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Zimmermann J, Lang L, Calabrese G, Laporte H, Amponsah PS, Michalk C, Sukmann T, Oestreicher J, Tursch A, Peker E, Owusu TNE, Weith M, Roma LP, Deponte M, Riemer J, Morgan B. Tsa1 is the dominant peroxide scavenger and a source of H 2O 2-dependent GSSG production in yeast. Free Radic Biol Med 2025; 226:408-420. [PMID: 39515595 DOI: 10.1016/j.freeradbiomed.2024.11.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/03/2024] [Revised: 10/30/2024] [Accepted: 11/02/2024] [Indexed: 11/16/2024]
Abstract
Hydrogen peroxide (H2O2) is an important biological molecule, functioning both as a second messenger in cell signaling and, especially at higher concentrations, as a cause of cell damage. Cells harbor multiple enzymes that have peroxide reducing activity in vitro. However, the contribution of each of these enzymes towards peroxide scavenging in vivo is less clear. Therefore, to directly investigate in vivo peroxide scavenging, we used the genetically encoded peroxide probes, roGFP2-Tsa2ΔCR and HyPer7, to systematically screen the peroxide scavenging capacity of baker's yeast thiol and heme peroxidase mutants. We show that the 2-Cys peroxiredoxin Tsa1 alone is responsible for almost all exogenous H2O2 and tert-butyl hydroperoxide scavenging. Furthermore, Tsa1 can become an important source of H2O2-dependent cytosolic glutathione disulfide production. The two catalases and cytochrome c peroxidase only produce observable scavenging defects at higher H2O2 concentrations when these three heme peroxidases are removed in combination. We also analyzed the reduction of Tsa1 in vitro, revealing that the enzyme is efficiently reduced by thioredoxin-1 with a rate constant of 2.8 × 106 M-1s-1 but not by glutaredoxin-2. Tsa1 reduction by reduced glutathione occurs nonenzymatically with a rate constant of 2.9 M-1s-1. Hence, the observed Tsa1-dependent glutathione disulfide production in yeast probably requires the oxidation of thioredoxins. Our findings clarify the importance of the various thiol and heme peroxidases for peroxide removal and suggest that most thiol peroxidases have alternative or specialized functions in specific subcellular compartments.
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Affiliation(s)
- Jannik Zimmermann
- Institute of Biochemistry, Centre for Human and Molecular Biology (ZHMB), Saarland University, 66123, Saarbrücken, Germany
| | - Lukas Lang
- Faculty of Chemistry, Comparative Biochemistry, RPTU Kaiserslautern, D-67663, Kaiserslautern, Germany
| | - Gaetano Calabrese
- Institute for Biochemistry, Redox Biochemistry, University of Cologne, Zuelpicher Str. 47a/R. 3.49, 50674, Cologne, Germany
| | - Hugo Laporte
- Institute of Biochemistry, Centre for Human and Molecular Biology (ZHMB), Saarland University, 66123, Saarbrücken, Germany
| | - Prince S Amponsah
- Institute of Biochemistry, Centre for Human and Molecular Biology (ZHMB), Saarland University, 66123, Saarbrücken, Germany; Cellular Biochemistry, RPTU Kaiserslautern, 67663, Kaiserslautern, Germany
| | - Christoph Michalk
- Cellular Biochemistry, RPTU Kaiserslautern, 67663, Kaiserslautern, Germany
| | - Tobias Sukmann
- Institute of Biochemistry, Centre for Human and Molecular Biology (ZHMB), Saarland University, 66123, Saarbrücken, Germany
| | - Julian Oestreicher
- Institute of Biochemistry, Centre for Human and Molecular Biology (ZHMB), Saarland University, 66123, Saarbrücken, Germany
| | - Anja Tursch
- Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany
| | - Esra Peker
- Institute for Biochemistry, Redox Biochemistry, University of Cologne, Zuelpicher Str. 47a/R. 3.49, 50674, Cologne, Germany
| | - Theresa N E Owusu
- Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany
| | - Matthias Weith
- Institute for Biochemistry, Redox Biochemistry, University of Cologne, Zuelpicher Str. 47a/R. 3.49, 50674, Cologne, Germany
| | - Leticia Prates Roma
- Institute of Biophysics, Centre for Human and Molecular Biology (ZHMB), Saarland University, 66424, Homburg, Germany
| | - Marcel Deponte
- Faculty of Chemistry, Comparative Biochemistry, RPTU Kaiserslautern, D-67663, Kaiserslautern, Germany.
| | - Jan Riemer
- Institute for Biochemistry, Redox Biochemistry, University of Cologne, Zuelpicher Str. 47a/R. 3.49, 50674, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931, Cologne, Germany.
| | - Bruce Morgan
- Institute of Biochemistry, Centre for Human and Molecular Biology (ZHMB), Saarland University, 66123, Saarbrücken, Germany.
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Lang L, Reinert P, Diaz C, Deponte M. The dithiol mechanism of class I glutaredoxins promotes specificity for glutathione as a reducing agent. Redox Biol 2024; 78:103410. [PMID: 39488995 PMCID: PMC11567954 DOI: 10.1016/j.redox.2024.103410] [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: 09/27/2024] [Revised: 10/22/2024] [Accepted: 10/22/2024] [Indexed: 11/05/2024] Open
Abstract
Class I glutaredoxins reversibly reduce glutathione- and nonglutathione disulfides with the help of reduced glutathione (GSH) using either a monothiol mechanism or a dithiol mechanism. The monothiol mechanism exclusively involves a single glutathionylated active-site cysteinyl residue, whereas the dithiol mechanism requires the additional formation of an intramolecular disulfide bond between the active-site cysteinyl residue and a resolving cysteinyl residue. While the oxidation of glutaredoxins by glutathione disulfide substrates has been extensively characterized, the enzyme-substrate interactions for the reduction of S-glutathionylated glutaredoxins or intramolecular glutaredoxin disulfides are still poorly characterized. Here we compared the thiol-specificity for the reduction of S-glutathionylated glutaredoxins and the intramolecular glutaredoxin disulfide. We show that S-glutathionylated glutaredoxins rapidly react with a plethora of thiols and that the 2nd glutathione-interaction site of class I glutaredoxins lacks specificity for GSH as a reducing agent. In contrast, the slower reduction of the partially strained intramolecular glutaredoxin disulfide involves specific interactions with both carboxylate groups of GSH at the 1st glutathione-interaction site. Thus, the dithiol mechanism of class I glutaredoxins promotes specificity for GSH as a reducing agent, which might explain the prevalence of dithiol glutaredoxins in pro- and eukaryotes.
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Affiliation(s)
- Lukas Lang
- Faculty of Chemistry, Comparative Biochemistry, RPTU Kaiserslautern, D-67663, Kaiserslautern, Germany
| | - Philipp Reinert
- Faculty of Chemistry, Comparative Biochemistry, RPTU Kaiserslautern, D-67663, Kaiserslautern, Germany
| | - Cedric Diaz
- Faculty of Chemistry, Comparative Biochemistry, RPTU Kaiserslautern, D-67663, Kaiserslautern, Germany
| | - Marcel Deponte
- Faculty of Chemistry, Comparative Biochemistry, RPTU Kaiserslautern, D-67663, Kaiserslautern, Germany.
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Schlößer M, Moseler A, Bodnar Y, Homagk M, Wagner S, Pedroletti L, Gellert M, Ugalde JM, Lillig CH, Meyer AJ. Localization of four class I glutaredoxins in the cytosol and the secretory pathway and characterization of their biochemical diversification. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2024; 118:1455-1474. [PMID: 38394181 DOI: 10.1111/tpj.16687] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/06/2023] [Revised: 01/31/2024] [Accepted: 02/07/2024] [Indexed: 02/25/2024]
Abstract
Class I glutaredoxins (GRXs) are catalytically active oxidoreductases and considered key proteins mediating reversible glutathionylation and deglutathionylation of protein thiols during development and stress responses. To narrow in on putative target proteins, it is mandatory to know the subcellular localization of the respective GRXs and to understand their catalytic activities and putative redundancy between isoforms in the same compartment. We show that in Arabidopsis thaliana, GRXC1 and GRXC2 are cytosolic proteins with GRXC1 being attached to membranes through myristoylation. GRXC3 and GRXC4 are identified as type II membrane proteins along the early secretory pathway with their enzymatic function on the luminal side. Unexpectedly, neither single nor double mutants lacking both GRXs isoforms in the cytosol or the ER show phenotypes that differ from wild-type controls. Analysis of electrostatic surface potentials and clustering of GRXs based on their electrostatic interaction with roGFP2 mirrors the phylogenetic classification of class I GRXs, which clearly separates the cytosolic GRXC1 and GRXC2 from the luminal GRXC3 and GRXC4. Comparison of all four studied GRXs for their oxidoreductase function highlights biochemical diversification with GRXC3 and GRXC4 being better catalysts than GRXC1 and GRXC2 for the reduction of bis(2-hydroxyethyl) disulfide. With oxidized roGFP2 as an alternative substrate, GRXC1 and GRXC2 catalyze the reduction faster than GRXC3 and GRXC4, which suggests that catalytic efficiency of GRXs in reductive reactions depends on the respective substrate. Vice versa, GRXC3 and GRXC4 are faster than GRXC1 and GRXC2 in catalyzing the oxidation of pre-reduced roGFP2 in the reverse reaction.
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Affiliation(s)
- Michelle Schlößer
- INRES-Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113, Bonn, Germany
| | - Anna Moseler
- INRES-Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113, Bonn, Germany
| | - Yana Bodnar
- Institute for Medical Biochemistry and Molecular Biology, University Medicine, University of Greifswald, Ferdinand-Sauerbruch-Straße, D-17475, Greifswald, Germany
| | - Maria Homagk
- INRES-Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113, Bonn, Germany
| | - Stephan Wagner
- INRES-Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113, Bonn, Germany
| | - Luca Pedroletti
- INRES-Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113, Bonn, Germany
| | - Manuela Gellert
- Institute for Medical Biochemistry and Molecular Biology, University Medicine, University of Greifswald, Ferdinand-Sauerbruch-Straße, D-17475, Greifswald, Germany
| | - José M Ugalde
- INRES-Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113, Bonn, Germany
| | - Christopher H Lillig
- Institute for Medical Biochemistry and Molecular Biology, University Medicine, University of Greifswald, Ferdinand-Sauerbruch-Straße, D-17475, Greifswald, Germany
| | - Andreas J Meyer
- INRES-Chemical Signalling, University of Bonn, Friedrich-Ebert-Allee 144, D-53113, Bonn, Germany
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Tossounian MA, Zhao Y, Yu BYK, Markey SA, Malanchuk O, Zhu Y, Cain A, Gout I. Low-molecular-weight thiol transferases in redox regulation and antioxidant defence. Redox Biol 2024; 71:103094. [PMID: 38479221 PMCID: PMC10950700 DOI: 10.1016/j.redox.2024.103094] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2023] [Revised: 02/08/2024] [Accepted: 02/18/2024] [Indexed: 03/24/2024] Open
Abstract
Low-molecular-weight (LMW) thiols are produced in all living cells in different forms and concentrations. Glutathione (GSH), coenzyme A (CoA), bacillithiol (BSH), mycothiol (MSH), ergothioneine (ET) and trypanothione T(SH)2 are the main LMW thiols in eukaryotes and prokaryotes. LMW thiols serve as electron donors for thiol-dependent enzymes in redox-mediated metabolic and signaling processes, protect cellular macromolecules from oxidative and xenobiotic stress, and participate in the reduction of oxidative modifications. The level and function of LMW thiols, their oxidized disulfides and mixed disulfide conjugates in cells and tissues is tightly controlled by dedicated oxidoreductases, such as peroxiredoxins, glutaredoxins, disulfide reductases and LMW thiol transferases. This review provides the first summary of the current knowledge of structural and functional diversity of transferases for LMW thiols, including GSH, BSH, MSH and T(SH)2. Their role in maintaining redox homeostasis in single-cell and multicellular organisms is discussed, focusing in particular on the conjugation of specific thiols to exogenous and endogenous electrophiles, or oxidized protein substrates. Advances in the development of new research tools, analytical methodologies, and genetic models for the analysis of known LMW thiol transferases will expand our knowledge and understanding of their function in cell growth and survival under oxidative stress, nutrient deprivation, and during the detoxification of xenobiotics and harmful metabolites. The antioxidant function of CoA has been recently discovered and the breakthrough in defining the identity and functional characteristics of CoA S-transferase(s) is soon expected.
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Affiliation(s)
- Maria-Armineh Tossounian
- Department of Structural and Molecular Biology, University College London, London, WC1E 6BT, United Kingdom
| | - Yuhan Zhao
- Department of Structural and Molecular Biology, University College London, London, WC1E 6BT, United Kingdom
| | - Bess Yi Kun Yu
- Department of Structural and Molecular Biology, University College London, London, WC1E 6BT, United Kingdom
| | - Samuel A Markey
- Department of Structural and Molecular Biology, University College London, London, WC1E 6BT, United Kingdom
| | - Oksana Malanchuk
- Department of Structural and Molecular Biology, University College London, London, WC1E 6BT, United Kingdom; Department of Cell Signaling, Institute of Molecular Biology and Genetics, Kyiv, 143, Ukraine
| | - Yuejia Zhu
- Department of Structural and Molecular Biology, University College London, London, WC1E 6BT, United Kingdom
| | - Amanda Cain
- Department of Structural and Molecular Biology, University College London, London, WC1E 6BT, United Kingdom
| | - Ivan Gout
- Department of Structural and Molecular Biology, University College London, London, WC1E 6BT, United Kingdom; Department of Cell Signaling, Institute of Molecular Biology and Genetics, Kyiv, 143, Ukraine.
