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Zhou M, Hanschmann EM, Römer A, Linn T, Petry SF. The significance of glutaredoxins for diabetes mellitus and its complications. Redox Biol 2024; 71:103043. [PMID: 38377787 PMCID: PMC10891345 DOI: 10.1016/j.redox.2024.103043] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2023] [Accepted: 01/13/2024] [Indexed: 02/22/2024] Open
Abstract
Diabetes mellitus is a non-communicable metabolic disease hallmarked by chronic hyperglycemia caused by beta-cell failure. Diabetic complications affect the vasculature and result in macro- and microangiopathies, which account for a significantly increased morbidity and mortality. The rising incidence and prevalence of diabetes is a major global health burden. There are no feasible strategies for beta-cell preservation available in daily clinical practice. Therefore, patients rely on antidiabetic drugs or the application of exogenous insulin. Glutaredoxins (Grxs) are ubiquitously expressed and highly conserved members of the thioredoxin family of proteins. They have specific functions in redox-mediated signal transduction, iron homeostasis and biosynthesis of iron-sulfur (FeS) proteins, and the regulation of cell proliferation, survival, and function. The involvement of Grxs in chronic diseases has been a topic of research for several decades, suggesting them as therapeutic targets. Little is known about their role in diabetes and its complications. Therefore, this review summarizes the available literature on the significance of Grxs in diabetes and its complications. In conclusion, Grxs are differentially expressed in the endocrine pancreas and in tissues affected by diabetic complications, such as the heart, the kidneys, the eye, and the vasculature. They are involved in several pathways essential for insulin signaling, metabolic inflammation, glucose and fatty acid uptake and processing, cell survival, and iron and mitochondrial metabolism. Most studies describe significant changes in glutaredoxin expression and/or activity in response to the diabetic metabolism. In general, mitigated levels of Grxs are associated with oxidative distress, cell damage, and even cell death. The induced overexpression is considered a potential part of the cellular stress-response, counteracting oxidative distress and exerting beneficial impact on cell function such as insulin secretion, cytokine expression, and enzyme activity.
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Affiliation(s)
- Mengmeng Zhou
- Clinical Research Unit, Medical Clinic and Polyclinic III, Center of Internal Medicine, Justus Liebig University, Giessen, Germany
| | - Eva-Maria Hanschmann
- Experimental and Translational Research, Department of Otorhinolaryngology, University Hospital Essen, Essen, Germany
| | - Axel Römer
- Clinical Research Unit, Medical Clinic and Polyclinic III, Center of Internal Medicine, Justus Liebig University, Giessen, Germany
| | - Thomas Linn
- Clinical Research Unit, Medical Clinic and Polyclinic III, Center of Internal Medicine, Justus Liebig University, Giessen, Germany
| | - Sebastian Friedrich Petry
- Clinical Research Unit, Medical Clinic and Polyclinic III, Center of Internal Medicine, Justus Liebig University, Giessen, Germany.
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2
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Jo JH, Kim SA, Lee JH, Park YR, Kim C, Park SB, Jung DE, Lee HS, Chung MJ, Song SY. GLRX3, a novel cancer stem cell-related secretory biomarker of pancreatic ductal adenocarcinoma. BMC Cancer 2021; 21:1241. [PMID: 34794402 PMCID: PMC8603516 DOI: 10.1186/s12885-021-08898-y] [Citation(s) in RCA: 7] [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: 06/22/2021] [Accepted: 10/13/2021] [Indexed: 12/02/2022] Open
Abstract
BACKGROUND Cancer stem cells (CSCs) are implicated in carcinogenesis, cancer progression, and recurrence. Several biomarkers have been described for pancreatic ductal adenocarcinoma (PDAC) CSCs; however, their function and mechanism remain unclear. METHOD In this study, secretome analysis was performed in pancreatic CSC-enriched spheres and control adherent cells for biomarker discovery. Glutaredoxin3 (GLRX3), a novel candidate upregulated in spheres, was evaluated for its function and clinical implication. RESULTS PDAC CSC populations, cell lines, patient tissues, and blood samples demonstrated GLRX3 overexpression. In contrast, GLRX3 silencing decreased the in vitro proliferation, migration, clonogenicity, and sphere formation of cells. GLRX3 knockdown also reduced tumor formation and growth in vivo. GLRX3 was found to regulate Met/PI3K/AKT signaling and stemness-related molecules. ELISA results indicated GLRX3 overexpression in the serum of patients with PDAC compared to that in healthy controls. The sensitivity and specificity of GLRX3 for PDAC diagnosis were 80.0 and 100%, respectively. When GLRX3 and CA19-9 were combined, sensitivity was significantly increased to 98.3% compared to that with GLRX3 or CA19-9 alone. High GLRX3 expression was also associated with poor disease-free survival in patients receiving curative surgery. CONCLUSION Overall, these results indicate GLRX3 as a novel diagnostic marker and therapeutic target for PDAC targeting CSCs.
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MESH Headings
- Animals
- CA-19-9 Antigen/metabolism
- Carcinoma, Pancreatic Ductal/diagnosis
- Carcinoma, Pancreatic Ductal/metabolism
- Carcinoma, Pancreatic Ductal/mortality
- Carcinoma, Pancreatic Ductal/pathology
- Carrier Proteins/genetics
- Carrier Proteins/metabolism
- Cell Line, Tumor
- Cell Movement
- Cell Proliferation
- Disease-Free Survival
- Gene Silencing
- Humans
- Male
- Mice
- Mice, Inbred NOD
- Mice, SCID
- Neoplasm Proteins/metabolism
- Neoplastic Stem Cells/metabolism
- Pancreatic Neoplasms/diagnosis
- Pancreatic Neoplasms/metabolism
- Pancreatic Neoplasms/mortality
- Pancreatic Neoplasms/pathology
- Phosphatidylinositol 3-Kinases/metabolism
- Proto-Oncogene Proteins c-akt/metabolism
- Proto-Oncogene Proteins c-met/metabolism
- RNA, Small Interfering
- Secretome
- Sensitivity and Specificity
- Spheroids, Cellular/metabolism
- Spheroids, Cellular/pathology
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Affiliation(s)
- Jung Hyun Jo
- Division of Gastroenterology, Department of Internal Medicine, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, South Korea
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, 03722, South Korea
| | - Sun A Kim
- Cowell Biodigm Co., Ltd, Seoul, South Korea
| | - Jeong Hoon Lee
- Department of Biomedical Systems Informatics, Yonsei University College of Medicine, Seoul, 03722, South Korea
| | - Yu Rang Park
- Department of Biomedical Systems Informatics, Yonsei University College of Medicine, Seoul, 03722, South Korea
| | - Chanyang Kim
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, 03722, South Korea
| | - Soo Been Park
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, 03722, South Korea
| | - Dawoon E Jung
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, 03722, South Korea
| | - Hee Seung Lee
- Division of Gastroenterology, Department of Internal Medicine, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, South Korea
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, 03722, South Korea
| | - Moon Jae Chung
- Division of Gastroenterology, Department of Internal Medicine, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, South Korea
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, 03722, South Korea
| | - Si Young Song
- Division of Gastroenterology, Department of Internal Medicine, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, South Korea.
