Thioredoxin and glutaredoxin systems under oxidative and nitrosative stress
Author: Ren, Xiaoyuan
Date: 2017-04-07
Location: Samuelssonsalen, Scheelelaboratoriet, Tomtebodavägen 6, Karolinska Instutet, Solna
Time: 09.00
Department: Inst för medicinsk biokemi och biofysik / Dept of Medical Biochemistry and Biophysics
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Thesis (3.171Mb)
Abstract
Our knowledge about reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been changed from simple damaging molecules to redox signaling mediators. ROS/RNS mediated signaling is mainly based on the reversible post-translational modifications of cysteine which works as a switch for protein functions. A proper amount of ROS/RNS is necessary to trigger such signaling, while excessive amount will lead to oxidative and nitrosative stress, which was recently defined as the disruption of redox signaling and control. In order to maintain the redox homeostasis, mammalian cells are equipped with two major antioxidant systems: the thioredoxin (Trx) system, which is composed of Trx, thioredoxin reductase (TrxR) and NADPH, and the glutaredoxin (Grx) system, which is grouped by Grx, glutathione (GSH), glutathione reductase (GR) and NADPH. Both systems play important roles in counteracting ROS/RNS and regulating redox signaling.
In Paper I, we investigated the toxicity of several arsenic compounds on mammalian cells. We found that arsenical-induced cytotoxicity was related to inhibition of TrxR, suggesting an important role of TrxR for cell survival and potential usage as an anti-cancer target. Among the compounds, As6 and As7 exhibited higher cytotoxicity by directly oxidizing Trx1 and leading to the formation of a structural disulfide between Cys63 and Cys69. The formation of Cys63-Cys69 disulfide blocked the electron transfer from TrxR to peroxiredoxin (Prx) via Trx1, which allowed H2O2 to accumulate and activate the Nrf2 antioxidant pathway. This study highlighted the importance of the structural cysteines in human Trx1 and provided a potential rational design of new anticancer agents.
In Paper II, we studied the effect of Apatone, a vitamin C and vitamin K3 combination used for cancer treatment, on antioxidant systems. We found that Apatone induced oxidative stress in various cancer cell lines which is characterized by GSH depletion, protein glutathionylation, and Trx1 oxidation. In addition, it inhibited ribonucleotide reductase (RNR), which is essential for DNA replication and repair, and caused replicative stress. A caspase-independent cell death pathway was also elucidated that Apatone elevated lipid peroxidation which triggered the nuclear translocation of apoptosis-inducing factor (AIF). We conclude that Apatone works by dramatically disturbing the redox balance in cancer cells.
In Paper III, the role of nitric oxide (NO) during trypanosome infection was studied by using a Trypanosoma Brucei infected inducible nitric oxide synthase knocked (inos-/-) mice model. NO exhibited a protective role by maintaining the integrity of blood-brain-barrier (BBB). We found that macrophage-derived NO curbed the inflammatory effect of TNF-α by S-nitrosylating the p65 subunit of NF-κB, a transcription factor staying in the center of inflammation. Matrix metalloproteinase 9 (MMP9), one of the targets of NF-κB degrading BBB, was also decreased by NO. Thus we conclude that NO plays a protective role during parasite infection by serving as a negative feedback for neuronal inflammatory signaling.
In Paper IV, we characterized Grxs as S-denitrosylases catalyzing the reversible S-nitrosylation. We observed that reduced human dithiol Grx1 and Grx2a denitrosylated S-nitrosothiols (SNOs) directly by the active site dithiol. GSH can denitrosylate part of protein SNOs, while some of them are stable in the presence of high concentration of GSH. Both dithiol and monothiol Grxs exhibited denitrosylation ability to GSH-stable SNOs. We proposed Grxs catalyze S-denitrosylation via both dithiol and monothiol mechanisms.
To summarize, this thesis consolidated the importance of Trx and Grx systems in fighting against ROS/RNS and mediating redox signaling in mammalian cells.
In Paper I, we investigated the toxicity of several arsenic compounds on mammalian cells. We found that arsenical-induced cytotoxicity was related to inhibition of TrxR, suggesting an important role of TrxR for cell survival and potential usage as an anti-cancer target. Among the compounds, As6 and As7 exhibited higher cytotoxicity by directly oxidizing Trx1 and leading to the formation of a structural disulfide between Cys63 and Cys69. The formation of Cys63-Cys69 disulfide blocked the electron transfer from TrxR to peroxiredoxin (Prx) via Trx1, which allowed H2O2 to accumulate and activate the Nrf2 antioxidant pathway. This study highlighted the importance of the structural cysteines in human Trx1 and provided a potential rational design of new anticancer agents.
