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1
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Wu T, Hossain MN, Hattori M, Nagai T. Ratiometric bioluminescent detection of Cu(II) ion based on differences in enzymatic reaction kinetics of two luciferase variants. Talanta 2025; 287:127576. [PMID: 39818047 DOI: 10.1016/j.talanta.2025.127576] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2024] [Revised: 01/08/2025] [Accepted: 01/10/2025] [Indexed: 01/18/2025]
Abstract
Heavy metal contamination in water bodies has raised global concerns due to its significant threats to both public health and ecosystem. Copper (Cu), one of the most widely used metals, is also an essential trace element in physiological systems. Excessive intake of Cu from water can cause toxicity, potentially resulting in serious health risks. Ensuring water safety necessitates the critical detection of copper ion (Cu2+). Here, we report a ratiometric bioluminescent detection method for Cu2+, DERK-Cu(II), which is based on the Distinct Enzymatic Reaction Kinetics of two luciferase variants. In DERK-Cu(II), the blue luminescent luciferase exhibits lower catalytic efficiency than the green luminescent luciferase, thus it is less inhibited by Cu2+. Consequently, the luminescence color of their mixture is dependent on the Cu2+ concentrations, enabling us to find an optimal mixing ratio at which luminescence color changes evidently from green to blue. Building on this, we established a quantitative detection method for Cu2+ using a smartphone and successfully measured the Cu2+ concentrations in several water samples. The method we developed, using the difference in reaction kinetics of two enzymes with the same substrate specificity, would become a versatile approach applicable to the development of other enzyme-based indicators.
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Affiliation(s)
- Ti Wu
- Graduate School of Pharmaceutical Science, Osaka University, Suita, Osaka, 565-0871, Japan; SANKEN (The Institute of Scientific and Industrial Research), Osaka University, Ibaraki, Osaka, 567-0047, Japan
| | - Md Nadim Hossain
- SANKEN (The Institute of Scientific and Industrial Research), Osaka University, Ibaraki, Osaka, 567-0047, Japan
| | - Mitsuru Hattori
- SANKEN (The Institute of Scientific and Industrial Research), Osaka University, Ibaraki, Osaka, 567-0047, Japan
| | - Takeharu Nagai
- Graduate School of Pharmaceutical Science, Osaka University, Suita, Osaka, 565-0871, Japan; SANKEN (The Institute of Scientific and Industrial Research), Osaka University, Ibaraki, Osaka, 567-0047, Japan; Transdimensional Life Imaging Division, Institute for Open and Transdisciplinary Research Initiative, Osaka University, Suita, Osaka, 565-0871, Japan; Research Institute for Electronic Science, Hokkaido University, Sapporo, Hokkaido, 001-0020, Japan.
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2
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Nogami A, Amemiya HJ, Fujiwara H, Umezawa Y, Tohda S, Nagao T. Targeting USP14/UCHL5: A Breakthrough Approach to Overcoming Treatment-Resistant FLT3-ITD-Positive AML. Int J Mol Sci 2024; 25:10372. [PMID: 39408703 PMCID: PMC11476563 DOI: 10.3390/ijms251910372] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2024] [Revised: 09/17/2024] [Accepted: 09/24/2024] [Indexed: 10/20/2024] Open
Abstract
FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations in acute myeloid leukemia (AML) are associated with poor prognosis and therapy resistance. This study aimed to demonstrate that inhibiting the deubiquitinating enzymes ubiquitin-specific peptidase 14 (USP14) and ubiquitin C-terminal hydrolase L5 (UCHL5) (USP14/UCHL5) with b-AP15 or the organogold compound auranofin (AUR) induces apoptosis in the ITD-transformed human leukemia cell line MV4-11 and mononuclear leukocytes derived from patients with FLT3-ITD-positive AML. This study included patients diagnosed with AML at Tokyo Medical and Dental University Hospital between January 2018 and July 2024. Both treatments blocked downstream FLT3 pathway events, with the effects potentiated by USP14 knockdown. Both treatments inhibited FLT3 deubiquitination via K48 and disrupted translation initiation via 4EBP1, a downstream FLT3 target. FLT3 was downregulated in the leukemic cells, with the associated activation of stress-related MAP kinase pathways and increased NF-E2-related factor 2. Furthermore, the overexpression of B-cell lymphoma-extra-large and myeloid cell leukemia-1 prevented the cell death caused by b-AP15 and AUR. These results suggest that inhibiting USP14/UCHL5, which involves multiple regulatory mechanisms, is a promising target for novel therapies for treatment-resistant FLT3-ITD-positive AML.