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, 03722, South Korea.
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3
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Ogata FT, Branco V, Vale FF, Coppo L. Glutaredoxin: Discovery, redox defense and much more. Redox Biol 2021; 43:101975. [PMID: 33932870 PMCID: PMC8102999 DOI: 10.1016/j.redox.2021.101975] [Citation(s) in RCA: 83] [Impact Index Per Article: 20.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2020] [Revised: 04/07/2021] [Accepted: 04/10/2021] [Indexed: 01/15/2023] Open
Abstract
Glutaredoxin, Grx, is a small protein containing an active site cysteine pair and was discovered in 1976 by Arne Holmgren. The Grx system, comprised of Grx, glutathione, glutathione reductase, and NADPH, was first described as an electron donor for Ribonucleotide Reductase but, from the first discovery in E.coli, the Grx family has impressively grown, particularly in the last two decades. Several isoforms have been described in different organisms (from bacteria to humans) and with different functions. The unique characteristic of Grxs is their ability to catalyse glutathione-dependent redox regulation via glutathionylation, the conjugation of glutathione to a substrate, and its reverse reaction, deglutathionylation. Grxs have also recently been enrolled in iron sulphur cluster formation. These functions have been implied in various physiological and pathological conditions, from immune defense to neurodegeneration and cancer development thus making Grx a possible drug target. This review aims to give an overview on Grxs, starting by a phylogenetic analysis of vertebrate Grxs, followed by an analysis of the mechanisms of action, the specific characteristics of the different human isoforms and a discussion on aspects related to human physiology and diseases.
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Affiliation(s)
- Fernando T Ogata
- Department of Biochemistry/Molecular Biology, CTCMol, Universidade Federal de São Paulo, Rua Mirassol, 207. 04044-010, São Paulo - SP, Brazil
| | - Vasco Branco
- Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisboa, Portugal
| | - Filipa F Vale
- Host-Pathogen Interactions Unit, Research Institute for Medicines (iMed-ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisboa, Portugal
| | - Lucia Coppo
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Solnavägen 9, SE-17165, Stockholm, Sweden.
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Son S, Kim H, Lee KS, Kim S, Park SR. Rice glutaredoxin GRXS15 confers broad-spectrum resistance to Xanthomonas oryzae pv. oryzae and Fusarium fujikuroi. Biochem Biophys Res Commun 2020; 533:1385-1392. [DOI: 10.1016/j.bbrc.2020.10.027] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Accepted: 10/12/2020] [Indexed: 02/04/2023]
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Pandya P, Isakov N. PICOT promotes T lymphocyte proliferation by down-regulating cyclin D2 expression. World J Immunol 2020; 10:1-12. [DOI: 10.5411/wji.v10.i1.1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/25/2020] [Revised: 05/09/2020] [Accepted: 05/13/2020] [Indexed: 02/06/2023] Open
Abstract
The mammalian protein kinase C-interacting cousin of thioredoxin (PICOT; also termed glutaredoxin 3) is a multi-domain monothiol glutaredoxin that is involved in a wide variety of signaling pathways and biological processes. PICOT is required for normal and transformed cell growth and is critical for embryonic development. Recent studies in T lymphocytes demonstrated that PICOT can translocate to the nucleus and interact with embryonic ectoderm development, a polycomb group protein and a core component of the polycomb repressive complex 2, which contributes to the maintenance of transcriptional repression and chromatin remodeling. Furthermore, PICOT was found to interact with chromatin-bound embryonic ectoderm development and alter the extent of histone 3 lysine 27 trimethylation at the promoter region of selected polycomb repressive complex 2 target genes. PICOT knockdown in Jurkat T cells led to increased histone 3 lysine 27 trimethylation at the promoter region of CCND2, a cell cycle-regulating gene which encodes the cyclin D2 protein. As a result, the expression levels of CCND2 mRNA and protein levels were reduced, concomitantly with inhibition of the cell growth rate. Analysis of multiple data sets from the Cancer Genome Atlas revealed that a high expression of PICOT correlated with a low expression of CCND2 in a large number of human cancers. In addition, this parameter correlated with poor patient survival, suggesting that the ratio between PICOT/CCND2 mRNA levels might serve as a predictor of patient survival in selected types of human cancer.
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Affiliation(s)
- Pinakin Pandya
- The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, Beer Sheva 84105, Israel
- Department of Computational and System biology, UPMC Hillman Cancer Center, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15232, United States
| | - Noah Isakov
- The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, Beer Sheva 84105, Israel
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6
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Pandya P, Jethva M, Rubin E, Birnbaum RY, Braiman A, Isakov N. PICOT binding to chromatin-associated EED negatively regulates cyclin D2 expression by increasing H3K27me3 at the CCND2 gene promoter. Cell Death Dis 2019; 10:685. [PMID: 31527584 PMCID: PMC6746821 DOI: 10.1038/s41419-019-1935-0] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2019] [Revised: 08/08/2019] [Accepted: 08/27/2019] [Indexed: 12/14/2022]
Abstract
Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT; also termed glutaredoxin 3 (Grx3; Glrx3)) is a ubiquitous protein that can interact with the embryonic ectoderm development (EED) protein via each of its two C-terminal PICOT/Grx homology domains. Since EED is a Polycomb-Group protein and a core component of the polycomb repressive complex 2 (PRC2), we tested the involvement of PICOT in the regulation of PRC2-mediated H3 lysine 27 trimethylation (H3K27me3), transcription and translation of selected PRC2 target genes. A fraction of the cellular PICOT protein was found in the nuclei of leukemia cell lines, where it was associated with the chromatin. In addition, PICOT coimmunoprecipitated with chromatin-residing EED derived from Jurkat and COS-7 cell nuclei. PICOT knockdown led to a reduced H3K27me3 mark and a decrease in EED and EZH2 at the CCND2 gene promoter. In agreement, PICOT-deficient T cells exhibited a significant increase in CCND2 mRNA and protein expression. Since elevated expression levels of PICOT were reported in several different tumors and correlated in the current studies with decreased transcription and translation of the CCND2 gene, we tested whether this opposite correlation exists in human cancers. Data from the Cancer Genome Atlas (TCGA) database indicated statistically significant negative correlation between PICOT and CCND2 in eight different human tumors where the highest correlation was in lung (p = 8.67E−10) and pancreatic (p = 1.06E−5) adenocarcinoma. Furthermore, high expression of PICOT and low expression of CCND2 correlated with poor patient survival in five different types of human tumors. The results suggest that PICOT binding to chromatin-associated EED modulates the H3K27me3 level at the CCND2 gene promoter which may be one of the potential mechanisms for regulation of cyclin D2 expression in tumors. These findings also indicate that a low PICOT/CCND2 expression ratio might serve as a good predictor of patient survival in selected human cancers.
