massive hepatic necrosis was present in the IR group, while only few necroses were present in the IR-GGsTop group. Treatment with GGsTop increased hepatic GSH content, which was significantly reduced in the IR group. Furthermore, GGsTop prevented increase of hepatic γ-GT, malondialdehyde, 4-hydroxynonenal, and TNF-α while all these molecules significantly increased in the IR group. In conclusion, treatment with GGsTop increased glutathione levels and prevented formation of free radicals in the hepatic tissue that led to decreased IR-induced liver injury. GGsTop could be used as a pharmacological agent to prevent IR-induced liver injury and the related adverse events.
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Affiliation(s)
- Kaneto Tamura
- Department of Hepatology, Kanazawa Medical University, Uchinada, Ishikawa, Japan
| | - Nobuhiko Hayashi
- Department of Hepatology, Kanazawa Medical University, Uchinada, Ishikawa, Japan
| | - Joseph George
- Department of Hepatology, Kanazawa Medical University, Uchinada, Ishikawa, Japan
| | - Nobuyuki Toshikuni
- Department of Hepatology, Kanazawa Medical University, Uchinada, Ishikawa, Japan
| | - Tomiyasu Arisawa
- Department of Gastroenterology, Kanazawa Medical University, Uchinada, Ishikawa, Japan; and
| | - Jun Hiratake
- Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto, Japan
| | - Mutsumi Tsuchishima
- Department of Hepatology, Kanazawa Medical University, Uchinada, Ishikawa, Japan
| | - Mikihiro Tsutsumi
- Department of Hepatology, Kanazawa Medical University, Uchinada, Ishikawa, Japan;
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Park S, Lim SY, Bae SM, Kim SY, Myung SJ, Kim HJ. Indocyanine-Based Activatable Fluorescence Turn-On Probe for γ-Glutamyltranspeptidase and Its Application to the Mouse Model of Colon Cancer. ACS Sens 2016. [DOI: 10.1021/acssensors.5b00249] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Affiliation(s)
- Seokan Park
- Department
of Chemistry, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea
| | - Soo-Yeon Lim
- Department
of Chemistry, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea
| | | | | | | | - Hae-Jo Kim
- Department
of Chemistry, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea
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Jiang Y, Wang X, Li Y, Mu S, Zhou S, Liu Y, Zhang B. GGsTOP increases migration of human periodontal ligament cells in vitro via reactive oxygen species pathway. Mol Med Rep 2016; 13:3813-20. [PMID: 27035100 PMCID: PMC4838071 DOI: 10.3892/mmr.2016.5038] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2015] [Accepted: 03/07/2016] [Indexed: 01/01/2023] Open
Abstract
GGsTOP is a novel and selective inhibitor of gamma-glutamyl transferase (GGT), a cell-surface enzyme that has a key role in glutathione homeostasis and the maintenance of cellular reactive oxygen species (ROS). ROS are essential for wound healing. However, little is known about the molecular mechanisms underlying the inhibition of GGT by GGsTOP in human periodontal ligament cells (hPLCs). The present study assessed GGT expression in mouse periodontal ligament tissues, GGT activity in hPLCs, and the potential physiological effect of GGsTOP on hPLC migration. Immunohistochemical analysis confirmed that GGT was widely expressed in mouse periodontal ligament tissue. Treatment with GGsTOP was associated with greater proliferation and migration of hPLCs, and higher levels of cellular ROS compared with untreated hPLCs. However, the increase in intracellular ROS was attenuated in hPLCs co-cultured with the anti-oxidant N-acetylcysteine (NAC), a precursor of glutathione. The higher ROS levels associated with GGsTOP treatment were in parallel with increases in the levels of type I collagen and alpha smooth muscle actin, which was inhibited in hPLCs co-cultured with NAC. Thus, GGsTOP may promote hPLC migration and participate in the maintenance of the periodontal ligament apparatus via the ROS pathway.
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Affiliation(s)
- Ying Jiang
- Institute of Hard Tissue Development and Regeneration, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, P.R. China
| | - Xiang Wang
- Institute of Hard Tissue Development and Regeneration, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, P.R. China
| | - Ying Li
- Institute of Hard Tissue Development and Regeneration, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, P.R. China
| | - Sen Mu
- Institute of Hard Tissue Development and Regeneration, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, P.R. China
| | - Shuang Zhou
- Institute of Hard Tissue Development and Regeneration, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, P.R. China
| | - Yi Liu
- College of Stomatology, Tongji University, Shanghai 200072, P.R. China
| | - Bin Zhang
- Institute of Hard Tissue Development and Regeneration, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, P.R. China
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Park S, Bae DJ, Ryu YM, Kim SY, Myung SJ, Kim HJ. Mitochondria-targeting ratiometric fluorescent probe for γ-glutamyltranspeptidase and its application to colon cancer. Chem Commun (Camb) 2016; 52:10400-2. [DOI: 10.1039/c6cc05573g] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
An indocyanine-based probe for γ-glutamyltranspeptidase exhibited ratiometric fluorescence in mitochondria through an enzyme-mediated amide-to-amine transformation reaction applicable for colon cancer detection.
