|
1
|
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.
Collapse
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.
| |
Collapse
|
|
2
|
Nagasawa M. Pathophysiology of acute graft-versus-host disease from the perspective of hemodynamics determined by dielectric analysis. World J Transplant 2023; 13:379-390. [PMID: 38174146 PMCID: PMC10758686 DOI: 10.5500/wjt.v13.i6.379] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/22/2023] [Revised: 11/01/2023] [Accepted: 12/01/2023] [Indexed: 12/15/2023] Open
Abstract
BACKGROUND Numerous reports have demonstrated that the pathophysiology of graft-versus-host disease (GVHD) during hematopoietic stem cell transplantation (HSCT) is closely related to vascular endothelial disorders and coagulation abnormalities. We previously presented the discovery of a principle and the development of a novel instrument for measuring whole blood coagulation. This was achieved by assessing the variations in the dielectric properties of whole blood. AIM To investigate how GVHD affects the changes of dielectric properties of whole blood in patients with HSCT. METHODS We examined the changes of dielectric properties of whole blood and erythrocyte proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis sequentially in patients with HSCT and compared it with clinical symptoms and inflammatory parameters of GVHD. RESULTS During severe GVHD, the dielectric relaxation strength markedly increased and expression of band3 decreased. The dielectric relaxation strength normalized with the improvement of GVHD. In vitro analysis confirmed that the increase of relaxation strength was associated with severe erythrocyte aggregates, but not with decreased expression of band3. CONCLUSION Severe erythrocyte aggregates observed in GVHD may cause coagulation abnor malities and circulatory failure, which, together with the irreversible erythrocyte dysfunction we recently reported, could lead to organ failure.
Collapse
Affiliation(s)
- Masayuki Nagasawa
- Department of Pediatrics, Musashino Red Cross Hospital, Musashino City 180-8610, Tokyo, Japan
- Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental University, Tokyo 113-8519, Tokyo, Japan
| |
Collapse
|
|
3
|
Piñeyro MD, Chiribao ML, Arias DG, Robello C, Parodi-Talice A. Overoxidation and Oligomerization of Trypanosoma cruzi Cytosolic and Mitochondrial Peroxiredoxins. Pathogens 2023; 12:1273. [PMID: 37887789 PMCID: PMC10610341 DOI: 10.3390/pathogens12101273] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2023] [Revised: 10/11/2023] [Accepted: 10/13/2023] [Indexed: 10/28/2023] Open
Abstract
Peroxiredoxins (Prxs) have been shown to be important enzymes for trypanosomatids, counteracting oxidative stress and promoting cell infection and intracellular survival. In this work, we investigate the in vitro sensitivity to overoxidation and the overoxidation dynamics of Trypanosoma cruzi Prxs in parasites in culture and in the infection context. We showed that recombinant m-TXNPx, in contrast to what was observed for c-TXNPx, exists as low molecular mass forms in the overoxidized state. We observed that T. cruzi Prxs were overoxidized in epimastigotes treated with oxidants, and a significant proportion of the overoxidized forms were still present at least 24 h after treatment suggesting that these forms are not actively reversed. In in vitro infection experiments, we observed that Prxs are overoxidized in amastigotes residing in infected macrophages, demonstrating that inactivation of at least part of the Prxs by overoxidation occurs in a physiological context. We have shown that m-TXNPx has a redox-state-dependent chaperone activity. This function may be related to the increased thermotolerance observed in m-TXNPx-overexpressing parasites. This study suggests that despite the similarity between protozoan and mammalian Prxs, T. cruzi Prxs have different oligomerization dynamics and sensitivities to overoxidation, which may have implications for their function in the parasite life cycle and infection process.
Collapse
Affiliation(s)
- María Dolores Piñeyro
- Laboratorio de Interacciones Hospedero Patógeno, Unidad de Biología Molecular, Institut Pasteur de Montevideo, Montevideo 11400, Uruguay; (M.D.P.); (M.L.C.); (C.R.)
- Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay
| | - María Laura Chiribao
- Laboratorio de Interacciones Hospedero Patógeno, Unidad de Biología Molecular, Institut Pasteur de Montevideo, Montevideo 11400, Uruguay; (M.D.P.); (M.L.C.); (C.R.)
- Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay
| | - Diego G. Arias
- Laboratorio de Enzimología Molecular, Instituto de Agrobiotecnología del Litoral, UNL-CONICET, Santa Fe 3000, Argentina;
- Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe 3000, Argentina
| | - Carlos Robello
- Laboratorio de Interacciones Hospedero Patógeno, Unidad de Biología Molecular, Institut Pasteur de Montevideo, Montevideo 11400, Uruguay; (M.D.P.); (M.L.C.); (C.R.)
- Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay
| | - Adriana Parodi-Talice
- Laboratorio de Interacciones Hospedero Patógeno, Unidad de Biología Molecular, Institut Pasteur de Montevideo, Montevideo 11400, Uruguay; (M.D.P.); (M.L.C.); (C.R.)
- Sección Genética Evolutiva, Instituto de Biología, Facultad de Ciencias, Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay
| |
Collapse
|
|
4
|
Nagasawa M. Degradation of band3 and PRDX2 in erythrocytes during severe acute GVHD. EJHAEM 2023; 4:459-462. [PMID: 37206257 PMCID: PMC10188505 DOI: 10.1002/jha2.660] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 12/29/2022] [Revised: 02/01/2023] [Accepted: 02/03/2023] [Indexed: 05/21/2023]
Abstract
We investigated the proteins of erythrocytes from stem cell transplantation patients and found decreased expression of band3 and C-terminal-truncated peroxiredoxin 2 (PRDX2) only during severe graft-versus-host disease (GVHD), using time-of-flight mass spectrometry (TOF-MS) analysis and Western blotting. During the same period, PRDX2 dimerization and calpain-1 activation were observed, indicating severe oxidative stress. We also found a putative cleavage site for calpain-1 in the C-terminal-truncated site of PRDX2. Decreased band3 expression impairs the plasticity and stability of erythrocytes, and C-terminal-truncated PRDX2 induces irreversible dysfunction of antioxidant activity. These effects may exacerbate microcirculation disorders and the progression of organ dysfunction.
Collapse
Affiliation(s)
- Masayuki Nagasawa
- Department of PediatricsMusashino Red Cross HospitalMusashinoTokyoJapan
- Department of Pediatrics and Developmental BiologyTokyo Medical and Dental UniversityTokyoJapan
| |
Collapse
|
|
5
|
Yu QQ, Zhang H, Guo Y, Han B, Jiang P. The Intestinal Redox System and Its Significance in Chemotherapy-Induced Intestinal Mucositis. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2022; 2022:7255497. [PMID: 35585883 PMCID: PMC9110227 DOI: 10.1155/2022/7255497] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/02/2022] [Revised: 04/04/2022] [Accepted: 04/09/2022] [Indexed: 12/12/2022]
Abstract
Chemotherapy-induced intestinal mucositis (CIM) is a significant dose-limiting adverse reaction brought on by the cancer treatment. Multiple studies reported that reactive oxygen species (ROS) is rapidly produced during the initial stages of chemotherapy, when the drugs elicit direct damage to intestinal mucosal cells, which, in turn, results in necrosis, mitochondrial dysfunction, and ROS production. However, the mechanism behind the intestinal redox system-based induction of intestinal mucosal injury and necrosis of CIM is still undetermined. In this article, we summarized relevant information regarding the intestinal redox system, including the composition and regulation of redox enzymes, ROS generation, and its regulation in the intestine. We innovatively proposed the intestinal redox "Tai Chi" theory and revealed its significance in the pathogenesis of CIM. We also conducted an extensive review of the English language-based literatures involving oxidative stress (OS) and its involvement in the pathological mechanisms of CIM. From the date of inception till July 31, 2021, 51 related articles were selected. Based on our analysis of these articles, only five chemotherapeutic drugs, namely, MTX, 5-FU, cisplatin, CPT-11, and oxaliplatin were shown to trigger the ROS-based pathological mechanisms of CIM. We also discussed the redox system-mediated modulation of CIM pathogenesis via elaboration of the relationship between chemotherapeutic drugs and the redox system. It is our belief that this overview of the intestinal redox system and its role in CIM pathogenesis will greatly enhance research direction and improve CIM management in the future.
Collapse
Affiliation(s)
- Qing-Qing Yu
- Laboratory of Biochemistry and Biomedical Materials, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China
- Jining First People's Hospital, Jining Medical College, Jining 272000, China
| | - Heng Zhang
- Department of Laboratory, Shandong Daizhuang Hospital, Jining 272051, China
| | - Yujin Guo
- Jining First People's Hospital, Jining Medical College, Jining 272000, China
| | - Baoqin Han
- Laboratory of Biochemistry and Biomedical Materials, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China
- Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao 266235, China
| | - Pei Jiang
- Jining First People's Hospital, Jining Medical College, Jining 272000, China
| |
Collapse
|
|
6
|
Beaussart A, Canonico F, Mazon H, Hidalgo J, Cianférani S, Le Cordier H, Kriznik A, Rahuel-Clermont S. Probing the mechanism of the peroxiredoxin decamer interaction with its reductase sulfiredoxin from the single molecule to the solution scale. NANOSCALE HORIZONS 2022; 7:515-525. [PMID: 35234779 DOI: 10.1039/d2nh00037g] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Peroxiredoxins from the Prx1 subfamily (Prx) are highly regulated multifunctional proteins involved in oxidative stress response, redox signaling and cell protection. Prx is a homodimer that associates into a decamer. The monomer C-terminus plays intricate roles in Prx catalytic functions, decamer stability and interaction with its redox partner, the small reductase sulfiredoxin (Srx), that regulates the switching between Prx cellular functions. As only static structures of covalent Prx-Srx complexes have been reported, whether Srx binding dissociates the decameric assembly and how Prx subunit flexibility impacts complex formation are unknown. Here, we assessed the non-covalent interaction mechanism and dynamics in the solution of Saccharomyces cerevisiae Srx with the ten subunits of Prx Tsa1 at the decamer level via a combination of multiscale biophysical approaches including native mass spectrometry. We show that the ten subunits of the decamer can be saturated by ten Srx molecules and that the Tsa1 decamer in complex with Srx does not dissociate in solution. Furthermore, the binding events of atomic force microscopy (AFM) tip-grafted Srx molecules to Tsa1 individual subunits were relevant to the interactions between free molecules in solution. Combined with protein engineering and rapid kinetics, the observation of peculiar AFM force-distance signatures revealed that Tsa1 C-terminus flexibility controls Tsa1/Srx two-step binding and dynamics and determines the force-induced dissociation of Srx from each subunit of the decameric complex in a sequential or concerted mode. This combined approach from the solution to the single-molecule level offers promising prospects for understanding oligomeric protein interactions with their partners.