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Affiliation(s)
- Pinakin Pandya
- The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, 84105, Beer Sheva, Israel
| | - Minesh Jethva
- The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, 84105, Beer Sheva, Israel
| | - Eitan Rubin
- The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, 84105, Beer Sheva, Israel
| | - Ramon Y Birnbaum
- Department of Life Sciences, Ben-Gurion University of the Negev, 84105, Beer-Sheva, Israel
| | - Alex Braiman
- The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, 84105, Beer Sheva, Israel
| | - Noah Isakov
- The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, 84105, Beer Sheva, Israel.
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7
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Pandya P, Braiman A, Isakov N. PICOT (GLRX3) is a positive regulator of stress-induced DNA-damage response. Cell Signal 2019; 62:109340. [PMID: 31176019 DOI: 10.1016/j.cellsig.2019.06.005] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2019] [Revised: 05/15/2019] [Accepted: 06/04/2019] [Indexed: 12/14/2022]
Abstract
Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT; also termed glutaredoxin 3 (Glrx3)) is a ubiquitously expressed protein that possesses an N-terminal monothiol thioredoxin (Trx) domain and two C-terminal tandem copies of a monothiol Glrx domain. It has an overall highly conserved amino acid sequence and is encoded by a unique gene, both in humans and mice, without having other functional gene homologs in the entire genome. Despite being discovered almost two decades ago, the biological function of PICOT remains largely ill-defined and its ramifications are underestimated considering the fact that PICOT-deficiency in mice results in embryonic lethality. Since classical Glrxs are important regulators of the cellular redox homeostasis, we tested whether PICOT participate in the stress-induced DNA-damage response, focusing on nuclear proteins that function as integral components of the DNA repair machinery. Using wild type versus PICOT-deficient (PICOT-KD) Jurkat T cells we found that the anti-oxidant mechanism in PICOT-deficient cells is impaired, and that these cells respond to genotoxic drugs, such as etoposide and camptothecin, by increased caspase-3 activity, a reduced survival and a slower and diminished phosphorylation of the histone protein, H2AX. Nevertheless, the effect of PICOT on the drug-induced phosphorylation of H2AX was independent of the cellular levels of reactive oxygen species. PICOT-deficient cells also demonstrated reduced and slower γH2AX foci formation in response to radiation. Furthermore, immunofluorescence staining using PICOT- and γH2AX-specific Abs followed by confocal microscopy demonstrated partial localization of PICOT at the γH2AX-containing foci at the site of the DNA double strand breaks. In addition, PICOT knockdown resulted in inhibition of phosphorylation of ATR, Chk1 and Chk2 kinases, which play an essential role in the DNA-damage response and serve as upstream regulators of γH2AX. The present data suggest that PICOT protects cells from DNA damage-inducing agents by operating as an upstream positive regulator of ATR-dependent signaling pathways. By promoting the activity of ATR, PICOT indirectly regulates the phosphorylation and activation of Chk1, Chk2, and γH2AX, which are critical components of the DNA damage repair mechanism and thereby attenuate the stress- and replication-induced genome instability.
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Affiliation(s)
- Pinakin Pandya
- The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, Beer Sheva 84105, Israel
| | - Alex Braiman
- The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, Beer Sheva 84105, Israel
| | - Noah Isakov
- The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, Beer Sheva 84105, Israel..
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8
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PICOT binding to the polycomb group protein, EED, alters H3K27 methylation at the MYT1 PRC2 target gene. Biochem Biophys Res Commun 2018; 509:469-475. [PMID: 30595380 DOI: 10.1016/j.bbrc.2018.12.153] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2018] [Accepted: 12/21/2018] [Indexed: 01/09/2023]
Abstract
PICOT is a ubiquitous protein that has no functional redundant ortholog and is critical for mouse embryonic development. It is involved in the regulation of signal transduction in T lymphocytes and cardiac muscle, and in cellular iron metabolism and biogenesis of Fe/S proteins. However, very little is known about the physiological role of PICOT and its mechanism of action, and on its upstream regulators or downstream target molecules. In attempt to identify new PICOT interaction partners, we adopted the yeast two-hybrid system and screened a Jurkat T cell cDNA library using the full-length human PICOT cDNA as a bait. We found that PICOT interacts with embryonic ectoderm development (EED), a Polycomb Group (PcG) protein that serves as a core component of the Polycomb repressive complex 2 (PRC2) and contributes to the regulation of chromatin remodeling and cell differentiation. Using bead immobilized GST-PICOT and GST-EED fusion proteins in a pull-down assay and reciprocal coimmunoprecipitation studies we demonstrated that the interaction between PICOT and EED also occurs in human Jurkat T cells. In addition, immunofluorescence staining of Jurkat T cells revealed partial colocalization of PICOT and EED, predominantly in the cell nuclei. A pull-down assay using the GST-EED fusion protein and lysates of cells expressing different Myc-tagged truncation products of PICOT revealed that binding of EED is mediated by each of the two C-terminal PICOT homology domains and suggests that simultaneous interaction via both domains increases the binding affinity. Furthermore, PICOT knock-down in Jurkat T cells resulted in a reduced histone H3 lysine 27 trimethylation (H3K27me3) at the PRC2 target gene, myelin transcription factor 1 (MYT1), suggesting that PICOT binding to EED alters PRC2-regulated transcriptional repression, and potentially contributes to the epigenetic regulation of chromatin silencing and remodeling.