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Affiliation(s)
- Seokan Park
- Department of Chemistry
- Hankuk University of Foreign Studies
- Yongin 449-791
- Korea
| | - Dong Jun Bae
- ASAN Institute for Life Sciences
- ASAN Medical Center
- University of Ulsan College of Medicine
- Seoul 138-736
- Republic of Korea
| | - Yeon-Mi Ryu
- ASAN Institute for Life Sciences
- ASAN Medical Center
- University of Ulsan College of Medicine
- Seoul 138-736
- Republic of Korea
| | - Sang-Yeob Kim
- ASAN Institute for Life Sciences
- ASAN Medical Center
- University of Ulsan College of Medicine
- Seoul 138-736
- Republic of Korea
| | - Seung-Jae Myung
- ASAN Institute for Life Sciences
- ASAN Medical Center
- University of Ulsan College of Medicine
- Seoul 138-736
- Republic of Korea
| | - Hae-Jo Kim
- Department of Chemistry
- Hankuk University of Foreign Studies
- Yongin 449-791
- Korea
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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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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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Lin LL, Chen YY, Chi MC, Merlino A. Low resolution X-ray structure of γ-glutamyltranspeptidase from Bacillus licheniformis: Opened active site cleft and a cluster of acid residues potentially involved in the recognition of a metal ion. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2014; 1844:1523-9. [DOI: 10.1016/j.bbapap.2014.04.016] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/25/2014] [Revised: 04/12/2014] [Accepted: 04/21/2014] [Indexed: 12/21/2022]
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Zabot GP, Carvalhal GF, Marroni NP, Hartmann RM, Silva VDD, Fillmann HS. Glutamine prevents oxidative stress in a model of mesenteric ischemia and reperfusion. World J Gastroenterol 2014; 20:11406-11414. [PMID: 25170229 PMCID: PMC4145783 DOI: 10.3748/wjg.v20.i32.11406] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/17/2014] [Revised: 03/04/2014] [Accepted: 05/05/2014] [Indexed: 02/06/2023] Open
Abstract
AIM: To evaluate preventative effects of glutamine in an animal model of gut ischemia/reperfusion (I/R).
METHODS: Male Wistar rats were housed in a controlled environment and allowed access to food and water ad libitum. Twenty male Wistar rats were divided into four experimental groups: (1) control group (control) - rats underwent exploratory laparotomy; (2) control + glutamine group (control-GLU) - rats were subjected to laparotomy and treated intraperitoneally with glutamine 24 and 48 h prior to surgery; (3) I/R group - rats were subjected to occlusion of the superior mesenteric artery for 30 min followed by 15 min of reperfusion; and (4) ischemia/reperfusion + glutamine group (G + I/R) - rats were treated intraperitoneally with glutamine 24 and 48 h before I/R. Local and systemic injuries were determined by evaluating intestinal and lung segments for oxidative stress using lipid peroxidation and the activity of superoxide dismutase (SOD), interleukin-6 (IL-6) and nuclear factor kappa beta (NF-κB) after mesenteric I/R.
RESULTS: Lipid peroxidation of the membrane was increased in the animals subjected to I/R (P < 0.05). However, the group that received glutamine 24 and 48 h before the I/R procedure showed levels of lipid peroxidation similar to the control groups (P < 0.05). The activity of the antioxidant enzyme SOD was decreased in the gut of animals subjected to I/R when compared with the control group of animals not subjected to I/R (P < 0.05). However, the group that received glutamine 24 and 48 h before I/R showed similar SOD activity to both control groups not subjected to I/R (P < 0.05). The mean area of NF-κB staining for each of the control groups was similar. The I/R group showed the largest area of staining for NF-κB. The G + I/R group had the second highest amount of staining, but the mean value was much lower than that of the I/R group (P < 0.05). For IL-6, control and control-GLU groups showed similar areas of staining. The I/R group contained the largest area of IL-6 staining, followed by the G + I/R animals; however, this area was significantly lower than that of the group that underwent I/R without glutamine (P < 0.05).
