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1
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Villar SF, Möller MN, Denicola A. Biophysical tools to study the oligomerization dynamics of Prx1-class peroxiredoxins. Biophys Rev 2023; 15:601-609. [PMID: 37681093 PMCID: PMC10480382 DOI: 10.1007/s12551-023-01076-3] [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: 04/14/2023] [Accepted: 06/04/2023] [Indexed: 09/09/2023] Open
Abstract
Peroxiredoxins (Prx) are ubiquitous, highly conserved peroxidases whose activity depends on catalytic cysteine residues. The Prx1-class of the peroxiredoxin family, also called typical 2-Cys Prx, organize as head-to-tail homodimers containing two active sites. The peroxidatic cysteine CP of one monomer reacts with the peroxide substrate to form sulfenic acid that reacts with the resolving cysteine (CR) of the adjacent subunit to form an intermolecular disulfide, that is reduced back by the thioredoxin/thioredoxin reductase/NADPH system. Although the minimal catalytic unit is the dimer, these Prx oligomerize into (do)decamers. In addition, these ring-shaped decamers can pile-up into high molecular weight structures. Prx not only display peroxidase activity reducing H2O2, peroxynitrous acid and lipid hydroperoxides (antioxidant enzymes), but also exhibit holdase activity protecting other proteins from unfolding (molecular chaperones). Highly relevant is their participation in redox cellular signaling that is currently under active investigation. The different activities attributed to Prx are strongly ligated to their quaternary structure. In this review, we will describe different biophysical approaches used to characterize the oligomerization dynamics of Prx that include the classical size-exclusion chromatography, analytical ultracentrifugation, calorimetry, and also fluorescence anisotropy and lifetime measurements, as well as mass photometry.
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Affiliation(s)
- Sebastián F. Villar
- Laboratorio Fisicoquímica Biológica, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
| | - Matías N. Möller
- Laboratorio Fisicoquímica Biológica, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
| | - Ana Denicola
- Laboratorio Fisicoquímica Biológica, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
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2
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Marsan ES, Dreab A, Bayse CA. In silico insights into the dimer structure and deiodinase activity of type III iodothyronine deiodinase from bioinformatics, molecular dynamics simulations, and QM/MM calculations. J Biomol Struct Dyn 2023; 41:4819-4829. [PMID: 35579922 PMCID: PMC9878935 DOI: 10.1080/07391102.2022.2073271] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2022] [Accepted: 04/27/2022] [Indexed: 01/28/2023]
Abstract
The homodimeric family of iodothyronine deiodinases (Dios) regioselectively remove iodine from thyroid hormones. Currently, structural data has only been reported for the monomer of the mus type III thioredoxin (Trx) fold catalytic domain (Dio3Trx), but the mode of dimerization has not yet been determined. Various groups have proposed dimer structures that are similar to the A-type and B-type dimerization modes of peroxiredoxins. Computational methods are used to compare the sequence of Dio3Trx to related proteins known to form A-type and B-type dimers. Sequence analysis and in silico protein-protein docking methods suggest that Dio3Trx is more consistent with proteins that adopt B-type dimerization. Molecular dynamics (MD) simulations of the refined Dio3Trx dimer constructed using the SymmDock and GalaxyRefineComplex databases indicate stable dimer formation along the β4α3 interface consistent with other Trx fold B-type dimers. Free energy calculations show that the dimer is stabilized by interdimer interactions between the β-sheets and α-helices. A comparison of MD simulations of the apo and thyroxine-bound dimers suggests that the active site binding pocket is not affected by dimerization. Determination of the transition state for deiodination of thyroxine from the monomer structure using QM/MM methods provides an activation barrier consistent with previous small model DFT studies.Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Eric S Marsan
- Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA
| | - Ana Dreab
- Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA
| | - Craig A Bayse
- Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA
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3
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Amoscato AA, Anthonymuthu T, Kapralov O, Sparvero LJ, Shrivastava IH, Mikulska-Ruminska K, Tyurin VA, Shvedova AA, Tyurina YY, Bahar I, Wenzel S, Bayir H, Kagan VE. Formation of protein adducts with Hydroperoxy-PE electrophilic cleavage products during ferroptosis. Redox Biol 2023; 63:102758. [PMID: 37245287 PMCID: PMC10238881 DOI: 10.1016/j.redox.2023.102758] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2023] [Revised: 05/17/2023] [Accepted: 05/21/2023] [Indexed: 05/30/2023] Open
Abstract
Ferroptosis is an iron dependent form of cell death, that is triggered by the discoordination of iron, lipids, and thiols. Its unique signature that distinguishes it from other forms of cell death is the formation and accumulation of lipid hydroperoxides, particularly oxidized forms of polyunsaturated phosphatidylethanolamines (PEs), which drives cell death. These readily undergo iron-catalyzed secondary free radical reactions leading to truncated products which retain the signature PE headgroup and which can readily react with nucleophilic moieties in proteins via their truncated electrophilic acyl chains. Using a redox lipidomics approach, we have identified oxidatively-truncated PE species (trPEox) in enzymatic and non-enzymatic model systems. Further, using a model peptide we demonstrate adduct formation with Cys as the preferred nucleophilic residue and PE(26:2) +2 oxygens, as one of the most reactive truncated PE-electrophiles produced. In cells stimulated to undergo ferroptosis we identified PE-truncated species with sn-2 truncations ranging from 5 to 9 carbons. Taking advantage of the free PE headgroup, we have developed a new technology using the lantibiotic duramycin, to enrich and identify the PE-lipoxidated proteins. Our results indicate that several dozens of proteins for each cell type, are PE-lipoxidated in HT-22, MLE, and H9c2 cells and M2 macrophages after they were induced to undergo ferroptosis. Pretreatment of cells with the strong nucleophile, 2-mercaptoethanol, prevented the formation of PE-lipoxidated proteins and blocked ferroptotic death. Finally, our docking simulations showed that the truncated PE species bound at least as good to several of the lantibiotic-identified proteins, as compared to the non-truncated parent molecule, stearoyl-arachidonoyl PE (SAPE), indicating that these oxidatively-truncated species favor/promote the formation of PEox-protein adducts. The identification of PEox-protein adducts during ferroptosis suggests that they are participants in the ferroptotic process preventable by 2-mercaptoethanol and may contribute to a point of no return in the ferroptotic death process.
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Affiliation(s)
- A A Amoscato
- Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA.
| | - T Anthonymuthu
- Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA; Adeptrix Corp, 100 Cummings Center, Suite 339c, Beverly, MA, 01915, USA
| | - O Kapralov
- Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA
| | - L J Sparvero
- Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA
| | - I H Shrivastava
- NIOSH/HELD/EAB, 1095 Willowdale Road, Morgantown, WV, 26505, USA
| | - K Mikulska-Ruminska
- Institute of Physics, Faculty of Physics Astronomy and Informatics, Nicolaus Copernicus University in Toruń, PL87100, Toruń, Poland
| | - V A Tyurin
- Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA
| | - A A Shvedova
- NIOSH/HELD/EAB, 1095 Willowdale Road, Morgantown, WV, 26505, USA; Department of Physiology and Pharmacology, School of Medicine, West Virginia University, Morgantown, WV, USA
| | - Y Y Tyurina
- Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA
| | - I Bahar
- Department of Computational and Systems Biology, University of Pittsburgh, 800 Murdoch I Bldg., 3420 Forbes Avenue, Pittsburgh, PA, 15213, USA; Laufer Center for Physical and Quantitative Biology, Laufer Center, Z-5252, Stony Brook University, Stony Brook, NY, 11794, USA
| | - S Wenzel
- Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh Asthma and Environmental Lung Health Institute at UPMC, University of Pittsburgh, Pittsburgh, PA, 15261, USA
| | - H Bayir
- Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA; Safar Center for Resuscitation Research, Department of Critical Care Medicine, University of Pittsburgh Medical Center, 4401 Penn Ave, Pittsburgh, PA, 15224, USA; Department of Pediatrics Critical Care, Columbia University, 3959 Broadway, CHN-10, New York, NY, 10032, USA
| | - V E Kagan
- Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh School of Public Health, 130 Desoto St, Pittsburgh, PA, 15261, USA; Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, 8-2 Trubetskaya Str, 11999, Moscow, Russia.
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Montanhero Cabrera VI, do Nascimento Sividanes G, Quintiliano NF, Hikari Toyama M, Ghilardi Lago JH, de Oliveira MA. Exploring functional and structural features of chemically related natural prenylated hydroquinone and benzoic acid from Piper crassinervium (Piperaceae) on bacterial peroxiredoxin inhibition. PLoS One 2023; 18:e0281322. [PMID: 36827425 PMCID: PMC9956870 DOI: 10.1371/journal.pone.0281322] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2023] [Accepted: 01/19/2023] [Indexed: 02/26/2023] Open
Abstract
Multiple drug resistance (MDR) bacterial strains are responsible by 1.2 million of human deaths all over the world. The pathogens possess efficient enzymes which are able to mitigate the toxicity of reactive oxygen species (ROS) produced by some antibiotics and the host immune cells. Among them, the bacterial peroxiredoxin alkyl hydroperoxide reductase C (AhpC) is able to decompose efficiently several kinds of hydroperoxides. To decompose their substrates AhpC use a reactive cysteine residue (peroxidatic cysteine-CysP) that together with two other polar residues (Thr/Ser and Arg) comprise the catalytic triad of these enzymes and are involved in the substrate targeting/stabilization to allow a bimolecular nucleophilic substitution (SN2) reaction. Additionally to the high efficiency the AhpC is very abundant in the cells and present virulent properties in some bacterial species. Despite the importance of AhpC in bacteria, few studies aimed at using natural compounds as inhibitors of this class of enzymes. Some natural products were identified as human isoforms, presenting as common characteristics a bulk hydrophobic moiety and an α, β-unsaturated carbonylic system able to perform a thiol-Michael reaction. In this work, we evaluated two chemically related natural products: 1,4-dihydroxy-2-(3',7'-dimethyl-1'-oxo-2'E,6'-octadienyl) benzene (C1) and 4-hydroxy-2-(3',7'-dimethyl-1'-oxo-2'E,6'-octadienyl) benzoic acid (C2), both were isolated from branches Piper crassinervium (Piperaceae), over the peroxidase activity of AhpC from Pseudomonas aeruginosa (PaAhpC) and Staphylococcus epidermidis (SeAhpC). By biochemical assays we show that although both compounds can perform the Michael addition reaction, only compound C2 was able to inhibit the PaAhpC peroxidase activity but not SeAhpC, presenting IC50 = 20.3 μM. SDS-PAGE analysis revealed that the compound was not able to perform a thiol-Michael addition, suggesting another inhibition behavior. Using computer-assisted simulations, we also show that an acidic group present in the structure of compound C2 may be involved in the stabilization by polar interactions with the Thr and Arg residues from the catalytic triad and several apolar interactions with hydrophobic residues. Finally, C2 was not able to interfere in the peroxidase activity of the isoform Prx2 from humans or even the thiol proteins of the Trx reducing system from Escherichia coli (EcTrx and EcTrxR), indicating specificity for P. aeruginosa AhpC.