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He F, Wei L, Luo W, Liao Z, Li B, Zhou X, Xiao X, You J, Chen Y, Zheng S, Li P, Murata M, Huang G, Zhang Z. Glutaredoxin 3 promotes nasopharyngeal carcinoma growth and metastasis via EGFR/Akt pathway and independent of ROS. Oncotarget 2018; 7:37000-37012. [PMID: 27203742 PMCID: PMC5095054 DOI: 10.18632/oncotarget.9454] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2016] [Accepted: 04/16/2016] [Indexed: 12/14/2022] Open
Abstract
Glutaredoxin 3 (GLRX3) is antioxidant enzyme, maintaining a low level of ROS, thus contributing to the survival and metastasis of several types of cancer. However, the expression and functions of GLRX3 have not been addressed in nasopharyngeal carcinoma (NPC). In this study, we found that GLRX3 was overexpressed in NPC. Knockdown of GLRX3 in NPC cell lines inhibited proliferation in vitro, tumorignesis in vivo, and colony formation. In addition, GLRX3 knockdown decreased the migration and invasion capacity of NPC cells by reversing the epithelial-mesenchymal transition (EMT). Furthermore, stabilization of GLRX3 was positively related to with epidermal growth factor receptor (EGFR) expression and negatively with ROS generation. Phosphorylation of Akt, a key downstream effector, was induced by EGFR signaling but did not rely on increasing ROS level in NPC cells. GLRX3 might be an oncoprotein in NPC, playing important roles in increasing redox reaction and activating EGFR/ Akt signals, so it may be a therapeutic target for NPC.
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Affiliation(s)
- Feng He
- Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Lili Wei
- Department of Pathology, First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Wenqi Luo
- Department of Pathology, First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Zhipeng Liao
- Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Bo Li
- Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Xiaoying Zhou
- Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Xue Xiao
- Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Jingping You
- Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Yufeng Chen
- Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Shixing Zheng
- Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Ping Li
- Department of Pathology, First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Mariko Murata
- Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie, Japan
| | - Guangwu Huang
- Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Zhe Zhang
- Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, China
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Pujol-Carrion N, Torre-Ruiz MADL. Physical interaction between the MAPK Slt2 of the PKC1-MAPK pathway and Grx3/Grx4 glutaredoxins is required for the oxidative stress response in budding yeast. Free Radic Biol Med 2017; 103:107-120. [PMID: 28007574 DOI: 10.1016/j.freeradbiomed.2016.12.023] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/04/2016] [Revised: 12/02/2016] [Accepted: 12/18/2016] [Indexed: 01/20/2023]
Abstract
This study demonstrates that both monothiol glutaredoxins Grx3 and Grx4 physically interact with the MAPK Slt2 forming a complex involved in the cellular response to oxidative stress. The simultaneous absence of Grx3 and Grx4 provokes a serious impairment in cell viability, Slt2 activation and Rlm1 transcription in response to oxidative stress. Both in vivo and in vitro results clearly show that Slt2 can independently bind either Grx3 or Grx4 proteins. Our results suggest that Slt2 form iron/sulphur bridged clusters with Grx3 and Grx4. For the assembly of this complex, cysteines of the active site of each Grx3/4 glutaredoxins, glutathione and specific cysteine residues from Slt2 provide the ligands. One of the ligands of Slt2 is required for its dimerisation upon oxidative treatment and iron repletion. These interactions are relevant for the oxidative response, given that mutants in the cysteine ligands identified in the complex show a severe impairment of both cell viability and Slt2 phosphorylation upon oxidative stress. Grx4 is the relevant glutaredoxin that regulates Slt2 phosphorylation under oxidative conditions precluding cell survival. Our studies contribute to extend the functions of both monothiol glutaredoxins to the regulation of a MAPK in the context of the oxidative stress response.
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Affiliation(s)
- Nuria Pujol-Carrion
- Department of Basic Medical Sciences, IRB-Lleida. University of Lleida, Av. Alcalde Rovira Roure no 80, 25198 Lleida, Spain
| | - Maria Angeles de la Torre-Ruiz
- Department of Basic Medical Sciences, IRB-Lleida. University of Lleida, Av. Alcalde Rovira Roure no 80, 25198 Lleida, Spain.
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11
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Dai X, Li Y, Sun X, Cai K, Mao Q, Xia H. Generation of Domain-Specific Monoclonal Antibodies Against Human Glutaredoxin3. Monoclon Antib Immunodiagn Immunother 2016; 35:285-292. [PMID: 27923109 DOI: 10.1089/mab.2016.0032] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Human Glutaredoxin3 (hGLRX3), which encodes a 37.4 kDa protein, possesses an N-terminal Trx homology domain followed by two tandem repeats of Grx domains. GLRX3 is expressed in many tissues and plays important roles in iron metabolism, antioxidant effect, cell proliferation and development, regulation of immune reaction, and tumorigenesis. The mechanisms underlying the biological function of GLRX3 are still not clear. To facilitate the functional research of GLRX3, in this study, monoclonal antibodies (MAbs) against hGLRX3 were produced by using purified prokaryotic recombinant 6His-hGLRX3 fusion protein as the immunogen. Five MAbs were obtained after preliminary screening by indirect enzyme-linked immunosorbent assay, then further characterized by Western blot analysis and immunocytochemistry. The domain specificity of these MAbs was also evaluated. Owing to the high conservation of protein sequences among different species, anti-GLRX3 MAbs produced in this study were shown to be immunoactive for GLRX3 in the cells from other species, such as mice, rats, Chinese hamster, and zebrafish. These domain-specific anti-GLRX3 MAbs will be an essential tool to investigate the roles of GLRX3 in normal physiological or pathological conditions.
