Characterization of thioredoxin related protein of 14 kDa and its role in redox signaling
Author: Pader, Irina
Date: 2016-03-18
Location: Samuelssonsalen, Scheelelaboratoriet, Karolinska Institutet, Solna
Time: 09.00
Department: Inst för medicinsk biokemi och biofysik / Dept of Medical Biochemistry and Biophysics
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Thesis (3.734Mb)
Abstract
Reversible reduction/oxidation (redox) reactions play key roles in cellular signaling pathways.
Particularly cysteine residues in proteins can be modified by reactive oxygen-, nitrogen- or sulfur
species (ROS, RNS, RSS), thereby altering the functions of the respective proteins. These
modifications can be reversed by two major reductive systems in mammalian cells – the thioredoxin
(Trx) and glutathione (GSH) systems. Both contain various representatives of the Trx fold family of
proteins, among them the name-giving Trxs being the most prominent. In the cytosolic Trx system,
electrons are transferred from NADPH to Trx reductase 1 (TrxR1) and subsequently to Trx1, which
reduces a multitude of cellular substrates. Thioredoxin-related protein of 14 kDa (TRP14, TXNDC17)
is a sparsely characterized, but evolutionarily well-conserved member of the Trx system. The studies
comprising this thesis examined TRP14 in several aspects of redox signaling.
In Paper I we investigated the inhibition of TrxR1 by noble metal compounds and their effect on cancer cell survival. Inhibition of the Trx system as anti-cancer strategy is thought to attenuate the antioxidant capacity of cancer cells, thereby leading to cell death. We found that gold (Au), platinum (Pt), and palladium (Pd) compounds all inhibited TrxR1 in vitro, but in a cellular context, the inhibition and cytotoxicity were mainly dependent on the ligand substituents and cellular uptake. Furthermore, we found a covalent crosslink between TrxR1 and TRP14 upon treatment of cells with the antitumor agent cisplatin. We concluded that noble metals are potent TrxR1 inhibitors but Pt compounds, especially cisplatin, trigger highly specific cellular effects, including the covalent complex formation.
In Paper II we studied the role of the Trx system in reactivation of oxidized protein tyrosine phoshatases (PTPs) in platelet derived growth factor (PDGF) signaling. Using fibroblasts that lacked TrxR1 (Txnrd1 -/-), we found both an increased oxidation of PTP1B and phosphorylation of the PDGF β receptor (PDGF βR). Consequently, we showed that both Trx1 and TRP14, coupled to TrxR1, are able to reduce oxidized PTP1B in vitro. This study demonstrated that the Trx system, including both Trx1 and TRP14, impacts the oxidation of specific PTPs and can thereby modulate PDGF signaling.
In Paper III we established TRP14 as an efficient TrxR1-dependent reductase and denitrosylase. Using several low molecular weight disulfide compounds, we found that, dependent on the substrate, TRP14 can be at least as efficient as Trx1. We also suggested TRP14 instead of Trx1 to be a major intracellular cystine reductase, because Trx1 does not reduce cystine once a preferred substrate such as insulin is present. Acting in parallel with Trx1, we also provide evidence of TRP14 being an efficient cellular reductase for nitrosylated proteins and concluded that TRP14 should be considered as an integral part of the Trx system.
In Paper IV we developed a novel method for the detection of protein persulfides named Protein Persulfide Detection Protocol, ProPerDP. The formation of persulfide (-SSH) moieties at regulatory cysteine residues is emerging as a major pathway of hydrogen sulfide (H2S) mediated redox signaling. Using ProPerDP we discovered that both the Trx and the GSH system are potent reduction pathways for poly- and persulfides in cells.
These studies reinforce the notion that TrxR1-dependent pathways are not only mediated via its wellknown substrate Trx1. We show that TRP14 is yet another cytosolic oxidoreductase with various intracellular functions, including reduction of PTPs, disulfides, nitrosothiols and persulfides. TRP14 is thereby potentially involved in a variety of different redox signaling pathways.
In Paper I we investigated the inhibition of TrxR1 by noble metal compounds and their effect on cancer cell survival. Inhibition of the Trx system as anti-cancer strategy is thought to attenuate the antioxidant capacity of cancer cells, thereby leading to cell death. We found that gold (Au), platinum (Pt), and palladium (Pd) compounds all inhibited TrxR1 in vitro, but in a cellular context, the inhibition and cytotoxicity were mainly dependent on the ligand substituents and cellular uptake. Furthermore, we found a covalent crosslink between TrxR1 and TRP14 upon treatment of cells with the antitumor agent cisplatin. We concluded that noble metals are potent TrxR1 inhibitors but Pt compounds, especially cisplatin, trigger highly specific cellular effects, including the covalent complex formation.
In Paper II we studied the role of the Trx system in reactivation of oxidized protein tyrosine phoshatases (PTPs) in platelet derived growth factor (PDGF) signaling. Using fibroblasts that lacked TrxR1 (Txnrd1 -/-), we found both an increased oxidation of PTP1B and phosphorylation of the PDGF β receptor (PDGF βR). Consequently, we showed that both Trx1 and TRP14, coupled to TrxR1, are able to reduce oxidized PTP1B in vitro. This study demonstrated that the Trx system, including both Trx1 and TRP14, impacts the oxidation of specific PTPs and can thereby modulate PDGF signaling.