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5
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Geissel F, Lang L, Husemann B, Morgan B, Deponte M. Deciphering the mechanism of glutaredoxin-catalyzed roGFP2 redox sensing reveals a ternary complex with glutathione for protein disulfide reduction. Nat Commun 2024; 15:1733. [PMID: 38409212 PMCID: PMC10897161 DOI: 10.1038/s41467-024-45808-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2023] [Accepted: 02/06/2024] [Indexed: 02/28/2024] Open
Abstract
Glutaredoxins catalyze the reduction of disulfides and are key players in redox metabolism and regulation. While important insights were gained regarding the reduction of glutathione disulfide substrates, the mechanism of non-glutathione disulfide reduction remains highly debated. Here we determined the rate constants for the individual redox reactions between PfGrx, a model glutaredoxin from Plasmodium falciparum, and redox-sensitive green fluorescent protein 2 (roGFP2), a model substrate and versatile tool for intracellular redox measurements. We show that the PfGrx-catalyzed oxidation of roGFP2 occurs via a monothiol mechanism and is up to three orders of magnitude faster when roGFP2 and PfGrx are fused. The oxidation kinetics of roGFP2-PfGrx fusion constructs reflect at physiological GSSG concentrations the glutathionylation kinetics of the glutaredoxin moiety, thus allowing intracellular structure-function analysis. Reduction of the roGFP2 disulfide occurs via a monothiol mechanism and involves a ternary complex with GSH and PfGrx. Our study provides the mechanistic basis for understanding roGFP2 redox sensing and challenges previous mechanisms for protein disulfide reduction.
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Affiliation(s)
- Fabian Geissel
- Faculty of Chemistry, Comparative Biochemistry, RPTU Kaiserslautern, D-67663, Kaiserslautern, Germany
| | - Lukas Lang
- Faculty of Chemistry, Comparative Biochemistry, RPTU Kaiserslautern, D-67663, Kaiserslautern, Germany
| | - Britta Husemann
- Faculty of Chemistry, Comparative Biochemistry, RPTU Kaiserslautern, D-67663, Kaiserslautern, Germany
| | - Bruce Morgan
- Institute of Biochemistry, Centre for Human and Molecular Biology (ZHMB), Saarland University, D-66123, Saarbrücken, Germany
| | - Marcel Deponte
- Faculty of Chemistry, Comparative Biochemistry, RPTU Kaiserslautern, D-67663, Kaiserslautern, Germany.
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6
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Amason ME, Li L, Harvest CK, Lacey CA, Miao EA. Validation of the Intermolecular Disulfide Bond in Caspase-2. BIOLOGY 2024; 13:49. [PMID: 38248479 PMCID: PMC10813798 DOI: 10.3390/biology13010049] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/12/2023] [Revised: 01/05/2024] [Accepted: 01/15/2024] [Indexed: 01/23/2024]
Abstract
Caspases are a family of proteins involved in cell death. Although several caspase members have been well characterized, caspase-2 remains enigmatic. Caspase-2 has been implicated in several phenotypes, but there has been no consensus in the field about its upstream activating signals or its downstream protein targets. In addition, the unique ability of caspase-2 to form a disulfide-bonded dimer has not been studied in depth. Herein, we investigate the disulfide bond in the context of inducible dimerization, showing that disulfide bond formation is dimerization dependent. We also explore and review several stimuli published in the caspase-2 field, test ferroptosis-inducing stimuli, and study in vivo infection models. We hypothesize that the disulfide bond will ultimately prove to be essential for the evolved function of caspase-2. Proving this will require the discovery of cell death phenotypes where caspase-2 is definitively essential.
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Affiliation(s)
- Megan E. Amason
- Department of Integrative Immunobiology, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
- Department of Pathology, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Cell Biology, Duke University School of Medicine, Durham, NC 27710, USA
| | - Lupeng Li
- Department of Integrative Immunobiology, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Pathology, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Cell Biology, Duke University School of Medicine, Durham, NC 27710, USA
| | - Carissa K. Harvest
- Department of Integrative Immunobiology, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
- Department of Pathology, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Cell Biology, Duke University School of Medicine, Durham, NC 27710, USA
| | - Carolyn A. Lacey
- Department of Integrative Immunobiology, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Pathology, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Cell Biology, Duke University School of Medicine, Durham, NC 27710, USA
| | - Edward A. Miao
- Department of Integrative Immunobiology, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Pathology, Duke University School of Medicine, Durham, NC 27710, USA
- Department of Cell Biology, Duke University School of Medicine, Durham, NC 27710, USA
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Bischoff E, Lang L, Zimmermann J, Luczak M, Kiefer AM, Niedner-Schatteburg G, Manolikakes G, Morgan B, Deponte M. Glutathione kinetically outcompetes reactions between dimedone and a cyclic sulfenamide or physiological sulfenic acids. Free Radic Biol Med 2023; 208:165-177. [PMID: 37541455 DOI: 10.1016/j.freeradbiomed.2023.08.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/07/2023] [Revised: 07/31/2023] [Accepted: 08/01/2023] [Indexed: 08/06/2023]
Abstract
Dimedone and its derivates are used as selective probes for the nucleophilic detection of sulfenic acids in biological samples. Qualitative analyses suggested that dimedone also reacts with cyclic sulfenamides. Furthermore, under physiological conditions, dimedone must compete with the highly concentrated nucleophile glutathione. We therefore quantified the reaction kinetics for a cyclic sulfenamide model peptide and the sulfenic acids of glutathione and a model peroxiredoxin in the presence or absence of dimedone and glutathione. We show that the cyclic sulfenamide is stabilized at lower pH and that it reacts with dimedone. While reactions between dimedone and sulfenic acids or the cyclic sulfenamide have similar rate constants, glutathione kinetically outcompetes dimedone as a nucleophile by several orders of magnitude. Our comparative in vitro and intracellular analyses challenge the selectivity of dimedone. Consequently, the dimedone labeling of cysteinyl residues inside living cells points towards unidentified reaction pathways or unknown, kinetically competitive redox species.
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Affiliation(s)
- Eileen Bischoff
- Fachbereich Chemie & Landesforschungszentrum OPTIMAS, RPTU Kaiserslautern, Erwin-Schrödinger Straße 54, D-67663, Kaiserslautern, Germany
| | - Lukas Lang
- Fachbereich Chemie & Landesforschungszentrum OPTIMAS, RPTU Kaiserslautern, Erwin-Schrödinger Straße 54, D-67663, Kaiserslautern, Germany
| | - Jannik Zimmermann
- Zentrum für Human- und Molekularbiologie (ZHMB), Universität des Saarlandes, Biochemie Campus, Geb. B2.2, D-66123, Saarbrücken, Germany
| | - Maximilian Luczak
- Fachbereich Chemie & Landesforschungszentrum OPTIMAS, RPTU Kaiserslautern, Erwin-Schrödinger Straße 54, D-67663, Kaiserslautern, Germany
| | - Anna Maria Kiefer
- Fachbereich Biologie, RPTU Kaiserslautern, Paul-Ehrlich Straße 23, D-67663, Kaiserslautern, Germany
| | - Gereon Niedner-Schatteburg
- Fachbereich Chemie & Landesforschungszentrum OPTIMAS, RPTU Kaiserslautern, Erwin-Schrödinger Straße 54, D-67663, Kaiserslautern, Germany
| | - Georg Manolikakes
- Fachbereich Chemie & Landesforschungszentrum OPTIMAS, RPTU Kaiserslautern, Erwin-Schrödinger Straße 54, D-67663, Kaiserslautern, Germany
| | - Bruce Morgan
- Zentrum für Human- und Molekularbiologie (ZHMB), Universität des Saarlandes, Biochemie Campus, Geb. B2.2, D-66123, Saarbrücken, Germany
| | - Marcel Deponte
- Fachbereich Chemie & Landesforschungszentrum OPTIMAS, RPTU Kaiserslautern, Erwin-Schrödinger Straße 54, D-67663, Kaiserslautern, Germany.
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8
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Bodnar Y, Gellert M, Hossain FM, Lillig CH. Breakdown of Arabidopsis thaliana thioredoxins and glutaredoxins based on electrostatic similarity-Leads to common and unique interaction partners and functions. PLoS One 2023; 18:e0291272. [PMID: 37695767 PMCID: PMC10495010 DOI: 10.1371/journal.pone.0291272] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Accepted: 08/24/2023] [Indexed: 09/13/2023] Open
Abstract
The reversible reduction and oxidation of protein thiols was first described as mechanism to control light/dark-dependent metabolic regulation in photosynthetic organisms. Today, it is recognized as an essential mechanism of regulation and signal transduction in all kingdoms of life. Proteins of the thioredoxin (Trx) family, Trxs and glutaredoxins (Grxs) in particular, catalyze thiol-disulfide exchange reactions and are vital players in the operation of thiol switches. Various Trx and Grx isoforms are present in all compartments of the cell. These proteins have a rather broad but at the same time distinct substrate specificity. Understanding the molecular basis of their target specificity is central to the understanding of physiological and pathological redox signaling. Electrostatic complementarity of the redoxins with their target proteins has been proposed as a major reason. Here, we analyzed the electrostatic similarity of all Arabidopsis thaliana Trxs, Grxs, and proteins containing such domains. Clustering of the redoxins based on this comparison suggests overlapping and also distant target specificities and thus functions of the different sub-classes including all Trx isoforms as well as the three classes of Grxs, i.e. CxxC-, CGFS-, and CC-type Grxs. Our analysis also provides a rationale for the tuned substrate specificities of both the ferredoxin- and NADPH-dependent Trx reductases.
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Affiliation(s)
- Yana Bodnar
- Institute for Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Greifswald, Germany
- Institute for Physics, University of Greifswald, Greifswald, Germany
| | - Manuela Gellert
- Institute for Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Greifswald, Germany
| | - Faruq Mohammed Hossain
- Institute for Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Greifswald, Germany
| | - Christopher Horst Lillig
- Institute for Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Greifswald, Germany
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Bodnar Y, Lillig CH. Cysteinyl and methionyl redox switches: Structural prerequisites and consequences. Redox Biol 2023; 65:102832. [PMID: 37536083 PMCID: PMC10412846 DOI: 10.1016/j.redox.2023.102832] [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: 07/12/2023] [Accepted: 07/27/2023] [Indexed: 08/05/2023] Open
Abstract
Redox modifications of specific cysteinyl and methionyl residues regulate key enzymes and signal-transducing proteins in various pathways. Here, we analyzed the effect of redox modifications on protein structure screening the RCSB protein data bank for oxidative modifications of proteins, i.e. protein disulfides, mixed disulfides with glutathione, cysteinyl sulfenic acids, cysteinyl S-nitrosylation, and methionyl sulfoxide residues. When available, these structures were compared to the structures of the same proteins in the reduced state with respect to both pre-requirements for the oxidative modifications as well as the structural consequences of the modifications. In general, the conformational changes induced by the redox modification are small, i.e. within the range of normal fluctuations. Some redox modifications, disulfides in particular, induces alterations in the electrostatic properties of the proteins. Solvent accessibility does not seem to be a strict pre-requirement for the redox modification of a particular residue. We identified an enrichment of certain other amino acid residues in the vicinity of the susceptible residues, for disulfide and sulfenic acid modifications, for instance, histidyl and tyrosyl residues. These motifs, as well as the specific features of the susceptible sulfur-containing amino acids, may become helpful for the prediction of redox modifications.
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Affiliation(s)
- Yana Bodnar
- Institut for Physics, University of Greifswald, Germany; Institute for Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Germany
| | - Christopher Horst Lillig
- Institute for Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Germany.
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10
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Weiser A, Hermant A, Bermont F, Sizzano F, Karaz S, Alvarez-Illera P, Santo-Domingo J, Sorrentino V, Feige JN, De Marchi U. The mitochondrial calcium uniporter (MCU) activates mitochondrial respiration and enhances mobility by regulating mitochondrial redox state. Redox Biol 2023; 64:102759. [PMID: 37302345 PMCID: PMC10363449 DOI: 10.1016/j.redox.2023.102759] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2023] [Revised: 05/14/2023] [Accepted: 05/21/2023] [Indexed: 06/13/2023] Open
Abstract
Regulation of mitochondrial redox balance is emerging as a key event for cell signaling in both physiological and pathological conditions. However, the link between the mitochondrial redox state and the modulation of these conditions remains poorly defined. Here, we discovered that activation of the evolutionary conserved mitochondrial calcium uniporter (MCU) modulates mitochondrial redox state. By using mitochondria-targeted redox and calcium sensors and genetic MCU-ablated models, we provide evidence of the causality between MCU activation and net reduction of mitochondrial (but not cytosolic) redox state. Redox modulation of redox-sensitive groups via MCU stimulation is required for maintaining respiratory capacity in primary human myotubes and C. elegans, and boosts mobility in worms. The same benefits are obtained bypassing MCU via direct pharmacological reduction of mitochondrial proteins. Collectively, our results demonstrate that MCU regulates mitochondria redox balance and that this process is required to promote the MCU-dependent effects on mitochondrial respiration and mobility.