Collapse
Affiliation(s)
| | | | - Hortense Mazon
- Université de Lorraine, CNRS, IMoPA, F-54000 Nancy, France
| | - Jorge Hidalgo
- Université de Lorraine, CNRS, IMoPA, F-54000 Nancy, France
| | - Sarah Cianférani
- Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France
- Infrastructure Nationale de Protéomique ProFI - FR2048 CNRS CEA, 67087 Strasbourg, France
| | | | - Alexandre Kriznik
- Université de Lorraine, CNRS, IMoPA, F-54000 Nancy, France
- Université de Lorraine, CNRS, INSERM, UMS2008 IBSLor, Biophysics and Structural Biology core facility, F-54000 Nancy, France.
| | - Sophie Rahuel-Clermont
- Université de Lorraine, CNRS, IMoPA, F-54000 Nancy, France
- Université de Lorraine, CNRS, INSERM, UMS2008 IBSLor, Biophysics and Structural Biology core facility, F-54000 Nancy, France.
| |
Collapse
|
|
7
|
Kriznik A, Libiad M, Le Cordier H, Boukhenouna S, Toledano MB, Rahuel-Clermont S. Dynamics of a Key Conformational Transition in the Mechanism of Peroxiredoxin Sulfinylation. ACS Catal 2020; 10:3326-3339. [PMID: 32363077 PMCID: PMC7189429 DOI: 10.1021/acscatal.9b04471] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2019] [Revised: 01/14/2020] [Indexed: 12/11/2022]
Abstract
![]()
Peroxiredoxins from
the Prx1 subfamily (Prx) are moonlighting peroxidases
that operate in peroxide signaling and are regulated by sulfinylation.
Prxs offer a major model of protein–thiol oxidative modification.
They react with H2O2 to form a sulfenic acid
intermediate that either engages into a disulfide bond, committing
the enzyme into its peroxidase cycle, or again reacts with peroxide
to produce a sulfinic acid that inactivates the enzyme. Sensitivity
to sulfinylation depends on the kinetics of these two competing reactions
and is critically influenced by a structural transition from a fully
folded (FF) to locally unfolded (LU) conformation. Analysis of the
reaction of the Tsa1 Saccharomyces cerevisiae Prx with H2O2 by Trp fluorescence-based rapid
kinetics revealed a process linked to the FF/LU transition that is
kinetically distinct from disulfide formation and suggested that sulfenate
formation facilitates local unfolding. Use of mutants of distinctive
sensitivities and of different peroxide substrates showed that sulfinylation
sensitivity is not coupled to the resolving step kinetics but depends
only on the sulfenic acid oxidation and FF-to-LU transition rate constants.
In addition, stabilization of the active site FF conformation, the
determinant of sulfinylation kinetics, is only moderately influenced
by the Prx C-terminal tail dynamics that determine the FF →
LU kinetics. From these two parameters, the relative sensitivities
of Prxs toward hyperoxidation with different substrates can be predicted,
as confirmed by in vitro and in vivo patterns of sulfinylation.
Collapse
Affiliation(s)
- Alexandre Kriznik
- IMoPA, Université de Lorraine, CNRS, Biopole, Campus Biologie Sante′, F-54000 Nancy, France
- UMS2008 IBSLor, Biophysics and Structural Biology Core Facility, Université de Lorraine, CNRS, INSERM, Biopole, Campus Biologie Sante′, F-54000 Nancy, France
| | - Marouane Libiad
- Laboratoire Stress oxydant et Cancer, Institute for Integrative Biology of the Cell (I2BC), UMR9198, CNRS, CEA-Saclay, Université Paris-Saclay, iBiTecS/SBIGEM, Bat 142, F-91198 Gif-sur-Yvette Cedex, France
| | - Hélène Le Cordier
- IMoPA, Université de Lorraine, CNRS, Biopole, Campus Biologie Sante′, F-54000 Nancy, France
| | - Samia Boukhenouna
- IMoPA, Université de Lorraine, CNRS, Biopole, Campus Biologie Sante′, F-54000 Nancy, France
| | - Michel B. Toledano
- Laboratoire Stress oxydant et Cancer, Institute for Integrative Biology of the Cell (I2BC), UMR9198, CNRS, CEA-Saclay, Université Paris-Saclay, iBiTecS/SBIGEM, Bat 142, F-91198 Gif-sur-Yvette Cedex, France
| | - Sophie Rahuel-Clermont
- IMoPA, Université de Lorraine, CNRS, Biopole, Campus Biologie Sante′, F-54000 Nancy, France
- UMS2008 IBSLor, Biophysics and Structural Biology Core Facility, Université de Lorraine, CNRS, INSERM, Biopole, Campus Biologie Sante′, F-54000 Nancy, France
| |
Collapse
|
|
8
|
Li M, Wang J, Xu W, Wang Y, Zhang M, Wang M. Crystal structure of
Akkermansia muciniphila
peroxiredoxin reveals a novel regulatory mechanism of typical 2‐Cys Prxs by a distinct loop. FEBS Lett 2020; 594:1550-1563. [DOI: 10.1002/1873-3468.13753] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2019] [Revised: 01/12/2020] [Accepted: 01/17/2020] [Indexed: 01/05/2023]
Affiliation(s)
- Mengyu Li
- School of Life Sciences Anhui University Hefei China
| | - Junchao Wang
- School of Life Sciences Anhui University Hefei China
- Institutes of Physical Science and Information Technology Anhui University Hefei China
- Key Laboratory of Human Microenvironment and Precision Medicine of Anhui Higher Education Institutes Anhui University Hefei China
| | - Wenjuan Xu
- School of Life Sciences Anhui University Hefei China
| | - Yongzhong Wang
- School of Life Sciences Anhui University Hefei China
- Key Laboratory of Human Microenvironment and Precision Medicine of Anhui Higher Education Institutes Anhui University Hefei China
| | - Min Zhang
- School of Life Sciences Anhui University Hefei China
- Key Laboratory of Human Microenvironment and Precision Medicine of Anhui Higher Education Institutes Anhui University Hefei China
| | - Mingzhu Wang
- School of Life Sciences Anhui University Hefei China
- Institutes of Physical Science and Information Technology Anhui University Hefei China
- Key Laboratory of Human Microenvironment and Precision Medicine of Anhui Higher Education Institutes Anhui University Hefei China
| |
Collapse
|
|
9
|
Al-Asadi S, Malik A, Bakiu R, Santovito G, Menz I, Schuller K. Characterization of the peroxiredoxin 1 subfamily from Tetrahymena thermophila. Cell Mol Life Sci 2019; 76:4745-4768. [PMID: 31129858 PMCID: PMC11105310 DOI: 10.1007/s00018-019-03131-3] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2019] [Revised: 04/02/2019] [Accepted: 05/02/2019] [Indexed: 12/16/2022]
Abstract
Peroxiredoxins are antioxidant enzymes that use redox active Cys residues to reduce H2O2 and various organic hydroperoxides to less reactive products, and thereby protect cells against oxidative stress. In yeasts and mammals, the Prx1 proteins are sensitive to hyperoxidation and consequent loss of their peroxidase activity whereas in most bacteria they are not. In this paper we report the characterization of the Prx1 family in the non-parasitic protist Tetrahymena thermophila. In this organism, four genes potentially encoding Prx1 have been identified. In particular, we show that the mitochondrial Prx1 protein (Prx1m) from T. thermophila is relatively robust to hyperoxidation. This is surprising given that T. thermophila is a eukaryote like yeasts and mammals. In addition, the proliferation of the T. thermophila cells was relatively robust to inhibition by H2O2, cumene hydroperoxide and plant natural products that are known to promote the production of H2O2. In the presence of these agents, the abundance of the T. thermophila Prx1m protein was shown to increase. This suggested that the Prx1m protein may be protecting the cells against oxidative stress. There was no evidence for any increase in Prx1m gene expression in the stressed cells. Thus, increasing protein stability rather than increasing gene expression may explain the increasing Prx1m protein abundance we observed.
Collapse
Affiliation(s)
- Sarmad Al-Asadi
- College of Science and Engineering, Flinders University, GPO Box 2100, Adelaide, SA, 5001, Australia
- Department of Biology, College of Education for Pure Sciences, University of Basrah, Basrah, Iraq
| | - Arif Malik
- College of Science and Engineering, Flinders University, GPO Box 2100, Adelaide, SA, 5001, Australia
| | - Rigers Bakiu
- Department of Aquaculture and Fisheries, Agricultural University of Tirana, Tirana, Albania
| | | | - Ian Menz
- College of Science and Engineering, Flinders University, GPO Box 2100, Adelaide, SA, 5001, Australia
| | - Kathryn Schuller
- College of Science and Engineering, Flinders University, GPO Box 2100, Adelaide, SA, 5001, Australia.
| |
Collapse
|
|
10
|
Oligomerization dynamics and functionality of Trypanosoma cruzi cytosolic tryparedoxin peroxidase as peroxidase and molecular chaperone. Biochim Biophys Acta Gen Subj 2019; 1863:1583-1594. [DOI: 10.1016/j.bbagen.2019.06.013] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2019] [Revised: 06/26/2019] [Accepted: 06/27/2019] [Indexed: 12/26/2022]
|
|
11
|
Son YW, Cheon MG, Kim Y, Jang HH. Prx2 links ROS homeostasis to stemness of cancer stem cells. Free Radic Biol Med 2019; 134:260-267. [PMID: 30611866 DOI: 10.1016/j.freeradbiomed.2019.01.001] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/12/2018] [Revised: 12/21/2018] [Accepted: 01/03/2019] [Indexed: 01/03/2023]
Abstract
Cancer stem cells (CSC) with low levels of reactive oxygen species (ROS) are resistant to conventional chemotherapy or radiation therapy. Peroxiredoxin 2 (Prx2) is a redox regulatory protein that plays a key role in maintaining ROS homeostasis in the tumor microenvironment. However, despite the role of Prx2 in ROS-mediated signal transduction, the association of Prx2 with stemness via ROS in CSC has not been thoroughly investigated. In this study, we investigated the link between Prx2 and CSC stemness through regulation of ROS levels in hepatocellular carcinoma (HCC) cells. ROS induced CSC stemness reduction and downregulated stem cell markers in Huh7 and SK-HEP1 cells. Prx2 knockdown decreased CSC sphere formation and expression of stem cell makers with increasing intracellular ROS levels. This effect was reversed by the ROS scavengers NAC and GSH in Prx2 knockdown cells. Conversely, we found that Prx2 overexpression promotes CSC stemness and the peroxidase activity of Prx2 is essential for CSC stemness using peroxidase inactive mutant, Prx2C51/172S. More importantly, the hyperoxidation-resistant mutant (Prx2ΔYF), which has a constant ROS scavenging activity even at high concentrations of ROS, increased the CSC stemness and expression of stem cell markers more than Prx2WT under oxidative stress. Taken together, our findings demonstrate that Prx2 links ROS homeostasis to CSC stemness; Prx2 is a mediator between ROS homeostasis and CSC stemness.
Collapse
Affiliation(s)
- Ye Won Son
- Department of Biochemistry, College of Medicine, Gachon University, Incheon, South Korea
| | - Min Gyeong Cheon
- Department of Biochemistry, College of Medicine, Gachon University, Incheon, South Korea
| | - Yosup Kim
- Department of Health Sciences and Technology, Graduate School of Medicine, Gachon University, Incheon, South Korea
| | - Ho Hee Jang
- Department of Biochemistry, College of Medicine, Gachon University, Incheon, South Korea; Department of Health Sciences and Technology, Graduate School of Medicine, Gachon University, Incheon, South Korea.
| |
Collapse
|
|
12
|
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.