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Affiliation(s)
| | | | | | - Marcos Hikari Toyama
- Instituto de Biociências, Universidade Estadual Paulista, UNESP, São Vicente, SP, Brazil
| | - João Henrique Ghilardi Lago
- Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, SP, Brazil
- * E-mail: (MAO); (JHGL)
| | - Marcos Antonio de Oliveira
- Instituto de Biociências, Universidade Estadual Paulista, UNESP, São Vicente, SP, Brazil
- * E-mail: (MAO); (JHGL)
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Aki T, Unuma K, Uemura K. The Role of Peroxiredoxins in the Regulation of Sepsis. Antioxidants (Basel) 2022; 11:antiox11010126. [PMID: 35052630 PMCID: PMC8773135 DOI: 10.3390/antiox11010126] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2021] [Revised: 01/05/2022] [Accepted: 01/05/2022] [Indexed: 02/01/2023] Open
Abstract
Oxidative stress, a result of a disturbance in redox homeostasis, is considered to be one of the main aggravating events in the pathogenesis of immune disorders. Peroxiredoxins (Prdxs) are an enzyme family that catalyzes the reduction of peroxides, including hydrogen peroxide, lipid peroxides, and nitrogen peroxides. Although the maintenance of cellular redox homeostasis through Prdxs is essential for surviving in adverse environments, Prdxs also participate in the regulation of cellular signal transduction by modulating the activities of a panel of molecules involved in the signal transduction process. Although Prdxs were discovered as intracellular anti-oxidative enzymes, recent research has revealed that Prdxs also play important roles in the extracellular milieu. Indeed, Prdxs have been shown to have the capacity to activate immune cells through ligation with innate immune receptors such as toll-like receptors (TLRs). In this review, we will summarize the intracellular as well as extracellular roles of Prdxs for and against the pathogenesis of inflammatory disorders including sepsis, hemorrhagic shock, and drug-induced liver injury.
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Fuentes-Lemus E, Hägglund P, López-Alarcón C, Davies MJ. Oxidative Crosslinking of Peptides and Proteins: Mechanisms of Formation, Detection, Characterization and Quantification. Molecules 2021; 27:15. [PMID: 35011250 PMCID: PMC8746199 DOI: 10.3390/molecules27010015] [Citation(s) in RCA: 48] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2021] [Revised: 12/17/2021] [Accepted: 12/18/2021] [Indexed: 12/14/2022] Open
Abstract
Covalent crosslinks within or between proteins play a key role in determining the structure and function of proteins. Some of these are formed intentionally by either enzymatic or molecular reactions and are critical to normal physiological function. Others are generated as a consequence of exposure to oxidants (radicals, excited states or two-electron species) and other endogenous or external stimuli, or as a result of the actions of a number of enzymes (e.g., oxidases and peroxidases). Increasing evidence indicates that the accumulation of unwanted crosslinks, as is seen in ageing and multiple pathologies, has adverse effects on biological function. In this article, we review the spectrum of crosslinks, both reducible and non-reducible, currently known to be formed on proteins; the mechanisms of their formation; and experimental approaches to the detection, identification and characterization of these species.
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Affiliation(s)
- Eduardo Fuentes-Lemus
- Department of Biomedical Sciences, Panum Institute, University of Copenhagen, 2200 Copenhagen, Denmark; (E.F.-L.); (P.H.)
| | - Per Hägglund
- Department of Biomedical Sciences, Panum Institute, University of Copenhagen, 2200 Copenhagen, Denmark; (E.F.-L.); (P.H.)
| | - Camilo López-Alarcón
- Departamento de Química Física, Facultad de Química y de Farmacia, Pontificia Universidad Catolica de Chile, Santiago 7820436, Chile;
| | - Michael J. Davies
- Department of Biomedical Sciences, Panum Institute, University of Copenhagen, 2200 Copenhagen, Denmark; (E.F.-L.); (P.H.)
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Role of the Redox State of Human Peroxiredoxin-5 on Its TLR4-Activating DAMP Function. Antioxidants (Basel) 2021; 10:antiox10121902. [PMID: 34943005 PMCID: PMC8750366 DOI: 10.3390/antiox10121902] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2021] [Revised: 11/23/2021] [Accepted: 11/24/2021] [Indexed: 11/17/2022] Open
Abstract
Human peroxiredoxin-5 (PRDX5) is a unique redox-sensitive protein that plays a dual role in brain ischemia-reperfusion injury. While intracellular PRDX5 has been reported to act as a neuroprotective antioxidative enzyme by scavenging peroxides, once released extracellularly from necrotic brain cells, the protein aggravates neural cell death by inducing expression of proinflammatory cytokines in macrophages through activation of Toll-like receptor (TLR) 2 (TLR2) and 4 (TLR4). Although recent evidence showed that PRDX5 was able to interact directly with TLR4, little is known regarding the role of the cysteine redox state of PRDX5 on its DAMP function. To gain insights into the role of PRDX5 redox-active cysteine residues in the TLR4-dependent proinflammatory activity of the protein, we used a recombinant human PRDX5 in the disulfide (oxidized) form and a mutant version lacking the peroxidatic cysteine, as well as chemically reduced and hyperoxidized PRDX5 proteins. We first analyzed the oxidation state and oligomerization profile by Western blot, mass spectrometry, and SEC-MALS. Using ELISA, we demonstrate that the disulfide bridge between the enzymatic cysteines is required to allow improved TLR4-dependent IL-8 secretion. Moreover, single-molecule force spectroscopy experiments revealed that TLR4 alone is not sufficient to discriminate the different PRDX5 redox forms. Finally, flow cytometry binding assays show that disulfide PRDX5 has a higher propensity to bind to the surface of living TLR4-expressing cells than the mutant protein. Taken together, these results demonstrate the importance of the redox state of PRDX5 cysteine residues on TLR4-induced inflammation.
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Bolduc J, Koruza K, Luo T, Malo Pueyo J, Vo TN, Ezeriņa D, Messens J. Peroxiredoxins wear many hats: Factors that fashion their peroxide sensing personalities. Redox Biol 2021; 42:101959. [PMID: 33895094 PMCID: PMC8113037 DOI: 10.1016/j.redox.2021.101959] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2020] [Revised: 03/07/2021] [Accepted: 03/25/2021] [Indexed: 02/07/2023] Open
Abstract
Peroxiredoxins (Prdxs) sense and assess peroxide levels, and signal through protein interactions. Understanding the role of the multiple structural and post-translational modification (PTM) layers that tunes the peroxiredoxin specificities is still a challenge. In this review, we give a tabulated overview on what is known about human and bacterial peroxiredoxins with a focus on structure, PTMs, and protein-protein interactions. Armed with numerous cellular and atomic level experimental techniques, we look at the future and ask ourselves what is still needed to give us a clearer view on the cellular operating power of Prdxs in both stress and non-stress conditions.
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Affiliation(s)
- Jesalyn Bolduc
- VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, B-1050, Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium
| | - Katarina Koruza
- VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, B-1050, Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium
| | - Ting Luo
- VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, B-1050, Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium
| | - Julia Malo Pueyo
- VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, B-1050, Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium
| | - Trung Nghia Vo
- VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, B-1050, Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium
| | - Daria Ezeriņa
- VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, B-1050, Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium
| | - Joris Messens
- VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, B-1050, Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium.
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9
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Neuroprotective Effect of 3-[(4-Chlorophenyl)selanyl]-1-methyl-1H-indole on Hydrogen Peroxide-Induced Oxidative Stress in SH-SY5Y Cells. Neurochem Res 2021; 46:535-549. [PMID: 33548035 DOI: 10.1007/s11064-020-03190-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2020] [Revised: 11/11/2020] [Accepted: 11/27/2020] [Indexed: 10/22/2022]
Abstract
Extensive data have reported the involvement of oxidative stress in the pathogenesis of neuropsychiatric disorders, prompting the pursuit of antioxidant molecules that could become adjuvant pharmacological agents for the management of oxidative stress-associated disorders. The 3-[(4-chlorophenyl)selanyl]-1-methyl-1H-indole (CMI) has been reported as an antioxidant and immunomodulatory compound that improves depression-like behavior and cognitive impairment in mice. However, the exact effect of CMI on specific brain cells is yet to be studied. In this context, the present study aimed to evaluate the antioxidant activity of CMI in H2O2-induced oxidative stress on human dopaminergic neuroblastoma cells (SH-SY5Y) and to shed some light into its possible mechanism of action. Our results demonstrated that the treatment of SH-SY5Y cells with 4 µM CMI protected them against H2O2 (343 μM)-induced oxidative stress. Specifically, CMI prevented the increased number of reactive oxygen species (ROS)-positive cells induced by H2O2 exposure. Furthermore, CMI treatment increased the levels of reduced glutathione in SH-SY5Y cells. Molecular docking studies demonstrated that CMI might interact with enzymes involved in glutathione metabolism (i.e., glutathione peroxidase and glutathione reductase) and H2O2 scavenging (i.e., catalase). In silico pharmacokinetics analysis predicted that CMI might be well absorbed, metabolized, and excreted, and able to cross the blood-brain barrier. Also, CMI was not considered toxic overall. Taken together, our results suggest that CMI protects dopaminergic neurons from H2O2-induced stress by lowering ROS levels and boosting the glutathione system. These results will facilitate the clinical application of CMI to treat nervous system diseases associated with oxidative stress.
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10
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Chowhan RK, Rahaman H, Singh LR. Structural basis of peroxidase catalytic cycle of human Prdx6. Sci Rep 2020; 10:17416. [PMID: 33060708 PMCID: PMC7566464 DOI: 10.1038/s41598-020-74052-6] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2019] [Accepted: 09/24/2020] [Indexed: 11/23/2022] Open
Abstract
Peroxiredoxin 6 (Prdx6) is a ubiquitously expressed antioxidant non-selenium glutathione peroxidase that is known to play a major role in various physiological and pathological processes. It belongs to the family of peroxidases (referred to as Peroxiredoxins, Prdx's) that work independently of any prosthetic groups or co-factors, and instead utilize a peroxidatic thiol residue for peroxide reduction. Mammalian Prdx's are classified according to the number of Cys implicated in their catalytic activity by the formation of either inter-molecular (typical 2-Cys, Prdx1-4) or intra-molecular (atypical 2-Cys, Prdx5) disulfide bond, or non-covalent interactions (1-Cys, Prdx6). The typical and atypical 2-Prdx's have been identified to show decamer/dimer and monomer/dimer transition, respectively, upon oxidation of their peroxidatic cysteine. However, the alterations in the oligomeric status of Prdx6 as a function of peroxidatic thiol's redox state are still ambiguous. While the crystal structure of recombinant human Prdx6 is resolved as a dimer, the solution structures are reported to have both monomers and dimers. In the present study, we have employed several spectroscopic and electrophoretic probes to discern the impact of change in the redox status of peroxidatic cysteine on conformation and oligomeric status of Prdx6. Our study indicates Prdx6's peroxidase activity to be a redox-based conformation driven process which essentially involves monomer-dimer transition.