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Affiliation(s)
- Anna Weiser
- Nestlé Institute of Health Sciences, Nestlé Research, EPFL Innovation Park, CH-1015 Lausanne, Switzerland; Molecular Nutritional Medicine, Else Kröner Fresenius Center for Nutritional Medicine, Technische Universität München, 85354 Freising, Germany
| | - Aurélie Hermant
- Nestlé Institute of Health Sciences, Nestlé Research, EPFL Innovation Park, CH-1015 Lausanne, Switzerland
| | - Flavien Bermont
- Nestlé Institute of Health Sciences, Nestlé Research, EPFL Innovation Park, CH-1015 Lausanne, Switzerland
| | - Federico Sizzano
- Nestlé Institute of Health Sciences, Nestlé Research, EPFL Innovation Park, CH-1015 Lausanne, Switzerland
| | - Sonia Karaz
- Nestlé Institute of Health Sciences, Nestlé Research, EPFL Innovation Park, CH-1015 Lausanne, Switzerland
| | - Pilar Alvarez-Illera
- Department of Biochemistry and Molecular Biology, University of Valladolid, Unidad de Excelencia Instituto de Biología y Genética Molecular (IBGM), 47003 Valladolid, Spain
| | - Jaime Santo-Domingo
- Department of Biochemistry and Molecular Biology, University of Valladolid, Unidad de Excelencia Instituto de Biología y Genética Molecular (IBGM), 47003 Valladolid, Spain
| | - Vincenzo Sorrentino
- Nestlé Institute of Health Sciences, Nestlé Research, EPFL Innovation Park, CH-1015 Lausanne, Switzerland; Healthy Longevity Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, 119228, Singapore; Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 117596, Singapore
| | - Jerome N Feige
- Nestlé Institute of Health Sciences, Nestlé Research, EPFL Innovation Park, CH-1015 Lausanne, Switzerland
| | - Umberto De Marchi
- Nestlé Institute of Health Sciences, Nestlé Research, EPFL Innovation Park, CH-1015 Lausanne, Switzerland.
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11
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Jobson J, Tsegay PS, Beltran MT, Taher EA, Rein SR, Liu Y, Rein KS. Brevetoxin induces a shift in the redox state of the proteome and unfolded protein response in human lymphoblast cells that can be alleviated with the acrolein scavenger MESNA. ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY 2023; 100:104137. [PMID: 37127110 DOI: 10.1016/j.etap.2023.104137] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/19/2022] [Revised: 04/24/2023] [Accepted: 04/29/2023] [Indexed: 05/03/2023]
Abstract
Human lymphoblast cells were treated with the marine algal toxin, brevetoxin-2 (PbTx-2), and its effects on the proteome were assessed by redox proteomics using cysteine reactive tandem mass tags (TMT). Additionally, cells were simultaneously treated with PbTx-2 and the antioxidant and acrolein scavenger sodium 2-mercaptoethylsulfonate (MESNA) to determine if MESNA could prevent the proteomic effects of brevetoxin-2. A massive shift in the redox state of the proteome of brevetoxin-2 treated cells was observed. The main pathway affected was genetic information processing. Significantly oxidized proteins included Trx-1, peroxyredoxins (Prxs), ribosomal proteins, and the eukaryotic initiation factor 2 β subunit (eIF2β). Proteins that were overexpressed in brevetoxin-treated cells included four folding chaperones. These effects were diminished in the presence of MESNA indicating that MESNA may act through its antioxidant properties or as a brevetoxin scavenger. These studies provide novel insights into new prophylactics for brevetoxicosis in humans and wildlife.
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Affiliation(s)
- Jordan Jobson
- Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA
| | - Pawlos S Tsegay
- Biochemistry Ph.D. Program, Florida International University, Miami, FL 33199, USA
| | - Mayra Tabares Beltran
- Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA
| | - Eman A Taher
- Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA
| | - Samuel R Rein
- The School District of Philadelphia, Philadelphia, PA 19130, USA
| | - Yuan Liu
- Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA; Biochemistry Ph.D. Program, Florida International University, Miami, FL 33199, USA; Biomolecular Sciences Institute, Florida International University, Miami, FL 33199, USA
| | - Kathleen S Rein
- Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA; Current address: The Water School, Department of Marine and Earth Science and Department of Chemistry and Physics, Florida Gulf Coast University, Fort Myers, FL 33965.
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12
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Tossounian MA, Baczynska M, Dalton W, Peak-Chew SY, Undzenas K, Korza G, Filonenko V, Skehel M, Setlow P, Gout I. Bacillus subtilis YtpP and Thioredoxin A Are New Players in the Coenzyme-A-Mediated Defense Mechanism against Cellular Stress. Antioxidants (Basel) 2023; 12:antiox12040938. [PMID: 37107313 PMCID: PMC10136147 DOI: 10.3390/antiox12040938] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2023] [Revised: 04/06/2023] [Accepted: 04/12/2023] [Indexed: 04/29/2023] Open
Abstract
Coenzyme A (CoA) is an important cellular metabolite that is critical for metabolic processes and the regulation of gene expression. Recent discovery of the antioxidant function of CoA has highlighted its protective role that leads to the formation of a mixed disulfide bond with protein cysteines, which is termed protein CoAlation. To date, more than 2000 CoAlated bacterial and mammalian proteins have been identified in cellular responses to oxidative stress, with the majority being involved in metabolic pathways (60%). Studies have shown that protein CoAlation is a widespread post-translational modification which modulates the activity and conformation of the modified proteins. The induction of protein CoAlation by oxidative stress was found to be rapidly reversed after the removal of oxidizing agents from the medium of cultured cells. In this study, we developed an enzyme-linked immunosorbent assay (ELISA)-based deCoAlation assay to detect deCoAlation activity from Bacillus subtilis and Bacillus megaterium lysates. We then used a combination of ELISA-based assay and purification strategies to show that deCoAlation is an enzyme-driven mechanism. Using mass-spectrometry and deCoAlation assays, we identified B. subtilis YtpP (thioredoxin-like protein) and thioredoxin A (TrxA) as enzymes that can remove CoA from different substrates. With mutagenesis studies, we identified YtpP and TrxA catalytic cysteine residues and proposed a possible deCoAlation mechanism for CoAlated methionine sulfoxide reducatse A (MsrA) and peroxiredoxin 5 (PRDX5) proteins, which results in the release of both CoA and the reduced form of MsrA or PRDX5. Overall, this paper reveals the deCoAlation activity of YtpP and TrxA and opens doors to future studies on the CoA-mediated redox regulation of CoAlated proteins under various cellular stress conditions.
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Affiliation(s)
| | - Maria Baczynska
- Department of Structural and Molecular Biology, University College London, London WC1E 6BT, UK
| | - William Dalton
- Department of Structural and Molecular Biology, University College London, London WC1E 6BT, UK
| | - Sew Yeu Peak-Chew
- MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK
| | - Kipras Undzenas
- Department of Structural and Molecular Biology, University College London, London WC1E 6BT, UK
| | - George Korza
- Department of Molecular Biology and Biophysics, UConn Health, Farmington, CT 06030, USA
| | - Valeriy Filonenko
- Department of Cell Signaling, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 03680 Kyiv, Ukraine
| | - Mark Skehel
- The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
| | - Peter Setlow
- Department of Molecular Biology and Biophysics, UConn Health, Farmington, CT 06030, USA
| | - Ivan Gout
- Department of Structural and Molecular Biology, University College London, London WC1E 6BT, UK
- Department of Cell Signaling, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 03680 Kyiv, Ukraine
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13
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A redox switch regulates the assembly and anti-CRISPR activity of AcrIIC1. Nat Commun 2022; 13:7071. [PMID: 36400778 PMCID: PMC9674691 DOI: 10.1038/s41467-022-34551-8] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2022] [Accepted: 10/25/2022] [Indexed: 11/19/2022] Open
Abstract
Anti-CRISPRs (Acrs) are natural inhibitors of bacteria's CRISPR-Cas systems, and have been developed as a safeguard to reduce the off-target effects of CRISPR gene-editing technology. Acrs can directly bind to CRISPR-Cas complexes and inhibit their activities. However, whether this process is under regulation in diverse eukaryotic cellular environments is poorly understood. In this work, we report the discovery of a redox switch for NmeAcrIIC1, which regulates NmeAcrIIC1's monomer-dimer interconversion and inhibitory activity on Cas9. Further structural studies reveal that a pair of conserved cysteines mediates the formation of inactive NmeAcrIIC1 dimer and directs the redox cycle. The redox switch also applies to the other two AcrIIC1 orthologs. Moreover, by replacing the redox-sensitive cysteines, we generated a robust AcrIIC1 variant that maintains potent inhibitory activity under various redox conditions. Our results reveal a redox-dependent regulation mechanism of Acr, and shed light on the design of superior Acr for CRISPR-Cas systems.
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14
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Chen Q, Xiao Y, Ming Y, Peng R, Hu J, Wang HB, Jin HL. Quantitative proteomics reveals redox-based functional regulation of photosynthesis under fluctuating light in plants. JOURNAL OF INTEGRATIVE PLANT BIOLOGY 2022; 64:2168-2186. [PMID: 35980302 DOI: 10.1111/jipb.13348] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/12/2022] [Accepted: 08/16/2022] [Indexed: 06/15/2023]
Abstract
Photosynthesis involves a series of redox reactions and is the major source of reactive oxygen species in plant cells. Fluctuating light (FL) levels, which occur commonly in natural environments, affect photosynthesis; however, little is known about the specific effects of FL on the redox regulation of photosynthesis. Here, we performed global quantitative mapping of the Arabidopsis thaliana cysteine thiol redox proteome under constant light and FL conditions. We identified 8857 redox-switched thiols in 4350 proteins, and 1501 proteins that are differentially modified depending on light conditions. Notably, proteins related to photosynthesis, especially photosystem I (PSI), are operational thiol-switching hotspots. Exposure of wild-type A. thaliana to FL resulted in decreased PSI abundance, stability, and activity. Interestingly, in response to PSI photodamage, more of the PSI assembly factor PSA3 dynamically switches to the reduced state. Furthermore, the Cys199 and Cys200 sites in PSA3 are necessary for its full function. Moreover, thioredoxin m (Trx m) proteins play roles in redox switching of PSA3, and are required for PSI activity and photosynthesis. This study thus reveals a mechanism for redox-based regulation of PSI under FL, and provides insight into the dynamic acclimation of photosynthesis in a changing environment.
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Affiliation(s)
- Qi Chen
- Institute of Medical Plant Physiology and Ecology, School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, China
| | - Yixian Xiao
- Institute of Medical Plant Physiology and Ecology, School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, China
| | - Yu Ming
- Institute of Medical Plant Physiology and Ecology, School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, China
| | - Rong Peng
- Institute of Medical Plant Physiology and Ecology, School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, China
| | - Jiliang Hu
- Institute of Medical Plant Physiology and Ecology, School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, China
| | - Hong-Bin Wang
- Institute of Medical Plant Physiology and Ecology, School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, China
| | - Hong-Lei Jin
- Institute of Medical Plant Physiology and Ecology, School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, China
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15
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Morellon-Sterling R, Bolivar JM, Fernandez-Lafuente R. Switch off/switch on of a cysteinyl protease as a way to preserve the active catalytic group by modification with a reversible covalent thiol modifier: Immobilization of ficin on vinyl-sulfone activated supports. Int J Biol Macromol 2022; 220:1155-1162. [PMID: 36037909 DOI: 10.1016/j.ijbiomac.2022.08.155] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2022] [Revised: 08/11/2022] [Accepted: 08/23/2022] [Indexed: 11/05/2022]
Abstract
The immobilization of ficin (a cysteinyl proteases) on vinyl sulfone agarose produced its almost full inactivation. It was observed that the incubation of the free and immobilized enzyme in β-mercaptoethanol produced a 20 % of enzyme activity recovery, suggesting that the inactivation due to the immobilization could be a consequence of the modification of the catalytic Cys. To prevent the enzyme inactivation during the immobilization, switching off of ficin via Cys reaction with dipyridyl-disulfide was implemented, giving a reversible disulfide bond that produced a fully inactive enzyme. The switch on of ficin activity was implemented by incubation in 1 M β-mercaptoethanol. Using this strategy to immobilize the enzyme on vinyl sulfone agarose beads, the expressed activity of the immobilized ficin could be boosted up to 80 %. The immobilized enzyme presented a thermal stabilization similar to that obtained using ficin-glyoxyl-agarose beads. This procedure may be extended to many enzymes containing critical Cys, to permit their immobilization or chemical modification.