Collapse
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
| |
Collapse
|
|
13
|
Bolduc JA, Nelson KJ, Haynes AC, Lee J, Reisz JA, Graff AH, Clodfelter JE, Parsonage D, Poole LB, Furdui CM, Lowther WT. Novel hyperoxidation resistance motifs in 2-Cys peroxiredoxins. J Biol Chem 2018; 293:11901-11912. [PMID: 29884768 PMCID: PMC6066324 DOI: 10.1074/jbc.ra117.001690] [Citation(s) in RCA: 44] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2017] [Revised: 05/29/2018] [Indexed: 01/07/2023] Open
Abstract
2-Cys peroxiredoxins (Prxs) modulate hydrogen peroxide (H2O2)-mediated cell signaling. At high H2O2 levels, eukaryotic Prxs can be inactivated by hyperoxidation and are classified as sensitive Prxs. In contrast, prokaryotic Prxs are categorized as being resistant to hyperoxidation and lack the GGLG and C-terminal YF motifs present in the sensitive Prxs. Additional molecular determinants that account for the subtle differences in the susceptibility to hyperoxidation remain to be identified. A comparison of a new, 2.15-Å-resolution crystal structure of Prx2 in the oxidized, disulfide-bonded state with the hyperoxidized structure of Prx2 and Prx1 in complex with sulfiredoxin revealed three structural regions that rearrange during catalysis. With these regions in hand, focused sequence analyses were performed comparing sensitive and resistant Prx groups. From this combinatorial approach, we discovered two novel hyperoxidation resistance motifs, motifs A and B, which were validated using mutagenesis of sensitive human Prxs and resistant Salmonella enterica serovar Typhimurium AhpC. Introduction and removal of these motifs, respectively, resulted in drastic changes in the sensitivity to hyperoxidation with Prx1 becoming 100-fold more resistant to hyperoxidation and AhpC becoming 800-fold more sensitive to hyperoxidation. The increased sensitivity of the latter AhpC variant was also confirmed in vivo These results support the function of motifs A and B as primary drivers for tuning the sensitivity of Prxs to different levels of H2O2, thus enabling the initiation of variable signaling or antioxidant responses in cells.
Collapse
Affiliation(s)
| | | | | | - Jingyun Lee
- Wake Forest Baptist Comprehensive Cancer Center, and
| | - Julie A. Reisz
- Section on Molecular Medicine, Department of Internal Medicine
| | - Aaron H. Graff
- From the Center for Structural Biology, Department of Biochemistry
| | | | - Derek Parsonage
- From the Center for Structural Biology, Department of Biochemistry
| | - Leslie B. Poole
- From the Center for Structural Biology, Department of Biochemistry, ,Wake Forest Baptist Comprehensive Cancer Center, and ,Center for Redox Biology and Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina 27157 and ,Center for Molecular Signaling, Wake Forest University, Winston-Salem, North Carolina 27101
| | - Cristina M. Furdui
- Section on Molecular Medicine, Department of Internal Medicine, ,Wake Forest Baptist Comprehensive Cancer Center, and ,Center for Redox Biology and Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina 27157 and ,Center for Molecular Signaling, Wake Forest University, Winston-Salem, North Carolina 27101
| | - W. Todd Lowther
- From the Center for Structural Biology, Department of Biochemistry, ,Wake Forest Baptist Comprehensive Cancer Center, and ,Center for Redox Biology and Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina 27157 and ,Center for Molecular Signaling, Wake Forest University, Winston-Salem, North Carolina 27101, To whom correspondence should be addressed:
Center for Structural Biology, Dept. of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27157. Tel.:
336-716-7230; Fax:
336-713-1283; E-mail:
| |
Collapse
|
|
14
|
Structural and biochemical analyses reveal ubiquitin C-terminal hydrolase-L1 as a specific client of the peroxiredoxin II chaperone. Arch Biochem Biophys 2018; 640:61-74. [PMID: 29339092 DOI: 10.1016/j.abb.2018.01.003] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2017] [Revised: 01/02/2018] [Accepted: 01/07/2018] [Indexed: 01/30/2023]
Abstract
Peroxiredoxins (Prxs) play dual roles as both thiol-peroxidases and molecular chaperones. Peroxidase activity enables various intracellular functions, however, the physiological roles of Prxs as chaperones are not well established. To study the chaperoning function of Prx, we previously sought to identify heat-induced Prx-binding proteins as the clients of a Prx chaperone. By using His-tagged Prx I as a bait, we separated ubiquitin C-terminal hydrolase-L1 (UCH-L1) as a heat-induced Prx I binding protein from rat brain crude extracts. Protein complex immunoprecipitation with HeLa cell lysates revealed that both Prx I and Prx II interact with UCH-L1. However, Prx II interacted considerably more favorably with UCH-L1 than Prx I. Prx II exhibited more effective molecular chaperone activity than Prx I when UCH-L1 was the client. Prx II interacted with UCH-L1 through its C-terminal region to protect UCH-L1 from thermal or oxidative inactivation. We found that chaperoning via interaction through C-terminal region (specific-client chaperoning) is more efficient than that involving oligomeric structural change (general-client chaperoning). Prx II binds either thermally or oxidatively unfolding early intermediates of specific clients and thereby shifted the equilibrium towards their native state. We conclude that this chaperoning mechanism provides a very effective and selective chaperoning activity.
Collapse
|
|
15
|
Interaction of tankyrase and peroxiredoxin II is indispensable for the survival of colorectal cancer cells. Nat Commun 2017; 8:40. [PMID: 28659575 PMCID: PMC5489516 DOI: 10.1038/s41467-017-00054-0] [Citation(s) in RCA: 35] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2016] [Accepted: 05/02/2017] [Indexed: 12/16/2022] Open
Abstract
Mammalian 2-Cys peroxiredoxin (Prx) enzymes are overexpressed in most cancer tissues, but their specific signaling role in cancer progression is poorly understood. Here we demonstrate that Prx type II (PrxII) plays a tumor-promoting role in colorectal cancer by interacting with a poly(ADP-ribose) polymerase (PARP) tankyrase. PrxII deletion in mice with inactivating mutation of adenomatous polyposis coli (APC) gene reduces intestinal adenomatous polyposis via Axin/β-catenin axis and thereby promotes survival. In human colorectal cancer cells with APC mutations, PrxII depletion consistently reduces the β-catenin levels and the expression of β-catenin target genes. Essentially, PrxII depletion hampers the PARP-dependent Axin1 degradation through tankyrase inactivation. Direct binding of PrxII to tankyrase ARC4/5 domains seems to be crucial for protecting tankyrase from oxidative inactivation. Furthermore, a chemical compound targeting PrxII inhibits the expansion of APC-mutant colorectal cancer cells in vitro and in vivo tumor xenografts. Collectively, this study reveals a redox mechanism for regulating tankyrase activity and implicates PrxII as a targetable antioxidant enzyme in APC-mutation-positive colorectal cancer. 2-Cys peroxiredoxin (Prx) enzymes are highly expressed in most cancers but how they promote cancer progression is unclear. Here the authors show that in colorectal cancers with APC mutation, PrxII binds to tankyrase and prevents its oxidative inactivation, thereby preventing Axin1-dependent degradation of ²b-catenin.
Collapse
|
|
16
|
Abstract
Peroxiredoxins (Prxs) constitute a major family of peroxidases, with mammalian cells expressing six Prx isoforms (PrxI to PrxVI). Cells produce hydrogen peroxide (H2O2) at various intracellular locations where it can serve as a signaling molecule. Given that Prxs are abundant and possess a structure that renders the cysteine (Cys) residue at the active site highly sensitive to oxidation by H2O2, the signaling function of this oxidant requires extensive and highly localized regulation. Recent findings on the reversible regulation of PrxI through phosphorylation at the centrosome and on the hyperoxidation of the Cys at the active site of PrxIII in mitochondria are described in this review as examples of such local regulation of H2O2 signaling. Moreover, their high affinity for and sensitivity to oxidation by H2O2 confer on Prxs the ability to serve as sensors and transducers of H2O2 signaling through transfer of their oxidation state to bound effector proteins.
Collapse
Affiliation(s)
- Sue Goo Rhee
- Yonsei Biomedical Research Institute, Yonsei University College of Medicine, Seoul 120-752, Korea;
| | - In Sup Kil
- Yonsei Biomedical Research Institute, Yonsei University College of Medicine, Seoul 120-752, Korea;
| |
Collapse
|
|
17
|
Tomalin LE, Day AM, Underwood ZE, Smith GR, Dalle Pezze P, Rallis C, Patel W, Dickinson BC, Bähler J, Brewer TF, Chang CJL, Shanley DP, Veal EA. Increasing extracellular H2O2 produces a bi-phasic response in intracellular H2O2, with peroxiredoxin hyperoxidation only triggered once the cellular H2O2-buffering capacity is overwhelmed. Free Radic Biol Med 2016; 95:333-48. [PMID: 26944189 PMCID: PMC4891068 DOI: 10.1016/j.freeradbiomed.2016.02.035] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/14/2015] [Revised: 02/25/2016] [Accepted: 02/29/2016] [Indexed: 11/30/2022]
Abstract
Reactive oxygen species, such as H2O2, can damage cells but also promote fundamental processes, including growth, differentiation and migration. The mechanisms allowing cells to differentially respond to toxic or signaling H2O2 levels are poorly defined. Here we reveal that increasing external H2O2 produces a bi-phasic response in intracellular H2O2. Peroxiredoxins (Prx) are abundant peroxidases which protect against genome instability, ageing and cancer. We have developed a dynamic model simulating in vivo changes in Prx oxidation. Remarkably, we show that the thioredoxin peroxidase activity of Prx does not provide any significant protection against external rises in H2O2. Instead, our model and experimental data are consistent with low levels of extracellular H2O2 being efficiently buffered by other thioredoxin-dependent activities, including H2O2-reactive cysteines in the thiol-proteome. We show that when extracellular H2O2 levels overwhelm this buffering capacity, the consequent rise in intracellular H2O2 triggers hyperoxidation of Prx to thioredoxin-resistant, peroxidase-inactive form/s. Accordingly, Prx hyperoxidation signals that H2O2 defenses are breached, diverting thioredoxin to repair damage.