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MESH Headings
- Humans
- fms-Like Tyrosine Kinase 3/genetics
- fms-Like Tyrosine Kinase 3/metabolism
- Leukemia, Myeloid, Acute/genetics
- Leukemia, Myeloid, Acute/metabolism
- Leukemia, Myeloid, Acute/drug therapy
- Leukemia, Myeloid, Acute/pathology
- Ubiquitin Thiolesterase/metabolism
- Ubiquitin Thiolesterase/genetics
- Ubiquitin Thiolesterase/antagonists & inhibitors
- Drug Resistance, Neoplasm/drug effects
- Drug Resistance, Neoplasm/genetics
- Female
- Male
- Cell Line, Tumor
- Middle Aged
- Aged
- Apoptosis/drug effects
- Adult
- Mutation
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Affiliation(s)
- Ayako Nogami
- Department of Laboratory Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyoku, Tokyo 113-8510, Japan
- Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo 113-8510, Japan
| | - Hideki Jose Amemiya
- Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo 113-8510, Japan
| | - Hiroki Fujiwara
- Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo 113-8510, Japan
| | - Yoshihiro Umezawa
- Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo 113-8510, Japan
| | - Shuji Tohda
- Department of Laboratory Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyoku, Tokyo 113-8510, Japan
| | - Toshikage Nagao
- Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo 113-8510, Japan
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3
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Gencheva R, Cheng Q, Arnér ESJ. Thioredoxin reductase selenoproteins from different organisms as potential drug targets for treatment of human diseases. Free Radic Biol Med 2022; 190:320-338. [PMID: 35987423 DOI: 10.1016/j.freeradbiomed.2022.07.020] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/30/2022] [Revised: 06/25/2022] [Accepted: 07/26/2022] [Indexed: 11/15/2022]
Abstract
Human thioredoxin reductase (TrxR) is a selenoprotein with a central role in cellular redox homeostasis, utilizing a highly reactive and solvent-exposed selenocysteine (Sec) residue in its active site. Pharmacological modulation of TrxR can be obtained with several classes of small compounds showing different mechanisms of action, but most often dependent upon interactions with its Sec residue. The clinical implications of TrxR modulation as mediated by small compounds have been studied in diverse diseases, from rheumatoid arthritis and ischemia to cancer and parasitic infections. The possible involvement of TrxR in these diseases was in some cases serendipitously discovered, by finding that existing clinically used drugs are also TrxR inhibitors. Inhibiting isoforms of human TrxR is, however, not the only strategy for human disease treatment, as some pathogenic parasites also depend upon Sec-containing TrxR variants, including S. mansoni, B. malayi or O. volvulus. Inhibiting parasite TrxR has been shown to selectively kill parasites and can thus become a promising treatment strategy, especially in the context of quickly emerging resistance towards other drugs. Here we have summarized the basis for the targeting of selenoprotein TrxR variants with small molecules for therapeutic purposes in different human disease contexts. We discuss how Sec engagement appears to be an indispensable part of treatment efficacy and how some therapeutically promising compounds have been evaluated in preclinical or clinical studies. Several research questions remain before a wider application of selenoprotein TrxR inhibition as a first-line treatment strategy might be developed. These include further mechanistic studies of downstream effects that may mediate treatment efficacy, identification of isoform-specific enzyme inhibition patterns for some given therapeutic compounds, and the further elucidation of cell-specific effects in disease contexts such as in the tumor microenvironment or in host-parasite interactions, and which of these effects may be dependent upon the specific targeting of Sec in distinct TrxR isoforms.
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Affiliation(s)
- Radosveta Gencheva
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, 17177, Sweden
| | - Qing Cheng
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, 17177, Sweden
| | - Elias S J Arnér
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, 17177, Sweden; Department of Selenoprotein Research, National Tumor Biology Laboratory, National Institute of Oncology, 1122, Budapest, Hungary.
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4
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Superoxide Radicals in the Execution of Cell Death. Antioxidants (Basel) 2022; 11:antiox11030501. [PMID: 35326151 PMCID: PMC8944419 DOI: 10.3390/antiox11030501] [Citation(s) in RCA: 114] [Impact Index Per Article: 38.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2022] [Revised: 03/01/2022] [Accepted: 03/01/2022] [Indexed: 12/24/2022] Open
Abstract
Superoxide is a primary oxygen radical that is produced when an oxygen molecule receives one electron. Superoxide dismutase (SOD) plays a primary role in the cellular defense against an oxidative insult by ROS. However, the resulting hydrogen peroxide is still reactive and, in the presence of free ferrous iron, may produce hydroxyl radicals and exacerbate diseases. Polyunsaturated fatty acids are the preferred target of hydroxyl radicals. Ferroptosis, a type of necrotic cell death induced by lipid peroxides in the presence of free iron, has attracted considerable interest because of its role in the pathogenesis of many diseases. Radical electrons, namely those released from mitochondrial electron transfer complexes, and those produced by enzymatic reactions, such as lipoxygenases, appear to cause lipid peroxidation. While GPX4 is the most potent anti-ferroptotic enzyme that is known to reduce lipid peroxides to alcohols, other antioxidative enzymes are also indirectly involved in protection against ferroptosis. Moreover, several low molecular weight compounds that include α-tocopherol, ascorbate, and nitric oxide also efficiently neutralize radical electrons, thereby suppressing ferroptosis. The removal of radical electrons in the early stages is of primary importance in protecting against ferroptosis and other diseases that are related to oxidative stress.
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Waddell J, Banerjee A, Kristian T. Acetylation in Mitochondria Dynamics and Neurodegeneration. Cells 2021; 10:cells10113031. [PMID: 34831252 PMCID: PMC8616140 DOI: 10.3390/cells10113031] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2021] [Revised: 11/03/2021] [Accepted: 11/03/2021] [Indexed: 12/23/2022] Open
Abstract
Mitochondria are a unique intracellular organelle due to their evolutionary origin and multifunctional role in overall cellular physiology and pathophysiology. To meet the specific spatial metabolic demands within the cell, mitochondria are actively moving, dividing, or fusing. This process of mitochondrial dynamics is fine-tuned by a specific group of proteins and their complex post-translational modifications. In this review, we discuss the mitochondrial dynamics regulatory enzymes, their adaptor proteins, and the effect of acetylation on the activity of fusion and fission machinery as a ubiquitous response to metabolic stresses. Further, we discuss the role of intracellular cytoskeleton structures and their post-translational modifications in the modulation of mitochondrial fusion and fission. Finally, we review the role of mitochondrial dynamics dysregulation in the pathophysiology of acute brain injury and the treatment strategies based on modulation of NAD+-dependent deacetylation.