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Affiliation(s)
- Roberto Morellon-Sterling
- Departamento de Biocatálisis, ICP-CSIC, Marie Curie 2, Campus UAM-CSIC Cantoblanco, 28049 Madrid, Spain; Student of Departamento de Biología Molecular, Universidad Autónoma de Madrid, Darwin 2, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain
| | - Juan M Bolivar
- FQPIMA Group, Chemical and Materials Engineering Department, Faculty of Chemical Sciences, Complutense University of Madrid, Complutense Ave., Madrid 28040, Spain
| | - Roberto Fernandez-Lafuente
- Departamento de Biocatálisis, ICP-CSIC, Marie Curie 2, Campus UAM-CSIC Cantoblanco, 28049 Madrid, Spain; Center of Excellence in Bionanoscience Research, External Scientific Advisory Academics, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
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16
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Schumann R, Lang L, Deponte M. Characterization of the glutathione-dependent reduction of the peroxiredoxin 5 homolog PfAOP from Plasmodium falciparum. Protein Sci 2022; 31:e4290. [PMID: 35481660 PMCID: PMC8994508 DOI: 10.1002/pro.4290] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2021] [Revised: 02/07/2022] [Accepted: 02/17/2022] [Indexed: 11/30/2022]
Abstract
Peroxiredoxins use a variety of thiols to rapidly reduce hydroperoxides and peroxynitrite. While the oxidation kinetics of peroxiredoxins have been studied in great detail, enzyme-specific differences regarding peroxiredoxin reduction and the overall rate-limiting step under physiological conditions often remain to be deciphered. The 1-Cys peroxiredoxin 5 homolog PfAOP from the malaria parasite Plasmodium falciparum is an established model enzyme for glutathione/glutaredoxin-dependent peroxiredoxins. Here, we reconstituted the catalytic cycle of PfAOP in vitro and analyzed the reaction between oxidized PfAOP and reduced glutathione (GSH) using molecular docking and stopped-flow measurements. Molecular docking revealed that oxidized PfAOP has to adopt a locally unfolded conformation to react with GSH. Furthermore, we determined a second-order rate constant of 6 × 105 M-1 s-1 at 25°C and thermodynamic activation parameters ΔH‡ , ΔS‡ , and ΔG‡ of 39.8 kJ/mol, -0.8 J/mol, and 40.0 kJ/mol, respectively. The gain-of-function mutant PfAOPL109M had almost identical reaction parameters. Taking into account physiological hydroperoxide and GSH concentrations, we suggest (a) that the reaction between oxidized PfAOP and GSH might be even faster than the formation of the sulfenic acid in vivo, and (b) that conformational changes are likely rate limiting for PfAOP catalysis. In summary, we characterized and quantified the reaction between GSH and the model enzyme PfAOP, thus providing detailed insights regarding the reactivity of its sulfenic acid and the versatile chemistry of peroxiredoxins.
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Affiliation(s)
- Robin Schumann
- Faculty of ChemistryTU KaiserslauternKaiserslauternGermany
| | - Lukas Lang
- Faculty of ChemistryTU KaiserslauternKaiserslauternGermany
| | - Marcel Deponte
- Faculty of ChemistryTU KaiserslauternKaiserslauternGermany
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17
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Abstract
Import and oxidative folding of proteins in the mitochondrial intermembrane space differ among eukaryotic lineages. While opisthokonts such as yeast rely on the receptor and oxidoreductase Mia40 in combination with the Mia40:cytochrome c oxidoreductase Erv, kinetoplastid parasites and other Excavata/Discoba lack Mia40 but have a functional Erv homologue. Whether excavate Erv homologues rely on a Mia40 replacement or directly interact with imported protein substrates remains controversial. Here, we used the CRISPR-Cas9 system to generate a set of tagged and untagged homozygous mutants of LTERV from the kinetoplastid model parasite Leishmania tarentolae. Modifications of the shuttle cysteine motif of LtErv were lethal, whereas replacement of clamp residue Cys17 or removal of the kinetoplastida-specific second (KISS) domain had no impact on parasite viability under standard growth conditions. However, removal of the KISS domain rendered parasites sensitive to heat stress and led to the accumulation of homodimeric and mixed LtErv disulfides. We therefore determined and compared the redox interactomes of tagged wild-type LtErv and LtErvΔKISS using stable isotope labeling by amino acids in cell culture (SILAC) and quantitative mass spectrometry. While the Mia40-replacement candidate Mic20 and all but one typical substrate with twin Cx3/9C-motifs were absent in both redox interactomes, we identified a small set of alternative potential interaction partners with putative redox-active cysteine residues. In summary, our study reveals parasite-specific intracellular structure-function relationships and redox interactomes of LtErv with implications for current hypotheses on mitochondrial protein import in nonopisthokonts. IMPORTANCE The discovery of the redox proteins Mia40/CHCHD4 and Erv1/ALR, as well as the elucidation of their relevance for oxidative protein folding in the mitochondrial intermembrane space of yeast and mammals, founded a new research topic in redox biology and mitochondrial protein import. The lack of Mia40/CHCHD4 in protist lineages raises fundamental and controversial questions regarding the conservation and evolution of this essential pathway. Do protist Erv homologues act alone, or do they use the candidate Mic20 or another protein as a Mia40 replacement? Furthermore, we previously showed that Erv homologues in L. tarentolae and the human pathogen L. infantum are not only essential but also differ structurally and mechanistically from yeast and human Erv1/ALR. Here, we analyzed the relevance of such structural differences in vivo and determined the first redox interactomes of a nonopisthokont Erv homologue. Our data challenge recent hypotheses on mitochondrial protein import in nonopisthokonts.
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18
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Molecular Basis for the Interactions of Human Thioredoxins with Their Respective Reductases. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2021; 2021:6621292. [PMID: 34122725 PMCID: PMC8189816 DOI: 10.1155/2021/6621292] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/08/2020] [Revised: 04/20/2021] [Accepted: 05/20/2021] [Indexed: 12/03/2022]
Abstract
The mammalian cytosolic thioredoxin (Trx) system consists of Trx1 and its reductase, the NADPH-dependent seleno-enzyme TrxR1. These proteins function as electron donor for metabolic enzymes, for instance in DNA synthesis, and the redox regulation of numerous processes. In this work, we analysed the interactions between these two proteins. We proposed electrostatic complementarity as major force controlling the formation of encounter complexes between the proteins and thus the efficiency of the subsequent electron transfer reaction. If our hypothesis is valid, formation of the encounter complex should be independent of the redox reaction. In fact, we were able to confirm that also a redox inactive mutant of Trx1 lacking both active site cysteinyl residues (C32,35S) binds to TrxR1 in a similar manner and with similar kinetics as the wild-type protein. We have generated a number of mutants with alterations in electrostatic properties and characterised their interaction with TrxR1 in kinetic assays. For human Trx1 and TrxR1, complementary electrostatic surfaces within the area covered in the encounter complex appear to control the affinity of the reductase for its substrate Trx. Electrostatic compatibility was even observed in areas that do not form direct molecular interactions in the encounter complex, and our results suggest that the electrostatic complementarity in these areas influences the catalytic efficiency of the reduction. The human genome encodes ten cytosolic Trx-like or Trx domain-containing proteins. In agreement with our hypothesis, the proteins that have been characterised as TrxR1 substrates also show the highest similarity in their electrostatic properties.
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19
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Signal-regulated oxidation of proteins via MICAL. Biochem Soc Trans 2021; 48:613-620. [PMID: 32219383 DOI: 10.1042/bst20190866] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2019] [Revised: 03/09/2020] [Accepted: 03/11/2020] [Indexed: 12/12/2022]
Abstract
Processing of and responding to various signals is an essential cellular function that influences survival, homeostasis, development, and cell death. Extra- or intracellular signals are perceived via specific receptors and transduced in a particular signalling pathway that results in a precise response. Reversible post-translational redox modifications of cysteinyl and methionyl residues have been characterised in countless signal transduction pathways. Due to the low reactivity of most sulfur-containing amino acid side chains with hydrogen peroxide, for instance, and also to ensure specificity, redox signalling requires catalysis, just like phosphorylation signalling requires kinases and phosphatases. While reducing enzymes of both cysteinyl- and methionyl-derivates have been characterised in great detail before, the discovery and characterisation of MICAL proteins evinced the first examples of specific oxidases in signal transduction. This article provides an overview of the functions of MICAL proteins in the redox regulation of cellular functions.
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20
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Tossounian MA, Zhang B, Gout I. The Writers, Readers, and Erasers in Redox Regulation of GAPDH. Antioxidants (Basel) 2020; 9:antiox9121288. [PMID: 33339386 PMCID: PMC7765867 DOI: 10.3390/antiox9121288] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2020] [Revised: 11/28/2020] [Accepted: 12/14/2020] [Indexed: 12/16/2022] Open
Abstract
Glyceraldehyde 3–phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme, which is crucial for the breakdown of glucose to provide cellular energy. Over the past decade, GAPDH has been reported to be one of the most prominent cellular targets of post-translational modifications (PTMs), which divert GAPDH toward different non-glycolytic functions. Hence, it is termed a moonlighting protein. During metabolic and oxidative stress, GAPDH is a target of different oxidative PTMs (oxPTM), e.g., sulfenylation, S-thiolation, nitrosylation, and sulfhydration. These modifications alter the enzyme’s conformation, subcellular localization, and regulatory interactions with downstream partners, which impact its glycolytic and non-glycolytic functions. In this review, we discuss the redox regulation of GAPDH by different redox writers, which introduce the oxPTM code on GAPDH to instruct a redox response; the GAPDH readers, which decipher the oxPTM code through regulatory interactions and coordinate cellular response via the formation of multi-enzyme signaling complexes; and the redox erasers, which are the reducing systems that regenerate the GAPDH catalytic activity. Human pathologies associated with the oxidation-induced dysregulation of GAPDH are also discussed, featuring the importance of the redox regulation of GAPDH in neurodegeneration and metabolic disorders.
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21
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Abstract
Significance: Oxidative stress in moderation positively affects homeostasis through signaling, while in excess it is associated with adverse health outcomes. Both activities are generally attributed to reactive oxygen species (ROS); hydrogen peroxide as the signal, and cysteines on regulatory proteins as the target. However, using antioxidants to affect signaling or benefit health has not consistently translated into expected outcomes, or when it does, the mechanism is often unclear. Recent Advances: Reactive sulfur species (RSS) were integral in the origin of life and throughout much of evolution. Sophisticated metabolic pathways that evolved to regulate RSS were easily "tweaked" to deal with ROS due to the remarkable similarities between the two. However, unlike ROS, RSS are stored, recycled, and chemically more versatile. Despite these observations, the relevance and regulatory functions of RSS in extant organisms are generally underappreciated. Critical Issues: A number of factors bias observations in favor of ROS over RSS. Research conducted in room air is hyperoxic to cells, and promotes ROS production and RSS oxidation. Metabolic rates of rodent models greatly exceed those of humans; does this favor ROS? Analytical methods designed to detect ROS also respond to RSS. Do these disguise the contributions of RSS? Future Directions: Resolving the ROS/RSS issue is vital to understand biology in general and human health in particular. Improvements in experimental design and analytical methods are crucial. Perhaps the most important is an appreciation of all the attributes of RSS and keeping an open mind.
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Affiliation(s)
- Kenneth R Olson
- Department of Physiology, Indiana University School of Medicine-South Bend, South Bend, Indiana, USA
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22
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Molecular basis for the distinct functions of redox-active and FeS-transfering glutaredoxins. Nat Commun 2020; 11:3445. [PMID: 32651396 PMCID: PMC7351949 DOI: 10.1038/s41467-020-17323-0] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2019] [Accepted: 06/18/2020] [Indexed: 02/08/2023] Open
Abstract
Despite their very close structural similarity, CxxC/S-type (class I) glutaredoxins (Grxs) act as oxidoreductases, while CGFS-type (class II) Grxs act as FeS cluster transferases. Here we show that the key determinant of Grx function is a distinct loop structure adjacent to the active site. Engineering of a CxxC/S-type Grx with a CGFS-type loop switched its function from oxidoreductase to FeS transferase. Engineering of a CGFS-type Grx with a CxxC/S-type loop abolished FeS transferase activity and activated the oxidative half reaction of the oxidoreductase. The reductive half-reaction, requiring the interaction with a second GSH molecule, was enabled by switching additional residues in the active site. We explain how subtle structural differences, mostly depending on the structure of one particular loop, act in concert to determine Grx function.