Collapse
Affiliation(s)
- Lewis Elwood Tomalin
- Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
| | - Alison Michelle Day
- Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
| | - Zoe Elizabeth Underwood
- Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
| | - Graham Robert Smith
- Bioinformatics Support Unit, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
| | - Piero Dalle Pezze
- Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
| | - Charalampos Rallis
- University College London, Department of Genetics, Evolution & Environment and Institute of Healthy Ageing, Gower Street - Darwin Building, London WC1E 6BT, UK
| | - Waseema Patel
- Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
| | | | - Jürg Bähler
- University College London, Department of Genetics, Evolution & Environment and Institute of Healthy Ageing, Gower Street - Darwin Building, London WC1E 6BT, UK
| | - Thomas Francis Brewer
- Howard Hughes Medical Institute and Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
| | - Christopher Joh-Leung Chang
- Howard Hughes Medical Institute and Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
| | - Daryl Pierson Shanley
- Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
| | - Elizabeth Ann Veal
- Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
| |
Collapse
|
|
18
|
Van Laer K, Dick TP. Utilizing Natural and Engineered Peroxiredoxins As Intracellular Peroxide Reporters. Mol Cells 2016; 39:46-52. [PMID: 26810074 PMCID: PMC4749873 DOI: 10.14348/molcells.2016.2328] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2015] [Accepted: 12/06/2015] [Indexed: 01/27/2023] Open
Abstract
It is increasingly apparent that nature evolved peroxiredoxins not only as H2O2 scavengers but also as highly sensitive H2O2 sensors and signal transducers. Here we ask whether the H2O2 sensing role of Prx can be exploited to develop probes that allow to monitor intracellular H2O2 levels with unprecedented sensitivity. Indeed, simple gel shift assays visualizing the oxidation of endogenous 2-Cys peroxiredoxins have already been used to detect subtle changes in intracellular H2O2 concentration. The challenge however is to create a genetically encoded probe that offers real-time measurements of H2O2 levels in intact cells via the Prx oxidation state. We discuss potential design strategies for Prx-based probes based on either the redox-sensitive fluorophore roGFP or the conformation-sensitive fluorophore cpYFP. Furthermore, we outline the structural and chemical complexities which need to be addressed when using Prx as a sensing moiety for H2O2 probes. We suggest experimental strategies to investigate the influence of these complexities on probe behavior. In doing so, we hope to stimulate the development of Prx-based probes which may spearhead the further study of cellular H2O2 homeostasis and Prx signaling.
Collapse
Affiliation(s)
- Koen Van Laer
- Division of Redox Regulation, DKFZ–ZMBH Alliance, German Cancer Research Center, Heidelberg,
Germany
| | - Tobias P. Dick
- Division of Redox Regulation, DKFZ–ZMBH Alliance, German Cancer Research Center, Heidelberg,
Germany
| |
Collapse
|
|
19
|
Cruzen S, Pearce S, Baumgard L, Gabler N, Huff-Lonergan E, Lonergan S. Proteomic changes to the sarcoplasmic fraction of predominantly red or white muscle following acute heat stress. J Proteomics 2015; 128:141-53. [DOI: 10.1016/j.jprot.2015.07.032] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2015] [Revised: 07/10/2015] [Accepted: 07/28/2015] [Indexed: 01/08/2023]
|
|
20
|
Noichri Y, Palais G, Ruby V, D'Autreaux B, Delaunay-Moisan A, Nyström T, Molin M, Toledano MB. In vivo parameters influencing 2-Cys Prx oligomerization: The role of enzyme sulfinylation. Redox Biol 2015; 6:326-333. [PMID: 26335398 PMCID: PMC4556779 DOI: 10.1016/j.redox.2015.08.011] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2015] [Revised: 08/10/2015] [Accepted: 08/11/2015] [Indexed: 11/28/2022] Open
Abstract
2-Cys Prxs are H2O2-specific antioxidants that become inactivated by enzyme hyperoxidation at elevated H2O2 levels. Although hyperoxidation restricts the antioxidant physiological role of these enzymes, it also allows the enzyme to become an efficient chaperone holdase. The critical molecular event allowing the peroxidase to chaperone switch is thought to be the enzyme assembly into high molecular weight (HMW) structures brought about by enzyme hyperoxidation. How hyperoxidation promotes HMW assembly is not well understood and Prx mutants allowing disentangling its peroxidase and chaperone functions are lacking. To begin addressing the link between enzyme hyperoxidation and HMW structures formation, we have evaluated the in vivo 2-Cys Prxs quaternary structure changes induced by H2O2 by size exclusion chromatography (SEC) on crude lysates, using wild type (Wt) untagged and Myc-tagged S. cerevisiae 2-Cys Prx Tsa1 and derivative Tsa1 mutants or genetic conditions known to inactivate peroxidase or chaperone activity or altering the enzyme sensitivity to hyperoxidation. Our data confirm the strict causative link between H2O2-induced hyperoxidation and HMW formation/stabilization, also raising the question of whether CP hyperoxidation triggers the assembly of HMW structures by the stacking of decamers, which is the prevalent view of the literature, or rather, the stabilization of preassembled stacked decamers.
Collapse
Affiliation(s)
- Y Noichri
- Oxidative Stress and Cancer, IBITECS, SBIGEM, CEA-Saclay, 91191 Gif-sur-Yvette, France
| | - G Palais
- Oxidative Stress and Cancer, IBITECS, SBIGEM, CEA-Saclay, 91191 Gif-sur-Yvette, France
| | - V Ruby
- Oxidative Stress and Cancer, IBITECS, SBIGEM, CEA-Saclay, 91191 Gif-sur-Yvette, France
| | - B D'Autreaux
- Oxidative Stress and Cancer, IBITECS, SBIGEM, CEA-Saclay, 91191 Gif-sur-Yvette, France
| | - A Delaunay-Moisan
- Oxidative Stress and Cancer, IBITECS, SBIGEM, CEA-Saclay, 91191 Gif-sur-Yvette, France
| | - T Nyström
- Department of Chemistry and Molecular Biology (CMB), University of Gothenburg, Medicinaregatan 9C, S-413 90 Göteborg, Sweden
| | - M Molin
- Department of Chemistry and Molecular Biology (CMB), University of Gothenburg, Medicinaregatan 9C, S-413 90 Göteborg, Sweden
| | - M B Toledano
- Oxidative Stress and Cancer, IBITECS, SBIGEM, CEA-Saclay, 91191 Gif-sur-Yvette, France.
| |
Collapse
|
|
21
|
Staudacher V, Djuika CF, Koduka J, Schlossarek S, Kopp J, Büchler M, Lanzer M, Deponte M. Plasmodium falciparum antioxidant protein reveals a novel mechanism for balancing turnover and inactivation of peroxiredoxins. Free Radic Biol Med 2015; 85:228-36. [PMID: 25952724 DOI: 10.1016/j.freeradbiomed.2015.04.030] [Citation(s) in RCA: 16] [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: 03/11/2015] [Revised: 04/20/2015] [Accepted: 04/24/2015] [Indexed: 12/12/2022]
Abstract
Life under aerobic conditions has shaped peroxiredoxins (Prx) as ubiquitous thiol-dependent hydroperoxidases and redox sensors. Structural features that balance the catalytically active or inactive redox states of Prx, and, therefore, their hydroperoxidase or sensor function, have so far been analyzed predominantly for Prx1-type enzymes. Here we identify and characterize two modulatory residues of the Prx5-type model enzyme PfAOP from the malaria parasite Plasmodium falciparum. Gain- and loss-of-function mutants reveal a correlation between the enzyme parameters and the inactivation susceptibility of PfAOP with the size of residue 109 and the presence or absence of a catalytically relevant but nonessential cysteine residue. Based on our kinetic data and the crystal structure of PfAOP(L109M), we suggest a novel mechanism for balancing the hydroperoxidase activity and inactivation susceptibility of Prx5-type enzymes. Our study provides unexpected insights into Prx structure-function relationships and contributes to our understanding of what makes Prx good enzymes or redox sensors.
Collapse
Affiliation(s)
- Verena Staudacher
- Department of Parasitology, Ruprecht-Karls University, D-69120 Heidelberg, Germany
| | - Carine F Djuika
- Department of Parasitology, Ruprecht-Karls University, D-69120 Heidelberg, Germany
| | - Joshua Koduka
- Department of Parasitology, Ruprecht-Karls University, D-69120 Heidelberg, Germany
| | - Sarah Schlossarek
- Department of Parasitology, Ruprecht-Karls University, D-69120 Heidelberg, Germany
| | - Jürgen Kopp
- Biochemistry Center (BZH), Ruprecht-Karls University, D-69120 Heidelberg, Germany; Cellnetworks Excellence Cluster, Ruprecht-Karls University, D-69120 Heidelberg, Germany
| | - Marleen Büchler
- Department of Parasitology, Ruprecht-Karls University, D-69120 Heidelberg, Germany
| | - Michael Lanzer
- Department of Parasitology, Ruprecht-Karls University, D-69120 Heidelberg, Germany
| | - Marcel Deponte
- Department of Parasitology, Ruprecht-Karls University, D-69120 Heidelberg, Germany.
| |
Collapse
|
|
22
|
Kil IS, Ryu KW, Lee SK, Kim JY, Chu SY, Kim JH, Park S, Rhee SG. Circadian Oscillation of Sulfiredoxin in the Mitochondria. Mol Cell 2015; 59:651-63. [PMID: 26236015 DOI: 10.1016/j.molcel.2015.06.031] [Citation(s) in RCA: 70] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2015] [Revised: 06/12/2015] [Accepted: 06/23/2015] [Indexed: 10/23/2022]
Abstract
Hydrogen peroxide (H2O2) released from mitochondria regulates various cell signaling pathways. Given that H2O2-eliminating enzymes such as peroxiredoxin III (PrxIII) are abundant in mitochondria, however, it has remained unknown how such release can occur. Active PrxIII-SH undergoes reversible inactivation via hyperoxidation to PrxIII-SO2, which is then reduced by sulfiredoxin. We now show that the amounts of PrxIII-SO2 and sulfiredoxin undergo antiphasic circadian oscillation in the mitochondria of specific tissues of mice maintained under normal conditions. Cytosolic sulfiredoxin was found to be imported into the mitochondria via a mechanism that requires formation of a disulfide-linked complex with heat shock protein 90, which is promoted by H2O2 released from mitochondria. The imported sulfiredoxin is degraded by Lon in a manner dependent on PrxIII hyperoxidation state. The coordinated import and degradation of sulfiredoxin provide the basis for sulfiredoxin oscillation and consequent PrxIII-SO2 oscillation in mitochondria and likely result in an oscillatory H2O2 release.
Collapse
Affiliation(s)
- In Sup Kil
- Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea.
| | - Keun Woo Ryu
- Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea
| | - Se Kyoung Lee
- Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea
| | - Jeong Yeon Kim
- Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea
| | - Sei Yoon Chu
- Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea
| | - Ju Hee Kim
- Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea
| | - Sunjoo Park
- Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea
| | - Sue Goo Rhee
- Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea.
| |
Collapse
|
|
23
|
Camejo D, Ortiz-Espín A, Lázaro JJ, Romero-Puertas MC, Lázaro-Payo A, Sevilla F, Jiménez A. Functional and structural changes in plant mitochondrial PrxII F caused by NO. J Proteomics 2015; 119:112-25. [PMID: 25682994 DOI: 10.1016/j.jprot.2015.01.022] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2014] [Revised: 01/16/2015] [Accepted: 01/29/2015] [Indexed: 11/17/2022]
Abstract
Peroxiredoxins (Prxs) have emerged as important factors linking reactive oxygen species (ROS) metabolism to redox-dependent signaling events. Together with ROS, nitric oxide (NO) is a free radical product of the cell metabolism that is essential in the signal transduction. S-Nitrosylation is emerging as a fundamental protein modification for the transduction of NO bioactivity. Using recombinant pea mitochondrial PsPrxII F (PrxII F), the effect of S-nitrosoglutathione (GSNO) and sodium nitroprusside dehydrate (SNP), which are known to mediate protein S-nitrosylation processes, was studied. S-Nitrosylation of the PrxII F was demonstrated using the biotin switch method and LC ESI-QTOF tandem MS analysis. S-nitrosylated PrxII F decreased its peroxidase activity and acquired a new transnitrosylase activity, preventing the thermal aggregation of citrate synthase (CS). For the first time, we demonstrate the dual function for PrxII F as peroxidase and transnitrosylase. This switch was accompanied by a conformational change of the protein that could favor the protein-protein interaction CS-PrxII F. The observed in vivo S-nitrosylation of PrxII F could probably function as a protective mechanism under oxidative and nitrosative stress, such as occurs under salinity. We conclude that we are dealing with a novel regulatory mechanism for this protein by NO. BIOLOGICAL SIGNIFICANCE S-Nitrosylation is a post-translational modification that is increasingly viewed as fundamental for the signal transduction role of NO in plants. This study demonstrates that S-nitrosylation of the mitochondrial peroxiredoxin PrxII F induces a conformational change in the protein and provokes a reduction in its peroxidase activity, while acquiring a novel function as transnitrosylase. The implication of this mechanism will increase our understanding of the role of posttranslational modifications in the protein function in plants under stress situations such as salinity, in which NO could act as signaling molecule.