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Affiliation(s)
- Jaylyn Waddell
- Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD 21201, USA; (J.W.); (A.B.)
| | - Aditi Banerjee
- Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD 21201, USA; (J.W.); (A.B.)
| | - Tibor Kristian
- Veterans Affairs Maryland Health Center System, 10 North Greene Street, Baltimore, MD 21201, USA
- Department of Anesthesiology and the Center for Shock, Trauma, and Anesthesiology Research (S.T.A.R.), University of Maryland School of Medicine, Baltimore, MD 21201, USA
- Correspondence: ; Tel.: +1-410-706-3418
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6
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Auranofin: Past to Present, and repurposing. Int Immunopharmacol 2021; 101:108272. [PMID: 34731781 DOI: 10.1016/j.intimp.2021.108272] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2021] [Revised: 10/09/2021] [Accepted: 10/12/2021] [Indexed: 01/15/2023]
Abstract
Auranofin (AF), a gold compound, has been used to treat rheumatoid arthritis (RA) for more than 40 years; however, its mechanism of action remains unknown. We revealed that AF inhibited the induction of proinflammatory proteins and their mRNAs by the inflammatory stimulants, cyclooxygenase-2 and inducible nitric oxide synthase, and their upstream regulator, NF-κB. AF also activated the proteins peroxyredoxin-1, Kelch-like ECH-associated protein 1, and NF-E2-related factor 2, and inhibited thioredoxin reductase, all of which are involved in oxidative or electrophilic stress under physiological conditions. Although the cell membrane was previously considered to be permeable to AF because of its hydrophobicity, the mechanisms responsible for transporting AF into and out of cells as well as its effects on the uptake and excretion of other drugs have not yet been elucidated. Antibodies for cytokines have recently been employed in the treatment of RA, which has had an impact on the use of AF. Trials to repurpose AF as a risk-controlled agent to treat cancers or infectious diseases, including severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019, are ongoing. Novel gold compounds are also under development as anti-cancer and anti-infection agents.
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7
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Geldon S, Fernández-Vizarra E, Tokatlidis K. Redox-Mediated Regulation of Mitochondrial Biogenesis, Dynamics, and Respiratory Chain Assembly in Yeast and Human Cells. Front Cell Dev Biol 2021; 9:720656. [PMID: 34557489 PMCID: PMC8452992 DOI: 10.3389/fcell.2021.720656] [Citation(s) in RCA: 33] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Accepted: 08/04/2021] [Indexed: 12/24/2022] Open
Abstract
Mitochondria are double-membrane organelles that contain their own genome, the mitochondrial DNA (mtDNA), and reminiscent of its endosymbiotic origin. Mitochondria are responsible for cellular respiration via the function of the electron oxidative phosphorylation system (OXPHOS), located in the mitochondrial inner membrane and composed of the four electron transport chain (ETC) enzymes (complexes I-IV), and the ATP synthase (complex V). Even though the mtDNA encodes essential OXPHOS components, the large majority of the structural subunits and additional biogenetical factors (more than seventy proteins) are encoded in the nucleus and translated in the cytoplasm. To incorporate these proteins and the rest of the mitochondrial proteome, mitochondria have evolved varied, and sophisticated import machineries that specifically target proteins to the different compartments defined by the two membranes. The intermembrane space (IMS) contains a high number of cysteine-rich proteins, which are mostly imported via the MIA40 oxidative folding system, dependent on the reduction, and oxidation of key Cys residues. Several of these proteins are structural components or assembly factors necessary for the correct maturation and function of the ETC complexes. Interestingly, many of these proteins are involved in the metalation of the active redox centers of complex IV, the terminal oxidase of the mitochondrial ETC. Due to their function in oxygen reduction, mitochondria are the main generators of reactive oxygen species (ROS), on both sides of the inner membrane, i.e., in the matrix and the IMS. ROS generation is important due to their role as signaling molecules, but an excessive production is detrimental due to unwanted oxidation reactions that impact on the function of different types of biomolecules contained in mitochondria. Therefore, the maintenance of the redox balance in the IMS is essential for mitochondrial function. In this review, we will discuss the role that redox regulation plays in the maintenance of IMS homeostasis as well as how mitochondrial ROS generation may be a key regulatory factor for ETC biogenesis, especially for complex IV.