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23
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Olson KR, Briggs A, Devireddy M, Xian M, Gao Y. Are the beneficial effects of 'antioxidant' lipoic acid mediated through metabolism of reactive sulfur species? Free Radic Biol Med 2020; 146:139-149. [PMID: 31676393 DOI: 10.1016/j.freeradbiomed.2019.10.410] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/24/2019] [Revised: 10/08/2019] [Accepted: 10/21/2019] [Indexed: 12/28/2022]
Abstract
The health benefits of lipoic acid (LA) are generally attributed to mitigating the harmful effects of reactive oxygen species (ROS). ROS are chemically similar to reactive sulfur species (RSS) and signal through identical mechanisms. Here we examined the effects of LA on RSS in HEK293 cells using H2S and polysulfide (PS) specific fluorophores, AzMC and SSP4. We show that LA concentration-dependently increased both H2S and PS. Physioxia (5% O2) augmented the effects of LA on H2S production but decreased PS production. Thiosulfate, a known substrate for reduced LA, and an intermediate in the catabolism of H2S enhanced the effects of LA on H2S and PS production. Inhibiting peroxiredoxins with conoidin A and gluraredoxins with tiopronin augmented the effects of LA on PS and H2S, respectively while decreasing glutathione with buthionine-sulfoximine (BSO) or diethyl maleate (DEM) decreased the stimulatory effect of LA on H2S production but augmented LA's effect on PS. Aminooxyacetate (AOA) and propargylglycine (PPG), inhibitors of H2S production from cysteine partially inhibited LA augmentation of H2S production and further decreased the LA effect when applied concurrently with BSO and DEM. The selective and cell-permeable H2S scavenger, SS20, inhibited the effects of LA on cellular H2S. Estimates of single-cell H2S production suggest that 0.1-0.2% of O2 consumption is used to metabolize H2S and these requirements may increase to 1-2% with 1 mM LA. Collectively, these results suggest that LA rescues H2S from irreversible oxidation and that the effects of LA on RSS directly confer antioxidant, anti-inflammatory and cytoprotective responses. They also suggest that TS may be an effective supplement to increase the efficacy of LA in clinical settings.
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Affiliation(s)
- Kenneth R Olson
- Indiana University School of Medicine, South Bend Center, South Bend, IN, 46617, USA; Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, 46556, USA.
| | - Austin Briggs
- Indiana University School of Medicine, South Bend Center, South Bend, IN, 46617, USA
| | - Monesh Devireddy
- Indiana University School of Medicine, South Bend Center, South Bend, IN, 46617, USA
| | - Ming Xian
- Department of Chemistry, Washington State University, Pullman, WA, 99164, USA
| | - Yan Gao
- Indiana University School of Medicine, South Bend Center, South Bend, IN, 46617, USA
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24
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Padayachee L, Rohwer JM, Pillay CS. The thioredoxin redox potential and redox charge are surrogate measures for flux in the thioredoxin system. Arch Biochem Biophys 2019; 680:108231. [PMID: 31877266 DOI: 10.1016/j.abb.2019.108231] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2019] [Accepted: 12/19/2019] [Indexed: 11/19/2022]
Abstract
The thioredoxin system plays a central role in intracellular redox regulation and its dysregulation is associated with a number of pathologies. However, the connectivity within this system poses a significant challenge for quantification and consequently several disparate measures have been used to characterize the system. For in vitro studies, the thioredoxin system flux has been measured by NADPH oxidation while the thioredoxin redox state has been used to estimate the activity of the system in vivo. The connection between these measures has been obscure although substrate saturation in the thioredoxin system results from the saturation of the thioredoxin redox cycle. We used computational modeling and in vitro kinetic assays to clarify the relationship between flux and the current in vivo measures of the thioredoxin system together with a novel measure, the thioredoxin redox charge (reduced thioredoxin/total thioredoxin). Our results revealed that the thioredoxin redox potential and redox charge closely tracked flux perturbations showing that these indices could be used as surrogate measures of the flux in vivo and, provide a mechanistic explanation for the previously observed correlations between thioredoxin oxidation and certain pathologies. While we found no significant difference in the linear correlations obtained for the thioredoxin redox potential and redox charge with the flux, the redox charge may be preferred because it is bounded between zero and one and can be determined over a wider range of conditions allowing for quantitative flux comparisons between cell types and conditions.
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Affiliation(s)
- Letrisha Padayachee
- School of Life Sciences, University of KwaZulu-Natal, Scottsville, South Africa.
| | - Johann M Rohwer
- Laboratory for Molecular Systems Biology, Department of Biochemistry, Stellenbosch University, Stellenbosch, South Africa.
| | - Ché S Pillay
- School of Life Sciences, University of KwaZulu-Natal, Scottsville, South Africa.
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25
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Henríquez-Olguín C, Boronat S, Cabello-Verrugio C, Jaimovich E, Hidalgo E, Jensen TE. The Emerging Roles of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 2 in Skeletal Muscle Redox Signaling and Metabolism. Antioxid Redox Signal 2019; 31:1371-1410. [PMID: 31588777 PMCID: PMC6859696 DOI: 10.1089/ars.2018.7678] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Significance: Skeletal muscle is a crucial tissue to whole-body locomotion and metabolic health. Reactive oxygen species (ROS) have emerged as intracellular messengers participating in both physiological and pathological adaptations in skeletal muscle. A complex interplay between ROS-producing enzymes and antioxidant networks exists in different subcellular compartments of mature skeletal muscle. Recent evidence suggests that nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are a major source of contraction- and insulin-stimulated oxidants production, but they may paradoxically also contribute to muscle insulin resistance and atrophy. Recent Advances: Pharmacological and molecular biological tools, including redox-sensitive probes and transgenic mouse models, have generated novel insights into compartmentalized redox signaling and suggested that NOX2 contributes to redox control of skeletal muscle metabolism. Critical Issues: Major outstanding questions in skeletal muscle include where NOX2 activation occurs under different conditions in health and disease, how NOX2 activation is regulated, how superoxide/hydrogen peroxide generated by NOX2 reaches the cytosol, what the signaling mediators are downstream of NOX2, and the role of NOX2 for different physiological and pathophysiological processes. Future Directions: Future research should utilize and expand the current redox-signaling toolbox to clarify the NOX2-dependent mechanisms in skeletal muscle and determine whether the proposed functions of NOX2 in cells and animal models are conserved into humans.
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Affiliation(s)
- Carlos Henríquez-Olguín
- Section of Molecular Physiology, Department of Nutrition, Exercise and Sports (NEXS), Faculty of Science, University of Copenhagen, Copenhagen, Denmark.,Muscle Cell Physiology Laboratory, Center for Exercise, Metabolism, and Cancer, Instituto de Ciencias Biomédicas, Universidad de Chile, Santiago, Chile
| | - Susanna Boronat
- Oxidative Stress and Cell Cycle Group, Universitat Pompeu Fabra, Barcelona, Spain
| | - Claudio Cabello-Verrugio
- Laboratory of Muscle Pathology, Fragility and Aging, Department of Biological Sciences, Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile.,Millennium Institute on Immunology and Immunotherapy, Santiago, Chile.,Center for the Development of Nanoscience and Nanotechnology (CEDENNA), Universidad de Santiago de Chile, Santiago, Chile
| | - Enrique Jaimovich
- Muscle Cell Physiology Laboratory, Center for Exercise, Metabolism, and Cancer, Instituto de Ciencias Biomédicas, Universidad de Chile, Santiago, Chile
| | - Elena Hidalgo
- Oxidative Stress and Cell Cycle Group, Universitat Pompeu Fabra, Barcelona, Spain
| | - Thomas E Jensen
- Section of Molecular Physiology, Department of Nutrition, Exercise and Sports (NEXS), Faculty of Science, University of Copenhagen, Copenhagen, Denmark
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26
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Ismail T, Kim Y, Lee H, Lee DS, Lee HS. Interplay Between Mitochondrial Peroxiredoxins and ROS in Cancer Development and Progression. Int J Mol Sci 2019; 20:ijms20184407. [PMID: 31500275 PMCID: PMC6770548 DOI: 10.3390/ijms20184407] [Citation(s) in RCA: 101] [Impact Index Per Article: 16.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2019] [Revised: 09/04/2019] [Accepted: 09/05/2019] [Indexed: 12/14/2022] Open
Abstract
Mitochondria are multifunctional cellular organelles that are major producers of reactive oxygen species (ROS) in eukaryotes; to maintain the redox balance, they are supplemented with different ROS scavengers, including mitochondrial peroxiredoxins (Prdxs). Mitochondrial Prdxs have physiological and pathological significance and are associated with the initiation and progression of various cancer types. In this review, we have focused on signaling involving ROS and mitochondrial Prdxs that is associated with cancer development and progression. An upregulated expression of Prdx3 and Prdx5 has been reported in different cancer types, such as breast, ovarian, endometrial, and lung cancers, as well as in Hodgkin's lymphoma and hepatocellular carcinoma. The expression of Prdx3 and Prdx5 in different types of malignancies involves their association with different factors, such as transcription factors, micro RNAs, tumor suppressors, response elements, and oncogenic genes. The microenvironment of mitochondrial Prdxs plays an important role in cancer development, as cancerous cells are equipped with a high level of antioxidants to overcome excessive ROS production. However, an increased production of Prdx3 and Prdx5 is associated with the development of chemoresistance in certain types of cancers and it leads to further complications in cancer treatment. Understanding the interplay between mitochondrial Prdxs and ROS in carcinogenesis can be useful in the development of anticancer drugs with better proficiency and decreased resistance. However, more targeted studies are required for exploring the tumor microenvironment in association with mitochondrial Prdxs to improve the existing cancer therapies and drug development.
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Affiliation(s)
- Tayaba Ismail
- KNU-Center for Nonlinear Dynamics, CMRI, School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, College of Natural Sciences, Kyungpook National University, Daegu 41566, Korea.
| | - Youni Kim
- KNU-Center for Nonlinear Dynamics, CMRI, School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, College of Natural Sciences, Kyungpook National University, Daegu 41566, Korea.
| | - Hongchan Lee
- KNU-Center for Nonlinear Dynamics, CMRI, School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, College of Natural Sciences, Kyungpook National University, Daegu 41566, Korea.
| | - Dong-Seok Lee
- KNU-Center for Nonlinear Dynamics, CMRI, School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, College of Natural Sciences, Kyungpook National University, Daegu 41566, Korea.
| | - Hyun-Shik Lee
- KNU-Center for Nonlinear Dynamics, CMRI, School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, College of Natural Sciences, Kyungpook National University, Daegu 41566, Korea.
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27
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Ferguson GD, Bridge WJ. The glutathione system and the related thiol network in Caenorhabditis elegans. Redox Biol 2019. [DOI: 10.1110.1016/j.redox.2019.101171] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
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28
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Olson KR, Gao Y. Effects of inhibiting antioxidant pathways on cellular hydrogen sulfide and polysulfide metabolism. Free Radic Biol Med 2019; 135:1-14. [PMID: 30790656 DOI: 10.1016/j.freeradbiomed.2019.02.011] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/17/2018] [Revised: 02/12/2019] [Accepted: 02/12/2019] [Indexed: 12/18/2022]
Abstract
Elaborate antioxidant pathways have evolved to minimize the threat of excessive reactive oxygen species (ROS) and to regulate ROS as signaling entities. ROS are chemically and functionally similar to reactive sulfur species (RSS) and both ROS and RSS have been shown to be metabolized by the antioxidant enzymes, superoxide dismutase and catalase. Here we use fluorophores to examine the effects of a variety of inhibitors of antioxidant pathways on metabolism of two important RSS, hydrogen sulfide (H2S with AzMC) and polysulfides (H2Sn, where n = 2-7, with SSP4) in HEK293 cells. Cells were exposed to inhibitors for up to 5 days in normoxia (21% O2) and hypoxia (5% O2), conditions also known to affect ROS production. Decreasing intracellular glutathione (GSH) with l-buthionine-sulfoximine (BSO) or diethyl maleate (DEM) decreased H2S production for 5 days but did not affect H2Sn. The glutathione reductase inhibitor, auranofin, initially decreased H2S and H2Sn but after two days H2Sn increased over controls. Inhibition of peroxiredoxins with conoidin A decreased H2S and increased H2Sn, whereas the glutathione peroxidase inhibitor, tiopronin, increased H2S. Aminoadipic acid, an inhibitor of cystine uptake did not affect either H2S or H2Sn. In buffer, the glutathione reductase and thioredoxin reductase inhibitor, 2-AAPA, the glutathione peroxidase mimetic, ebselen, and tiopronin variously reacted directly with AzMC and SSP4, reacted with H2S and H2S2, or optically interfered with AzMC or SSP4 fluorescence. Collectively these results show that antioxidant inhibitors, generally known for their ability to increase cellular ROS, have various effects on cellular RSS. These findings suggest that the inhibitors may affect cellular sulfur metabolism pathways that are not related to ROS production and in some instances they may directly affect RSS or the methods used to measure them. They also illustrate the importance of carefully evaluating RSS metabolism when biologically or pharmacologically attempting to manipulate ROS.