Collapse
Affiliation(s)
- Daymi Camejo
- CEBAS-CSIC, Department of Stress Biology and Plant Pathology, E-30100 Murcia, Spain.
| | - Ana Ortiz-Espín
- CEBAS-CSIC, Department of Stress Biology and Plant Pathology, E-30100 Murcia, Spain.
| | - Juan J Lázaro
- EEZ-CSIC, Department of Biochemistry, Cellular and Molecular Biology of Plants, E-18080 Granada, Spain.
| | - María C Romero-Puertas
- EEZ-CSIC, Department of Biochemistry, Cellular and Molecular Biology of Plants, E-18080 Granada, Spain.
| | - Alfonso Lázaro-Payo
- EEZ-CSIC, Department of Biochemistry, Cellular and Molecular Biology of Plants, E-18080 Granada, Spain.
| | - Francisca Sevilla
- CEBAS-CSIC, Department of Stress Biology and Plant Pathology, E-30100 Murcia, Spain.
| | - Ana Jiménez
- CEBAS-CSIC, Department of Stress Biology and Plant Pathology, E-30100 Murcia, Spain.
| |
Collapse
|
|
24
|
Abstract
Peroxiredoxins were not recognized as a family of enzymes until the 1990s but are now known to be the dominant peroxidases in most organisms. Here, the history and fundamental properties of peroxiredoxins are briefly reviewed, with a special focus on describing how an exquisitely tunable balance between fully folded and locally unfolded conformations plays a large role in peroxiredoxin catalytic properties.
Collapse
Affiliation(s)
- P Andrew Karplus
- Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA.
| |
Collapse
|
|
25
|
Bak DW, Weerapana E. Cysteine-mediated redox signalling in the mitochondria. MOLECULAR BIOSYSTEMS 2015; 11:678-97. [DOI: 10.1039/c4mb00571f] [Citation(s) in RCA: 63] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
This review represents a novel look at the many sources, cysteine targets, and signaling processes of ROS in the mitochondria.
Collapse
Affiliation(s)
- D. W. Bak
- Department of Chemistry
- Merkert Chemistry Center
- Boston College
- Massachusetts 02467
- USA
| | - E. Weerapana
- Department of Chemistry
- Merkert Chemistry Center
- Boston College
- Massachusetts 02467
- USA
| |
Collapse
|
|
26
|
|
|
27
|
The fission yeast Schizosaccharomyces pombe as a model to understand how peroxiredoxins influence cell responses to hydrogen peroxide. Biochem Soc Trans 2014; 42:909-16. [DOI: 10.1042/bst20140059] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
As a more selectively reactive oxygen species, H2O2 (hydrogen peroxide) has been co-opted as a signalling molecule, but high levels can still lead to lethal amounts of cell damage. 2-Cys Prxs (peroxiredoxins) are ubiquitous thioredoxin peroxidases which utilize reversibly oxidized catalytic cysteine residues to reduce peroxides. As such, Prxs potentially make an important contribution to the repertoire of cell defences against oxidative damage. Although the abundance of eukaryotic 2-Cys Prxs suggests an important role in maintaining cell redox, the surprising sensitivity of their thioredoxin peroxidase activity to inactivation by H2O2 has raised questions as to their role as an oxidative stress defence. Indeed, work in model yeast has led the way in revealing that Prxs do much more than simply remove peroxides and have even uncovered circumstances where their thioredoxin peroxidase activity is detrimental. In the present paper, we focus on what we have learned from studies in the fission yeast Schizosaccharomyces pombe about the different roles of 2-Cys Prxs in responses to H2O2 and discuss the general implications of these findings for other systems.
Collapse
|
|
28
|
Randall LM, Manta B, Hugo M, Gil M, Batthyàny C, Trujillo M, Poole LB, Denicola A. Nitration transforms a sensitive peroxiredoxin 2 into a more active and robust peroxidase. J Biol Chem 2014; 289:15536-43. [PMID: 24719319 DOI: 10.1074/jbc.m113.539213] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Peroxiredoxins (Prx) are efficient thiol-dependent peroxidases and key players in the mechanism of H2O2-induced redox signaling. Any structural change that could affect their redox state, oligomeric structure, and/or interaction with other proteins could have a significant impact on the cascade of signaling events. Several post-translational modifications have been reported to modulate Prx activity. One of these, overoxidation of the peroxidatic cysteine to the sulfinic derivative, inactivates the enzyme and has been proposed as a mechanism of H2O2 accumulation in redox signaling (the floodgate hypothesis). Nitration of Prx has been reported in vitro as well as in vivo; in particular, nitrated Prx2 was identified in brains of Alzheimer disease patients. In this work we characterize Prx2 tyrosine nitration, a post-translational modification on a noncatalytic residue that increases its peroxidase activity and its resistance to overoxidation. Mass spectrometry analysis revealed that treatment of disulfide-oxidized Prx2 with excess peroxynitrite renders mainly mononitrated and dinitrated species. Tyrosine 193 of the YF motif at the C terminus, associated with the susceptibility toward overoxidation of eukaryotic Prx, was identified as nitrated and is most likely responsible for the protection of the peroxidatic cysteine against oxidative inactivation. Kinetic analyses suggest that tyrosine nitration facilitates the intermolecular disulfide formation, transforming a sensitive Prx into a robust one. Thus, tyrosine nitration appears as another mechanism to modulate these enzymes in the complex network of redox signaling.
Collapse
Affiliation(s)
- Lía M Randall
- From the Laboratorio de Fisicoquímica Biológica, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, 11400 Montevideo, Uruguay, the Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo 11100, Uruguay
| | - Bruno Manta
- From the Laboratorio de Fisicoquímica Biológica, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, 11400 Montevideo, Uruguay, the Laboratorio de Biología Redox de Tripanosomas, Institut Pasteur de Montevideo, 11400 Montevideo, Uruguay
| | - Martín Hugo
- the Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo 11100, Uruguay, the Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, 11100 Montevideo, Uruguay
| | - Magdalena Gil
- the Unidad de Bioquímica y Proteómica Analíticas, Institut Pasteur de Montevideo, 11400 Montevideo, Uruguay, the Unidad de Bioquímica y Proteómica Analíticas, Instituto de Investigaciones Biológicas Clemente Estable, Ministerio de Educación y Cultura, 11600 Montevideo, Uruguay, and
| | - Carlos Batthyàny
- the Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo 11100, Uruguay, the Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, 11100 Montevideo, Uruguay, the Unidad de Bioquímica y Proteómica Analíticas, Institut Pasteur de Montevideo, 11400 Montevideo, Uruguay
| | - Madia Trujillo
- the Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo 11100, Uruguay, the Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, 11100 Montevideo, Uruguay
| | - Leslie B Poole
- the Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, North Carolina 27157
| | - Ana Denicola
- From the Laboratorio de Fisicoquímica Biológica, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, 11400 Montevideo, Uruguay, the Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo 11100, Uruguay,
| |
Collapse
|
|
29
|
Perkins A, Nelson KJ, Williams JR, Parsonage D, Poole LB, Karplus PA. The sensitive balance between the fully folded and locally unfolded conformations of a model peroxiredoxin. Biochemistry 2013; 52:8708-21. [PMID: 24175952 DOI: 10.1021/bi4011573] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
To reduce peroxides, peroxiredoxins (Prxs) require a key "peroxidatic" Cys that, in a substrate-ready fully folded (FF) conformation, is oxidized to sulfenic acid and then, after a local unfolding (LU) of the active site, forms a disulfide bond with a second "resolving" Cys. For Salmonella typhimurium alkyl hydroperoxide reductase C (StAhpC) and some other Prxs, the FF structure is only known for a peroxidatic Cys→Ser variant, which may not accurately represent the wild-type enzyme. Here, we obtain the structure of authentic reduced wild-type StAhpC by dithiothreitol treatment of disulfide form crystals that fortuitously accommodate both the LU and FF conformations. The unique environment of one molecule in the crystal reveals a thermodynamic linkage between the folding of the active site loop and C-terminal regions, and comparisons with the Ser variant show structural and mobility differences from which we infer that the Cys→Ser mutation stabilizes the FF active site. A structure for the C165A variant (a resolving Cys to Ala mutant) in the same crystal form reveals that this mutation destabilizes the folding of the C-terminal region. These structures prove that subtle modifications to Prx structures can substantially influence enzymatic properties. We also present a simple thermodynamic framework for understanding the various mixtures of FF and LU conformations seen in these structures. On the basis of this framework, we rationalize how physiologically relevant regulatory post-translational modifications may modulate activity, and we propose a nonconventional strategy for designing selective Prx inhibitors.
Collapse
Affiliation(s)
- Arden Perkins
- Department of Biochemistry and Biophysics, Oregon State University , Corvallis, Oregon 97331, United States
| | | | | | | | | | | |
Collapse
|
|
30
|
Haynes AC, Qian J, Reisz JA, Furdui CM, Lowther WT. Molecular basis for the resistance of human mitochondrial 2-Cys peroxiredoxin 3 to hyperoxidation. J Biol Chem 2013; 288:29714-23. [PMID: 24003226 DOI: 10.1074/jbc.m113.473470] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Peroxiredoxins (Prxs) detoxify peroxides and modulate H2O2-mediated cell signaling in normal and numerous pathophysiological contexts. The typical 2-Cys subclass of Prxs (human Prx1-4) utilizes a Cys sulfenic acid (Cys-SOH) intermediate and disulfide bond formation across two subunits during catalysis. During oxidative stress, however, the Cys-SOH moiety can react with H2O2 to form Cys sulfinic acid (Cys-SO2H), resulting in inactivation. The propensity to hyperoxidize varies greatly among human Prxs. Mitochondrial Prx3 is the most resistant to inactivation, but the molecular basis for this property is unknown. A panel of chimeras and Cys variants of Prx2 and Prx3 were treated with H2O2 and analyzed by rapid chemical quench and time-resolved electrospray ionization-TOF mass spectrometry. The latter utilized an on-line rapid-mixing setup to collect data on the low seconds time scale. These approaches enabled the first direct observation of the Cys-SOH intermediate and a putative Cys sulfenamide (Cys-SN) for Prx2 and Prx3 during catalysis. The substitution of C-terminal residues in Prx3, residues adjacent to the resolving Cys residue, resulted in a Prx2-like protein with increased sensitivity to hyperoxidation and decreased ability to form the intermolecular disulfide bond between subunits. The corresponding Prx2 chimera became more resistant to hyperoxidation. Taken together, the results of this study support that the kinetics of the Cys-SOH intermediate is key to determine the probability of hyperoxidation or disulfide formation. Given the oxidizing environment of the mitochondrion, it makes sense that Prx3 would favor disulfide bond formation as a protection mechanism against hyperoxidation and inactivation.