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Affiliation(s)
- Rimpy Kaur Chowhan
- Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, 110007, India
| | - Hamidur Rahaman
- Department of Biotechnology, Manipur University, Imphal, 795003, India
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11
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Vander Zanden CM, Czarny RS, Ho EN, Robertson AB, Ho PS. Structural adaptation of vertebrate endonuclease G for 5-hydroxymethylcytosine recognition and function. Nucleic Acids Res 2020; 48:3962-3974. [PMID: 32095813 PMCID: PMC7144941 DOI: 10.1093/nar/gkaa117] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2019] [Revised: 02/09/2020] [Accepted: 02/19/2020] [Indexed: 01/07/2023] Open
Abstract
Modified DNA bases functionally distinguish the taxonomic forms of life—5-methylcytosine separates prokaryotes from eukaryotes and 5-hydroxymethylcytosine (5hmC) invertebrates from vertebrates. We demonstrate here that mouse endonuclease G (mEndoG) shows specificity for both 5hmC and Holliday junctions. The enzyme has higher affinity (>50-fold) for junctions over duplex DNAs. A 5hmC-modification shifts the position of the cut site and increases the rate of DNA cleavage in modified versus unmodified junctions. The crystal structure of mEndoG shows that a cysteine (Cys69) is positioned to recognize 5hmC through a thiol-hydroxyl hydrogen bond. Although this Cys is conserved from worms to mammals, a two amino acid deletion in the vertebrate relative to the invertebrate sequence unwinds an α-helix, placing the thiol of Cys69 into the mEndoG active site. Mutations of Cys69 with alanine or serine show 5hmC-specificity that mirrors the hydrogen bonding potential of the side chain (C–H < S–H < O–H). A second orthogonal DNA binding site identified in the mEndoG structure accommodates a second arm of a junction. Thus, the specificity of mEndoG for 5hmC and junctions derives from structural adaptations that distinguish the vertebrate from the invertebrate enzyme, thereby thereby supporting a role for 5hmC in recombination processes.
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Affiliation(s)
- Crystal M Vander Zanden
- Department of Biochemistry & Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA
| | - Ryan S Czarny
- Department of Biochemistry & Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA
| | - Ethan N Ho
- Department of Biochemistry & Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA
| | - Adam B Robertson
- Department of Molecular Microbiology, Oslo University Hospital, Sognsvannsveien 20, NO-0027 Oslo, Norway
| | - P Shing Ho
- Department of Biochemistry & Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA
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12
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Hawkins CL, Davies MJ. Detection, identification, and quantification of oxidative protein modifications. J Biol Chem 2019; 294:19683-19708. [PMID: 31672919 PMCID: PMC6926449 DOI: 10.1074/jbc.rev119.006217] [Citation(s) in RCA: 249] [Impact Index Per Article: 41.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Exposure of biological molecules to oxidants is inevitable and therefore commonplace. Oxidative stress in cells arises from both external agents and endogenous processes that generate reactive species, either purposely (e.g. during pathogen killing or enzymatic reactions) or accidentally (e.g. exposure to radiation, pollutants, drugs, or chemicals). As proteins are highly abundant and react rapidly with many oxidants, they are highly susceptible to, and major targets of, oxidative damage. This can result in changes to protein structure, function, and turnover and to loss or (occasional) gain of activity. Accumulation of oxidatively-modified proteins, due to either increased generation or decreased removal, has been associated with both aging and multiple diseases. Different oxidants generate a broad, and sometimes characteristic, spectrum of post-translational modifications. The kinetics (rates) of damage formation also vary dramatically. There is a pressing need for reliable and robust methods that can detect, identify, and quantify the products formed on amino acids, peptides, and proteins, especially in complex systems. This review summarizes several advances in our understanding of this complex chemistry and highlights methods that are available to detect oxidative modifications-at the amino acid, peptide, or protein level-and their nature, quantity, and position within a peptide sequence. Although considerable progress has been made in the development and application of new techniques, it is clear that further development is required to fully assess the relative importance of protein oxidation and to determine whether an oxidation is a cause, or merely a consequence, of injurious processes.
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Affiliation(s)
- Clare L Hawkins
- Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Copenhagen 2200, Denmark
| | - Michael J Davies
- Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Copenhagen 2200, Denmark
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Elko EA, Cunniff B, Seward DJ, Chia SB, Aboushousha R, van de Wetering C, van der Velden J, Manuel A, Shukla A, Heintz NH, Anathy V, van der Vliet A, Janssen-Heininger YMW. Peroxiredoxins and Beyond; Redox Systems Regulating Lung Physiology and Disease. Antioxid Redox Signal 2019; 31:1070-1091. [PMID: 30799628 PMCID: PMC6767868 DOI: 10.1089/ars.2019.7752] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Significance: The lung is a unique organ, as it is constantly exposed to air, and thus it requires a robust antioxidant defense system to prevent the potential damage from exposure to an array of environmental insults, including oxidants. The peroxiredoxin (PRDX) family plays an important role in scavenging peroxides and is critical to the cellular antioxidant defense system. Recent Advances: Exciting discoveries have been made to highlight the key features of PRDXs that regulate the redox tone. PRDXs do not act in isolation as they require the thioredoxin/thioredoxin reductase/NADPH, sulfiredoxin (SRXN1) redox system, and in some cases glutaredoxin/glutathione, for their reduction. Furthermore, the chaperone function of PRDXs, controlled by the oxidation state, demonstrates the versatility in redox regulation and control of cellular biology exerted by this class of proteins. Critical Issues: Despite the long-known observations that redox perturbations accompany a number of pulmonary diseases, surprisingly little is known about the role of PRDXs in the etiology of these diseases. In this perspective, we review the studies that have been conducted thus far to address the roles of PRDXs in lung disease, or experimental models used to study these diseases. Intriguing findings, such as the secretion of PRDXs and the formation of autoantibodies, raise a number of questions about the pathways that regulate secretion, redox status, and immune response to PRDXs. Future Directions: Further understanding of the mechanisms by which individual PRDXs control lung inflammation, injury, repair, chronic remodeling, and cancer, and the importance of PRDX oxidation state, configuration, and client proteins that govern these processes is needed.
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Affiliation(s)
- Evan A Elko
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Brian Cunniff
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - David J Seward
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Shi Biao Chia
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Reem Aboushousha
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Cheryl van de Wetering
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Jos van der Velden
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Allison Manuel
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Arti Shukla
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Nicholas H Heintz
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Vikas Anathy
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Albert van der Vliet
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont
| | - Yvonne M W Janssen-Heininger
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont
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14
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Pacifici F, Della Morte D, Capuani B, Pastore D, Bellia A, Sbraccia P, Di Daniele N, Lauro R, Lauro D. Peroxiredoxin6, a Multitask Antioxidant Enzyme Involved in the Pathophysiology of Chronic Noncommunicable Diseases. Antioxid Redox Signal 2019; 30:399-414. [PMID: 29160110 DOI: 10.1089/ars.2017.7427] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
SIGNIFICANCE Chronic noncommunicable diseases (NCDs) are the leading causes of disability and death worldwide. NCDs mainly comprise diabetes mellitus, cardiovascular diseases, chronic obstructive pulmonary disease, cancer, and neurological degenerative diseases, which kill more than 80% of population, especially the elderly, worldwide. Recent Advances: Several recent theories established NCDs as multifactorial diseases, where a combination of genetic, epigenetic, and environmental factors contributes to their pathogenesis. Nevertheless, recent findings suggest that the common factor linking all these pathologies is an increase in oxidative stress and the age-related loss of the antioxidant mechanisms of defense against it. Impairment in mitochondrial homeostasis with consequent deregulation in oxidative stress balance has also been suggested. CRITICAL ISSUES Therefore, antioxidant proteins deserve particular attention for their potential role against NCDs. In particular, peroxiredoxin(Prdx)6 is a unique antioxidant enzyme, belonging to the Prdx family, with double properties, peroxidase and phospholipase activities. Through these activities, Prdx6 has been shown to be a powerful antioxidant enzyme, implicated in the pathogenesis of different NCDs. Recently, we described a phenotype of diabetes mellitus in Prdx6 knockout mice, suggesting a pivotal role of Prdx6 in the pathogenesis of cardiometabolic diseases. FUTURE DIRECTIONS Increasing awareness on the role of antioxidant defenses in the pathogenesis of NCDs may open novel therapeutic approaches to reduce the burden of this pandemic phenomenon. However, knowledge of the role of Prdx6 in NCD prevention and pathogenesis is still not clarified.
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Affiliation(s)
- Francesca Pacifici
- 1 Department of Systems Medicine, University of Rome Tor Vergata , Rome, Italy
| | - David Della Morte
- 1 Department of Systems Medicine, University of Rome Tor Vergata , Rome, Italy
- 2 Department of Human Sciences and Quality of Life Promotion, San Raffaele Roma Open University , Rome, Italy
| | - Barbara Capuani
- 1 Department of Systems Medicine, University of Rome Tor Vergata , Rome, Italy
| | - Donatella Pastore
- 1 Department of Systems Medicine, University of Rome Tor Vergata , Rome, Italy
| | - Alfonso Bellia
- 1 Department of Systems Medicine, University of Rome Tor Vergata , Rome, Italy
- 3 Policlinico Tor Vergata Foundation, University Hospital , Rome, Italy
| | - Paolo Sbraccia
- 1 Department of Systems Medicine, University of Rome Tor Vergata , Rome, Italy
- 3 Policlinico Tor Vergata Foundation, University Hospital , Rome, Italy
| | - Nicola Di Daniele
- 1 Department of Systems Medicine, University of Rome Tor Vergata , Rome, Italy
- 3 Policlinico Tor Vergata Foundation, University Hospital , Rome, Italy
| | - Renato Lauro
- 1 Department of Systems Medicine, University of Rome Tor Vergata , Rome, Italy
| | - Davide Lauro
- 1 Department of Systems Medicine, University of Rome Tor Vergata , Rome, Italy
- 3 Policlinico Tor Vergata Foundation, University Hospital , Rome, Italy
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15
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Lismont C, Nordgren M, Brees C, Knoops B, Van Veldhoven PP, Fransen M. Peroxisomes as Modulators of Cellular Protein Thiol Oxidation: A New Model System. Antioxid Redox Signal 2019; 30:22-39. [PMID: 28594286 DOI: 10.1089/ars.2017.6997] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
AIMS Peroxisomes are ubiquitous, single-membrane-bounded organelles that contain considerable amounts of enzymes involved in the production or breakdown of hydrogen peroxide (H2O2), a key signaling molecule in multiple biological processes and disease states. Despite this, the role of this organelle in cross-compartmental H2O2 signaling remains largely unclear, mainly because of the difficulty to modulate peroxisomal H2O2 production in a selective manner. This study aimed at establishing and validating a cellular model suitable to decipher the complex signaling processes associated with peroxisomal H2O2 release. RESULTS Here, we report the development of a human cell line that can be used to selectively generate H2O2 inside peroxisomes in a time- and dose-controlled manner. In addition, we provide evidence that peroxisome-derived H2O2 can oxidize redox-sensitive cysteine residues in multiple proteins within (e.g., peroxiredoxin-5 [PRDX5]) and outside (e.g., nuclear factor kappa B subunit 1 [NFKB1] and subunit RELA proto-oncogene [RELA], phosphatase and tensin homolog [PTEN], forkhead box O3 [FOXO3], and peroxin 5 [PEX5]) the peroxisomal compartment. Furthermore, we show that the extent of protein oxidation depends on the subcellular location of the target protein and is inversely correlated to catalase activity and cellular glutathione content. Finally, we demonstrate that excessive H2O2 production inside peroxisomes does not induce their selective degradation, at least not under the conditions examined. INNOVATION This study describes for the first time a powerful model system that can be used to examine the role of peroxisome-derived H2O2 in redox-regulated (patho)physiological processes, a research area in need of further investigation and innovative approaches. CONCLUSION Our results provide unambiguous evidence that peroxisomes can serve as regulatory hubs in thiol-based signaling networks.