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Affiliation(s)
- Kenneth R Olson
- Indiana University School of Medicine - South Bend, South Bend, IN, 46617, USA; Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, 46556, USA.
| | - Yan Gao
- Indiana University School of Medicine - South Bend, South Bend, IN, 46617, USA
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29
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Ferguson GD, Bridge WJ. The glutathione system and the related thiol network in Caenorhabditis elegans. Redox Biol 2019; 24:101171. [PMID: 30901603 PMCID: PMC6429583 DOI: 10.1016/j.redox.2019.101171] [Citation(s) in RCA: 70] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2018] [Revised: 03/07/2019] [Accepted: 03/13/2019] [Indexed: 01/09/2023] Open
Abstract
Advances in the field of redox biology have contributed to the understanding of the complexity of the thiol-based system in mediating signal transduction. The redox environment is the overall spatiotemporal balance of oxidation-reduction systems within the integrated compartments of the cell, tissues and whole organisms. The ratio of the reduced to disulfide glutathione redox couple (GSH:GSSG) is a key indicator of the redox environment and its associated cellular health. The reaction mechanisms of glutathione-dependent and related thiol-based enzymes play a fundamental role in the function of GSH as a redox regulator. Glutathione homeostasis is maintained by the balance of GSH synthesis (de novo and salvage pathways) and its utilization through its detoxification, thiol signalling, and antioxidant defence functions via GSH-dependent enzymes and free radical scavenging. As such, GSH acts in concert with the entire redox network to maintain reducing conditions in the cell. Caenorhabditis elegans offers a simple model to facilitate further understanding at the multicellular level of the physiological functions of GSH and the GSH-dependent redox network. This review discusses the C. elegans studies that have investigated glutathione and related systems of the redox network including; orthologs to the protein-encoding genes of GSH synthesis; glutathione peroxidases; glutathione-S-transferases; and the glutaredoxin, thioredoxin and peroxiredoxin systems.
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Affiliation(s)
- Gavin Douglas Ferguson
- School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, 2052, Australia
| | - Wallace John Bridge
- School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, 2052, Australia.
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30
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Khurana A, Tekula S, Saifi MA, Venkatesh P, Godugu C. Therapeutic applications of selenium nanoparticles. Biomed Pharmacother 2019; 111:802-812. [DOI: 10.1016/j.biopha.2018.12.146] [Citation(s) in RCA: 292] [Impact Index Per Article: 48.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2018] [Revised: 12/18/2018] [Accepted: 12/31/2018] [Indexed: 12/12/2022] Open
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31
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Nucleoredoxin-Dependent Targets and Processes in Neuronal Cells. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2018; 2018:4829872. [PMID: 30584462 PMCID: PMC6280245 DOI: 10.1155/2018/4829872] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/29/2018] [Revised: 08/24/2018] [Accepted: 09/12/2018] [Indexed: 12/15/2022]
Abstract
Nucleoredoxin (Nrx) is an oxidoreductase of the thioredoxin family of proteins. It was shown to act as a signal transducer in some pathways; however, so far, no comprehensive analysis of its regulated substrates and functions was available. Here, we used a combination of two different strategies to fill this gap. First, we analyzed the thiol-redox state of the proteome of SH-SY5Y neuroblastoma cells depleted of Nrx compared to control cells using a differential thiol-labeling technique and quantitative mass spectrometry. 171 proteins were identified with an altered redox state; 161 of these were more reduced in the absence of Nrx. This suggests functions of Nrx in the oxidation of protein thiols. Second, we utilized the active site mutant Cys208Ser of Nrx, which stabilizes a mixed disulfide intermediate with its substrates and therefore trapped interacting proteins from the mouse brain (identifying 1710 proteins) and neuronal cell culture extracts (identifying 609 proteins). Profiling of the affected biological processes and molecular functions in cells of neuronal origin suggests numerous functions of Nrx in the redox regulation of metabolic pathways, cellular morphology, and signal transduction. These results characterize Nrx as a cellular oxidase that itself may be oxidized by the formation of disulfide relays with peroxiredoxins.
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32
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Roma LP, Deponte M, Riemer J, Morgan B. Mechanisms and Applications of Redox-Sensitive Green Fluorescent Protein-Based Hydrogen Peroxide Probes. Antioxid Redox Signal 2018; 29:552-568. [PMID: 29160083 DOI: 10.1089/ars.2017.7449] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
SIGNIFICANCE Genetically encoded hydrogen peroxide (H2O2) sensors, based on fusions between thiol peroxidases and redox-sensitive green fluorescent protein 2 (roGFP2), have dramatically broadened the available "toolbox" for monitoring cellular H2O2 changes. Recent Advances: Recently developed peroxiredoxin-based probes such as roGFP2-Tsa2ΔCR offer considerably improved H2O2 sensitivity compared with previously available genetically encoded sensors and now permit dynamic, real-time, monitoring of changes in endogenous H2O2 levels. CRITICAL ISSUES The correct understanding and interpretation of probe read-outs is crucial for their meaningful use. We discuss probe mechanisms, potential pitfalls, and best practices for application and interpretation of probe responses and highlight where gaps in our knowledge remain. FUTURE DIRECTIONS The full potential of the newly available sensors remains far from being fully realized and exploited. We discuss how the ability to monitor basal H2O2 levels in real time now allows us to re-visit long-held ideas in redox biology such as the response to ischemia-reperfusion and hypoxia-induced reactive oxygen species production. Further, recently proposed circadian cycles of peroxiredoxin hyperoxidation might now be rigorously tested. Beyond their application as H2O2 probes, roGFP2-based H2O2 sensors hold exciting potential for studying thiol peroxidase mechanisms, inactivation properties, and the impact of post-translational modifications, in vivo. Antioxid. Redox Signal. 29, 552-568.
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Affiliation(s)
- Leticia Prates Roma
- 1 Biophysics Department, Center for Human and Molecular Biology, Universität des Saarlandes , Homburg/Saar, Germany
| | - Marcel Deponte
- 2 Faculty of Chemistry/Biochemistry, University of Kaiserslautern , Kaiserslautern, Germany
| | - Jan Riemer
- 3 Institute of Biochemistry, University of Cologne , Cologne, Germany
| | - Bruce Morgan
- 4 Department of Cellular Biochemistry, University of Kaiserslautern , Kaiserslautern, Germany
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33
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Mullineaux PM, Exposito-Rodriguez M, Laissue PP, Smirnoff N. ROS-dependent signalling pathways in plants and algae exposed to high light: Comparisons with other eukaryotes. Free Radic Biol Med 2018; 122:52-64. [PMID: 29410363 DOI: 10.1016/j.freeradbiomed.2018.01.033] [Citation(s) in RCA: 96] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/01/2017] [Revised: 01/27/2018] [Accepted: 01/31/2018] [Indexed: 01/09/2023]
Abstract
Like all aerobic organisms, plants and algae co-opt reactive oxygen species (ROS) as signalling molecules to drive cellular responses to changes in their environment. In this respect, there is considerable commonality between all eukaryotes imposed by the constraints of ROS chemistry, similar metabolism in many subcellular compartments, the requirement for a high degree of signal specificity and the deployment of thiol peroxidases as transducers of oxidising equivalents to regulatory proteins. Nevertheless, plants and algae carry out specialised signalling arising from oxygenic photosynthesis in chloroplasts and photoautotropism, which often induce an imbalance between absorption of light energy and the capacity to use it productively. A key means of responding to this imbalance is through communication of chloroplasts with the nucleus to adjust cellular metabolism. Two ROS, singlet oxygen (1O2) and hydrogen peroxide (H2O2), initiate distinct signalling pathways when photosynthesis is perturbed. 1O2, because of its potent reactivity means that it initiates but does not transduce signalling. In contrast, the lower reactivity of H2O2 means that it can also be a mobile messenger in a spatially-defined signalling pathway. How plants translate a H2O2 message to bring about changes in gene expression is unknown and therefore, we draw on information from other eukaryotes to propose a working hypothesis. The role of these ROS generated in other subcellular compartments of plant cells in response to HL is critically considered alongside other eukaryotes. Finally, the responses of animal cells to oxidative stress upon high irradiance exposure is considered for new comparisons between plant and animal cells.
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Affiliation(s)
- Philip M Mullineaux
- School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK.
| | | | | | - Nicholas Smirnoff
- Biosciences, College of Life and Environmental Sciences, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK
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34
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Systemic concentrations of asymmetric dimethylarginine (ADMA) in chronic obstructive pulmonary disease (COPD): state of the art. Amino Acids 2018; 50:1169-1176. [PMID: 29951704 DOI: 10.1007/s00726-018-2606-7] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2018] [Accepted: 06/20/2018] [Indexed: 01/08/2023]
Abstract
Experimental evidence suggests that oxidative stress (OS) may increase the activity of arginine methylating enzymes that produce the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). In addition, it is well documented that OS can significantly decrease the synthesis and/or activity of ADMA degrading enzymes, thus causing ADMA accumulation in biological fluids. Recent reports have focused on circulating methylated arginine concentrations in chronic obstructive pulmonary disease, a disease characterized by a significant increase in OS. This review discusses the results of these studies and the opportunities for further research in this area.
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Abstract
SIGNIFICANCE Numerous studies have demonstrated the actions of reactive oxygen species (ROS) as regulators of several physiological processes. In this study, we discuss how redox signaling mechanisms operate to control different processes such as neuronal differentiation, oligodendrocyte differentiation, dendritic growth, and axonal growth. Recent Advances: Redox homeostasis regulates the physiology of neural stem cells (NSCs). Notably, the neuronal differentiation process of NSCs is determined by a change toward oxidative metabolism, increased levels of mitochondrial ROS, increased activity of NADPH oxidase (NOX) enzymes, decreased levels of Nrf2, and differential regulation of different redoxins. Furthermore, during the neuronal maturation processes, NOX and MICAL produce ROS to regulate cytoskeletal dynamics, which control the dendritic and axonal growth, as well as the axonal guidance. CRITICAL ISSUES The redox homeostasis changes are, in part, attributed to cell metabolism and compartmentalized production of ROS, which is regulated, sensed, and transduced by different molecules such as thioredoxins, glutaredoxins, peroxiredoxins, and nucleoredoxin to control different signaling pathways in different subcellular regions. The study of how these elements cooperatively act is essential for the understanding of nervous system development, as well as the application of regenerative therapies that recapitulate these processes. FUTURE DIRECTIONS The information about these topics in the last two decades leads us to the conclusion that the role of ROS signaling in development of the nervous system is more important than it was previously believed and makes clear the importance of exploring in more detail the mechanisms of redox signaling. Antioxid. Redox Signal. 28, 1603-1625.
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Affiliation(s)
- Mauricio Olguín-Albuerne
- División de Neurociencias, Instituto de Fisiología Celular , Universidad Nacional Autónoma de México, Ciudad de México, México
| | - Julio Morán
- División de Neurociencias, Instituto de Fisiología Celular , Universidad Nacional Autónoma de México, Ciudad de México, México
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36
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Kritsiligkou P, Rand JD, Weids AJ, Wang X, Kershaw CJ, Grant CM. Endoplasmic reticulum (ER) stress-induced reactive oxygen species (ROS) are detrimental for the fitness of a thioredoxin reductase mutant. J Biol Chem 2018; 293:11984-11995. [PMID: 29871930 DOI: 10.1074/jbc.ra118.001824] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2018] [Revised: 05/16/2018] [Indexed: 12/16/2022] Open
Abstract
The unfolded protein response (UPR) is constitutively active in yeast thioredoxin reductase mutants, suggesting a link between cytoplasmic thiol redox control and endoplasmic reticulum (ER) oxidative protein folding. The unique oxidative environment of the ER lumen requires tight regulatory control, and we show that the active UPR depends on the presence of oxidized thioredoxins rather than arising because of a loss of thioredoxin function. Preventing activation of the UPR by deletion of HAC1, encoding the UPR transcription factor, rescues a number of thioredoxin reductase mutant phenotypes, including slow growth, shortened longevity, and oxidation of the cytoplasmic GSH pool. This is because the constitutive UPR in a thioredoxin reductase mutant results in the generation of hydrogen peroxide. The oxidation of thioredoxins in a thioredoxin reductase mutant requires aerobic metabolism and the presence of the Tsa1 and Tsa2 peroxiredoxins, indicating that a complete cytoplasmic thioredoxin system is crucial for maintaining ER redox homeostasis.
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Affiliation(s)
- Paraskevi Kritsiligkou
- Faculty of Biology, Medicine, and Health, University of Manchester, Manchester M13 9PT, United Kingdom
| | - Jonathan D Rand
- Faculty of Biology, Medicine, and Health, University of Manchester, Manchester M13 9PT, United Kingdom
| | - Alan J Weids
- Faculty of Biology, Medicine, and Health, University of Manchester, Manchester M13 9PT, United Kingdom
| | - Ximeng Wang
- Faculty of Biology, Medicine, and Health, University of Manchester, Manchester M13 9PT, United Kingdom
| | - Chris J Kershaw
- Faculty of Biology, Medicine, and Health, University of Manchester, Manchester M13 9PT, United Kingdom
| | - Chris M Grant
- Faculty of Biology, Medicine, and Health, University of Manchester, Manchester M13 9PT, United Kingdom.