Collapse
Affiliation(s)
- Alexina C Haynes
- From the Center for Structural Biology and Department of Biochemistry
| | | | | | | | | |
Collapse
|
|
31
|
König J, Galliardt H, Jütte P, Schäper S, Dittmann L, Dietz KJ. The conformational bases for the two functionalities of 2-cysteine peroxiredoxins as peroxidase and chaperone. JOURNAL OF EXPERIMENTAL BOTANY 2013; 64:3483-97. [PMID: 23828546 PMCID: PMC3733160 DOI: 10.1093/jxb/ert184] [Citation(s) in RCA: 80] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/08/2023]
Abstract
2-Cysteine peroxiredoxins (2-CysPrxs) are ubiquitous and highly abundant proteins that serve multiple functions as peroxidases, chaperones, and thiol oxidases and in redox-dependent cell signalling. The chloroplast protein plays a role in seedling development and protection of the photosynthetic apparatus. This study aimed to unequivocally link conformation and function. To this end, a set of non-tagged site-directed mutagenized At2-CysPrx variants was engineered, which mimicked the conformational states and their specific functions: hyperoxidized form (C54D), reduced form (C54S, C176S), oxidized form (C54DC176K), phosphorylated form (T92D), reduced ability for oligomerization by interfering with the dimer-dimer interface (F84R) and a C-terminally truncated form [ΔC (-20 aa)]. These variants were fully or partly fixed in their quaternary structure and function, respectively, and were analysed for their conformational state and peroxidase and chaperone activity, as well as for their sensitivity to hyperoxidation. The presence of a His6-tag strongly influenced the properties of the protein. The ΔC variant became insensitive to hyperoxidation, while T92D and F84R became more sensitive. The C54D variant revealed the highest chaperone activity. The highest peroxidase activity was observed for the F84R and ΔC variants. Efficient interaction with NADP-dependent thioredoxin reductase C depended on the presence of Cys residues and the C-terminal tail. The results suggest that the structural flexibility is important for the switch between peroxidase and chaperone function and that evolution has conserved the functional switch instead of maximizing a single function. These variants are ideal tools for future conformation-specific studies in vivo and in vitro.
Collapse
|
|
32
|
Hyperoxidized peroxiredoxin 2 interacts with the protein disulfide- isomerase ERp46. Biochem J 2013; 453:475-85. [DOI: 10.1042/bj20130030] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Prx (peroxiredoxin) 2 protects cells from deleterious oxidative damage. It catalyses the breakdown of hydroperoxides through a highly reactive cysteine residue and has been linked to chaperone activity that promotes cell survival under conditions of oxidative stress. It may also be involved in redox signalling by binding to other proteins. In the present study we have searched for binding partners of Prx2 in H2O2-treated Jurkat and human umbilical vein endothelial cells and discovered that the hyperoxidized form selectively co-precipitated with the protein disulfide-isomerase ERp46. Mutant analyses revealed that loss of the peroxidative cysteine residue of Prx2 also facilitated complex formation with ERp46, even without H2O2 treatment, whereas the resolving cysteine residue of Prx2 was indispensible for the interaction to occur. The complex involved a stable non-covalent interaction that was disassociated by the reduction of intramolecular disulfides in ERp46, or by disruption of the decameric structure of hyperoxidized Prx2. This is the first example of a protein interaction dependent on the hyperoxidized status of a Prx.
Collapse
|
|
33
|
De Franceschi L, Franco RS, Bertoldi M, Brugnara C, Matté A, Siciliano A, Wieschhaus AJ, Chishti AH, Joiner CH. Pharmacological inhibition of calpain-1 prevents red cell dehydration and reduces Gardos channel activity in a mouse model of sickle cell disease. FASEB J 2012; 27:750-9. [PMID: 23085996 DOI: 10.1096/fj.12-217836] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Sickle cell disease (SCD) is a globally distributed hereditary red blood cell (RBC) disorder. One of the hallmarks of SCD is the presence of circulating dense RBCs, which are important in SCD-related clinical manifestations. In human dense sickle cells, we found reduced calpastatin activity and protein expression compared to either healthy RBCs or unfractionated sickle cells, suggesting an imbalance between activator and inhibitor of calpain-1 in favor of activator in dense sickle cells. Calpain-1 is a nonlysosomal cysteine proteinase that modulates multiple cell functions through the selective cleavage of proteins. To investigate the relevance of this observation in vivo, we evaluated the effects of the orally active inhibitor of calpain-1, BDA-410 (30 mg/kg/d), on RBCs from SAD mice, a mouse model for SCD. In SAD mice, BDA-410 improved RBC morphology, reduced RBC density (D(20); from 1106 ± 0.001 to 1100 ± 0.001 g/ml; P<0.05) and increased RBC-K(+) content (from 364 ± 10 to 429 ± 12.3 mmol/kg Hb; P<0.05), markedly reduced the activity of the Ca(2+)-activated K(+)channel (Gardos channel), and decreased membrane association of peroxiredoxin-2. The inhibitory effect of calphostin C, a specific inhibitor of protein kinase C (PKC), on the Gardos channel was eliminated after BDA-410 treatment, which suggests that calpain-1 inhibition affects the PKC-dependent fraction of the Gardos channel. BDA-410 prevented hypoxia-induced RBC dehydration and K(+) loss in SAD mice. These data suggest a potential role of BDA-410 as a novel therapeutic agent for treatment of SCD.
Collapse
|
|
34
|
Day AM, Brown JD, Taylor SR, Rand JD, Morgan BA, Veal EA. Inactivation of a peroxiredoxin by hydrogen peroxide is critical for thioredoxin-mediated repair of oxidized proteins and cell survival. Mol Cell 2012; 45:398-408. [PMID: 22245228 DOI: 10.1016/j.molcel.2011.11.027] [Citation(s) in RCA: 164] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2011] [Revised: 10/10/2011] [Accepted: 11/28/2011] [Indexed: 10/14/2022]
Abstract
Eukaryotic 2-Cys peroxiredoxins (Prx) are abundant antioxidant enzymes whose thioredoxin peroxidase activity plays an important role in protecting against oxidative stress, aging, and cancer. Paradoxically, this thioredoxin peroxidase activity is highly sensitive to inactivation by peroxide-induced Prx hyperoxidation. However, any possible advantage in preventing Prx from removing peroxides under oxidative stress conditions has remained obscure. Here we demonstrate that, in cells treated with hydrogen peroxide, the Prx Tpx1 is a major substrate for thioredoxin in the fission yeast Schizosaccharomyces pombe and, as such, competitively inhibits thioredoxin-mediated reduction of other oxidized proteins. Consequently, we reveal that the hyperoxidation of Tpx1 is critical to allow thioredoxin to act on other substrates ensuring repair of oxidized proteins and cell survival following exposure to toxic levels of hydrogen peroxide. We conclude that the inactivation of the thioredoxin peroxidase activity of Prx is important to maintain thioredoxin activity and cell viability under oxidative stress conditions.
Collapse
Affiliation(s)
- Alison M Day
- Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, Tyne and Wear, UK
| | | | | | | | | | | |
Collapse
|
|
35
|
Cao Z, Tavender TJ, Roszak AW, Cogdell RJ, Bulleid NJ. Crystal structure of reduced and of oxidized peroxiredoxin IV enzyme reveals a stable oxidized decamer and a non-disulfide-bonded intermediate in the catalytic cycle. J Biol Chem 2011; 286:42257-42266. [PMID: 21994946 PMCID: PMC3234919 DOI: 10.1074/jbc.m111.298810] [Citation(s) in RCA: 66] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2011] [Revised: 09/19/2011] [Indexed: 11/30/2022] Open
Abstract
Peroxiredoxin IV (PrxIV) is an endoplasmic reticulum-localized enzyme that metabolizes the hydrogen peroxide produced by endoplasmic reticulum oxidase 1 (Ero1). It has been shown to play a role in de novo disulfide formation, oxidizing members of the protein disulfide isomerase family of enzymes, and is a member of the typical 2-Cys peroxiredoxin family. We have determined the crystal structure of both reduced and disulfide-bonded, as well as a resolving cysteine mutant of human PrxIV. We show that PrxIV has a similar structure to other typical 2-Cys peroxiredoxins and undergoes a conformational change from a fully folded to a locally unfolded form following the formation of a disulfide between the peroxidatic and resolving cysteine residues. Unlike other mammalian typical 2-Cys peroxiredoxins, we show that human PrxIV forms a stable decameric structure even in its disulfide-bonded state. In addition, the structure of a resolving cysteine mutant reveals an intermediate in the reaction cycle that adopts the locally unfolded conformation. Interestingly the peroxidatic cysteine in the crystal structure is sulfenylated rather than sulfinylated or sulfonylated. In addition, the peroxidatic cysteine in the resolving cysteine mutant is resistant to hyper-oxidation following incubation with high concentrations of hydrogen peroxide. These results highlight some unique properties of PrxIV and suggest that the equilibrium between the fully folded and locally unfolded forms favors the locally unfolded conformation upon sulfenylation of the peroxidatic cysteine residue.
Collapse
Affiliation(s)
- Zhenbo Cao
- Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
| | - Timothy J Tavender
- Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
| | - Aleksander W Roszak
- WestCHEM, School of Chemistry, College of Science and Engineering, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
| | - Richard J Cogdell
- Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
| | - Neil J Bulleid
- Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
| |
Collapse
|
|
36
|
Rhee SG, Woo HA, Kil IS, Bae SH. Peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides. J Biol Chem 2011; 287:4403-10. [PMID: 22147704 DOI: 10.1074/jbc.r111.283432] [Citation(s) in RCA: 433] [Impact Index Per Article: 30.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Peroxiredoxins (Prxs) contain an active site cysteine that is sensitive to oxidation by H(2)O(2). Mammalian cells express six Prx isoforms that are localized to various cellular compartments. The oxidized active site cysteine of Prx can be reduced by a cellular thiol, thus enabling Prx to function as a locally constrained peroxidase. Regulation of Prx via phosphorylation in response to extracellular signals allows the local accumulation of H(2)O(2) and thereby enables its messenger function. The fact that the oxidation state of the active site cysteine of Prx can be transferred to other proteins that are less intrinsically susceptible to H(2)O(2) also allows Prx to function as an H(2)O(2) sensor.