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Affiliation(s)
| | - Erika Fernández-Vizarra
- Institute of Molecular Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
| | - Kostas Tokatlidis
- Institute of Molecular Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
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8
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Abstract
The cytosolic selenoprotein thioredoxin reductase 1 (TrxR1, TXNRD1), and to some extent mitochondrial TrxR2 (TXNRD2), can be inhibited by a wide range of electrophilic compounds. Many such compounds also yield cytotoxicity toward cancer cells in culture or in mouse models, and most compounds are likely to irreversibly modify the easily accessible selenocysteine residue in TrxR1, thereby inhibiting its normal activity to reduce cytosolic thioredoxin (Trx1, TXN) and other substrates of the enzyme. This leads to an oxidative challenge. In some cases, the inhibited forms of TrxR1 are not catalytically inert and are instead converted to prooxidant NADPH oxidases, named SecTRAPs, thus further aggravating the oxidative stress, particularly in cells expressing higher levels of the enzyme. In this review, the possible molecular and cellular consequences of these effects are discussed in relation to cancer therapy, with a focus on outstanding questions that should be addressed if targeted TrxR1 inhibition is to be further developed for therapeutic use. Expected final online publication date for the Annual Review of Pharmacology and Toxicology, Volume 62 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Affiliation(s)
- Radosveta Gencheva
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 Stockholm, Sweden;
| | - Elias S J Arnér
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 Stockholm, Sweden; .,Department of Selenoprotein Research, National Institute of Oncology, Budapest 1122, Hungary
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9
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Klimova N, Fearnow A, Kristian T. Role of NAD +-Modulated Mitochondrial Free Radical Generation in Mechanisms of Acute Brain Injury. Brain Sci 2020; 10:brainsci10070449. [PMID: 32674501 PMCID: PMC7408119 DOI: 10.3390/brainsci10070449] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2020] [Revised: 06/30/2020] [Accepted: 07/09/2020] [Indexed: 12/15/2022] Open
Abstract
It is commonly accepted that mitochondria represent a major source of free radicals following acute brain injury or during the progression of neurodegenerative diseases. The levels of reactive oxygen species (ROS) in cells are determined by two opposing mechanisms—the one that produces free radicals and the cellular antioxidant system that eliminates ROS. Thus, the balance between the rate of ROS production and the efficiency of the cellular detoxification process determines the levels of harmful reactive oxygen species. Consequently, increase in free radical levels can be a result of higher rates of ROS production or due to the inhibition of the enzymes that participate in the antioxidant mechanisms. The enzymes’ activity can be modulated by post-translational modifications that are commonly altered under pathologic conditions. In this review we will discuss the mechanisms of mitochondrial free radical production following ischemic insult, mechanisms that protect mitochondria against free radical damage, and the impact of post-ischemic nicotinamide adenine mononucleotide (NAD+) catabolism on mitochondrial protein acetylation that affects ROS generation and mitochondrial dynamics. We propose a mechanism of mitochondrial free radical generation due to a compromised mitochondrial antioxidant system caused by intra-mitochondrial NAD+ depletion. Finally, the interplay between different mechanisms of mitochondrial ROS generation and potential therapeutic approaches are reviewed.
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Affiliation(s)
- Nina Klimova
- Veterans Affairs Maryland Health Center System, 10 North Greene Street, Baltimore, MD 21201, USA; (N.K.); (A.F.)
- Department of Anesthesiology and the Center for Shock, Trauma, and Anesthesiology Research (S.T.A.R.), University of Maryland School of Medicine, Baltimore, MD 21201, USA
| | - Adam Fearnow
- Veterans Affairs Maryland Health Center System, 10 North Greene Street, Baltimore, MD 21201, USA; (N.K.); (A.F.)
| | - Tibor Kristian
- Veterans Affairs Maryland Health Center System, 10 North Greene Street, Baltimore, MD 21201, USA; (N.K.); (A.F.)
- Department of Anesthesiology and the Center for Shock, Trauma, and Anesthesiology Research (S.T.A.R.), University of Maryland School of Medicine, Baltimore, MD 21201, USA
- Correspondence:
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10
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Cheleschi S, Calamia V, Fernandez-Moreno M, Biava M, Giordani A, Fioravanti A, Anzini M, Blanco F. In vitro comprehensive analysis of VA692 a new chemical entity for the treatment of osteoarthritis. Int Immunopharmacol 2018; 64:86-100. [DOI: 10.1016/j.intimp.2018.08.025] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2018] [Revised: 07/03/2018] [Accepted: 08/19/2018] [Indexed: 12/21/2022]
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11
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Single-lung ventilation and oxidative stress: a different perspective on a common practice. Curr Opin Anaesthesiol 2018; 30:42-49. [PMID: 27783023 DOI: 10.1097/aco.0000000000000410] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
PURPOSE OF REVIEW To summarize what is currently known about the relationship between single-lung ventilation (SLV), oxidative stress, and postoperative disruption of organ function. RECENT FINDINGS SLV produces progressive alelectasis that is associated with hypoxic pulmonary vasoconstriction and redistribution of blood flow away from the nonventilated lung. This local tissue hypoxia induces the generation of reactive oxygen and reactive nitrogen species, an effect subsequently amplified by lung re-expansion consistent with well described hypoxia/reperfusion responses. Both experimental and clinical data indicate that the magnitude of oxidative and nitrosative stress is related to the duration of SLV and that these stresses affect not only the collapsed/re-expanded lung, but other organs as well. SUMMARY SLV and subsequent re-expansion of atelectatic lung are associated with the generation of reactive oxygen and nitrogen species that may modulate persistent systemic effects.
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12
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Broxton CN, Culotta VC. An Adaptation to Low Copper in Candida albicans Involving SOD Enzymes and the Alternative Oxidase. PLoS One 2016; 11:e0168400. [PMID: 28033429 PMCID: PMC5198983 DOI: 10.1371/journal.pone.0168400] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2016] [Accepted: 11/30/2016] [Indexed: 01/30/2023] Open
Abstract
In eukaryotes, the Cu/Zn superoxide dismutase (SOD1) is a major cytosolic cuproprotein with a small fraction residing in the mitochondrial intermembrane space (IMS) to protect against respiratory superoxide. Curiously, the opportunistic human fungal pathogen Candida albicans is predicted to express two cytosolic SODs including Cu/Zn containing SOD1 and manganese containing SOD3. As part of a copper starvation response, C. albicans represses SOD1 and induces the non-copper alternative SOD3. While both SOD1 and SOD3 are predicted to exist in the same cytosolic compartment, their potential role in mitochondrial oxidative stress had yet to be investigated. We show here that under copper replete conditions, a fraction of the Cu/Zn containing SOD1 localizes to the mitochondrial IMS to guard against mitochondrial superoxide. However in copper starved cells, localization of the manganese containing SOD3 is restricted to the cytosol leaving the mitochondrial IMS devoid of SOD. We observe that during copper starvation, an alternative oxidase (AOX) form of respiration is induced that is not coupled to ATP synthesis but maintains mitochondrial superoxide at low levels even in the absence of IMS SOD. Surprisingly, the copper-dependent cytochrome c oxidase (COX) form of respiration remains high with copper starvation. We provide evidence that repression of SOD1 during copper limitation serves to spare copper for COX and maintain COX respiration. Overall, the complex copper starvation response of C. albicans involving SOD1, SOD3 and AOX minimizes mitochondrial oxidative damage whilst maximizing COX respiration essential for fungal pathogenesis.