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Affiliation(s)
- Xin Dai
- 1 Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University , Xi'an, China
| | - Yanqing Li
- 1 Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University , Xi'an, China
| | - Xiaohong Sun
- 1 Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University , Xi'an, China
| | - Kai Cai
- 1 Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University , Xi'an, China
| | - Qinwen Mao
- 2 Department of Pathology, Northwestern University Feinberg School of Medicine , Chicago, Illinois
| | - Haibin Xia
- 1 Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University , Xi'an, China
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Song X, Yan M, Hu D, Wang Y, Wang N, Gu X, Peng X, Yang G. Molecular characterization and serodiagnostic potential of a novel dithiol glutaredoxin 1 from Echinococcus granulosus. Parasit Vectors 2016; 9:456. [PMID: 27535033 PMCID: PMC4989415 DOI: 10.1186/s13071-016-1741-9] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2016] [Accepted: 08/04/2016] [Indexed: 01/21/2023] Open
Abstract
Background The larval stage of Echinococcus granulosus is the etiological agent of cystic echinococcosis (CE), which causes serious morbidity and mortality in many areas. There is no reliable method to monitor sheep CE. Here, we characterize E. granulosus glutaredoxin 1 (Eg-Grx1) and report an improved immunodiagnostic method for CE. Methods We cloned and expressed recombinant Eg-Grx1 and generated antibodies. We analyzed the location of the protein in different parasite stages by fluorescence immunohistochemistry, detected the immunogenicity of recombinant Eg-Grx1, and developed an indirect ELISA (iELISA) for CE serodiagnosis. Results Eg-Grx1 is a classic dithiol Grx with several GSH-binding motifs. Native Eg-Grx1 protein was distributed in the tegument of protoscoleces, the whole germinal layer, and the parenchymatous tissue of adult worms. Recombinant Eg-Grx1 exhibited good immunoreactivity to CE-infected sheep serum. An iELISA using this antigen showed specificity of 64.3 % (9/14) and sensitivity of 1:3200, and the diagnostic accordance rate was 97.9 % (47/48) compared with the results of necropsy. Conclusion We characterized a novel Grx (Eg-Grx1) from a parasitic helminth and present a comprehensive analysis of the sequence and structure of this protein. The recombinant Eg-Grx1 protein showed good potential serodiagnostic performance, and we established an iELISA method, which may contribute to the surveillance of sheep CE in epidemic areas.
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Affiliation(s)
- Xingju Song
- Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Min Yan
- Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Dandan Hu
- Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Yu Wang
- Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Ning Wang
- Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Xiaobin Gu
- Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Xuerong Peng
- Department of Chemistry, College of Life and Basic Science, Sichuan Agricultural University, Ya'an, China
| | - Guangyou Yang
- Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.
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13
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Vall-Llaura N, Reverter-Branchat G, Vived C, Weertman N, Rodríguez-Colman MJ, Cabiscol E. Reversible glutathionylation of Sir2 by monothiol glutaredoxins Grx3/4 regulates stress resistance. Free Radic Biol Med 2016; 96:45-56. [PMID: 27085841 DOI: 10.1016/j.freeradbiomed.2016.04.008] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/28/2015] [Revised: 03/31/2016] [Accepted: 04/01/2016] [Indexed: 12/22/2022]
Abstract
The regulatory mechanisms of yeast Sir2, the founding member of the sirtuin family involved in oxidative stress and aging, are unknown. Redox signaling controls many cellular functions, especially under stress situations, with dithiol glutaredoxins (Grxs) playing an important role. However, monothiol Grxs are not considered to have major oxidoreductase activity. The present study investigated the redox regulation of yeast Sir2, together with the role and physiological impact of monothiol Grx3/4 as Sir2 thiol-reductases upon stress. S-glutathionylation of Sir2 upon disulfide stress was demonstrated both in vitro and in vivo, and decreased Sir2 deacetylase activity. Physiological levels of nuclear Grx3/4 can reverse the observed post-translational modification. Grx3/4 interacted with Sir2 and reduced it after stress, thereby restoring telomeric silencing activity. Using site-directed mutagenesis, key cysteine residues at the catalytic domain of Sir2 were identified as a target of S-glutathionylation. Mutation of these residues resulted in cells with increased resistance to disulfide stress. We provide new mechanistic insights into Grx3/4 regulation of Sir2 by S-deglutathionylation to increase cell resistance to stress. This finding offers news perspectives on monothiol Grxs in redox signaling, describing Sir2 as a physiological substrate regulated by S-glutathionylation. These results might have a relevant role in understanding aging and age-related diseases.
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Affiliation(s)
- Núria Vall-Llaura
- Departament de Ciències Mèdiques Bàsiques, IRBLleida, Universitat de Lleida, Edifici Biomedicina I, Av. Alcalde Rovira Roure, 80, 25198 Lleida, Catalonia, Spain
| | - Gemma Reverter-Branchat
- Departament de Ciències Mèdiques Bàsiques, IRBLleida, Universitat de Lleida, Edifici Biomedicina I, Av. Alcalde Rovira Roure, 80, 25198 Lleida, Catalonia, Spain
| | - Celia Vived
- Departament de Ciències Mèdiques Bàsiques, IRBLleida, Universitat de Lleida, Edifici Biomedicina I, Av. Alcalde Rovira Roure, 80, 25198 Lleida, Catalonia, Spain
| | - Naomi Weertman
- Departament de Ciències Mèdiques Bàsiques, IRBLleida, Universitat de Lleida, Edifici Biomedicina I, Av. Alcalde Rovira Roure, 80, 25198 Lleida, Catalonia, Spain
| | - María José Rodríguez-Colman
- Departament de Ciències Mèdiques Bàsiques, IRBLleida, Universitat de Lleida, Edifici Biomedicina I, Av. Alcalde Rovira Roure, 80, 25198 Lleida, Catalonia, Spain
| | - Elisa Cabiscol
- Departament de Ciències Mèdiques Bàsiques, IRBLleida, Universitat de Lleida, Edifici Biomedicina I, Av. Alcalde Rovira Roure, 80, 25198 Lleida, Catalonia, Spain.
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14
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Pham K, Pal R, Qu Y, Liu X, Yu H, Shiao SL, Wang X, O'Brian Smith E, Cui X, Rodney GG, Cheng N. Nuclear glutaredoxin 3 is critical for protection against oxidative stress-induced cell death. Free Radic Biol Med 2015; 85:197-206. [PMID: 25975981 PMCID: PMC4902114 DOI: 10.1016/j.freeradbiomed.2015.05.003] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/15/2014] [Revised: 04/17/2015] [Accepted: 05/01/2015] [Indexed: 01/12/2023]
Abstract
Mammalian glutaredoxin 3 (Grx3) has been shown to be critical in maintaining redox homeostasis and regulating cell survival pathways in cancer cells. However, the regulation of Grx3 is not fully understood. In the present study, we investigate the subcellular localization of Grx3 under normal growth and oxidative stress conditions. Both fluorescence imaging of Grx3-RFP fusion and Western blot analysis of cellular fractionation indicate that Grx3 is predominantly localized in the cytoplasm under normal growth conditions, whereas under oxidizing conditions, Grx3 is translocated into and accumulated in the nucleus. Grx3 nuclear accumulation was reversible in a redox-dependent fashion. Further analysis indicates that neither the N-terminal Trx-like domain nor the two catalytic cysteine residues in the active CGFS motif of Grx3 are involved in its nuclear translocation. Decreased levels of Grx3 render cells susceptible to cellular oxidative stress, whereas overexpression of nuclear-targeted Grx3 is sufficient to suppress cells' sensitivity to oxidant treatments and reduce reactive oxygen species production. These findings provide novel insights into the regulation of Grx3, which is crucial for cell survival against environmental insults.