Collapse
Affiliation(s)
- Sue Goo Rhee
- Division of Life and Pharmaceutical Sciences, Ewha Womans University,Seoul 120-750, Korea.
| | | | | | | |
Collapse
|
|
37
|
Rhee SG, Woo HA. Multiple functions of peroxiredoxins: peroxidases, sensors and regulators of the intracellular messenger H₂O₂, and protein chaperones. Antioxid Redox Signal 2011; 15:781-94. [PMID: 20919930 DOI: 10.1089/ars.2010.3393] [Citation(s) in RCA: 351] [Impact Index Per Article: 25.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Peroxiredoxins (Prxs) are a family of peroxidases that reduce peroxides, with a conserved cysteine residue (the peroxidatic Cys) serving as the site of oxidation by peroxides. Peroxides oxidize the peroxidatic Cys-SH to Cys-SOH, which then reacts with another cysteine residue (typically the resolving Cys [C(R)]) to form a disulfide that is subsequently reduced by an appropriate electron donor. On the basis of the location or absence of the C(R), Prxs are classified into 2-Cys, atypical 2-Cys, and 1-Cys Prx subfamilies. In addition to their peroxidase activity, members of the 2-Cys Prx subfamily appear to serve as peroxide sensors for other proteins and as molecular chaperones. During catalysis, the peroxidatic Cys-SOH of 2-Cys Prxs is occasionally further oxidized to Cys-SO(2)H before disulfide formation, resulting in inactivation of peroxidase activity. This hyperoxidation, which is reversed by the ATP-dependent enzyme sulfiredoxin, modulates the sensor and chaperone functions of 2-Cys Prxs. The peroxidase activity of 2-Cys Prxs is extensively regulated via tyrosine and threonine phosphorylation, which allows modulation of the local concentration of the intracellular messenger H(2)O(2). Finally, 2-Cys Prxs interact with a variety of proteins, with such interaction having been shown to modulate the function of the binding partners in a reciprocal manner.
Collapse
Affiliation(s)
- Sue Goo Rhee
- Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, Korea.
| | | |
Collapse
|
|
38
|
Poole LB, Hall A, Nelson KJ. Overview of peroxiredoxins in oxidant defense and redox regulation. CURRENT PROTOCOLS IN TOXICOLOGY 2011; Chapter 7:Unit7.9. [PMID: 21818754 PMCID: PMC3156475 DOI: 10.1002/0471140856.tx0709s49] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
Peroxiredoxins are important hydroperoxide detoxification enzymes, yet have only come to the fore in recent years relative to the other major players in peroxide detoxification, heme-containing catalases and peroxidases and glutathione peroxidases. These cysteine-dependent peroxidases exhibit high reactivity with hydrogen peroxide, organic hydroperoxides, and peroxynitrite and play major roles not only in peroxide defense, but also in regulating peroxide-mediated cell signaling. This overview focuses on important peroxiredoxin features that have emerged over the past several decades with an emphasis on catalytic mechanism, regulation, and biological function.
Collapse
Affiliation(s)
- Leslie B. Poole
- Dept. of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157
| | - Andrea Hall
- Dept. of Biochemistry and Biophysics, Oregon State University, Corvallis, OR
| | - Kimberly J. Nelson
- Dept. of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157
| |
Collapse
|
|
39
|
Abstract
Thiol peroxidases comprise glutathione peroxidases (GPx) and peroxiredoxins (Prx). The enzymes of both families reduce hydroperoxides with thiols by enzyme-substitution mechanisms. H(2)O(2) and organic hydroperoxides are reduced by all thiol peroxidases, most efficiently by SecGPxs, whereas fast peroxynitrite reduction is more common in Prxs. Reduction of lipid hydroperoxides is the domain of monomeric GPx4-type enzymes and of some Prxs. The catalysis starts with oxidation of an active-site selenocysteine (U(P)) or cysteine (C(P)). Activation of Cys (Sec) for hydroperoxide reduction in the GPx family is achieved by a typical tetrad composed of Cys (Sec), Asn, Gln, and Trp, whereas a triad of Cys Thr (or Ser) and Arg is the signature of Prx. In many of the CysGPxs and Prxs, a second Cys (C(R)) is required. In these 2-CysGPxs and 2-CysPrxs, the C(P) oxidized to a sulfenic acid forms an intra- or intermolecular disulfide (typical 2-CysPrx) with C(R), before a stepwise regeneration of ground-state enzyme by redoxin-type proteins can proceed. In SecGPxs and sporadically in Prxs, GSH is used as the reductant. Diversity combined with structural variability predestines thiol peroxidases for redox regulation via ROOH sensing and direct or indirect transduction of oxidant signals to specific protein targets.
Collapse
Affiliation(s)
- Leopold Flohé
- Otto-von-Guericke-Universität and MOLISA GmbH, Magdeburg, Germany.
| | | | | | | |
Collapse
|
|
40
|
Tomanek L, Zuzow MJ, Ivanina AV, Beniash E, Sokolova IM. Proteomic response to elevated PCO2 level in eastern oysters, Crassostrea virginica: evidence for oxidative stress. J Exp Biol 2011; 214:1836-44. [DOI: 10.1242/jeb.055475] [Citation(s) in RCA: 206] [Impact Index Per Article: 14.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
SUMMARY
Estuaries are characterized by extreme fluctuations in CO2 levels due to bouts of CO2 production by the resident biota that exceed its capacity of CO2 consumption and/or the rates of gas exchange with the atmosphere and open ocean waters. Elevated partial pressures of CO2 (PCO2; i.e. environmental hypercapnia) decrease the pH of estuarine waters and, ultimately, extracellular and intracellular pH levels of estuarine organisms such as mollusks that have limited capacity for pH regulation. We analyzed proteomic changes associated with exposure to elevated PCO2 in the mantle tissue of eastern oysters (Crassostrea virginica) after 2 weeks of exposure to control (∼39 Pa PCO2) and hypercapnic (∼357 Pa PCO2) conditions using two-dimensional gel electrophoresis and tandem mass spectrometry. Exposure to high PCO2 resulted in a significant proteome shift in the mantle tissue, with 12% of proteins (54 out of 456) differentially expressed under the high PCO2 compared with control conditions. Of the 54 differentially expressed proteins, we were able to identify 17. Among the identified proteins, two main functional categories were upregulated in response to hypercapnia: those associated with the cytoskeleton (e.g. several actin isoforms) and those associated with oxidative stress (e.g. superoxide dismutase and several peroxiredoxins as well as the thioredoxin-related nucleoredoxin). This indicates that exposure to high PCO2 (∼357 Pa) induces oxidative stress and suggests that the cytoskeleton is a major target of oxidative stress. We discuss how elevated CO2 levels may cause oxidative stress by increasing the production of reactive oxygen species (ROS) either indirectly by lowering organismal pH, which may enhance the Fenton reaction, and/or directly by CO2 interacting with other ROS to form more free radicals. Although estuarine species are already exposed to higher and more variable levels of CO2 than other marine species, climate change may further increase the extremes and thereby cause greater levels of oxidative stress.
Collapse
Affiliation(s)
- Lars Tomanek
- Department of Biological Sciences, Center for Coastal Marine Sciences and Environmental Proteomics Laboratory, California Polytechnic State University, San Luis Obispo, CA 93407-0401, USA
| | - Marcus J. Zuzow
- Department of Biological Sciences, Center for Coastal Marine Sciences and Environmental Proteomics Laboratory, California Polytechnic State University, San Luis Obispo, CA 93407-0401, USA
| | - Anna V. Ivanina
- Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223, USA
| | - Elia Beniash
- Department of Oral Biology, University of Pittsburgh, Pittsburgh, PA 15261, USA
| | - Inna M. Sokolova
- Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223, USA
| |
Collapse
|
|
41
|
Klomsiri C, Karplus PA, Poole LB. Cysteine-based redox switches in enzymes. Antioxid Redox Signal 2011; 14:1065-77. [PMID: 20799881 PMCID: PMC3064533 DOI: 10.1089/ars.2010.3376] [Citation(s) in RCA: 282] [Impact Index Per Article: 20.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/15/2010] [Accepted: 06/25/2010] [Indexed: 02/01/2023]
Abstract
The enzymes involved in metabolism and signaling are regulated by posttranslational modifications that influence their catalytic activity, rates of turnover, and targeting to subcellular locations. Most prominent among these has been phosphorylation/dephosphorylation, but now a distinct class of modification coming to the fore is a set of versatile redox modifications of key cysteine residues. Here we review the chemical, structural, and regulatory aspects of such redox regulation of enzymes and discuss examples of how these regulatory modifications often work in concert with phosphorylation/dephosphorylation events, making redox dependence an integral part of many cell signaling processes. Included are the emerging roles played by peroxiredoxins, a family of cysteine-based peroxidases that now appear to be major players in both antioxidant defense and cell signaling.
Collapse
Affiliation(s)
- Chananat Klomsiri
- Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina
| | - P. Andrew Karplus
- Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon
| | - Leslie B. Poole
- Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina
| |
Collapse
|
|
42
|
Ikeda Y, Nakano M, Ihara H, Ito R, Taniguchi N, Fujii J. Different consequences of reactions with hydrogen peroxide and t-butyl hydroperoxide in the hyperoxidative inactivation of rat peroxiredoxin-4. J Biochem 2011; 149:443-53. [PMID: 21212070 DOI: 10.1093/jb/mvq156] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Eukaryotic typical 2-Cys type peroxiredoxin (Prx) is inactivated by hyperoxidation of the peroxidatic cysteine to a sulphinic acid in a catalytic cycle-dependent manner. This inactivation process has been well documented for cytosolic isoforms of Prx. However, such a hyperoxidative inactivation has not fully been investigated in Prx-4, a secretable endoplasmic reticulum-resident isoform, in spite of being a typical 2-Cys type, and details of this process are reported herein. As has been observed in many peroxiredoxins, the peroxidase activity of Prx-4 was almost completely inhibited in the reaction with t-butyl hydroperoxide. On the other hand, when H(2)O(2) was used as the substrate, the peroxidase activity significantly remained after oxidative damage. In spite of these different consequences, mass spectrometric analyses indicated that both reactions resulted in the same oxidative damage, i.e. sulphinic acid formation at the peroxidatic cysteine, suggesting that another cysteine in the active site confers the peroxidase activity. As suggested by the analyses using cysteine-substituted mutants sulphinic acid formation at the peroxidatic cysteine may play a role in the development of the possible alternative mechanism, thereby sustaining the peroxidase activity that prefers H(2)O(2).