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Affiliation(s)
- Chynna N. Broxton
- Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, United States of America
| | - Valeria C. Culotta
- Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, United States of America
- * E-mail:
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13
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Yan J, Xu J, Fei Y, Jiang C, Zhu W, Han Y, Lu S. TrxR2 deficiencies promote chondrogenic differentiation and induce apoptosis of chondrocytes through mitochondrial reactive oxygen species. Exp Cell Res 2016; 344:67-75. [DOI: 10.1016/j.yexcr.2016.04.014] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2016] [Revised: 04/14/2016] [Accepted: 04/19/2016] [Indexed: 01/09/2023]
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14
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Nakao LS, Everley RA, Marino SM, Lo SM, de Souza LE, Gygi SP, Gladyshev VN. Mechanism-based proteomic screening identifies targets of thioredoxin-like proteins. J Biol Chem 2015; 290:5685-95. [PMID: 25561728 DOI: 10.1074/jbc.m114.597245] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes.
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Affiliation(s)
- Lia S Nakao
- From the Division of Genetics, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, the Universidade Federal do Paraná, Departamento de Patologia Básica, 81531-980, Curitiba, PR, Brazil, and
| | - Robert A Everley
- the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
| | - Stefano M Marino
- From the Division of Genetics, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
| | - Sze M Lo
- the Universidade Federal do Paraná, Departamento de Patologia Básica, 81531-980, Curitiba, PR, Brazil, and
| | - Luiz E de Souza
- the Universidade Federal do Paraná, Departamento de Patologia Básica, 81531-980, Curitiba, PR, Brazil, and
| | - Steven P Gygi
- the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
| | - Vadim N Gladyshev
- From the Division of Genetics, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115,
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15
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Shokolenko IN, Wilson GL, Alexeyev MF. Aging: A mitochondrial DNA perspective, critical analysis and an update. World J Exp Med 2014; 4:46-57. [PMID: 25414817 PMCID: PMC4237642 DOI: 10.5493/wjem.v4.i4.46] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/27/2014] [Revised: 07/15/2014] [Accepted: 08/31/2014] [Indexed: 02/06/2023] Open
Abstract
The mitochondrial theory of aging, a mainstream theory of aging which once included accumulation of mitochondrial DNA (mtDNA) damage by reactive oxygen species (ROS) as its cornerstone, has been increasingly losing ground and is undergoing extensive revision due to its inability to explain a growing body of emerging data. Concurrently, the notion of the central role for mtDNA in the aging process is being met with increased skepticism. Our progress in understanding the processes of mtDNA maintenance, repair, damage, and degradation in response to damage has largely refuted the view of mtDNA as being particularly susceptible to ROS-mediated mutagenesis due to its lack of “protective” histones and reduced complement of available DNA repair pathways. Recent research on mitochondrial ROS production has led to the appreciation that mitochondria, even in vitro, produce much less ROS than previously thought, automatically leading to a decreased expectation of physiologically achievable levels of mtDNA damage. New evidence suggests that both experimentally induced oxidative stress and radiation therapy result in very low levels of mtDNA mutagenesis. Recent advances provide evidence against the existence of the “vicious” cycle of mtDNA damage and ROS production. Meta-studies reveal no longevity benefit of increased antioxidant defenses. Simultaneously, exciting new observations from both comparative biology and experimental systems indicate that increased ROS production and oxidative damage to cellular macromolecules, including mtDNA, can be associated with extended longevity. A novel paradigm suggests that increased ROS production in aging may be the result of adaptive signaling rather than a detrimental byproduct of normal respiration that drives aging. Here, we review issues pertaining to the role of mtDNA in aging.
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Ullrich V, Schildknecht S. Sensing hypoxia by mitochondria: a unifying hypothesis involving S-nitrosation. Antioxid Redox Signal 2014; 20:325-38. [PMID: 22793377 DOI: 10.1089/ars.2012.4788] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
SIGNIFICANCE Sudden hypoxia requires a rapid response in tissues with high energy demand. Mitochondria are rapid sensors for a lack of oxygen, but no consistent mechanism for the sensing process and the subsequent counter-regulation has been described. RECENT ADVANCES In the present hypothesis review, we suggest an oxygen-sensing mechanism by mitochondria that is initiated at low oxygen tension by electrons from the respiratory chain, leading to the reduction of intracellular nitrite to nitric oxide ((•)NO) that would subsequently compete with oxygen for binding to cytochrome c oxidase. This allows superoxide ((•)O2(-)) formation in hypoxic areas, leading to S-nitrosation and the inhibition of mitochondrial Krebs cycle enzymes. With more formation of (•)O2(-), peroxynitrite is generated and known to damage the connection between the mitochondrial matrix and the outer membrane. CRITICAL ISSUES A fundamental question on a regulatory mechanism is its reversibility. Readmission of oxygen and opening of the mitochondrial KATP-channel would allow electrons from glycerol-3-phosphate to selectively reduce the ubiquinone pool to generate (•)O2(-) at both sides of the inner mitochondrial membrane. On the cytosolic side, superoxide is dismutated and will support H2O2/Fe(2+)-dependent transcription processes and on the mitochondrial matrix side, it could lead to the one-electron reduction and reactivation of S-nitrosated proteins. FUTURE DIRECTIONS It remains to be elucidated up to which stage the herein proposed silencing of mitochondria remains reversible and when irreversible changes that ultimately lead to classical reperfusion injury are initiated.