CONCLUSION: These results demonstrate that pretreatment with glutamine prevents mucosal injury and improves gut and lung recovery after I/R injury in rats.
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Tuzova M, Jean JC, Hughey RP, Brown LAS, Cruikshank WW, Hiratake J, Joyce-Brady M. Inhibiting lung lining fluid glutathione metabolism with GGsTop as a novel treatment for asthma. Front Pharmacol 2014; 5:179. [PMID: 25132819 PMCID: PMC4116799 DOI: 10.3389/fphar.2014.00179] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2014] [Accepted: 07/14/2014] [Indexed: 12/29/2022] Open
Abstract
Asthma is characterized by airway inflammation. Inflammation is associated with oxidant stress. Airway epithelial cells are shielded from this stress by a thin layer of lung lining fluid (LLF) which contains an abundance of the antioxidant glutathione. LLF glutathione metabolism is regulated by γ-glutamyl transferase (GGT). Loss of LLF GGT activity in the mutant GGTenu1 mouse causes an increase in baseline LLF glutathione content which is magnified in an IL-13 model of allergic airway inflammation and protective against asthma. Normal mice are susceptible to asthma in this model but can be protected with acivicin, a GGT inhibitor. GGT is a target to treat asthma but acivicin toxicity limits clinical use. GGsTop is a novel GGT inhibitor. GGsTop inhibits LLF GGT activity only when delivered through the airway. In the IL-13 model, mice treated with IL-13 and GGsTop exhibit a lung inflammatory response similar to that of mice treated with IL-13 alone. But mice treated with IL-13 and GGsTop show attenuation of methacholine-stimulated airway hyper-reactivity, inhibition of Muc5ac and Muc5b gene induction, decreased airway epithelial cell mucous accumulation and a fourfold increase in LLF glutathione content compared to mice treated with IL-13 alone. Mice treated with GGsTop alone are no different from that of mice treated with saline alone, and show no signs of toxicity. GGsTop could represent a valuable pharmacological tool to inhibit LLF GGT activity in pulmonary disease models. The associated increase in LLF glutathione can protect lung airway epithelial cells against oxidant injury associated with inflammation in asthma.
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Affiliation(s)
- Marina Tuzova
- The Pulmonary Center, Boston University School of Medicine Boston, MA, USA
| | - Jyh-Chang Jean
- The Pulmonary Center, Boston University School of Medicine Boston, MA, USA
| | - Rebecca P Hughey
- Department of Medicine, University of Pittsburgh Pittsburgh, PA, USA
| | - Lou Ann S Brown
- Department of Pediatrics, Emory University School of Medicine Atlanta, GA, USA
| | | | - Jun Hiratake
- Institute for Chemical Research, Kyoto University Kyoto, Japan
| | - Martin Joyce-Brady
- The Pulmonary Center, Boston University School of Medicine Boston, MA, USA
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The cyclin-dependent kinase inhibitor p21 is essential for the beneficial effects of renal ischemic preconditioning on renal ischemia/reperfusion injury in mice. Kidney Int 2014; 85:871-9. [DOI: 10.1038/ki.2013.496] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2013] [Revised: 07/23/2013] [Accepted: 08/22/2013] [Indexed: 01/02/2023]
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Sadek EM, Afifi NM, Elfattah LIA, Mohsen MAAE. Histological study on effect of mesenchymal stem cell therapy on experimental renal injury induced by ischemia/reperfusion in male albino rat. Int J Stem Cells 2013; 6:55-66. [PMID: 24298374 DOI: 10.15283/ijsc.2013.6.1.55] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/25/2013] [Indexed: 12/24/2022] Open
Abstract
BACKGROUND AND OBJECTIVES Acute kidney injury (AKI) represents a major clinical problem with high mortality and limited treatment protocols. This study was planned to evaluate the therapeutic effectiveness of bone marrow - derived mesenchymal stem cells (BM-MSCs) in a rat model of ischemia/reperfusion (I/R) AKI. METHODS AND RESULTS This study was carried out on thirty adult male albino rats. Animals were divided equally into three groups. Group I (control sham-operated group) (n=10), were subdivided equally into two subgroups; Ia and Ib. The experimental group (n=20) were all subjected to I/R injury by clamping both renal pedicles for 40 minutes. Half of the I/R animals did not receive MSC therapy (group II) [non-MSC treated group]. The other half of the I/R animals received single intravenous injection of PKH26 labelled BM-MSCs immediately after removal of the clamps and visual confirmation of reflow (group III) [MSC treated group]. Animals were sacrificed 24 hrs (subgroups IIa & IIIa) and 72 hrs (subgroups IIb & IIIb) after intervention. Serological measurements included serum urea and creatinine. Kidney specimens were processed for H&E, PAS and PCNA. Mean % of renal corpuscles with affected glomeruli, mean % of affected tubules, mean area % of PAS-positive reaction and mean area % of PCNA immunoreactivity were measured by histomorphometric studies and statistically compared. MSCs-treated group exhibited protection against renal injury serologically and histologically. CONCLUSIONS Results of the present study suggest a potential reno-protective capacity of MSCs which could be of considerable therapeutic promise for cell-based management of clinical AKI.