Collapse
Affiliation(s)
- Yoshitaka Ikeda
- Division of Molecular Cell Biology, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan.
| | | | | | | | | | | |
Collapse
|
|
43
|
Wu C, Liu T, Chen W, Oka SI, Fu C, Jain MR, Parrott AM, Baykal AT, Sadoshima J, Li H. Redox regulatory mechanism of transnitrosylation by thioredoxin. Mol Cell Proteomics 2010; 9:2262-75. [PMID: 20660346 PMCID: PMC2953919 DOI: 10.1074/mcp.m110.000034] [Citation(s) in RCA: 114] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2010] [Revised: 07/12/2010] [Indexed: 12/17/2022] Open
Abstract
Transnitrosylation and denitrosylation are emerging as key post-translational modification events in regulating both normal physiology and a wide spectrum of human diseases. Thioredoxin 1 (Trx1) is a conserved antioxidant that functions as a classic disulfide reductase. It also catalyzes the transnitrosylation or denitrosylation of caspase 3 (Casp3), underscoring its central role in determining Casp3 nitrosylation specificity. However, the mechanisms that regulate Trx1 transnitrosylation and denitrosylation of specific targets are unresolved. Here we used an optimized mass spectrometric method to demonstrate that Trx1 is itself nitrosylated by S-nitrosoglutathione at Cys(73) only after the formation of a Cys(32)-Cys(35) disulfide bond upon which the disulfide reductase and denitrosylase activities of Trx1 are attenuated. Following nitrosylation, Trx1 subsequently transnitrosylates Casp3. Overexpression of Trx1(C32S/C35S) (a mutant Trx1 with both Cys(32) and Cys(35) replaced by serine to mimic the disulfide reductase-inactive Trx1) in HeLa cells promoted the nitrosylation of specific target proteins. Using a global proteomics approach, we identified 47 novel Trx1 transnitrosylation target protein candidates. From further bioinformatics analysis of this set of nitrosylated peptides, we identified consensus motifs that are likely to be the determinants of Trx1-mediated transnitrosylation specificity. Among these proteins, we confirmed that Trx1 directly transnitrosylates peroxiredoxin 1 at Cys(173) and Cys(83) and protects it from H(2)O(2)-induced overoxidation. Functionally, we found that Cys(73)-mediated Trx1 transnitrosylation of target proteins is important for protecting HeLa cells from apoptosis. These data demonstrate that the ability of Trx1 to transnitrosylate target proteins is regulated by a crucial stepwise oxidative and nitrosative modification of specific cysteines, suggesting that Trx1, as a master regulator of redox signaling, can modulate target proteins via alternating modalities of reduction and nitrosylation.
Collapse
Affiliation(s)
- Changgong Wu
- From the ‡Center for Advanced Proteomics Research and Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School Cancer Center, Newark, New Jersey 07103
| | - Tong Liu
- From the ‡Center for Advanced Proteomics Research and Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School Cancer Center, Newark, New Jersey 07103
| | - Wei Chen
- From the ‡Center for Advanced Proteomics Research and Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School Cancer Center, Newark, New Jersey 07103
| | - Shin-ichi Oka
- §Cardiovascular Research Institute and Department of Cell Biology and Molecular Medicine, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103
| | - Cexiong Fu
- From the ‡Center for Advanced Proteomics Research and Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School Cancer Center, Newark, New Jersey 07103
- ¶Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11743, and
| | - Mohit Raja Jain
- From the ‡Center for Advanced Proteomics Research and Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School Cancer Center, Newark, New Jersey 07103
| | - Andrew Myles Parrott
- From the ‡Center for Advanced Proteomics Research and Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School Cancer Center, Newark, New Jersey 07103
| | - Ahmet Tarik Baykal
- From the ‡Center for Advanced Proteomics Research and Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School Cancer Center, Newark, New Jersey 07103
- ‖Research Institute for Genetic Engineering and Biotechnology, TUBITAK-Marmara Arastirma Merkezi, 41470 Gebze, Turkey
| | - Junichi Sadoshima
- §Cardiovascular Research Institute and Department of Cell Biology and Molecular Medicine, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103
| | - Hong Li
- From the ‡Center for Advanced Proteomics Research and Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School Cancer Center, Newark, New Jersey 07103
| |
Collapse
|
|
44
|
Nguyen HTM, Nam KH, Saleem Y, Kim KS. Characterization of Helicobacter pylori adhesin thiol peroxidase (HP0390) purified from Escherichia coli. J Biosci 2010; 35:241-8. [DOI: 10.1007/s12038-010-0028-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
|
|
45
|
Mitochondrial peroxiredoxin involvement in antioxidant defence and redox signalling. Biochem J 2009; 425:313-25. [PMID: 20025614 DOI: 10.1042/bj20091541] [Citation(s) in RCA: 387] [Impact Index Per Article: 24.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Prxs (peroxiredoxins) are a family of proteins that are extremely effective at scavenging peroxides. The Prxs exhibit a number of intriguing properties that distinguish them from conventional antioxidants, including a susceptibility to inactivation by hyperoxidation in the presence of excess peroxide and the ability to form complex oligomeric structures. These properties, combined with a high cellular abundance and reactivity with hydrogen peroxide, have led to speculation that the Prxs function as redox sensors that transmit signals as part of the cellular response to oxidative stress. Multicellular organisms express several different Prxs that can be categorized by their subcellular distribution. In mammals, Prx 3 and Prx 5 are targeted to the mitochondrial matrix. Mitochondria are a major source of hydrogen peroxide, and this oxidant is implicated in the damage associated with aging and a number of pathologies. Hydrogen peroxide can also act as a second messenger, and is linked with signalling events in mitochondria, including the induction of apoptosis. A simple kinetic competition analysis estimates that Prx 3 will be the target for up to 90% of hydrogen peroxide generated in the matrix. Therefore, mitochondrial Prxs have the potential to play a major role in mitochondrial redox signalling, but the extent of this role and the mechanisms involved are currently unclear.
Collapse
|
|
46
|
Shuvaeva TM, Novoselov VI, Fesenko EE, Lipkin VM. [Peroxiredoxins, a new family of antioxidant proteins]. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY 2009; 35:581-96. [PMID: 19915636 DOI: 10.1134/s106816200905001x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Current ideas are discussed about the structures and mechanisms of action of proteins that have been united at present into a family of thiol-specific antioxidants or peroxiredoxins, which protect the cells of different organisms from the action of hydrogen peroxide. Peroxiredoxins fulfill the same function as antioxidant enzymes such as catalases and glutathione-dependent peroxidases; however, their catalytic activity is lower than that of these enzymes. The level of expression of genes of peroxiredoxins is increased in many pathological states accompanied by oxidative stress, and today there is direct evidence for the important role of peroxiredoxins in the vital activity of cells.
Collapse
Affiliation(s)
- T M Shuvaeva
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
| | | | | | | |
Collapse
|
|
47
|
Bast A, Fischer K, Erttmann SF, Walther R. Induction of peroxiredoxin I gene expression by LPS involves the Src/PI3K/JNK signalling pathway. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2009; 1799:402-10. [PMID: 19941984 DOI: 10.1016/j.bbagrm.2009.11.015] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/18/2009] [Revised: 11/16/2009] [Accepted: 11/17/2009] [Indexed: 11/26/2022]
Abstract
Peroxiredoxin I (Prx I) belongs to a family of proteins with thiol-dependent peroxidase activity and is involved in the cellular protection against oxidative stress, the modulation of intracellular signalling cascades as well as the regulation of cell proliferation and apoptosis. In RAW 264.7 mouse macrophage cells Prx I was up-regulated on the mRNA and protein level by lipopolysaccharide (LPS). Treatment of cells with LPS increased the phosphorylation of c-Jun-NH(2) terminal kinase (JNK) and protein kinase B (PKB). Both SP600125, an inhibitor of JNK, and LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), dose-dependently decreased LPS-induced Prx I mRNA expression. Furthermore, up-regulation of Prx I mRNA by LPS was diminished by the Src tyrosine kinase inhibitor PP2 and the iNOS inhibitor L-NMMA. LPS-dependent induction of Prx I is likely mediated by an activator protein-1 site within the Prx I promoter region binding JunB and c-Fos. In contrast, NFkappaB was not involved in the activation of Prx I transcription. Our results suggest that the up-regulation of Prx I gene expression by LPS is part of the cellular response to stress and may protect against oxidative stress-related injury in RAW 264.7 cells.
Collapse
Affiliation(s)
- Antje Bast
- Department of Medical Biochemistry and Molecular Biology, Ernst-Moritz-Arndt University of Greifswald, 17487 Greifswald, Germany
| | | | | | | |
Collapse
|
|
48
|
The malarial parasite Plasmodium falciparum imports the human protein peroxiredoxin 2 for peroxide detoxification. Proc Natl Acad Sci U S A 2009; 106:13323-8. [PMID: 19666612 DOI: 10.1073/pnas.0905387106] [Citation(s) in RCA: 71] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Coevolution of the malarial parasite and its human host has resulted in a complex network of interactions contributing to the homeodynamics of the host-parasite unit. As a rapidly growing and multiplying organism, Plasmodium falciparum depends on an adequate antioxidant defense system that is efficient despite the absence of genuine catalase and glutathione peroxidase. Using different experimental approaches, we demonstrate that P. falciparum imports the human redox-active protein peroxiredoxin 2 (hPrx-2, hTPx1) into its cytosol. As shown by confocal microscopy and immunogold electron microscopy, hPrx-2 is also present in the Maurer's clefts, organelles that are described as being involved in parasite protein export. Enzyme kinetic analyses prove that hPrx-2 accepts Plasmodium cytosolic thioredoxin 1 as a reducing substrate. hPrx-2 accounts for roughly 50% of thioredoxin peroxidase activity in parasite extracts, thus indicating a functional role of hPrx-2 as an enzymatic scavenger of peroxides in the parasite. Under chloroquine treatment, a drug promoting oxidative stress, the abundance of hPrx-2 in the parasite increases significantly. P. falciparum has adapted to adopt the hPrx-2, thereby using the host protein for its own purposes.
Collapse
|
|
49
|
Mitochondrial peroxiredoxin 3 is more resilient to hyperoxidation than cytoplasmic peroxiredoxins. Biochem J 2009; 421:51-8. [PMID: 19356151 DOI: 10.1042/bj20090242] [Citation(s) in RCA: 91] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The Prxs (peroxiredoxins) are a family of cysteine-dependent peroxidases that decompose hydrogen peroxide. Prxs become hyperoxidized when a sulfenic acid formed during the catalytic cycle reacts with hydrogen peroxide. In the present study, Western blot methodology was developed to quantify hyperoxidation of individual 2-Cys Prxs in cells. It revealed that Prx 1 and 2 were hyperoxidized at lower doses of hydrogen peroxide than would be predicted from in vitro data, suggesting intracellular factors that promote hyperoxidation. In contrast, mitochondrial Prx 3 was considerably more resistant to hyperoxidation. The concentration of Prx 3 was estimated at 125 microM in the mitochondrial matrix of Jurkat T-lymphoma cells. Although the local cellular environment could influence susceptibility, purified Prx 3 was also more resistant to hyperoxidation, suggesting that despite having C-terminal motifs similar to sensitive eukaryote Prxs, other structural features must contribute to the innate resilience of Prx 3 to hyperoxidation.
Collapse
|
|
50
|
Barranco-Medina S, Lázaro JJ, Dietz KJ. The oligomeric conformation of peroxiredoxins links redox state to function. FEBS Lett 2009; 583:1809-16. [PMID: 19464293 DOI: 10.1016/j.febslet.2009.05.029] [Citation(s) in RCA: 173] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2009] [Revised: 05/08/2009] [Accepted: 05/12/2009] [Indexed: 12/25/2022]
Abstract
Protein-protein associations, i.e. formation of permanent or transient protein complexes, are essential for protein functionality and regulation within the cellular context. Peroxiredoxins (Prx) undergo major redox-dependent conformational changes and the dynamics are linked to functional switches. While a large number of investigations have addressed the principles and functions of Prx oligomerization, understanding of the diverse in vivo roles of this conserved redox-dependent feature of Prx is slowly emerging. The review summarizes studies on Prx oligomerization, its tight connection to the redox state, and the knowledge and hypotheses on its physiological function in the cell as peroxidase, chaperone, binding partner, enzyme activator and/or redox sensor.
Collapse
|