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Affiliation(s)
- Celien Lismont
- 1 Laboratory of Lipid Biochemistry and Protein Interactions, Department of Cellular and Molecular Medicine, KU Leuven-University of Leuven , Leuven, Belgium
| | - Marcus Nordgren
- 1 Laboratory of Lipid Biochemistry and Protein Interactions, Department of Cellular and Molecular Medicine, KU Leuven-University of Leuven , Leuven, Belgium
| | - Chantal Brees
- 1 Laboratory of Lipid Biochemistry and Protein Interactions, Department of Cellular and Molecular Medicine, KU Leuven-University of Leuven , Leuven, Belgium
| | - Bernard Knoops
- 2 Group of Animal Molecular and Cellular Biology, Institut des Sciences de la Vie (ISV), Université catholique de Louvain , Louvain-la-Neuve, Belgium
| | - Paul P Van Veldhoven
- 1 Laboratory of Lipid Biochemistry and Protein Interactions, Department of Cellular and Molecular Medicine, KU Leuven-University of Leuven , Leuven, Belgium
| | - Marc Fransen
- 1 Laboratory of Lipid Biochemistry and Protein Interactions, Department of Cellular and Molecular Medicine, KU Leuven-University of Leuven , Leuven, Belgium
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16
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Jain M, Munoz-Bodnar A, Zhang S, Gabriel DW. A Secreted 'Candidatus Liberibacter asiaticus' Peroxiredoxin Simultaneously Suppresses Both Localized and Systemic Innate Immune Responses In Planta. MOLECULAR PLANT-MICROBE INTERACTIONS : MPMI 2018; 31:1312-1322. [PMID: 29953333 DOI: 10.1094/mpmi-03-18-0068-r] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/08/2023]
Abstract
The oxidative (H2O2) burst is a seminal feature of the basal plant defense response to attempted pathogen invasions. In 'Candidatus Liberibacter asiaticus' UF506, expression of the SC2 prophage-encoded secreted peroxidase (F489_gp15) increases bacterial fitness and delays symptom progression in citrus. Two chromosomal 1-Cys peroxiredoxin genes, CLIBASIA_RS00940 (Lasprx5) and CLIBASIA_RS00445 (Lasbcp), are conserved among all sequenced 'Ca. L. asiaticus' strains, including those lacking prophages. Both LasBCP and LasdPrx5 have only a single conserved peroxidatic Cys (CP/SH) and lack the resolving Cys (CR/SH). Lasprx5 appeared to be a housekeeping gene with similar moderate transcript abundance in both 'Ca. L. asiaticus'-infected psyllids and citrus. By contrast, Lasbcp was expressed only in planta, similar to the expression of the SC2 peroxidase. Since 'Ca. L. asiaticus' is uncultured, Lasbcp and Lasprx5 were functionally validated in a cultured surrogate species, Liberibacter crescens, and both genes significantly increased oxidative stress tolerance and cell viability in culture. LasBCP was nonclassically secreted and, in L. crescens, conferred 214-fold more resistance to tert-butyl hydroperoxide (tBOOH) than wild type. Transient overexpression of Lasbcp in tobacco suppressed H2O2-mediated transcriptional activation of RbohB, the key gatekeeper of the systemic plant defense signaling cascade. Lasbcp expression did not interfere with the perception of 'Ca. L. asiaticus' flagellin (flg22Las) but interrupted the downstream activation of RbohB and stereotypical deposition of callose in tobacco. Critically, LasBCP also protected against tBOOH-induced peroxidative degradation of lipid membranes in planta, preventing subsequent accumulation of antimicrobial oxylipins that can also trigger the localized hypersensitive cell death response.
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Affiliation(s)
- Mukesh Jain
- Department of Plant Pathology, University of Florida, Gainesville, FL 32611, U.S.A
| | | | - Shujian Zhang
- Department of Plant Pathology, University of Florida, Gainesville, FL 32611, U.S.A
| | - Dean W Gabriel
- Department of Plant Pathology, University of Florida, Gainesville, FL 32611, U.S.A
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17
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Shen GN, Liu L, Feng L, Jin Y, Jin MH, Han YH, Jin CH, Jin YZ, Lee DS, Kwon TH, Cui YD, Sun HN. Knockdown of peroxiredoxin V increases glutamate‑induced apoptosis in HT22 hippocampal neuron cells. Mol Med Rep 2018; 17:7827-7834. [PMID: 29620243 DOI: 10.3892/mmr.2018.8826] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2017] [Accepted: 02/15/2018] [Indexed: 11/06/2022] Open
Abstract
High concentrations of glutamate may mediate neuronal cell apoptosis by increasing intracellular reactive oxygen species (ROS) levels. Peroxiredoxin V (Prx V), a member of the Prx family, serves crucial roles in protecting cells from oxidative stress. The present study investigated the regulatory effect of Prx V on glutamate‑induced effects on viability and apoptosis in HT22 cells. Western blotting was used for protein expression analysis and Annexin V/PI staining and flow cytometry for determination of apoptosis. The results demonstrated that glutamate may ROS‑dependently increase HT22 cell apoptosis and upregulate Prx V protein levels. Furthermore, knockdown of Prx V protein expression with a lentivirus significantly enhanced HT22 cell apoptosis mediated by glutamate, which was reversed by inhibition of ROS with N‑acetyl‑L‑cysteine. Inhibiting the extracellular signal‑regulated kinase (ERK) signaling pathway with PD98059, a specific inhibitor for ERK phosphorylation, markedly decreased glutamate‑induced HT22 cell apoptosis in Prx V knockdown cells, indicating the potential involvement of ERK signaling in glutamate‑induced HT22 cell apoptosis. In addition, an increase in nuclear apoptosis‑inducing factor was observed in Prx V knockdown HT22 cells following glutamate treatment, compared with mock cells, whereas no differences in B‑cell lymphoma‑2 and cleaved‑caspase‑3 protein expression levels were observed between mock and Prx V knockdown cells. The results of the present study indicated that Prx V may have potential as a therapeutic molecular target for glutamate‑induced neuronal cell death and provide novel insight into the role of Prx V in oxidative‑stress induced neuronal cell death.
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Affiliation(s)
- Gui-Nan Shen
- Department of Disease Model Animal Research Center, College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, P.R. China
| | - Lei Liu
- Department of Disease Model Animal Research Center, College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, P.R. China
| | - Li Feng
- Pharmaron Beijing Co., Ltd., Beijing 100176, P.R. China
| | - Yu Jin
- Laboratory of Anatomy and Histology, Yanbian University Health Science Center, Yanji, Jilin 133000, P.R. China
| | - Mei-Hua Jin
- Department of Disease Model Animal Research Center, College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, P.R. China
| | - Ying-Hao Han
- Department of Disease Model Animal Research Center, College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, P.R. China
| | - Cheng-Hao Jin
- Department of Disease Model Animal Research Center, College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, P.R. China
| | - Yong-Zhe Jin
- Laboratory of Anatomy and Histology, Yanbian University Health Science Center, Yanji, Jilin 133000, P.R. China
| | - Dong-Soek Lee
- Laboratory of Molecular Neurobiology, School of Life Sciences, KNU Creative Bio Research Group (BK21 Plus Project), Kyungpook National University, Daegu 41566, Republic of Korea
| | - Tae Ho Kwon
- Laboratory of Animal Genetic Engineering and Stem Cell Biology, Subtropical/Tropical Organism Gene Bank, Jeju National University, Jeju, Jeju 63243, Republic of Korea
| | - Yu-Dong Cui
- Department of Disease Model Animal Research Center, College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, P.R. China
| | - Hu-Nan Sun
- Department of Disease Model Animal Research Center, College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, P.R. China
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18
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Knoops B, Becker S, Poncin MA, Glibert J, Derclaye S, Clippe A, Alsteens D. Specific Interactions Measured by AFM on Living Cells between Peroxiredoxin-5 and TLR4: Relevance for Mechanisms of Innate Immunity. Cell Chem Biol 2018; 25:550-559.e3. [PMID: 29551349 DOI: 10.1016/j.chembiol.2018.02.006] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2017] [Revised: 01/08/2018] [Accepted: 02/07/2018] [Indexed: 12/14/2022]
Abstract
Inflammation is a pathophysiological response of innate immunity to infection or tissue damage. This response is among others triggered by factors released by damaged or dying cells, termed damage-associated molecular pattern (DAMP) molecules that act as danger signals. DAMPs interact with pattern recognition receptors (PRRs) to contribute to the induction of inflammation. However, how released peroxiredoxins (PRDXs) are able to activate PRRs, such as Toll-like receptors (TLRs), remains elusive. Here, we used force-distance curve-based atomic force microscopy to investigate the molecular mechanisms by which extracellular human PRDX5 can activate a proinflammatory response. Single-molecule experiments demonstrated that PRDX5 binds to purified TLR4 receptors, on macrophage-differentiated THP-1 cells, and on human TLR4-transfected CHO cells. These findings suggest that extracellular PRDX5 can specifically trigger a proinflammatory response. Moreover, our work also revealed that PRDX5 binding induces a cellular mechanoresponse. Collectively, this study provides insights into the role of extracellular PRDX5 in innate immunity.
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Affiliation(s)
- Bernard Knoops
- Université catholique de Louvain, Institut des Sciences de la Vie (ISV), 1348 Louvain-la-Neuve, Belgium.
| | - Sarah Becker
- Université catholique de Louvain, Institut des Sciences de la Vie (ISV), 1348 Louvain-la-Neuve, Belgium
| | - Mégane Anne Poncin
- Université catholique de Louvain, Institut des Sciences de la Vie (ISV), 1348 Louvain-la-Neuve, Belgium
| | - Julien Glibert
- Université catholique de Louvain, Institut des Sciences de la Vie (ISV), 1348 Louvain-la-Neuve, Belgium
| | - Sylvie Derclaye
- Université catholique de Louvain, Institut des Sciences de la Vie (ISV), 1348 Louvain-la-Neuve, Belgium
| | - André Clippe
- Université catholique de Louvain, Institut des Sciences de la Vie (ISV), 1348 Louvain-la-Neuve, Belgium
| | - David Alsteens
- Université catholique de Louvain, Institut des Sciences de la Vie (ISV), 1348 Louvain-la-Neuve, Belgium.
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19
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Ranawat P, Rawat S. Stress response physiology of thermophiles. Arch Microbiol 2017; 199:391-414. [DOI: 10.1007/s00203-016-1331-4] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2016] [Revised: 12/07/2016] [Accepted: 12/16/2016] [Indexed: 10/20/2022]
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20
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Cha MK, Bae YJ, Kim KJ, Park BJ, Kim IH. Characterization of two alkyl hydroperoxide reductase C homologs alkyl hydroperoxide reductase C_H1 and alkyl hydroperoxide reductase C_H2 in Bacillus subtilis. World J Biol Chem 2015; 6:249-64. [PMID: 26322180 PMCID: PMC4549766 DOI: 10.4331/wjbc.v6.i3.249] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/02/2015] [Revised: 05/21/2015] [Accepted: 06/09/2015] [Indexed: 02/05/2023] Open
Abstract
AIM To identify alkyl hydroperoxide reductase subunit C (AhpC) homologs in Bacillus subtilis (B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria. METHODS Two AhpC homologs (AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues (C37S, C47S, C166S, C37/47S, C37/166S, C47/166S, and C37/47/166S for AhpC_H1; C52S, C169S, and C52/169S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahpC genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined. RESULTS Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis. CONCLUSION AhpC_H1, a novel atypical 2-Cys AhpC, is functionally distinct from AhpC_H2, a typical 2-Cys AhpC.