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37
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Möller D, Gellert M, Langel W, Lillig CH. Molecular dynamics simulations and in vitro analysis of the CRMP2 thiol switch. MOLECULAR BIOSYSTEMS 2018; 13:1744-1753. [PMID: 28726921 DOI: 10.1039/c7mb00160f] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The collapsin response mediator protein CRMP2 (gene: DPYSL2) is crucial for neuronal development. The homotetrameric CRMP2 complex is regulated via two mechanisms: first by phosphorylation and second by the reduction and oxidation of the Cys504 residues of two adjacent subunits. Here, we have analysed the effects of this redox switch on the protein in vitro combined with force field molecular dynamics (MD). Earlier X-ray data reveal the structure of the rigid body of the molecule but lack the flexible C-terminus with the important sites for phosphorylation and redox regulation. An in silico model for this part was established by replica exchange simulations and homology modelling, which is consistent with the CD spectroscopy results of the recombinant protein. Thermofluor data indicated that the protein aggregates at bivalent ion concentrations below 200 mM. In simulations the protein surface was covered under these conditions by a large number of ions, which most likely prevent aggregation. A tryptophan residue (Trp295) in close proximity to the forming disulphide allowed the measurement of the structural relaxation of the rigid body upon reduction by fluorescence quenching. We were also able to determine the second-order rate constant of CRMP2 oxidation by H2O2. The simulated solvent accessible surface of the hydroxyl group of Ser518 significantly increased upon reduction of the disulphide bond. Our results give the first detailed insight into the profound structural changes of the tetrameric CRMP2 due to oxidation and indicate a tightly connected regulation by phosphorylation and redox modification.
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Affiliation(s)
- Daniel Möller
- Biophysical Chemistry, Institute of Biochemistry, University Greifswald, 17489 Greifswald, Germany
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Stöcker S, Van Laer K, Mijuskovic A, Dick TP. The Conundrum of Hydrogen Peroxide Signaling and the Emerging Role of Peroxiredoxins as Redox Relay Hubs. Antioxid Redox Signal 2018; 28:558-573. [PMID: 28587525 DOI: 10.1089/ars.2017.7162] [Citation(s) in RCA: 145] [Impact Index Per Article: 20.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
SIGNIFICANCE Hydrogen peroxide (H2O2) is known to act as a messenger in signal transduction. How H2O2 leads to selective and efficient oxidation of specific thiols on specific signaling proteins remains one of the most important open questions in redox biology. Recent Advances: Increasing evidence implicates thiol peroxidases as mediators of protein thiol oxidation. Recently, this evidence has been extended to include the peroxiredoxins (Prxs). Prxs are exceptionally sensitive to H2O2, abundantly expressed and capture most of the H2O2 that is generated inside cells. CRITICAL ISSUES The overall prevalence and importance of Prx-based redox signaling relays are still unknown. The same is true for alternative mechanisms of redox signaling. FUTURE DIRECTIONS It will be important to clarify the relative contributions of Prx-mediated and direct thiol oxidation to H2O2 signaling. Many questions relating to Prx-based redox relays remain to be answered, including their mechanism, structural organization, and the potential role of adaptor proteins. Antioxid. Redox Signal. 28, 558-573.
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Affiliation(s)
- Sarah Stöcker
- Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ) , Heidelberg, Germany
| | - Koen Van Laer
- Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ) , Heidelberg, Germany
| | - Ana Mijuskovic
- Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ) , Heidelberg, Germany
| | - Tobias P Dick
- Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ) , Heidelberg, Germany
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Topf U, Suppanz I, Samluk L, Wrobel L, Böser A, Sakowska P, Knapp B, Pietrzyk MK, Chacinska A, Warscheid B. Quantitative proteomics identifies redox switches for global translation modulation by mitochondrially produced reactive oxygen species. Nat Commun 2018; 9:324. [PMID: 29358734 PMCID: PMC5778013 DOI: 10.1038/s41467-017-02694-8] [Citation(s) in RCA: 151] [Impact Index Per Article: 21.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2017] [Accepted: 12/19/2017] [Indexed: 11/09/2022] Open
Abstract
The generation of reactive oxygen species (ROS) is inevitably linked to life. However, the precise role of ROS in signalling and specific targets is largely unknown. We perform a global proteomic analysis to delineate the yeast redoxome to a depth of more than 4,300 unique cysteine residues in over 2,200 proteins. Mapping of redox-active thiols in proteins exposed to exogenous or endogenous mitochondria-derived oxidative stress reveals ROS-sensitive sites in several components of the translation apparatus. Mitochondria are the major source of cellular ROS. We demonstrate that increased levels of intracellular ROS caused by dysfunctional mitochondria serve as a signal to attenuate global protein synthesis. Hence, we propose a universal mechanism that controls protein synthesis by inducing reversible changes in the translation machinery upon modulating the redox status of proteins involved in translation. This crosstalk between mitochondria and protein synthesis may have an important contribution to pathologies caused by dysfunctional mitochondria.
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Affiliation(s)
- Ulrike Topf
- International Institute of Molecular and Cell Biology, 4 Ks. Trojdena Street, 02-109, Warsaw, Poland.,Centre of New Technologies, University of Warsaw, S. Banacha 2c, 02-097, Warsaw, Poland
| | - Ida Suppanz
- Faculty of Biology, Institute of Biology II, Biochemistry-Functional Proteomics, University of Freiburg, Schänzlestrasse 1, 79104, Freiburg, Germany.,BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schänzlestrasse 18, 79104, Freiburg, Germany
| | - Lukasz Samluk
- International Institute of Molecular and Cell Biology, 4 Ks. Trojdena Street, 02-109, Warsaw, Poland.,Centre of New Technologies, University of Warsaw, S. Banacha 2c, 02-097, Warsaw, Poland
| | - Lidia Wrobel
- International Institute of Molecular and Cell Biology, 4 Ks. Trojdena Street, 02-109, Warsaw, Poland
| | - Alexander Böser
- Faculty of Biology, Institute of Biology II, Biochemistry-Functional Proteomics, University of Freiburg, Schänzlestrasse 1, 79104, Freiburg, Germany
| | - Paulina Sakowska
- International Institute of Molecular and Cell Biology, 4 Ks. Trojdena Street, 02-109, Warsaw, Poland
| | - Bettina Knapp
- Faculty of Biology, Institute of Biology II, Biochemistry-Functional Proteomics, University of Freiburg, Schänzlestrasse 1, 79104, Freiburg, Germany
| | - Martyna K Pietrzyk
- International Institute of Molecular and Cell Biology, 4 Ks. Trojdena Street, 02-109, Warsaw, Poland.,Centre of New Technologies, University of Warsaw, S. Banacha 2c, 02-097, Warsaw, Poland
| | - Agnieszka Chacinska
- International Institute of Molecular and Cell Biology, 4 Ks. Trojdena Street, 02-109, Warsaw, Poland. .,Centre of New Technologies, University of Warsaw, S. Banacha 2c, 02-097, Warsaw, Poland.
| | - Bettina Warscheid
- Faculty of Biology, Institute of Biology II, Biochemistry-Functional Proteomics, University of Freiburg, Schänzlestrasse 1, 79104, Freiburg, Germany. .,BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schänzlestrasse 18, 79104, Freiburg, Germany. .,ZBSA Centre for Biological Systems Analysis, University of Freiburg, Habsburgerstrasse 49, 79104, Freiburg, Germany.
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Abstract
SIGNIFICANCE Glutathione metabolism is comparable to a jigsaw puzzle with too many pieces. It is supposed to comprise (i) the reduction of disulfides, hydroperoxides, sulfenic acids, and nitrosothiols, (ii) the detoxification of aldehydes, xenobiotics, and heavy metals, and (iii) the synthesis of eicosanoids, steroids, and iron-sulfur clusters. In addition, glutathione affects oxidative protein folding and redox signaling. Here, I try to provide an overview on the relevance of glutathione-dependent pathways with an emphasis on quantitative data. Recent Advances: Intracellular redox measurements reveal that the cytosol, the nucleus, and mitochondria contain very little glutathione disulfide and that oxidative challenges are rapidly counterbalanced. Genetic approaches suggest that iron metabolism is the centerpiece of the glutathione puzzle in yeast. Furthermore, recent biochemical studies provide novel insights on glutathione transport processes and uncoupling mechanisms. CRITICAL ISSUES Which parts of the glutathione puzzle are most relevant? Does this explain the high intracellular concentrations of reduced glutathione? How can iron-sulfur cluster biogenesis, oxidative protein folding, or redox signaling occur at high glutathione concentrations? Answers to these questions not only seem to depend on the organism, cell type, and subcellular compartment but also on different ideologies among researchers. FUTURE DIRECTIONS A rational approach to compare the relevance of glutathione-dependent pathways is to combine genetic and quantitative kinetic data. However, there are still many missing pieces and too little is known about the compartment-specific repertoire and concentration of numerous metabolites, substrates, enzymes, and transporters as well as rate constants and enzyme kinetic patterns. Gathering this information might require the development of novel tools but is crucial to address potential kinetic competitions and to decipher uncoupling mechanisms to solve the glutathione puzzle. Antioxid. Redox Signal. 27, 1130-1161.
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Affiliation(s)
- Marcel Deponte
- Department of Parasitology, Ruprecht-Karls University , Heidelberg, Germany
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Abstract
SIGNIFICANCE Glutathione (GSH) is the most abundant cellular low-molecular-weight thiol in the majority of organisms in all kingdoms of life. Therefore, functions of GSH and disturbed regulation of its concentration are associated with numerous physiological and pathological situations. Recent Advances: The function of GSH as redox buffer or antioxidant is increasingly being questioned. New functions, especially functions connected to the cellular iron homeostasis, were elucidated. Via the formation of iron complexes, GSH is an important player in all aspects of iron metabolism: sensing and regulation of iron levels, iron trafficking, and biosynthesis of iron cofactors. The variety of GSH coordinated iron complexes and their functions with a special focus on FeS-glutaredoxins are summarized in this review. Interestingly, GSH analogues that function as major low-molecular-weight thiols in organisms lacking GSH resemble the functions in iron homeostasis. CRITICAL ISSUES Since these iron-related functions are most likely also connected to thiol redox chemistry, it is difficult to distinguish between mechanisms related to either redox or iron metabolisms. FUTURE DIRECTIONS The ability of GSH to coordinate iron in different complexes with or without proteins needs further investigation. The discovery of new Fe-GSH complexes and their physiological functions will significantly advance our understanding of cellular iron homeostasis. Antioxid. Redox Signal. 27, 1235-1251.
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Affiliation(s)
- Carsten Berndt
- 1 Department of Neurology, Medical Faculty, Life Science Center , Heinrich-Heine-Universität, Düsseldorf, Germany
| | - Christopher Horst Lillig
- 2 Institute for Medical Biochemistry and Molecular Biology, University Medicine Greifswald , Greifswald, Germany
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Redox Regulation of Inflammatory Processes Is Enzymatically Controlled. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2017; 2017:8459402. [PMID: 29118897 PMCID: PMC5651112 DOI: 10.1155/2017/8459402] [Citation(s) in RCA: 52] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/03/2017] [Revised: 07/06/2017] [Accepted: 07/25/2017] [Indexed: 12/11/2022]
Abstract
Redox regulation depends on the enzymatically controlled production and decay of redox active molecules. NADPH oxidases, superoxide dismutases, nitric oxide synthases, and others produce the redox active molecules superoxide, hydrogen peroxide, nitric oxide, and hydrogen sulfide. These react with target proteins inducing spatiotemporal modifications of cysteine residues within different signaling cascades. Thioredoxin family proteins are key regulators of the redox state of proteins. They regulate the formation and removal of oxidative modifications by specific thiol reduction and oxidation. All of these redox enzymes affect inflammatory processes and the innate and adaptive immune response. Interestingly, this regulation involves different mechanisms in different biological compartments and specialized cell types. The localization and activity of distinct proteins including, for instance, the transcription factor NFκB and the immune mediator HMGB1 are redox-regulated. The transmembrane protein ADAM17 releases proinflammatory mediators, such as TNFα, and is itself regulated by a thiol switch. Moreover, extracellular redox enzymes were shown to modulate the activity and migration behavior of various types of immune cells by acting as cytokines and/or chemokines. Within this review article, we will address the concept of redox signaling and the functions of both redox enzymes and redox active molecules in innate and adaptive immune responses.
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Bilan DS, Belousov VV. New tools for redox biology: From imaging to manipulation. Free Radic Biol Med 2017; 109:167-188. [PMID: 27939954 DOI: 10.1016/j.freeradbiomed.2016.12.004] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/22/2016] [Revised: 12/02/2016] [Accepted: 12/03/2016] [Indexed: 12/12/2022]
Abstract
Redox reactions play a key role in maintaining essential biological processes. Deviations in redox pathways result in the development of various pathologies at cellular and organismal levels. Until recently, studies on transformations in the intracellular redox state have been significantly hampered in living systems. The genetically encoded indicators, based on fluorescent proteins, have provided new opportunities in biomedical research. The existing indicators already enable monitoring of cellular redox parameters in different processes including embryogenesis, aging, inflammation, tissue regeneration, and pathogenesis of various diseases. In this review, we summarize information about all genetically encoded redox indicators developed to date. We provide the description of each indicator and discuss its advantages and limitations, as well as points that need to be considered when choosing an indicator for a particular experiment. One chapter is devoted to the important discoveries that have been made by using genetically encoded redox indicators.