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Affiliation(s)
- Khanh Pham
- USDA/ARS Children׳s Nutrition Research Center, Department of Pediatrics, Houston, TX 77030, USA
| | - Rituraj Pal
- Department of Molecular Physiology & Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
| | - Ying Qu
- Department of Surgery and Department of Obstetrics and Gynecology, Samuel Oschin Comprehensive Cancer Institute, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Xi Liu
- Ministry of Education Key Laboratory of Protein Science, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China
| | - Han Yu
- USDA/ARS Children׳s Nutrition Research Center, Department of Pediatrics, Houston, TX 77030, USA
| | - Stephen L Shiao
- Radiation Oncology and Biochemical Sciences, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Xinquan Wang
- Ministry of Education Key Laboratory of Protein Science, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China
| | - E O'Brian Smith
- USDA/ARS Children׳s Nutrition Research Center, Department of Pediatrics, Houston, TX 77030, USA
| | - Xiaojiang Cui
- Department of Surgery and Department of Obstetrics and Gynecology, Samuel Oschin Comprehensive Cancer Institute, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA.
| | - George G Rodney
- Department of Molecular Physiology & Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
| | - Ninghui Cheng
- USDA/ARS Children׳s Nutrition Research Center, Department of Pediatrics, Houston, TX 77030, USA.
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15
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Couturier J, Przybyla-Toscano J, Roret T, Didierjean C, Rouhier N. The roles of glutaredoxins ligating Fe–S clusters: Sensing, transfer or repair functions? BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2015; 1853:1513-27. [DOI: 10.1016/j.bbamcr.2014.09.018] [Citation(s) in RCA: 85] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/07/2014] [Revised: 09/17/2014] [Accepted: 09/18/2014] [Indexed: 01/05/2023]
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16
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Yeung N, Gold B, Liu NL, Prathapam R, Sterling HJ, Willams ER, Butland G. The E. coli monothiol glutaredoxin GrxD forms homodimeric and heterodimeric FeS cluster containing complexes. Biochemistry 2011; 50:8957-69. [PMID: 21899261 DOI: 10.1021/bi2008883] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
Monothiol glutaredoxins (mono-Grx) represent a highly evolutionarily conserved class of proteins present in organisms ranging from prokaryotes to humans. Mono-Grxs have been implicated in iron sulfur (FeS) cluster biosynthesis as potential scaffold proteins and in iron homeostasis via an FeS-containing complex with Fra2p (homologue of E. coli BolA) in yeast and are linked to signal transduction in mammalian systems. However, the function of the mono-Grx in prokaryotes and the nature of an interaction with BolA-like proteins have not been established. Recent genome-wide screens for E. coli genetic interactions reported the synthetic lethality (combination of mutations leading to cell death; mutation of only one of these genes does not) of a grxD mutation when combined with strains defective in FeS cluster biosynthesis (isc operon) functions [Butland, G., et al. (2008) Nature Methods 5, 789-795]. These data connected the only E. coli mono-Grx, GrxD to a potential role in FeS cluster biosynthesis. We investigated GrxD to uncover the molecular basis of this synthetic lethality and observed that GrxD can form FeS-bound homodimeric and BolA containing heterodimeric complexes. These complexes display substantially different spectroscopic and functional properties, including the ability to act as scaffold proteins for intact FeS cluster transfer to the model [2Fe-2S] acceptor protein E. coli apo-ferredoxin (Fdx), with the homodimer being significantly more efficient. In this work, we functionally dissect the potential cellular roles of GrxD as a component of both homodimeric and heterodimeric complexes to ultimately uncover if either of these complexes performs functions linked to FeS cluster biosynthesis.
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Affiliation(s)
- N Yeung
- Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
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17
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Glutaredoxins Grx4 and Grx3 of Saccharomyces cerevisiae play a role in actin dynamics through their Trx domains, which contributes to oxidative stress resistance. Appl Environ Microbiol 2010; 76:7826-35. [PMID: 20889785 DOI: 10.1128/aem.01755-10] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Grx3 and Grx4 are two monothiol glutaredoxins of Saccharomyces cerevisiae that have previously been characterized as regulators of Aft1 localization and therefore of iron homeostasis. In this study, we present data showing that both Grx3 and Grx4 have new roles in actin cytoskeleton remodeling and in cellular defenses against oxidative stress caused by reactive oxygen species (ROS) accumulation. The Grx4 protein plays a unique role in the maintenance of actin cable integrity, which is independent of its role in the transcriptional regulation of Aft1. Grx3 plays an additive and redundant role, in combination with Grx4, in the organization of the actin cytoskeleton, both under normal conditions and in response to external oxidative stress. Each Grx3 and Grx4 protein contains a thioredoxin domain sequence (Trx), followed by a glutaredoxin domain (Grx). We performed functional analyses of each of the two domains and characterized different functions for them. Each of the two Grx domains plays a role in ROS detoxification and cell viability. However, the Trx domain of each Grx4 and Grx3 protein acts independently of its respective Grx domain in a novel function that involves the polarization of the actin cytoskeleton, which also determines cell resistance against oxidative conditions. Finally, we present experimental evidence demonstrating that Grx4 behaves as an antioxidant protein increasing cell survival under conditions of oxidative stress.