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21
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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.
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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.
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22
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Perkins A, Poole L, Karplus PA. Tuning of peroxiredoxin catalysis for various physiological roles. Biochemistry 2014; 53:7693-705. [PMID: 25403613 PMCID: PMC4270387 DOI: 10.1021/bi5013222] [Citation(s) in RCA: 96] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2014] [Revised: 11/12/2014] [Indexed: 12/15/2022]
Abstract
Peroxiredoxins (Prxs) make up an ancient family of enzymes that are the predominant peroxidases for nearly all organisms and play essential roles in reducing hydrogen peroxide, organic hydroperoxides, and peroxynitrite. Even between distantly related organisms, the core protein fold and key catalytic residues related to its cysteine-based catalytic mechanism have been retained. Given that these enzymes appeared early in biology, Prxs have experienced more than 1 billion years of optimization for specific ecological niches. Although their basic enzymatic function remains the same, Prxs have diversified and are involved in roles such as protecting DNA against mutation, defending pathogens against host immune responses, suppressing tumor formation, and--for eukaryotes--helping regulate peroxide signaling via hyperoxidation of their catalytic Cys residues. Here, we review the current understanding of the physiological roles of Prxs by analyzing knockout and knockdown studies from ∼25 different species. We also review what is known about the structural basis for the sensitivity of some eukaryotic Prxs to inactivation by hyperoxidation. In considering the physiological relevance of hyperoxidation, we explore the distribution across species of sulfiredoxin (Srx), the enzyme responsible for rescuing hyperoxidized Prxs. We unexpectedly find that among eukaryotes appearing to have a "sensitive" Prx isoform, some do not contain Srx. Also, as Prxs are suggested to be promising targets for drug design, we discuss the rationale behind recently proposed strategies for their selective inhibition.
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Affiliation(s)
- Arden Perkins
- Department
of Biochemistry and Biophysics, Oregon State
University, Corvallis, Oregon 97331, United
States
| | - Leslie
B. Poole
- Department
of Biochemistry, Wake Forest School of Medicine, Winston-Salem, North Carolina 27157, United States
| | - P. Andrew Karplus
- Department
of Biochemistry and Biophysics, Oregon State
University, Corvallis, Oregon 97331, United
States
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23
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Portillo-Ledesma S, Sardi F, Manta B, Tourn MV, Clippe A, Knoops B, Alvarez B, Coitiño EL, Ferrer-Sueta G. Deconstructing the Catalytic Efficiency of Peroxiredoxin-5 Peroxidatic Cysteine. Biochemistry 2014; 53:6113-25. [DOI: 10.1021/bi500389m] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
| | - Florencia Sardi
- Laboratory
Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo, Uruguay
| | - Bruno Manta
- Laboratory
Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo, Uruguay
| | | | - André Clippe
- Laboratory
of Cell Biology, Institut des Sciences de la Vie, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium
| | - Bernard Knoops
- Laboratory
of Cell Biology, Institut des Sciences de la Vie, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium
| | - Beatriz Alvarez
- Center for
Free Radical and Biomedical Research, Universidad de la República, Montevideo, Uruguay
| | | | - Gerardo Ferrer-Sueta
- Center for
Free Radical and Biomedical Research, Universidad de la República, Montevideo, Uruguay
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24
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25
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Huang Y, Nam K, Westlund PO. The water R1(ω) NMRD profiles of a hydrated protein from molecular dynamics simulation. Phys Chem Chem Phys 2013; 15:14089-97. [PMID: 23868443 DOI: 10.1039/c3cp51147b] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The hydration of a protein, Peroxiredoxin 5, is obtained from a molecular dynamics simulation and compared with the picture of hydration which is obtained by analysing the water proton R1 NMRD profiles using a generally accepted relaxation model [K. Venu, V. P. Denisov and B. Halle, J. Am. Chem. Soc., 1997, 119, 3122]. The discrepancy between the hydration pictures derived from the water R1(ω0)-NMRD profiles and MD is relevant in a discussion of the factors behind the stretched NMRD profile, the distribution of orientational order parameters and residence times of buried water used in the NMRD model.
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Affiliation(s)
- Yang Huang
- Department of Chemistry, Umeå University, Umeå, Sweden
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26
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Chae HZ, Oubrahim H, Park JW, Rhee SG, Chock PB. Protein glutathionylation in the regulation of peroxiredoxins: a family of thiol-specific peroxidases that function as antioxidants, molecular chaperones, and signal modulators. Antioxid Redox Signal 2012; 16:506-23. [PMID: 22114845 PMCID: PMC3270059 DOI: 10.1089/ars.2011.4260] [Citation(s) in RCA: 85] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
SIGNIFICANCE Reversible protein glutathionylation plays an important role in cellular regulation, signaling transduction, and antioxidant defense. This redox-sensitive mechanism is involved in regulating the functions of peroxiredoxins (Prxs), a family of ubiquitously expressed thiol-specific peroxidase enzymes. Glutathionylation of certain Prxs at their active-site cysteines not only provides reducing equivalents to support their peroxidase activity but also protects Prxs from irreversible hyperoxidation. Typical 2-Cys Prx also functions as a molecular chaperone when it exists as a decamer and/or higher molecular weight complexes. The hyperoxidized sulfinic derivative of 2-Cys Prx is reactivated by sulfiredoxin (Srx). In this review, the roles of glutathionylation in the regulation of Prxs are discussed with respect to their molecular structure and functions as antioxidants, molecular chaperones, and signal modulators. RECENT ADVANCES Recent findings reveal that glutathionylation regulates the quaternary structure of Prx. Glutathionylation of Prx I at Cys(83) converts the decameric Prx to its dimers with the loss of chaperone activity. The findings that dimer/oligomer structure specific Prx I binding proteins, e.g., phosphatase and tensin homolog (PTEN) and mammalian Ste20-like kinase-1 (MST1), regulate cell cycle and apoptosis, respectively, suggest a possible link between glutathionylation and those signaling pathways. CRITICAL ISSUES Knowing how glutathionylation affects the interaction between Prx I and its nearly 20 known interacting proteins, e.g., PTEN and MST1 kinase, would reveal new insights on the physiological functions of Prx. FUTURE DIRECTIONS In vitro studies reveal that Prx oligomerization is linked to its functional changes. However, in vivo dynamics, including the effect by glutathionylation, and its physiological significance remain to be investigated.
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Affiliation(s)
- Ho Zoon Chae
- School of Biological Sciences and Technology, Chonnam National University, Gwangju, Korea
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27
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Hall A, Nelson K, Poole LB, Karplus PA. Structure-based insights into the catalytic power and conformational dexterity of peroxiredoxins. Antioxid Redox Signal 2011; 15:795-815. [PMID: 20969484 PMCID: PMC3125576 DOI: 10.1089/ars.2010.3624] [Citation(s) in RCA: 254] [Impact Index Per Article: 18.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/31/2010] [Revised: 10/01/2010] [Accepted: 10/24/2010] [Indexed: 12/25/2022]
Abstract
Peroxiredoxins (Prxs), some of nature's dominant peroxidases, use a conserved Cys residue to reduce peroxides. They are highly expressed in organisms from all kingdoms, and in eukaryotes they participate in hydrogen peroxide signaling. Seventy-two Prx structures have been determined that cover much of the diversity of the family. We review here the current knowledge and show that Prxs can be effectively classified by a structural/evolutionary organization into six subfamilies followed by specification of a 1-Cys or 2-Cys mechanism, and for 2-Cys Prxs, the structural location of the resolving Cys. We visualize the varied catalytic structural transitions and highlight how they differ depending on the location of the resolving Cys. We also review new insights into the question of how Prxs are such effective catalysts: the enzyme activates not only the conserved Cys thiolate but also the peroxide substrate. Moreover, the hydrogen-bonding network created by the four residues conserved in all Prx active sites stabilizes the transition state of the peroxidatic S(N)2 displacement reaction. Strict conservation of the peroxidatic active site along with the variation in structural transitions provides a fascinating picture of how the diverse Prxs function to break down peroxide substrates rapidly.
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Affiliation(s)
- Andrea Hall
- Department of Biochemistry & Biophysics, Oregon State University, Corvallis, Oregon
| | - Kimberly Nelson
- Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina
| | - Leslie B. Poole
- Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina
| | - P. Andrew Karplus
- Department of Biochemistry & Biophysics, Oregon State University, Corvallis, Oregon
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28
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Knoops B, Goemaere J, Van der Eecken V, Declercq JP. Peroxiredoxin 5: structure, mechanism, and function of the mammalian atypical 2-Cys peroxiredoxin. Antioxid Redox Signal 2011; 15:817-29. [PMID: 20977338 DOI: 10.1089/ars.2010.3584] [Citation(s) in RCA: 171] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Peroxiredoxin 5 (PRDX5) was the last member to be identified among the six mammalian peroxiredoxins. It is also the unique atypical 2-Cys peroxiredoxin in mammals. Like the other five members, PRDX5 is widely expressed in tissues but differs by its surprisingly large subcellular distribution. In human cells, it has been shown that PRDX5 can be addressed to mitochondria, peroxisomes, the cytosol, and the nucleus. PRDX5 is a peroxidase that can use cytosolic or mitochondrial thioredoxins to reduce alkyl hydroperoxides or peroxynitrite with high rate constants in the 10(6) to 10(7) M(-1)s(-1) range, whereas its reaction with hydrogen peroxide is more modest, in the 10(5) M(-1)s(-1) range. PRDX5 crystal structures confirmed the proposed enzymatic mechanisms based on biochemical data but revealed also some specific unexpected structural features. So far, PRDX5 has been viewed mainly as a cytoprotective antioxidant enzyme acting against endogenous or exogenous peroxide attacks rather than as a redox sensor. Accordingly, overexpression of the enzyme in different subcellular compartments protects cells against death caused by nitro-oxidative stresses, whereas gene silencing makes them more vulnerable. Thus, more than 10 years after its molecular cloning, mammalian PRDX5 appears to be a unique peroxiredoxin exhibiting specific functional and structural features.
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Affiliation(s)
- Bernard Knoops
- Institut des Sciences de Vie, Université catholique de Louvain, Louvain-la-Neuve, Belgium.
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29
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Hall A, Parsonage D, Poole LB, Karplus PA. Structural evidence that peroxiredoxin catalytic power is based on transition-state stabilization. J Mol Biol 2010; 402:194-209. [PMID: 20643143 PMCID: PMC2941395 DOI: 10.1016/j.jmb.2010.07.022] [Citation(s) in RCA: 138] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2010] [Revised: 07/08/2010] [Accepted: 07/08/2010] [Indexed: 11/18/2022]
Abstract
Peroxiredoxins (Prxs) are important peroxidases associated with both antioxidant protection and redox signaling. They use a conserved Cys residue to reduce peroxide substrates. The Prxs have a remarkably high catalytic efficiency that makes them a dominant player in cell-wide peroxide reduction, but the origins of their high activity have been mysterious. We present here a novel structure of human PrxV at 1.45 A resolution that has a dithiothreitol bound in the active site with its diol moiety mimicking the two oxygens of a peroxide substrate. This suggests diols and similar di-oxygen compounds as a novel class of competitive inhibitors for the Prxs. Common features of this and other structures containing peroxide, peroxide-mimicking ligands, or peroxide-mimicking water molecules reveal hydrogen bonding and steric factors that promote its high reactivity by creating an oxygen track along which the peroxide oxygens move as the reaction proceeds. Key insights include how the active-site microenvironment activates both the peroxidatic cysteine side chain and the peroxide substrate and how it is exquisitely well suited to stabilize the transition state of the in-line S(N)2 substitution reaction that is peroxidation.