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Affiliation(s)
- Volker Ullrich
- Department of Biology, University of Konstanz , Konstanz, Germany
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Sengupta D, Chowdhury KD, Sarkar A, Paul S, Sadhukhan GC. Berberine and S allyl cysteine mediated amelioration of DEN+CCl4 induced hepatocarcinoma. Biochim Biophys Acta Gen Subj 2014; 1840:219-44. [DOI: 10.1016/j.bbagen.2013.08.020] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2012] [Revised: 08/14/2013] [Accepted: 08/26/2013] [Indexed: 02/07/2023]
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Hardeland R. Melatonin and the theories of aging: a critical appraisal of melatonin's role in antiaging mechanisms. J Pineal Res 2013; 55:325-56. [PMID: 24112071 DOI: 10.1111/jpi.12090] [Citation(s) in RCA: 195] [Impact Index Per Article: 16.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/22/2013] [Accepted: 08/23/2013] [Indexed: 02/06/2023]
Abstract
The classic theories of aging such as the free radical theory, including its mitochondria-related versions, have largely focused on a few specific processes of senescence. Meanwhile, numerous interconnections have become apparent between age-dependent changes previously thought to proceed more or less independently. Increased damage by free radicals is not only linked to impairments of mitochondrial function, but also to inflammaging as it occurs during immune remodeling and by release of proinflammatory cytokines from mitotically arrested, DNA-damaged cells that exhibit the senescence-associated secretory phenotype (SASP). Among other effects, SASP can cause mutations in stem cells that reduce the capacity for tissue regeneration or, in worst case, lead to cancer stem cells. Oxidative stress has also been shown to promote telomere attrition. Moreover, damage by free radicals is connected to impaired circadian rhythmicity. Another nexus exists between cellular oscillators and metabolic sensing, in particular to the aging-suppressor SIRT1, which acts as an accessory clock protein. Melatonin, being a highly pleiotropic regulator molecule, interacts directly or indirectly with all the processes mentioned. These influences are critically reviewed, with emphasis on data from aged organisms and senescence-accelerated animals. The sometimes-controversial findings obtained either in a nongerontological context or in comparisons of tumor with nontumor cells are discussed in light of evidence obtained in senescent organisms. Although, in mammals, lifetime extension by melatonin has been rarely documented in a fully conclusive way, a support of healthy aging has been observed in rodents and is highly likely in humans.
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Affiliation(s)
- Rüdiger Hardeland
- Johann Friedrich Blumenbach Institute of Zoology and Anthropology, University of Göttingen, Göttingen, Germany
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Bindoli A, Rigobello MP. Principles in redox signaling: from chemistry to functional significance. Antioxid Redox Signal 2013; 18:1557-93. [PMID: 23244515 DOI: 10.1089/ars.2012.4655] [Citation(s) in RCA: 143] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Reactive oxygen and nitrogen species are currently considered not only harmful byproducts of aerobic respiration but also critical mediators of redox signaling. The molecules and the chemical principles sustaining the network of cellular redox regulated processes are described. Special emphasis is placed on hydrogen peroxide (H(2)O(2)), now considered as acting as a second messenger, and on sulfhydryl groups, which are the direct targets of the oxidant signal. Cysteine residues of some proteins, therefore, act as sensors of redox conditions and are oxidized in a reversible reaction. In particular, the formation of sulfenic acid and disulfide, the initial steps of thiol oxidation, are described in detail. The many cell pathways involved in reactive oxygen species formation are reported. Central to redox signaling processes are the glutathione and thioredoxin systems controlling H(2)O(2) levels and, hence, the thiol/disulfide balance. Lastly, some of the most important redox-regulated processes involving specific enzymes and organelles are described. The redox signaling area of research is rapidly expanding, and future work will examine new pathways and clarify their importance in cellular pathophysiology.
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Affiliation(s)
- Alberto Bindoli
- Institute of Neuroscience (CNR), Department of Biomedical Sciences, University of Padova, Padova, Italy.
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20
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Venditti P, Di Stefano L, Di Meo S. Mitochondrial metabolism of reactive oxygen species. Mitochondrion 2013; 13:71-82. [DOI: 10.1016/j.mito.2013.01.008] [Citation(s) in RCA: 219] [Impact Index Per Article: 18.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2012] [Revised: 01/16/2013] [Accepted: 01/22/2013] [Indexed: 11/16/2022]
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Antico Arciuch VG, Elguero ME, Poderoso JJ, Carreras MC. Mitochondrial regulation of cell cycle and proliferation. Antioxid Redox Signal 2012; 16:1150-80. [PMID: 21967640 PMCID: PMC3315176 DOI: 10.1089/ars.2011.4085] [Citation(s) in RCA: 317] [Impact Index Per Article: 24.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/24/2011] [Revised: 10/03/2011] [Accepted: 10/03/2011] [Indexed: 01/01/2023]
Abstract
Eukaryotic mitochondria resulted from symbiotic incorporation of α-proteobacteria into ancient archaea species. During evolution, mitochondria lost most of the prokaryotic bacterial genes and only conserved a small fraction including those encoding 13 proteins of the respiratory chain. In this process, many functions were transferred to the host cells, but mitochondria gained a central role in the regulation of cell proliferation and apoptosis, and in the modulation of metabolism; accordingly, defective organelles contribute to cell transformation and cancer, diabetes, and neurodegenerative diseases. Most cell and transcriptional effects of mitochondria depend on the modulation of respiratory rate and on the production of hydrogen peroxide released into the cytosol. The mitochondrial oxidative rate has to remain depressed for cell proliferation; even in the presence of O₂, energy is preferentially obtained from increased glycolysis (Warburg effect). In response to stress signals, traffic of pro- and antiapoptotic mitochondrial proteins in the intermembrane space (B-cell lymphoma-extra large, Bcl-2-associated death promoter, Bcl-2 associated X-protein and cytochrome c) is modulated by the redox condition determined by mitochondrial O₂ utilization and mitochondrial nitric oxide metabolism. In this article, we highlight the traffic of the different canonical signaling pathways to mitochondria and the contributions of organelles to redox regulation of kinases. Finally, we analyze the dynamics of the mitochondrial population in cell cycle and apoptosis.