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18
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Ohayon A, Babichev Y, Galperin M, Altman A, Isakov N. Widespread expression of PICOT in mouse and human tissues with predominant localization to epithelium. J Histochem Cytochem 2010; 58:799-806. [PMID: 20498481 DOI: 10.1369/jhc.2010.956532] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
The protein kinase C-interacting cousin of thioredoxin (PICOT; also termed glutaredoxin 3) protein was discovered a decade ago as a protein kinase C theta (PKCtheta)-binding protein in human T lymphocytes. PICOT possesses an amino-terminal monothiol thioredoxin-like domain and a carboxy-terminal tandem repeat of a monothiol glutaredoxin-like domain. Nevertheless, the enzymatic activities of PICOT and its potential substrates have not yet been characterized and its biological importance is unknown. Earlier studies reported the presence of PICOT in several different cell lines and tissues, but its expression pattern has not been thoroughly investigated. We performed Northern blot analysis of 19 different human organs and tissues and found the expression of PICOT mRNA in all organs and tissues tested. Western blot analysis confirmed the expression of PICOT at the protein level in all organs and tissues tested and showed, in addition, that PICOT and PKCtheta expression in different tissues only partially overlap. These findings support the involvement of PICOT in biological functions that are independent of PKCtheta. To analyze the in vivo expression pattern of PICOT within cells of different human organs, we performed immunohistochemical staining using PICOT-specific antibodies. Analysis of breast, pituitary, adrenal, pancreas, and kidney sections demonstrated a differential expression of PICOT in various cell types, with a predominant cytosolic staining of epithelial cells and low or undetectable levels of PICOT in the stroma.
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Affiliation(s)
- Ariel Ohayon
- Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel
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19
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Haunhorst P, Berndt C, Eitner S, Godoy JR, Lillig CH. Characterization of the human monothiol glutaredoxin 3 (PICOT) as iron-sulfur protein. Biochem Biophys Res Commun 2010; 394:372-6. [PMID: 20226171 DOI: 10.1016/j.bbrc.2010.03.016] [Citation(s) in RCA: 79] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2010] [Accepted: 03/03/2010] [Indexed: 11/16/2022]
Abstract
Mammalian glutaredoxin 3 (Grx3/PICOT) is an essential protein involved in the regulation of signal transduction, for instance during immune cell activation and development of cardiac hypertrophy, presumably in response to redox signals. This function requires the sensing of such stresses by a hitherto unknown mechanism. Here, we characterized Grx3/PICOT as iron-sulfur protein. The protein binds two bridging [2Fe-2S] clusters in a homodimeric complex with the active site cysteinyl residues of its two monothiol glutaredoxin domains and glutathione bound non-covalently to the Grx domains. Co-immunoprecipitation of 55-iron with Grx3/PICOT from Jurkat cells suggested the presence of these cofactors under physiological conditions. The [2Fe-2S]2+ clusters were not redox active, instead they were lost upon treatment of the holo protein with ferricyanide or S-nitroso glutathione. This redox-induced dissociation of the Grx3/PICOT holo complex may be a mechanism of Grx3/PICOT activation in response to reactive oxygen and nitrogen species.
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Affiliation(s)
- Petra Haunhorst
- Institut für Klinische Zytobiologie und Zytopathologie, Fachbereich Medizin, Philipps Universität Marburg, Germany
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20
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Ahsan MK, Lekli I, Ray D, Yodoi J, Das DK. Redox regulation of cell survival by the thioredoxin superfamily: an implication of redox gene therapy in the heart. Antioxid Redox Signal 2009; 11:2741-58. [PMID: 19583492 PMCID: PMC2821134 DOI: 10.1089/ars.2009.2683] [Citation(s) in RCA: 107] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Reactive oxygen species (ROS) are the key mediators of pathogenesis in cardiovascular diseases. Members of the thioredoxin superfamily take an active part in scavenging reactive oxygen species, thus playing an essential role in maintaining the intracellular redox status. The alteration in the expression levels of thioredoxin family members and related molecules constitute effective biomarkers in various diseases, including cardiovascular complications that involve oxidative stress. Thioredoxin, glutaredoxin, peroxiredoxin, and glutathione peroxidase, along with their isoforms, are involved in interaction with the members of metabolic and signaling pathways, thus making them attractive targets for clinical intervention. Studies with cells and transgenic animals have supported this notion and raised the hope for possible gene therapy as modern genetic medicine. Of all the molecules, thioredoxins, glutaredoxins, and peroxiredoxins are emphasized, because a growing body of evidence reveals their essential and regulatory role in several steps of redox regulation. In this review, we discuss some pertinent observations regarding their distribution, structure, functions, and interactions with the several survival- and death-signaling pathways, especially in the myocardium.
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Affiliation(s)
- Md Kaimul Ahsan
- Cardiovascular Research Center, Department of Surgery, School of Medicine, University of Connecticut Health Center , Farmington, CT 06030-1110, USA.
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21
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Cha MK, Kim IH. Preferential overexpression of glutaredoxin3 in human colon and lung carcinoma. Cancer Epidemiol 2009; 33:281-7. [PMID: 19797004 DOI: 10.1016/j.canep.2009.08.006] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2009] [Revised: 08/07/2009] [Accepted: 08/13/2009] [Indexed: 02/07/2023]
Abstract
Glutaredoxin (Glrx) uses the reducing power of glutathione to maintain and regulate the cellular redox state. Substantial evidence indicates that the alteration of cellular redox status is a critical factor involved in cell growth and death and results in tumorigenesis. We investigated levels of expression of all Glrx genes in a variety of cancers using a real-time polymerase chain reaction (RT-PCR). Among members of the Glrx, family, Glrx3 (PICOT: PKC-interacting cousin of thioredoxin) was preferentially induced in lung (55.3+/-30.1-fold induction) and colon (50.2+/-28.8-fold induction) cancer compared to their normal tissues (lung>or=colon>breast>ovary>bladder>prostate>thyroid>lymphoma>liver>or=kidney cancers). By contrast, the magnitude of induction folds in other cancer tissues was ranged from 0.83 to 4.0. Moreover, the induction folds of Glrx3 mRNA in colon and lung cancer tissues were significantly higher when compared to those of all thioredoxin (Trx) and peroxiredoxin (Prx) members. Western blot analysis of different and paired cancer tissues revealed the consistent and preferential expression of Glrx3 in lung and colon cancers. Taken together, these results suggest that Glrx3 could take a pivotal role in colon and lung cancer cells during the tumorigenesis.