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Affiliation(s)
- Andrea Hall
- Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331
| | - Derek Parsonage
- Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157
| | - Leslie B. Poole
- Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157
| | - P. Andrew Karplus
- Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331
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30
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Abstract
Proteins with oxidizable thiols are essential to many functions of cell nuclei, including transcription, chromatin stability, nuclear protein import and export, and DNA replication and repair. Control of the nuclear thiol-disulfide redox states involves both the elimination of oxidants to prevent oxidation and the reduction of oxidized thiols to restore function. These processes depend on the common thiol reductants, glutathione (GSH) and thioredoxin-1 (Trx1). Recent evidence shows that these systems are controlled independent of the cytoplasmic counterparts. In addition, the GSH and Trx1 couples are not in redox equilibrium, indicating that these reductants have nonredundant functions in their support of proteins involved in transcriptional regulation, nuclear protein trafficking, and DNA repair. Specific isoforms of glutathione peroxidases, glutathione S-transferases, and peroxiredoxins are enriched in nuclei, further supporting the interpretation that functions of the thiol-dependent systems in nuclei are at least quantitatively distinct, and probably also qualitatively distinct, from similar processes in the cytoplasm. Elucidation of the distinct nuclear functions and regulation of the thiol redox pathways in nuclei can be expected to improve understanding of nuclear processes and also to provide the basis for novel approaches to treat aging and disease processes associated with oxidative stress in the nuclei.
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Affiliation(s)
- Young-Mi Go
- Department of Medicine, Emory University, Atlanta, GA, USA
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31
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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.
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Affiliation(s)
- T M Shuvaeva
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
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32
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D'Ambrosio K, Limauro D, Pedone E, Galdi I, Pedone C, Bartolucci S, De Simone G. Insights into the catalytic mechanism of the Bcp family: functional and structural analysis of Bcp1 from Sulfolobus solfataricus. Proteins 2009; 76:995-1006. [PMID: 19338062 DOI: 10.1002/prot.22408] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Bcps constitute a group of antioxidant enzymes, belonging to the Prx family, that are widely distributed in bacteria, plants, and fungi. These proteins can contain two conserved cysteines within the CXXXXC motif. Recent studies demonstrated that though the role of the first cysteine is well defined, being the catalytic peroxidatic cysteine in all the members of this protein family, data on the function of the second cysteine are controversial and require further investigation. In this article, we report on the functional and structural characterization of Bcp1, an archaeal Bcp isolated from Sulfolobus solfataricus, which presents two conserved cysteine residues at positions 45 and 50. Functional studies revealed that this enzyme performs the catalytic reaction using an atypical 2-Cys mechanism, where Cys45 is the peroxidatic and Cys50 is the resolving cysteine. The X-ray structure of the double mutant C45S/C50S, representative of the fully reduced enzyme state, was determined at a resolution of 2.15 A, showing a Trx fold similar to that of other Prxs. Superposition with a structural homologue in the oxidized state provided, for the first time, a detailed description of the structural rearrangement necessary for a member of the Bcp family to perform the catalytic reaction. From this structural analysis, it emerges that a significant conformational change from a fully folded, to a locally unfolded form is required to form the intramolecular disulfide bond upon oxidation, according to the proposed reaction mechanism. Two residues, namely Arg53 and Asp54, which could play a role in this rearrangement, were also identified.
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Affiliation(s)
- Katia D'Ambrosio
- Istituto di Biostrutture e Bioimmagini-CNR, via Mezzocannone 16, 80134 Naples, Italy
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33
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Hall A, Sankaran B, Poole LB, Karplus PA. Structural changes common to catalysis in the Tpx peroxiredoxin subfamily. J Mol Biol 2009; 393:867-81. [PMID: 19699750 PMCID: PMC3664093 DOI: 10.1016/j.jmb.2009.08.040] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2009] [Revised: 08/14/2009] [Accepted: 08/15/2009] [Indexed: 11/28/2022]
Abstract
Thiol peroxidases (Tpxs) are dimeric 2-Cys peroxiredoxins from bacteria that preferentially reduce alkyl hydroperoxides. Catalysis requires two conserved residues, the peroxidatic cysteine and the resolving cysteine, which are located in helix alpha(2) and helix alpha(3), respectively. The partial unraveling of helices alpha(2) and alpha(3) during catalysis allows for the formation of an intramolecular disulfide between these two residues. Here, we present three structures of Escherichia coli Tpx representing the fully folded (peroxide binding site intact), locally unfolded (disulfide bond), and partially locally unfolded (transitional state) conformations. We also compare known Tpx crystal structures and analyze the sequence-conservation patterns among nearly 300 Tpx sequences. Twelve fully conserved Tpx-specific residues cluster at the active site and dimer interface, and an additional 37 highly conserved residues are mostly located in a cradle providing the environment for helix alpha(2). Using the structures determined here as representative fully folded, transitional, and locally unfolded Tpx conformations, we describe in detail the structural changes associated with catalysis in the Tpx subfamily. Key insights include the description of a conserved hydrophobic collar around the active site, a set of conserved packing interactions between helices alpha(2) and alpha(3) that allow the local unfolding of alpha(2) to trigger the partial unfolding of alpha(3), a conserved dimer interface that anchors the ends of helices alpha(2) and alpha(3) to stabilize the active site during structural transitions, and a conserved set of residues constituting a cradle that stabilizes the two discrete conformations of helix alpha(2) involved in catalysis. The involvement of the dimer interface in stabilizing active-site folding and in forming the hydrophobic collar implies that Tpx is an obligate homodimer and explains the high conservation of interface residues.
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Affiliation(s)
- Andrea Hall
- Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331
| | - Banumathi Sankaran
- Advance Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA 94720
| | - Leslie B. Poole
- Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157
| | - P. Andrew Karplus
- Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331
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34
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Abbas K, Breton J, Picot CR, Quesniaux V, Bouton C, Drapier JC. Signaling events leading to peroxiredoxin 5 up-regulation in immunostimulated macrophages. Free Radic Biol Med 2009; 47:794-802. [PMID: 19540914 DOI: 10.1016/j.freeradbiomed.2009.06.018] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/23/2009] [Revised: 06/05/2009] [Accepted: 06/15/2009] [Indexed: 12/20/2022]
Abstract
Peroxiredoxins (PRXs) are thiol peroxidases associated with many cellular functions including proliferation, cell cycle, apoptosis, and differentiation. There is also increasing evidence that these ubiquitous antioxidant enzymes control H(2)O(2) signaling in eukaryotes. Here, we provide evidence that the LPS/TLR4 and the Th1 cytokine IFN-gamma pathways induce expression of PRX5, a potent peroxide and peroxynitrite reductase, in primary macrophages. Furthermore, deletion of TRIF, MyD88, or type I IFN receptor revealed that the LPS/TLR4-dependent increase in PRX5 expression is mediated by a TRIF-dependent/IFN-beta-independent pathway. IFN-gamma-dependent induction of the PRX5 gene was markedly reduced in MyD88(-/-) and TNF(-/-) macrophages. Moreover, addition of exogenous TNF allowed the recovery of full PRX5 expression in both MyD88(-/-) and TNF(-/-) cells stimulated with IFN-gamma, suggesting that basal TNF produced in an MyD88-dependent manner contributes to PRX5 induction. Downstream of the TLR pathways, we have explored the role of MAPK activation and found that p38 and JNK mainly contribute to PRX5 up-regulation in immunostimulated macrophages. Expression of PRX5 is thus responsive to innate immunity signals, and we propose that PRX5 is an additional host defense weapon of activated macrophages.
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Affiliation(s)
- Kahina Abbas
- Institut de Chimie des Substances Naturelles, CNRS UPR2301, 91190 Gif-sur-Yvette, France
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35
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Zhang Q, Pi J, Woods CG, Andersen ME. A systems biology perspective on Nrf2-mediated antioxidant response. Toxicol Appl Pharmacol 2009; 244:84-97. [PMID: 19716833 DOI: 10.1016/j.taap.2009.08.018] [Citation(s) in RCA: 157] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2009] [Revised: 08/07/2009] [Accepted: 08/18/2009] [Indexed: 12/13/2022]
Abstract
Cells in vivo are constantly exposed to reactive oxygen species (ROS) generated endogenously and exogenously. To defend against the deleterious consequences of ROS, cells contain multiple antioxidant enzymes expressed in various cellular compartments to scavenge these toxic species. Under oxidative stresses, these antioxidant enzymes are upregulated to restore redox homeostasis. Such an adaptive response results from the activation of a redox-sensitive gene regulatory network mediated by nuclear factor E2-related factor 2. To more completely understand how the redox control system is designed by nature to meet homeostatic goals, we have examined the network from a systems perspective using engineering approaches. As with man-made control devices, the redox control system can be decomposed into distinct functional modules, including transducer, controller, actuator, and plant. Cells achieve specific performance objectives by utilizing nested feedback loops, feedforward control, and ultrasensitive signaling motifs, etc. Given that endogenously generated ROS are also used as signaling molecules, our analysis suggests a novel mode of action to explain oxidative stress-induced pathological conditions and diseases. Specifically, by adaptively upregulating antioxidant enzymes, oxidative stress may inadvertently attenuate ROS signals that mediate physiological processes, resulting in aberrations of cellular functions and adverse consequences. Lastly, by simultaneously considering the two competing cellular tasks-adaptive antioxidant defense and ROS signaling-we re-examine the premise that dietary antioxidant supplements is generally beneficial to human health. Our analysis highlights some possible adverse effects of these widely consumed antioxidants.
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Affiliation(s)
- Qiang Zhang
- Division of Computational Biology, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709, USA.
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36
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Liao SJ, Yang CY, Chin KH, Wang AHJ, Chou SH. Insights into the alkyl peroxide reduction pathway of Xanthomonas campestris bacterioferritin comigratory protein from the trapped intermediate-ligand complex structures. J Mol Biol 2009; 390:951-66. [PMID: 19477183 DOI: 10.1016/j.jmb.2009.05.030] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2009] [Revised: 05/14/2009] [Accepted: 05/16/2009] [Indexed: 11/18/2022]
Abstract
Considerable insights into the oxidoreduction activity of the Xanthomonas campestris bacterioferritin comigratory protein (XcBCP) have been obtained from trapped intermediate/ligand complex structures determined by X-ray crystallography. Multiple sequence alignment and enzyme assay indicate that XcBCP belongs to a subfamily of atypical 2-Cys peroxiredoxins (Prxs), containing a strictly conserved peroxidatic cysteine (C(P)48) and an unconserved resolving cysteine (C(R)84). Crystals at different states, i.e. Free_SH state, Intra_SS state, and Inter_SS state, were obtained by screening the XcBCP proteins from a double C48S/C84S mutant, a wild type, and a C48A mutant, respectively. A formate or an alkyl analog with two water molecules that mimic an alkyl peroxide substrate was found close to the active site of the Free_SH or Inter_SS state, respectively. Their global structures were found to contain a novel substrate-binding pocket capable of accommodating an alkyl chain of no less than 16 carbons. In addition, in the Intra_SS or Inter_SS state, substantial local unfolding or complete unfolding of the C(R)-helix was detected, with the C(P)-helix remaining essentially unchanged. This is in contrast to the earlier observation that the C(P)-helix exhibits local unfolding during disulfide bond formation in typical 2-Cys Prxs. These rich experimental data have enabled us to propose a pathway by which XcBCP carries out its oxidoreduction activity through the alternate opening and closing of the substrate entry channel and the disulfide-bond pocket.