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Affiliation(s)
| | - María Eugenia Elguero
- Laboratory of Oxygen Metabolism, University of Buenos Aires, University Hospital, Buenos Aires, Argentina
| | - Juan José Poderoso
- Laboratory of Oxygen Metabolism, University of Buenos Aires, University Hospital, Buenos Aires, Argentina
- Department of Internal Medicine, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina
- CONICET, Buenos Aires, Argentina
| | - María Cecilia Carreras
- Laboratory of Oxygen Metabolism, University of Buenos Aires, University Hospital, Buenos Aires, Argentina
- CONICET, Buenos Aires, Argentina
- Department of Clinical Biochemistry, INFIBIOC and School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina
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Aon MA, Stanley BA, Sivakumaran V, Kembro JM, O'Rourke B, Paolocci N, Cortassa S. Glutathione/thioredoxin systems modulate mitochondrial H2O2 emission: an experimental-computational study. ACTA ACUST UNITED AC 2012; 139:479-91. [PMID: 22585969 PMCID: PMC3362521 DOI: 10.1085/jgp.201210772] [Citation(s) in RCA: 158] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
The net emission of hydrogen peroxide (H(2)O(2)) from mitochondria results from the balance between reactive oxygen species (ROS) continuously generated in the respiratory chain and ROS scavenging. The relative contribution of the two major antioxidant systems in the mitochondrial matrix, glutathione (GSH) and thioredoxin (Trx), has not been assessed. In this paper, we examine this key question via combined experimental and theoretical approaches, using isolated heart mitochondria from mouse, rat, and guinea pig. As compared with untreated control mitochondria, selective inhibition of Trx reductase with auranofin along with depletion of GSH with 2,4-dinitrochlorobenzene led to a species-dependent increase in H(2)O(2) emission flux of 17, 11, and 6 fold in state 4 and 15, 7, and 8 fold in state 3 for mouse, rat, and guinea pig mitochondria, respectively. The maximal H(2)O(2) emission as a percentage of the total O(2) consumption flux was 11%/2.3% for mouse in states 4 and 3 followed by 2%/0.25% and 0.74%/0.29% in the rat and guinea pig, respectively. A minimal computational model accounting for the kinetics of GSH/Trx systems was developed and was able to simulate increase in H(2)O(2) emission fluxes when both scavenging systems were inhibited separately or together. Model simulations suggest that GSH/Trx systems act in concert. When the scavenging capacity of either one of them saturates during H(2)O(2) overload, they relieve each other until complete saturation, when maximal ROS emission occurs. Quantitatively, these results converge on the idea that GSH/Trx scavenging systems in mitochondria are both essential for keeping minimal levels of H(2)O(2) emission, especially during state 3 respiration, when the energetic output is maximal. This suggests that the very low levels of H(2)O(2) emission observed during forward electron transport in the respiratory chain are a result of the well-orchestrated actions of the two antioxidant systems working continuously to offset ROS production.
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Affiliation(s)
- Miguel Antonio Aon
- Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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Pereira CV, Nadanaciva S, Oliveira PJ, Will Y. The contribution of oxidative stress to drug-induced organ toxicity and its detection in vitro and in vivo. Expert Opin Drug Metab Toxicol 2012; 8:219-37. [PMID: 22248238 DOI: 10.1517/17425255.2012.645536] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
INTRODUCTION Nowadays the 'redox hypothesis' is based on the fact that thiol/disulfide couples such as glutathione (GSH/GSSG), cysteine (Cys/CySS) and thioredoxin ((Trx-(SH)2/Trx-SS)) are functionally organized in redox circuits controlled by glutathione pools, thioredoxins and other control nodes, and they are not in equilibrium relative to each other. Although ROS can be important intermediates of cellular signaling pathways, disturbances in the normal cellular redox can result in widespread damage to several cell components. Moreover, oxidative stress has been linked to a variety of age-related diseases. In recent years, oxidative stress has also been identified to contribute to drug-induced liver, heart, renal and brain toxicity. AREAS COVERED This review provides an overview of current in vitro and in vivo methods that can be deployed throughout the drug discovery process. In addition, animal models and noninvasive biomarkers are described. EXPERT OPINION Reducing post-market drug withdrawals is essential for all pharmaceutical companies in a time of increased patient welfare and tight budgets. Predictive screens positioned early in the drug discovery process will help to reduce such liabilities. Although new and more efficient assays and models are being developed, the hunt for biomarkers and noninvasive techniques is still in progress.