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Affiliation(s)
- Mee-Kyung Cha
- Department of Biochemistry, Paichai University, Daejeon 302-735, Republic of Korea
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22
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Lillig CH, Berndt C, Holmgren A. Glutaredoxin systems. Biochim Biophys Acta Gen Subj 2008; 1780:1304-17. [DOI: 10.1016/j.bbagen.2008.06.003] [Citation(s) in RCA: 482] [Impact Index Per Article: 28.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2008] [Revised: 06/11/2008] [Accepted: 06/11/2008] [Indexed: 12/15/2022]
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Jeong D, Kim JM, Cha H, Oh JG, Park J, Yun SH, Ju ES, Jeon ES, Hajjar RJ, Park WJ. PICOT attenuates cardiac hypertrophy by disrupting calcineurin-NFAT signaling. Circ Res 2008; 102:711-9. [PMID: 18258855 DOI: 10.1161/circresaha.107.165985] [Citation(s) in RCA: 71] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PICOT (protein kinase C-interacting cousin of thioredoxin) was previously shown to inhibit pressure overload-induced cardiac hypertrophy, concomitant with an increase in ventricular function and cardiomyocyte contractility. The combined analyses of glutathione S-transferase pull-down experiments and mass spectrometry enabled us to determine that PICOT directly interacts with muscle LIM protein (MLP) via its carboxyl-terminal half (PICOT-C). It was also shown that PICOT colocalizes with MLP in the Z-disc. MLP is known to play a role in anchoring calcineurin to the Z-disc in the sarcomere, which is critical for calcineurin-NFAT (nuclear factor of activated T cells) signaling. We, therefore, suggested that PICOT may affect calcineurin-NFAT signaling through its interaction with MLP. Consistent with this hypothesis, PICOT, or more specifically PICOT-C, abrogated phenylephrine-induced increases in calcineurin phosphatase activity, NFAT dephosphorylation/nuclear translocation, and NFAT-dependent transcriptional activation in neonatal cardiomyocytes. In addition, pressure overload-induced upregulation of NFAT target genes was significantly diminished in the hearts of PICOT-overexpressing transgenic mice. PICOT interfered with MLP-calcineurin interactions in a dose-dependent manner. Moreover, calcineurin was displaced from the Z-disc, concomitant with an abrogated interaction between calcineurin and MLP, in the hearts of PICOT transgenic mice. Replenishment of MLP restored the hypertrophic responses and the increase in calcineurin phosphatase activity that was inhibited by PICOT in phenylephrine-treated cardiomyocytes. Finally, PICOT-C inhibited cardiac hypertrophy to an extent that was comparable to that of full-length PICOT. Taken together, these data suggest that PICOT inhibits cardiac hypertrophy largely by negatively regulating calcineurin-NFAT signaling via disruption of the MLP-calcineurin interaction.
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Affiliation(s)
- Dongtak Jeong
- Global Research Laboratory and Department of Life Science, Gwangju Institute of Science and Technology, Gwangju, Korea
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Fladvad M, Bellanda M, Fernandes AP, Mammi S, Vlamis-Gardikas A, Holmgren A, Sunnerhagen M. Molecular Mapping of Functionalities in the Solution Structure of Reduced Grx4, a Monothiol Glutaredoxin from Escherichia coli. J Biol Chem 2005; 280:24553-61. [PMID: 15840565 DOI: 10.1074/jbc.m500679200] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The ubiquitous glutaredoxin protein family is present in both prokaryotes and eukaryotes, and is closely related to the thioredoxins, which reduce their substrates using a dithiol mechanism as part of the cellular defense against oxidative stress. Recently identified monothiol glutaredoxins, which must use a different functional mechanism, appear to be essential in both Escherichia coli and yeast and are well conserved in higher order genomes. We have employed high resolution NMR to determine the three-dimensional solution structure of a monothiol glutaredoxin, the reduced E. coli Grx4. The Grx4 structure comprises a glutaredoxin-like alpha-beta fold, founded on a limited set of strictly conserved and structurally critical residues. A tight hydrophobic core, together with a stringent set of secondary structure elements, is thus likely to be present in all monothiol glutaredoxins. A set of exposed and conserved residues form a surface region, implied in glutathione binding from a known structure of E. coli Grx3. The absence of glutaredoxin activity in E. coli Grx4 can be understood based on small but significant differences in the glutathione binding region, and through the lack of a conserved second GSH binding site. MALDI experiments suggest that disulfide formation on glutathionylation is accompanied by significant structural changes, in contrast with dithiol thioredoxins and glutaredoxins, where differences between oxidized and reduced forms are subtle and local. Structural and functional implications are discussed with particular emphasis on identifying common monothiol glutaredoxin properties in substrate specificity and ligand binding events, linking the thioredoxin and glutaredoxin systems.
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Affiliation(s)
- Malin Fladvad
- Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden
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Molina MM, Bellí G, de la Torre MA, Rodríguez-Manzaneque MT, Herrero E. Nuclear Monothiol Glutaredoxins of Saccharomyces cerevisiae Can Function as Mitochondrial Glutaredoxins. J Biol Chem 2004; 279:51923-30. [PMID: 15456753 DOI: 10.1074/jbc.m410219200] [Citation(s) in RCA: 79] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Glutaredoxins are thiol oxidoreductases that regulate protein redox state. In Saccharomyces cerevisiae, Grx1 and Grx2 are cytosolic dithiol glutaredoxins, whereas Grx3, Grx4, and Grx5 are monothiol glutaredoxins. Grx5 locates at the mitochondrial matrix and is needed for iron/sulfur cluster biogenesis. Its absence causes phenotypes such as inactivation of iron/sulfur enzymes and sensitivity to oxidative stress. Whereas Grx5 contains a single glutaredoxin domain, in Grx3 and Grx4 a thioredoxin-like domain is fused to the glutaredoxin domain. Here we have shown that Grx3 locates at the nucleus and that the thioredoxin-like domain is required for such location. We have addressed the functional divergence among glutaredoxins by targeting Grx2/3/4 molecules to the mitochondrial matrix using the Grx5 targeting sequence. The mitochondrial forms of Grx3 and Grx4 partially rescue the defects of a grx5 null mutant. On the contrary, mitochondrially targeted Grx2 does not suppress the mutant phenotype. Both the thioredoxin-like and glutaredoxin domains are needed for the mitochondrial activity of Grx3, although none of the cysteine residues at the thioredoxin-like domain is required for rescue of the grx5 phenotypes. We have concluded that dithiol glutaredoxins are functionally divergent from monothiol ones, but the latter can interchange their biological activities when compartment barriers are surpassed.
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Affiliation(s)
- María Micaela Molina
- Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, Montserrat Roig 2, 25008-Lleida, Spain
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