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Affiliation(s)
- Eman Mostafa Sadek
- Department of Histology, Faculty of Medicine, Cairo University, Cairo, Egypt
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Koyama T, Tawa M, Yamagishi N, Tsubota A, Sawano T, Ohkita M, Matsumura Y. Role of superoxide production in post-ischemic cardiac dysfunction and norepinephrine overflow in rat hearts. Eur J Pharmacol 2013; 711:36-41. [PMID: 23628722 DOI: 10.1016/j.ejphar.2013.04.019] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2012] [Revised: 04/04/2013] [Accepted: 04/04/2013] [Indexed: 10/26/2022]
Abstract
Reactive oxygen species and norepinephrine are known as physiological active substances which cause cell damage and cardiac dysfunction in myocardial ischemia/reperfusion injury. We investigated the role of reactive oxygen species, especially superoxide (O2(-)), in ischemia-induced norepinephrine overflow and cardiac dysfunction using superoxide scavengers tempol and tiron. According to the Langendorff technique, isolated rat hearts were subjected to 40-min global ischemia followed by 30-min reperfusion. Tempol (10 and 100 µM) and tiron (100 and 500 µM) were perfused 15 min before ischemia and during reperfusion. Cardiac levels of oxidative stress markers such as O2(-) and malondialdehyde were notably increased during ischemia and following reperfusion, which were suppressed by the administration of tempol or tiron. These agents significantly improved ischemia/reperfusion-induced cardiac dysfunction such as decreased left ventricular developed pressure and the maximum and minimum value of the first derivative of left ventricular pressure and increased left ventricular end-diastolic pressure. Furthermore, norepinephrine overflow in the coronary effluent after ischemia/reperfusion was significantly suppressed by the administration of each agent. These results suggest that endogenously increased O2(-) is involved in norepinephrine overflow and cardiac dysfunction after myocardial ischemia/reperfusion.
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Affiliation(s)
- Takeshi Koyama
- Laboratory of Pathological and Molecular Pharmacology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
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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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Liao ZX, Peng SF, Ho YC, Mi FL, Maiti B, Sung HW. Mechanistic study of transfection of chitosan/DNA complexes coated by anionic poly(γ-glutamic acid). Biomaterials 2012; 33:3306-15. [PMID: 22281422 DOI: 10.1016/j.biomaterials.2012.01.013] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2011] [Accepted: 01/05/2012] [Indexed: 11/24/2022]
Abstract
Chitosan (CS) has been investigated as a non-viral carrier for gene delivery, but resulting in a relatively low transfection. To address this concern, we developed a ternary system comprised the core of CS/DNA complex and the outer coating of an anionic polymer, poly(γ-glutamic acid) (γ-PGA). In molecular dynamic (MD) simulations, we found that γ-PGA was entangle tightly with the excess CS emanating from the surface of test complexes, thus making them more compact. With γ-PGA coating, the extent of test complexes internalized and their transfection efficiency were evidently enhanced. Trypsin treatment induced a concentration-dependent decrease in internalization of the γ-PGA-coated complexes, suggesting a specific protein-mediated endocytosis. The endocytosis inhibition study indicates that the γ-glutamyl transpeptidase (GGT) present on cell membranes was responsible for the uptake of test complexes. The amine group in the N-terminal γ-glutamyl unit on γ-PGA played an essential role in the interaction with GGT. When entangled with CS, the free N-terminal γ-glutamyl unit of γ-PGA on test complexes was exposed and might thus be accommodated within the γ-glutamyl binding pocket of the membrane GGT. Above results suggest that the γ-PGA coating on CS/DNA complexes can significantly enhance their cellular uptake via a specific GGT-mediated pathway. Knowledge of the uptake mechanism is crucial for the development of an efficient vector for gene transfection.
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Affiliation(s)
- Zi-Xian Liao
- Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan 30013, ROC
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