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Affiliation(s)
- Dmitry S Bilan
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia
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Knockout of the peroxiredoxin 5 homologue PFAOP does not affect the artemisinin susceptibility of Plasmodium falciparum. Sci Rep 2017; 7:4410. [PMID: 28667301 PMCID: PMC5493673 DOI: 10.1038/s41598-017-04277-5] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2016] [Accepted: 05/03/2017] [Indexed: 01/07/2023] Open
Abstract
Artemisinins are the current mainstay of malaria chemotherapy. Their exact mode of action is an ongoing matter of debate, and several factors have recently been reported to affect an early stage of artemisinin resistance of the most important human malaria parasite Plasmodium falciparum. Here, we identified a locus on chromosome 7 that affects the artemisinin susceptibility of P. falciparum in a quantitative trait locus analysis of a genetic cross between strains 7G8 and GB4. This locus includes the peroxiredoxin gene PFAOP. However, steady-state kinetic data with recombinant PfAOP do not support a direct interaction between this peroxidase and the endoperoxide artemisinin. Furthermore, neither the overexpression nor the deletion of the encoding gene affected the IC50 values for artemisinin or the oxidants diamide and tert-butyl hydroperoxide. Thus, PfAOP is dispensable for blood stage parasite survival, and the correlation between the artemisinin susceptibility and chromosome 7 is probably based on another gene within the identified locus.
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45
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Glutaredoxin catalysis requires two distinct glutathione interaction sites. Nat Commun 2017; 8:14835. [PMID: 28374771 PMCID: PMC5382279 DOI: 10.1038/ncomms14835] [Citation(s) in RCA: 71] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2016] [Accepted: 02/02/2017] [Indexed: 01/15/2023] Open
Abstract
Glutaredoxins are key players in cellular redox homoeostasis and exert a variety of essential functions ranging from glutathione-dependent catalysis to iron metabolism. The exact structure–function relationships and mechanistic differences among glutaredoxins that are active or inactive in standard enzyme assays have so far remained elusive despite numerous kinetic and structural studies. Here, we elucidate the enzymatic mechanism showing that glutaredoxins require two distinct glutathione interaction sites for efficient redox catalysis. The first site interacts with the glutathione moiety of glutathionylated disulfide substrates. The second site activates glutathione as the reducing agent. We propose that the requirement of two distinct glutathione interaction sites for the efficient reduction of glutathionylated disulfide substrates explains the deviating structure–function relationships, activities and substrate preferences of different glutaredoxin subfamilies as well as thioredoxins. Our model also provides crucial insights for the design or optimization of artificial glutaredoxins, transition-state inhibitors and glutaredoxin-coupled redox sensors. Glutaredoxins have important roles in redox processes. Here the authors show that the enzymatic activity of glutaredoxins requires two distinct glutathione interactions sites, one recognizing the glutathione disulfide substrate and one activating glutathione as a reducing agent.
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46
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Forred BJ, Daugaard DR, Titus BK, Wood RR, Floen MJ, Booze ML, Vitiello PF. Detoxification of Mitochondrial Oxidants and Apoptotic Signaling Are Facilitated by Thioredoxin-2 and Peroxiredoxin-3 during Hyperoxic Injury. PLoS One 2017; 12:e0168777. [PMID: 28045936 PMCID: PMC5207683 DOI: 10.1371/journal.pone.0168777] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2016] [Accepted: 12/06/2016] [Indexed: 01/22/2023] Open
Abstract
Mitochondria play a fundamental role in the regulation of cell death during accumulation of oxidants. High concentrations of atmospheric oxygen (hyperoxia), used clinically to treat tissue hypoxia in premature newborns, is known to elicit oxidative stress and mitochondrial injury to pulmonary epithelial cells. A consequence of oxidative stress in mitochondria is the accumulation of peroxides which are detoxified by the dedicated mitochondrial thioredoxin system. This system is comprised of the oxidoreductase activities of peroxiredoxin-3 (Prx3), thioredoxin-2 (Trx2), and thioredoxin reductase-2 (TrxR2). The goal of this study was to understand the role of the mitochondrial thioredoxin system and mitochondrial injuries during hyperoxic exposure. Flow analysis of the redox-sensitive, mitochondrial-specific fluorophore, MitoSOX, indicated increased levels of mitochondrial oxidant formation in human adenocarcinoma cells cultured in 95% oxygen. Increased expression of Trx2 and TrxR2 in response to hyperoxia were not attributable to changes in mitochondrial mass, suggesting that hyperoxic upregulation of mitochondrial thioredoxins prevents accumulation of oxidized Prx3. Mitochondrial oxidoreductase activities were modulated through pharmacological inhibition of TrxR2 with auranofin and genetically through shRNA knockdown of Trx2 and Prx3. Diminished Trx2 and Prx3 expression was associated with accumulation of mitochondrial superoxide; however, only shRNA knockdown of Trx2 increased susceptibility to hyperoxic cell death and increased phosphorylation of apoptosis signal-regulating kinase-1 (ASK1). In conclusion, the mitochondrial thioredoxin system regulates hyperoxic-mediated death of pulmonary epithelial cells through detoxification of oxidants and regulation of redox-dependent apoptotic signaling.
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Affiliation(s)
- Benjamin J. Forred
- Children’s Health Research Center, Sanford Research, Sioux Falls, South Dakota, United States of America
| | - Darwin R. Daugaard
- Children’s Health Research Center, Sanford Research, Sioux Falls, South Dakota, United States of America
| | - Brianna K. Titus
- Children’s Health Research Center, Sanford Research, Sioux Falls, South Dakota, United States of America
| | - Ryan R. Wood
- Children’s Health Research Center, Sanford Research, Sioux Falls, South Dakota, United States of America
| | - Miranda J. Floen
- Children’s Health Research Center, Sanford Research, Sioux Falls, South Dakota, United States of America
| | - Michelle L. Booze
- Children’s Health Research Center, Sanford Research, Sioux Falls, South Dakota, United States of America
| | - Peter F. Vitiello
- Children’s Health Research Center, Sanford Research, Sioux Falls, South Dakota, United States of America
- Department of Pediatrics, University of South Dakota Sanford School of Medicine, Sioux Falls, South Dakota, United States of America
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Booze ML, Hansen JM, Vitiello PF. A novel mouse model for the identification of thioredoxin-1 protein interactions. Free Radic Biol Med 2016; 99:533-543. [PMID: 27639450 PMCID: PMC5107173 DOI: 10.1016/j.freeradbiomed.2016.09.013] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/25/2016] [Revised: 09/07/2016] [Accepted: 09/13/2016] [Indexed: 12/19/2022]
Abstract
Thiol switches are important regulators of cellular signaling and are coordinated by several redox enzyme systems including thioredoxins, peroxiredoxins, and glutathione. Thioredoxin-1 (Trx1), in particular, is an important signaling molecule not only in response to redox perturbations, but also in cellular growth, regulation of gene expression, and apoptosis. The active site of this enzyme is a highly conserved C-G-P-C motif and the redox mechanism of Trx1 is rapid which presents a challenge in determining specific substrates. Numerous in vitro approaches have identified Trx1-dependent thiol switches; however, these findings may not be physiologically relevant and little is known about Trx1 interactions in vivo. In order to identify Trx1 targets in vivo, we generated a transgenic mouse with inducible expression of a mutant Trx1 transgene to stabilize intermolecular disulfides with protein substrates. Expression of the Trx1 "substrate trap" transgene did not interfere with endogenous thioredoxin or glutathione systems in brain, heart, lung, liver, and kidney. Following immunoprecipitation and proteomic analysis, we identified 41 homeostatic Trx1 interactions in perinatal lung, including previously described Trx1 substrates such as members of the peroxiredoxin family and collapsin response mediator protein 2. Using perinatal hyperoxia as a model of oxidative injury, we found 17 oxygen-induced interactions which included several cytoskeletal proteins which may be important to alveolar development. The data herein validates this novel mouse model for identification of tissue- and cell-specific Trx1-dependent pathways that regulate physiological signals in response to redox perturbations.
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Affiliation(s)
- Michelle L Booze
- Children's Health Research Center, Sanford Research, Sioux Falls, SD 57104, USA
| | - Jason M Hansen
- Department of Physiology and Developmental Biology, College of Life Sciences, Brigham Young University, Provo, UT 84602, USA
| | - Peter F Vitiello
- Children's Health Research Center, Sanford Research, Sioux Falls, SD 57104, USA; Department of Pediatrics, Sanford School of Medicine, The University of South Dakota, Sioux Falls, SD 57104, USA.
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Zinellu A, Fois AG, Sotgia S, Sotgiu E, Zinellu E, Bifulco F, Mangoni AA, Pirina P, Carru C. Arginines Plasma Concentration and Oxidative Stress in Mild to Moderate COPD. PLoS One 2016; 11:e0160237. [PMID: 27479314 PMCID: PMC4968788 DOI: 10.1371/journal.pone.0160237] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2016] [Accepted: 07/15/2016] [Indexed: 11/17/2022] Open
Abstract
Background Elevated plasma concentrations of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) have been observed in respiratory conditions such as asthma and cystic fibrosis. Since oxidative stress has been shown to increase the activity of arginine methylating enzymes, hence increased ADMA synthesis, and to reduce ADMA degrading enzymes, hence increased ADMA concentrations, we assessed methylated arginines concentrations in chronic obstructive pulmonary disease (COPD), a disease characterized by increased oxidative stress. Methods Plasma arginine, ADMA and symmetric dimethylarginine (SDMA), oxidative stress markers (thiobarbituric acid reactive substances, TBARS, and plasma proteins SH, PSH) and antioxidants (taurine and paraoxonase 1, PON1, activity) were measured in 43 COPD patients with mild (n = 29) or moderate (n = 14) disease and 43 age- and sex-matched controls. Results TBARS significantly increased with COPD presence and severity (median 2.93 vs 3.18 vs 3.64 μmol/L, respectively in controls, mild and moderate group, p<0.0001 by ANOVA) whereas PSH decreased (6.69±1.15 vs 6.04±0.85 vs 5.33±0.96 μmol/gr prot, p<0.0001 by ANOVA). Increased ADMA/arginine ratio, primarily due to reduced arginine concentrations, was also observed with COPD presence and severity (median 0.0067 vs 0.0075 vs 0.0100, p<0.0001 by ANOVA). In multiple logistic regression analysis, only TBARS (OR 0.44, 95% CI 0.25–0.77; p = 0.0045) and ADMA/Arginine ratio (OR 1.72, 95% CI 2.27–13.05; p = 0.02) were independently associated with COPD severity. Conclusion COPD presence and severity are associated with increased oxidative stress and alterations in arginine metabolism. The reduced arginine concentrations in COPD may offer a new target for therapeutic interventions increasing arginine availability.
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Affiliation(s)
- Angelo Zinellu
- Department of Biomedical Sciences, University of Sassari, Sassari, Italy
| | | | - Salvatore Sotgia
- Department of Biomedical Sciences, University of Sassari, Sassari, Italy
| | - Elisabetta Sotgiu
- Department of Biomedical Sciences, University of Sassari, Sassari, Italy
| | - Elisabetta Zinellu
- Department of Clinical and Experimental Medicine, University of Sassari, Sassari, Italy
| | - Fabiana Bifulco
- Department of Clinical and Experimental Medicine, University of Sassari, Sassari, Italy
| | - Arduino A Mangoni
- Department of Clinical Pharmacology, School of Medicine, Flinders University, Adelaide, Australia
| | - Pietro Pirina
- Department of Clinical and Experimental Medicine, University of Sassari, Sassari, Italy
| | - Ciriaco Carru
- Department of Biomedical Sciences, University of Sassari, Sassari, Italy.,Quality Control Unit, University Hospital Sassari (AOU), Sassari, Italy
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49
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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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50
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Pillay CS, Eagling BD, Driscoll SRE, Rohwer JM. Quantitative measures for redox signaling. Free Radic Biol Med 2016; 96:290-303. [PMID: 27151506 DOI: 10.1016/j.freeradbiomed.2016.04.199] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/22/2015] [Revised: 04/25/2016] [Accepted: 04/29/2016] [Indexed: 12/17/2022]
Abstract
Redox signaling is now recognized as an important regulatory mechanism for a number of cellular processes including the antioxidant response, phosphokinase signal transduction and redox metabolism. While there has been considerable progress in identifying the cellular machinery involved in redox signaling, quantitative measures of redox signals have been lacking, limiting efforts aimed at understanding and comparing redox signaling under normoxic and pathogenic conditions. Here we have outlined some of the accepted principles for redox signaling, including the description of hydrogen peroxide as a signaling molecule and the role of kinetics in conferring specificity to these signaling events. Based on these principles, we then develop a working definition for redox signaling and review a number of quantitative methods that have been employed to describe signaling in other systems. Using computational modeling and published data, we show how time- and concentration- dependent analyses, in particular, could be used to quantitatively describe redox signaling and therefore provide important insights into the functional organization of redox networks. Finally, we consider some of the key challenges with implementing these methods.
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Affiliation(s)
- Ché S Pillay
- School of Life Sciences, University of KwaZulu-Natal, Carbis Road, Pietermaritzburg 3201, South Africa.
| | - Beatrice D Eagling
- School of Life Sciences, University of KwaZulu-Natal, Carbis Road, Pietermaritzburg 3201, South Africa
| | - Scott R E Driscoll
- School of Life Sciences, University of KwaZulu-Natal, Carbis Road, Pietermaritzburg 3201, South Africa
| | - Johann M Rohwer
- Department of Biochemistry, Stellenbosch University, Private Bag X1, Matieland, 7602 Stellenbosch, South Africa
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