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Affiliation(s)
- Shu-Ju Liao
- Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, ROC
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37
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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.
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38
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Meir A, Helppolainen SH, Podoly E, Nordlund HR, Hytönen VP, Määttä JA, Wilchek M, Bayer EA, Kulomaa MS, Livnah O. Crystal Structure of Rhizavidin: Insights into the Enigmatic High-Affinity Interaction of an Innate Biotin-Binding Protein Dimer. J Mol Biol 2009; 386:379-90. [DOI: 10.1016/j.jmb.2008.11.061] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2008] [Revised: 11/25/2008] [Accepted: 11/26/2008] [Indexed: 10/21/2022]
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39
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Melchers J, Diechtierow M, Fehér K, Sinning I, Tews I, Krauth-Siegel RL, Muhle-Goll C. Structural basis for a distinct catalytic mechanism in Trypanosoma brucei tryparedoxin peroxidase. J Biol Chem 2008; 283:30401-11. [PMID: 18684708 PMCID: PMC2662087 DOI: 10.1074/jbc.m803563200] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2008] [Revised: 07/24/2008] [Indexed: 12/22/2022] Open
Abstract
Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three cysteine homologues (Px I-III) of classical selenocysteine-containing glutathione peroxidases. The enzymes obtain their reducing equivalents from the unique trypanothione (bis(glutathionyl)spermidine)/tryparedoxin system. During catalysis, these tryparedoxin peroxidases cycle between an oxidized form with an intramolecular disulfide bond between Cys(47) and Cys(95) and the reduced peroxidase with both residues in the thiol state. Here we report on the three-dimensional structures of oxidized T. brucei Px III at 1.4A resolution obtained by x-ray crystallography and of both the oxidized and the reduced protein determined by NMR spectroscopy. Px III is a monomeric protein unlike the homologous poplar thioredoxin peroxidase (TxP). The structures of oxidized and reduced Px III are essentially identical in contrast to what was recently found for TxP. In Px III, Cys(47), Gln(82), and Trp(137) do not form the catalytic triad observed in the selenoenzymes, and related proteins and the latter two residues are unaffected by the redox state of the protein. The mutational analysis of three conserved lysine residues in the vicinity of the catalytic cysteines revealed that exchange of Lys(107) against glutamate abrogates the reduction of hydrogen peroxide, whereas Lys(97) and Lys(99) play a crucial role in the interaction with tryparedoxin.
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Affiliation(s)
- Johannes Melchers
- Department of Structure and Biocomputing, EMBL, 69117 Heidelberg, Germany
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40
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Abstract
Thiol/selenol peroxidases are ubiquitous nonheme peroxidases. They are divided into two major subfamilies: peroxiredoxins (PRXs) and glutathione peroxidases (GPXs). PRXs are present in diverse subcellular compartments and divided into four types: 2-cys PRX, 1-cys PRX, PRX-Q, and type II PRX (PRXII). In mammals, most GPXs are selenoenzymes containing a highly reactive selenocysteine in their active site while yeast and land plants are devoid of selenoproteins but contain nonselenium GPXs. The presence of a chloroplastic 2-cys PRX, a nonselenium GPX, and two selenium-dependent GPXs has been reported in the unicellular green alga Chlamydomonas reinhardtii. The availability of the Chlamydomonas genome sequence offers the opportunity to complete our knowledge on thiol/selenol peroxidases in this organism. In this article, Chlamydomonas PRX and GPX families are presented and compared to their counterparts in Arabidopsis, human, yeast, and Synechocystis sp. A summary of the current knowledge on each family of peroxidases, especially in photosynthetic organisms, phylogenetic analyses, and investigations of the putative subcellular localization of each protein and its relative expression level, on the basis of EST data, are presented. We show that Chlamydomonas PRX and GPX families share some similarities with other photosynthetic organisms but also with human cells. The data are discussed in view of recent results suggesting that these enzymes are important scavengers of reactive oxygen species (ROS) and reactive nitrogen species (RNS) but also play a role in ROS signaling.
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41
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Schröder E, Brennan JP, Eaton P. Cardiac peroxiredoxins undergo complex modifications during cardiac oxidant stress. Am J Physiol Heart Circ Physiol 2008; 295:H425-33. [PMID: 18502910 PMCID: PMC2494773 DOI: 10.1152/ajpheart.00017.2008] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Peroxiredoxins (Prdxs), a family of antioxidant and redox-signaling proteins, are plentiful within the heart; however, their cardiac functions are poorly understood. These studies were designed to characterize the complex changes in Prdxs induced by oxidant stress in rat myocardium. Hydrogen peroxide, a Prdx substrate, was used as the model oxidant pertinent to redox signaling during health and to injury at higher concentrations. Rat hearts were aerobically perfused with a broad concentration range of hydrogen peroxide by the Langendorff method, homogenized, and analyzed by immunoblotting. Heart extracts were also analyzed by size-exclusion chromatography under nondenaturing conditions. Hydrogen peroxide-induced changes in disulfide bond formation, nonreversible oxidation of cysteine (hyperoxidation), and subcellular localization were determined. Hydrogen peroxide induced an array of changes in the myocardium, including formation of disulfide bonds that were intermolecular for Prdx1, Prdx2, and Prdx3 but intramolecular within Prdx5. For Prdx1, Prdx2, and Prdx5, disulfide bond formation can be approximated to an EC50 of 10–100, 1–10, and 100–1,000 μM peroxide, respectively. Hydrogen peroxide induced hyperoxidation, not just within monomeric Prdx (by SDS-PAGE), but also within Prdx disulfide dimers, and reflects a flexibility within the dimeric unit. Prdx oxidation was also associated with movement from the cytosolic to the membrane and myofilament-enriched fractions. In summary, Prdxs undergo a complex series of redox-dependent structural changes in the heart in response to oxidant challenge with its substrate hydrogen peroxide.
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Affiliation(s)
- Ewald Schröder
- Dept. of Cardiology, St. Thomas' Hospital, King's College London, London SE1 7EH, UK
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42
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Smeets A, Marchand C, Linard D, Knoops B, Declercq JP. The crystal structures of oxidized forms of human peroxiredoxin 5 with an intramolecular disulfide bond confirm the proposed enzymatic mechanism for atypical 2-Cys peroxiredoxins. Arch Biochem Biophys 2008; 477:98-104. [PMID: 18489898 DOI: 10.1016/j.abb.2008.04.036] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2008] [Revised: 04/21/2008] [Accepted: 04/22/2008] [Indexed: 10/22/2022]
Abstract
Peroxiredoxin 5 (PRDX5) belongs to the PRDX superfamily of thiol-dependent peroxidases able to reduce hydrogen peroxide, alkyl hydroperoxides and peroxynitrite. PRDX5 is classified in the atypical 2-Cys subfamily of PRDXs. In this subfamily, the oxidized form of the enzyme is characterized by the presence of an intramolecular disulfide bridge between the peroxidatic and the resolving cysteine residues. We report here three crystal forms in which this intramolecular disulfide bond is indeed observed. The structures are characterized by the expected local unfolding of the peroxidatic loop, but also by the unfolding of the resolving loop. A new type of interface between PRDX molecules is described. The three crystal forms were not oxidized in the same way and the influence of the oxidizing conditions is discussed.
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Affiliation(s)
- Aude Smeets
- Unit of Structural Chemistry (CSTR), Université catholique de Louvain, 1, place Louis Pasteur, B-1348 Louvain-la-Neuve, Belgium
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43
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Smeets A, Loumaye E, Clippe A, Rees JF, Knoops B, Declercq JP. The crystal structure of the C45S mutant of annelid Arenicola marina peroxiredoxin 6 supports its assignment to the mechanistically typical 2-Cys subfamily without any formation of toroid-shaped decamers. Protein Sci 2008; 17:700-10. [PMID: 18359859 DOI: 10.1110/ps.073399308] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
Abstract
The peroxiredoxins (PRDXs) define a superfamily of thiol-dependent peroxidases able to reduce hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. Besides their cytoprotective antioxidant function, PRDXs have been implicated in redox signaling and chaperone activity, the latter depending on the formation of decameric high-molecular-weight structures. PRDXs have been mechanistically divided into three major subfamilies, namely typical 2-Cys, atypical 2-Cys, and 1-Cys PRDXs, based on the number and position of cysteines involved in the catalysis. We report the structure of the C45S mutant of annelid worm Arenicola marina PRDX6 in three different crystal forms determined at 1.6, 2.0, and 2.4 A resolution. Although A. marina PRDX6 was cloned during the search of annelid homologs of mammalian 1-Cys PRDX6s, the crystal structures support its assignment to the mechanistically typical 2-Cys PRDX subfamily. The protein is composed of two distinct domains: a C-terminal domain and an N-terminal domain exhibiting a thioredoxin fold. The subunits are associated in dimers compatible with the formation of intersubunit disulfide bonds between the peroxidatic and the resolving cysteine residues in the wild-type enzyme. The packing of two crystal forms is very similar, with pairs of dimers associated as tetramers. The toroid-shaped decamers formed by dimer association and observed in most typical 2-Cys PRDXs is not present. Thus, A. marina PRDX6 presents structural features of typical 2-Cys PRDXs without any formation of toroid-shaped decamers, suggesting that it should function more like a cytoprotective antioxidant enzyme or a modulator of peroxide-dependent cell signaling rather than a molecular chaperone.
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Affiliation(s)
- Aude Smeets
- Unit of Structural Chemistry (CSTR), Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium
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44
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Trujillo M, Clippe A, Manta B, Ferrer-Sueta G, Smeets A, Declercq JP, Knoops B, Radi R. Pre-steady state kinetic characterization of human peroxiredoxin 5: Taking advantage of Trp84 fluorescence increase upon oxidation. Arch Biochem Biophys 2007; 467:95-106. [PMID: 17892856 DOI: 10.1016/j.abb.2007.08.008] [Citation(s) in RCA: 132] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2007] [Revised: 07/30/2007] [Accepted: 08/01/2007] [Indexed: 11/22/2022]
Abstract
Human peroxiredoxin 5 (PRDX5) catalyzes different peroxides reduction by enzymatic substitution mechanisms. Enzyme oxidation caused an increase in Trp84 fluorescence, allowing performing pre-steady state kinetic measurements. The technique was validated by comparing with data available from the literature or obtained herein by alternative approaches. PRDX5 reacted with organic hydroperoxides with rate constants in the 10(6)-10(7)M(-1)s(-1) range, similar to peroxynitrite-mediated PRDX5 oxidation, whereas its reaction with hydrogen peroxide was slower (10(5)M(-1)s(-1)). The method allowed determining the kinetics of intramolecular disulfide formation as well as thioredoxin 2-mediated reduction. The reactivities of PRDXs with peroxides were surprisingly high considering thiol pK(a), indicating that other protein determinants are involved in PRDXs specialization. The order of reactivities between PRDX5 towards oxidizing substrates differ from other PRDXs studied, pointing to a selective action of PRDXs with respect to peroxide detoxification, helping to rationalize the multiple enzyme isoforms present even in the same cellular compartment.