In Paper III we established TRP14 as an efficient TrxR1-dependent reductase and denitrosylase. Using several low molecular weight disulfide compounds, we found that, dependent on the substrate, TRP14 can be at least as efficient as Trx1. We also suggested TRP14 instead of Trx1 to be a major intracellular cystine reductase, because Trx1 does not reduce cystine once a preferred substrate such as insulin is present. Acting in parallel with Trx1, we also provide evidence of TRP14 being an efficient cellular reductase for nitrosylated proteins and concluded that TRP14 should be considered as an integral part of the Trx system.
In Paper IV we developed a novel method for the detection of protein persulfides named Protein Persulfide Detection Protocol, ProPerDP. The formation of persulfide (-SSH) moieties at regulatory cysteine residues is emerging as a major pathway of hydrogen sulfide (H2S) mediated redox signaling. Using ProPerDP we discovered that both the Trx and the GSH system are potent reduction pathways for poly- and persulfides in cells.
These studies reinforce the notion that TrxR1-dependent pathways are not only mediated via its wellknown substrate Trx1. We show that TRP14 is yet another cytosolic oxidoreductase with various intracellular functions, including reduction of PTPs, disulfides, nitrosothiols and persulfides. TRP14 is thereby potentially involved in a variety of different redox signaling pathways.
List of papers:
I. Stefanie Prast-Nielsen, Marcus Cebula, Irina Pader, Elias S J. Arnér. Noble metal targeting of thioredoxin reductase – covalent complexes with thioredoxin and thioredoxin-related protein of 14 kDa triggered by cisplatin. Free Radic Biol Med 49:1765-1778; 2010.
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II. Markus Dagnell, Jeroen Frijhoff, Irina Pader, Martin Augsten, Benoit Boivin, Jianqiang Xu, Pankaj Mandal, Nicholas K. Tonks, Carina Hellberg, Marcus Conrad, Elias S. J. Arnér, Arne Östman. Selective activation of oxidized PTP1B by the thioredoxin system modulates PDGF-beta receptor tyrosine kinase signaling. Proc Natl Acad Sci USA; 110:(33), 13398-13403; 2013.
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III. Irina Pader, Rajib Sengupta, Marcus Cebula, Jianqiang Xu, Jon O. Lundberg, Arne Holmgren, Katarina Johansson, Elias S. J. Arnér. Thioredoxin-related protein of 14 kDa is an efficient L-cystine reductase and S-denitrosylase. Proc Natl Acad Sci U S A; 111(19), 6964-6969; 2014.
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IV. Éva Dóka, Irina Pader, Adrienn Bíró, Katarina Johansson, Qing Cheng, Krisztina Ballagó, Justin R. Prigge, Daniel Pastor-Flores, Tobias P. Dick, Edward E. Schmidt, Elias S. J. Arnér, Péter Nagy. Novel persulfide detection method reveals protein persulfide and polysulfide reducing functions of thioredoxin- and glutathione systems. Science Advances 2:e1500968, 2016.
Fulltext (DOI)
Pubmed
I. Stefanie Prast-Nielsen, Marcus Cebula, Irina Pader, Elias S J. Arnér. Noble metal targeting of thioredoxin reductase – covalent complexes with thioredoxin and thioredoxin-related protein of 14 kDa triggered by cisplatin. Free Radic Biol Med 49:1765-1778; 2010.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Markus Dagnell, Jeroen Frijhoff, Irina Pader, Martin Augsten, Benoit Boivin, Jianqiang Xu, Pankaj Mandal, Nicholas K. Tonks, Carina Hellberg, Marcus Conrad, Elias S. J. Arnér, Arne Östman. Selective activation of oxidized PTP1B by the thioredoxin system modulates PDGF-beta receptor tyrosine kinase signaling. Proc Natl Acad Sci USA; 110:(33), 13398-13403; 2013.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Irina Pader, Rajib Sengupta, Marcus Cebula, Jianqiang Xu, Jon O. Lundberg, Arne Holmgren, Katarina Johansson, Elias S. J. Arnér. Thioredoxin-related protein of 14 kDa is an efficient L-cystine reductase and S-denitrosylase. Proc Natl Acad Sci U S A; 111(19), 6964-6969; 2014.
Fulltext (DOI)
Pubmed
View record in Web of Science®
IV. Éva Dóka, Irina Pader, Adrienn Bíró, Katarina Johansson, Qing Cheng, Krisztina Ballagó, Justin R. Prigge, Daniel Pastor-Flores, Tobias P. Dick, Edward E. Schmidt, Elias S. J. Arnér, Péter Nagy. Novel persulfide detection method reveals protein persulfide and polysulfide reducing functions of thioredoxin- and glutathione systems. Science Advances 2:e1500968, 2016.
Fulltext (DOI)
Pubmed
Institution: Karolinska Institutet
Supervisor: Arnér, Elias
Issue date: 2016-02-22
Rights:
Publication year: 2016
ISBN: 978-91-7676-214-1
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