In Paper II, we studied the effect of Apatone, a vitamin C and vitamin K3 combination used for cancer treatment, on antioxidant systems. We found that Apatone induced oxidative stress in various cancer cell lines which is characterized by GSH depletion, protein glutathionylation, and Trx1 oxidation. In addition, it inhibited ribonucleotide reductase (RNR), which is essential for DNA replication and repair, and caused replicative stress. A caspase-independent cell death pathway was also elucidated that Apatone elevated lipid peroxidation which triggered the nuclear translocation of apoptosis-inducing factor (AIF). We conclude that Apatone works by dramatically disturbing the redox balance in cancer cells.
In Paper III, the role of nitric oxide (NO) during trypanosome infection was studied by using a Trypanosoma Brucei infected inducible nitric oxide synthase knocked (inos-/-) mice model. NO exhibited a protective role by maintaining the integrity of blood-brain-barrier (BBB). We found that macrophage-derived NO curbed the inflammatory effect of TNF-α by S-nitrosylating the p65 subunit of NF-κB, a transcription factor staying in the center of inflammation. Matrix metalloproteinase 9 (MMP9), one of the targets of NF-κB degrading BBB, was also decreased by NO. Thus we conclude that NO plays a protective role during parasite infection by serving as a negative feedback for neuronal inflammatory signaling.
In Paper IV, we characterized Grxs as S-denitrosylases catalyzing the reversible S-nitrosylation. We observed that reduced human dithiol Grx1 and Grx2a denitrosylated S-nitrosothiols (SNOs) directly by the active site dithiol. GSH can denitrosylate part of protein SNOs, while some of them are stable in the presence of high concentration of GSH. Both dithiol and monothiol Grxs exhibited denitrosylation ability to GSH-stable SNOs. We proposed Grxs catalyze S-denitrosylation via both dithiol and monothiol mechanisms.
To summarize, this thesis consolidated the importance of Trx and Grx systems in fighting against ROS/RNS and mediating redox signaling in mammalian cells.
List of papers:
I. Xu Zhang, Jun Lu, Xiaoyuan Ren, Yatao Du, Yujuan Zheng, Panayiotis V. Ioannou, Arne Holmgren. Oxidation of structural cysteine residues in thioredoxin 1 by aromatic arsenicals enhances cancer cell cytotoxicity caused by the inhibition of thioredoxin reductase 1. Free Radical Biology and Medicine. 89 (2015) 192–200.
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II. Xiaoyuan Ren, Sebastin Santhosh, Lucia Coppo, Fernando Ogata, Jun Lu and Arne Holmgren. Vitamin C and K3 cause cancer cell death by oxidative stress and effects on ribonucleotide reductase and its electron donors. [Manuscript]
III. Gabriela C. Olivera, Xiaoyuan Ren, Suman K. Vodnala, Jun Lu, Lucia Coppo, Chaniya Leepiyasakulchai, Arne Holmgren, Krister Kristensson, Martin E. Rottenberg. Nitric Oxide Protects against Infection-Induced Neuroinflammation by Preserving the Stability of the Blood-Brain Barrier. PLoS Pathog. 2016 Feb; 12(2): e1005442.
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IV. Xiaoyuan Ren, Rajib Sengupta, Jun Lu, Jon O. Lundberg, Arne Holmgren. Characterization of mammalian glutaredoxin isoforms as S-denitrosylases. [Manuscript]
I. Xu Zhang, Jun Lu, Xiaoyuan Ren, Yatao Du, Yujuan Zheng, Panayiotis V. Ioannou, Arne Holmgren. Oxidation of structural cysteine residues in thioredoxin 1 by aromatic arsenicals enhances cancer cell cytotoxicity caused by the inhibition of thioredoxin reductase 1. Free Radical Biology and Medicine. 89 (2015) 192–200.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Xiaoyuan Ren, Sebastin Santhosh, Lucia Coppo, Fernando Ogata, Jun Lu and Arne Holmgren. Vitamin C and K3 cause cancer cell death by oxidative stress and effects on ribonucleotide reductase and its electron donors. [Manuscript]
III. Gabriela C. Olivera, Xiaoyuan Ren, Suman K. Vodnala, Jun Lu, Lucia Coppo, Chaniya Leepiyasakulchai, Arne Holmgren, Krister Kristensson, Martin E. Rottenberg. Nitric Oxide Protects against Infection-Induced Neuroinflammation by Preserving the Stability of the Blood-Brain Barrier. PLoS Pathog. 2016 Feb; 12(2): e1005442.
Fulltext (DOI)
Pubmed
View record in Web of Science®
IV. Xiaoyuan Ren, Rajib Sengupta, Jun Lu, Jon O. Lundberg, Arne Holmgren. Characterization of mammalian glutaredoxin isoforms as S-denitrosylases. [Manuscript]
Institution: Karolinska Institutet
Supervisor: Holmgren, Arne
Co-supervisor: Lu, Jun; Sengupta, Rajib; Arnér, Elias
Issue date: 2017-03-16
Rights:
Publication year: 2017
ISBN: 978-91-7676-615-6
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