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Affiliation(s)
- Madia Trujillo
- Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Avda. General Flores 2125, 11800 Montevideo, Uruguay.
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45
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Stanley WA, Fodor K, Marti-Renom MA, Schliebs W, Wilmanns M. Protein translocation into peroxisomes by ring-shaped import receptors. FEBS Lett 2007; 581:4795-802. [PMID: 17884042 DOI: 10.1016/j.febslet.2007.09.001] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2007] [Accepted: 09/04/2007] [Indexed: 12/27/2022]
Abstract
Folded and functional proteins destined for translocation from the cytosol into the peroxisomal matrix are recognized by two different peroxisomal import receptors, Pex5p and Pex7p. Both cargo-loaded receptors dock on the same translocon components, followed by cargo release and receptor recycling, as part of the complete translocation process. Recent structural and functional evidence on the Pex5p receptor has provided insight on the molecular requirements of specific cargo recognition, while the remaining processes still remain largely elusive. Comparison of experimental structures of Pex5p and a structural model of Pex7p reveal that both receptors are built by ring-like arrangements with cargo binding sites, central to the respective structures. Although, molecular insight into the complete peroxisomal translocon still remains to be determined, emerging data allow to deduce common molecular principles that may hold for other translocation systems as well.
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Affiliation(s)
- Will A Stanley
- ARC Plant Energy Biology Centre M316, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
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46
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Gherardini PF, Wass MN, Helmer-Citterich M, Sternberg MJE. Convergent Evolution of Enzyme Active Sites Is not a Rare Phenomenon. J Mol Biol 2007; 372:817-45. [PMID: 17681532 DOI: 10.1016/j.jmb.2007.06.017] [Citation(s) in RCA: 92] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2006] [Revised: 05/14/2007] [Accepted: 06/08/2007] [Indexed: 02/03/2023]
Abstract
Since convergent evolution of enzyme active sites was first identified in serine proteases, other individual instances of this phenomenon have been documented. However, a systematic analysis assessing the frequency of this phenomenon across enzyme space is still lacking. This work uses the Query3d structural comparison algorithm to integrate for the first time detailed knowledge about catalytic residues, available through the Catalytic Site Atlas (CSA), with the evolutionary information provided by the Structural Classification of Proteins (SCOP) database. This study considers two modes of convergent evolution: (i) mechanistic analogues which are enzymes that use the same mechanism to perform related, but possibly different, reactions (considered here as sharing the first three digits of the EC number); and (ii) transformational analogues which catalyse exactly the same reaction (identical EC numbers), but may use different mechanisms. Mechanistic analogues were identified in 15% (26 out of 169) of the three-digit EC groups considered, showing that this phenomenon is not rare. Furthermore 11 of these groups also contain transformational analogues. The catalytic triad is the most widespread active site; the results of the structural comparison show that this mechanism, or variations thereof, is present in 23 superfamilies. Transformational analogues were identified for 45 of the 951 four-digit EC numbers present within the CSA and about half of these were also mechanistic analogues exhibiting convergence of their active sites. This analysis has also been extended to the whole Protein Data Bank to provide a complete and manually curated list of the all the transformational analogues whose structure is classified in SCOP. The results of this work show that the phenomenon of convergent evolution is not rare, especially when considering large enzymatic families.
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Affiliation(s)
- Pier Federico Gherardini
- Biochemistry Building, Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK
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47
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Abstract
Peroxiredoxins (Prxs) are ubiquitous proteins that use an active site Cys residue to reduce hydroperoxides. Structural studies since the first Prx structure was determined in 1998 have produced 35 crystal structures of wild type and mutant Prxs with at least one representative structure from each of the five major evolutionary subfamilies of Prxs. These structures have yielded a great deal of knowledge about Prx structure and structure-function relations, revealing fascinating variations in quaternary structure and details of the fully-folded and locally-unfolded conformations that are involved in the catalytic cycle of all Prxs.
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Affiliation(s)
- P Andrew Karplus
- Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA
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48
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Schremmer B, Manevich Y, Feinstein SI, Fisher AB. Peroxiredoxins in the lung with emphasis on peroxiredoxin VI. Subcell Biochem 2007; 44:317-44. [PMID: 18084901 DOI: 10.1007/978-1-4020-6051-9_15] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
All six mammalian peroxiredoxins are expressed in the lung. Peroxiredoxin (Prx) VI is the isoform expressed at the highest level and its lung expression exceeds that for other organs. The predominant location of Prx VI is the cytosol and acidic organelles of Clara cells of the conducting airways and type II epithelial cells and macrophages in the alveoli. Prx I and VI show developmental induction of transcription at birth. PrxVI shares structural homology with other peroxiredoxins exhibiting a thioredoxin fold and a conserved catalytic Cys residue in the N-terminus of the protein. This enzyme is highly inducible by oxidative stress in both the neonatal and adult lung consistent with a role in antioxidant defense. Prx VI has several properties that distinguish its peroxidase activity from other peroxiredoxins: it can reduce phospholipid hydroperoxides in addition to other organic hydroperoxides and H2O2; the electron donor that serves to reduce the oxidized peroxidatic cysteine is not thioredoxin but GSH; instead of homodimerization, heterodimerization with pi-glutathione S-transferase is required for regeneration of the active enzyme. Prx VI also expresses a phospholipase A2 activity that is Ca2+-independent, maximal at acidic pH, and dependent on a serine-based catalytic triad and nucleophilic elbow at the surface of the protein. Models of altered Prx VI expression at the cellular, organ and whole animal levels have demonstrated that Prx VI functions as an important anti-oxidant enzyme with levels of protection that exceed those ascribed to GSH peroxidase (GPx1). The phospholipase A2 activity plays an important role in lung surfactant homeostasis and is responsible for the bulk of the degradation of internalized phosphatidylcholine and its resynthesis by the reacylation pathway. Expression of peroxiredoxins is elevated in several lung diseases including lung cancer, mesothelioma and sarcoidosis, although the mechanism for these alterations is not known. The unique properties of Prx VI enable it to play an important role in lung cell function.
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Affiliation(s)
- Bruno Schremmer
- Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
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49
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Abstract
Mitochondria are the major intracellular sites of oxygen consumption producing reactive oxygen species (ROS) as toxic by-products of oxidative phosphorylation, primarily via electron leakage from the respiratory chain. The resultant types of chemical damage to lipids, DNA and proteins are described as well as the broader implications for the involvement of ROS in disease onset and progression. The relative contributions of mitochondrial, enzyme-linked, antioxidant defence systems to tissue protection are also reviewed as is the emerging importance of the peroxiredoxin family in general to H2O2-mediated signalling The constituent enzymes of the mitochondrial PrxIII pathway are discussed in detail including the roles of PrxIII and PrxV in their capacities as typical 2-cys and atypical 2-cys thioredoxin-dependent hydroperoxide reductases, respectively. The structures and catalytic mechanisms of PrxIII and V are examined and some key properties of the reconstituted mitochondrial PrxIII pathway are highlighted with specific reference to the susceptibility of peroxiredoxins to inactivation at elevated H2O2 levels and their potential for participation in H2O2-mediated signalling responses. It is concluded that mitochondrial Prxs form a vital link in an integrated cellular antioxidant defence network that minimises ROS-mediated damage and ensures that cells mount appropriate responses to increased levels of oxidative stress via the upregulation of key cell signalling pathways.
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Affiliation(s)
- Zhenbo Cao
- Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, UK
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50
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Boucher IW, McMillan PJ, Gabrielsen M, Akerman SE, Brannigan JA, Schnick C, Brzozowski AM, Wilkinson AJ, Müller S. Structural and biochemical characterization of a mitochondrial peroxiredoxin from Plasmodium falciparum. Mol Microbiol 2006; 61:948-59. [PMID: 16879648 PMCID: PMC1618809 DOI: 10.1111/j.1365-2958.2006.05303.x] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Plasmodium falciparum possesses a single mitochondrion with a functional electron transport chain. During respiration, reactive oxygen species are generated that need to be removed to protect the organelle from oxidative damage. In the absence of catalase and glutathione peroxidase, the parasites rely primarily on peroxiredoxin-linked systems for protection. We have analysed the biochemical and structural features of the mitochondrial peroxiredoxin and thioredoxin of P. falciparum. The mitochondrial localization of both proteins was confirmed by expressing green fluorescent protein fusions in parasite erythrocytic stages. Recombinant protein was kinetically characterized using the cytosolic and the mitochondrial thioredoxin (PfTrx1 and PfTrx2 respectively). The peroxiredoxin clearly preferred PfTrx2 to PfTrx1 as a reducing partner, reflected by the KM values of 11.6 microM and 130.4 microM respectively. Substitution of the two dyads asparagine-62/tyrosine-63 and phenylalanine-139/alanine-140 residues by aspartate-phenylalaine and valine-serine, respectively, reduced the KM for Trx1 but had no effect on the KM of Trx2 suggesting some role for these residues in the discrimination between the two substrates. Solution studies suggest that the protein exists primarily in a homodecameric form. The crystal structure of the mitochondrial peroxiredoxin reveals a fold typical of the 2-Cys class peroxiredoxins and a dimeric form with an intermolecular disulphide bridge between Cys67 and Cys187. These results show that the mitochondrial peroxiredoxin of P. falciparum occurs in both dimeric and decameric forms when purified under non-reducing conditions.
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Affiliation(s)
- Ian W Boucher
- Structural Biology Laboratory, Department of Chemistry, University of YorkYork YO10 5YW, UK
| | - Paul J McMillan
- Institute of Biomedical and Life Sciences, Division of Infection and Immunity and Wellcome Centre for Molecular Parasitology, University of GlasgowGlasgow, UK
| | - Mads Gabrielsen
- Institute of Biomedical and Life Sciences, Division of Infection and Immunity and Wellcome Centre for Molecular Parasitology, University of GlasgowGlasgow, UK
| | - Susan E Akerman
- Institute of Biomedical and Life Sciences, Division of Infection and Immunity and Wellcome Centre for Molecular Parasitology, University of GlasgowGlasgow, UK
| | - James A Brannigan
- Structural Biology Laboratory, Department of Chemistry, University of YorkYork YO10 5YW, UK
| | - Claudia Schnick
- Structural Biology Laboratory, Department of Chemistry, University of YorkYork YO10 5YW, UK
| | - Andrzej M Brzozowski
- Structural Biology Laboratory, Department of Chemistry, University of YorkYork YO10 5YW, UK
| | - Anthony J Wilkinson
- Structural Biology Laboratory, Department of Chemistry, University of YorkYork YO10 5YW, UK
| | - Sylke Müller
- Institute of Biomedical and Life Sciences, Division of Infection and Immunity and Wellcome Centre for Molecular Parasitology, University of GlasgowGlasgow, UK
- *For correspondence. E-mail ; Tel. (+44) 141 330 2383; Fax (+44) 141 330 4600
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