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Affiliation(s)
- Claudia V Pereira
- Pfizer R&D, Compound Safety Prediction-WWMC, Cell Based Assays and Mitochondrial Biology, Eastern Point Rd, Groton, CT 06340, USA
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Lee HC, Wei YH. Mitochondria and Aging. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2012; 942:311-27. [DOI: 10.1007/978-94-007-2869-1_14] [Citation(s) in RCA: 143] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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Hirasaka K, Lago CU, Kenaston MA, Fathe K, Nowinski SM, Nikawa T, Mills EM. Identification of a redox-modulatory interaction between uncoupling protein 3 and thioredoxin 2 in the mitochondrial intermembrane space. Antioxid Redox Signal 2011; 15:2645-61. [PMID: 21619484 PMCID: PMC3183655 DOI: 10.1089/ars.2011.3888] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
UNLABELLED Uncoupling protein 3 (UCP3) is a member of the mitochondrial solute carrier superfamily that is enriched in skeletal muscle and controls mitochondrial reactive oxygen species (ROS) production, but the mechanisms underlying this function are unclear. AIMS The goal of this work focused on the identification of mechanisms underlying UCP3 functions. RESULTS Here we report that the N-terminal, intermembrane space (IMS)-localized hydrophilic domain of mouse UCP3 interacts with the N-terminal mitochondrial targeting signal of thioredoxin 2 (Trx2), a mitochondrial thiol reductase. Cellular immunoprecipitation and in vitro pull-down assays show that the UCP3-Trx2 complex forms directly, and that the Trx2 N-terminus is both necessary and sufficient to confer UCP3 binding. Mutation studies show that neither a catalytically inactivated Trx2 mutant, nor a mutant Trx2 bearing the N-terminal targeting sequence of cytochrome c oxidase (COXMTS-Trx2) bind UCP3. Biochemical analyses using permeabilized mitochondria, and live cell experiments using bimolecular fluorescence complementation show that the UCP3-Trx2 complex forms specifically in the IMS. Finally, studies in C2C12 myocytes stably overexpressing UCP3 (2.5-fold) and subjected to Trx2 knockdown show that Trx2 is required for the UCP3-dependent mitigation of complex III-driven mitochondrial ROS generation. UCP3 expression was increased in mice fed a high fat diet, leading to increased localization of Trx2 to the IMS. UCP3 overexpression also increased expression of the glucose transporter GLUT4 in a Trx2-dependent fashion. INNOVATION This is the first report of a mitochondrial protein-protein interaction with UCP3 and the first demonstration that UCP3 binds directly, and in cells and tissues with mitochondrial thioredoxin 2. CONCLUSION These studies identify a novel UCP3-Trx2 complex, a novel submitochondrial localization of Trx2, and a mechanism underlying UCP3-regulated mitochondrial ROS production.
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Affiliation(s)
- Katsuya Hirasaka
- Division of Pharmacology/Toxicology, University of Texas at Austin, Austin, Texas 78714, USA
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26
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Ghosh A, Bera S, Ray S, Banerjee T, Ray M. Methylglyoxal induces mitochondria-dependent apoptosis in sarcoma. BIOCHEMISTRY (MOSCOW) 2011; 76:1164-71. [DOI: 10.1134/s0006297911100105] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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27
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Suski JM, Karkucinska-Wieckowska A, Lebiedzinska M, Giorgi C, Szczepanowska J, Szabadkai G, Duszynski J, Pronicki M, Pinton P, Wieckowski MR. p66Shc aging protein in control of fibroblasts cell fate. Int J Mol Sci 2011; 12:5373-89. [PMID: 21954365 PMCID: PMC3179172 DOI: 10.3390/ijms12085373] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2011] [Revised: 08/02/2011] [Accepted: 08/15/2011] [Indexed: 02/05/2023] Open
Abstract
Reactive oxygen species (ROS) are wieldy accepted as one of the main factors of the aging process. These highly reactive compounds modify nucleic acids, proteins and lipids and affect the functionality of mitochondria in the first case and ultimately of the cell. Any agent or genetic modification that affects ROS production and detoxification can be expected to influence longevity. On the other hand, genetic manipulations leading to increased longevity can be expected to involve cellular changes that affect ROS metabolism. The 66-kDa isoform of the growth factor adaptor Shc (p66Shc) has been recognized as a relevant factor to the oxygen radical theory of aging. The most recent data indicate that p66Shc protein regulates life span in mammals and its phosphorylation on serine 36 is important for the initiation of cell death upon oxidative stress. Moreover, there is strong evidence that apart from aging, p66Shc may be implicated in many oxidative stress-associated pathologies, such as diabetes, mitochondrial and neurodegenerative disorders and tumorigenesis. This article summarizes recent knowledge about the role of p66Shc in aging and senescence and how this protein can influence ROS production and detoxification, focusing on studies performed on skin and skin fibroblasts.
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Affiliation(s)
- Jan M. Suski
- Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw 02-093, Poland; E-Mails: (J.M.S.); (M.L.); (J.S.); (J.D.)
- Department of Experimental and Diagnostic Medicine, Section of General Pathology, Interdisciplinary Center for the Study of Inflammation (ICSI) and LTTA Center, University of Ferrara, Ferrara 9-44121, Italy; E-Mails: (C.G.); (P.P.)
| | | | - Magdalena Lebiedzinska
- Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw 02-093, Poland; E-Mails: (J.M.S.); (M.L.); (J.S.); (J.D.)
| | - Carlotta Giorgi
- Department of Experimental and Diagnostic Medicine, Section of General Pathology, Interdisciplinary Center for the Study of Inflammation (ICSI) and LTTA Center, University of Ferrara, Ferrara 9-44121, Italy; E-Mails: (C.G.); (P.P.)
| | - Joanna Szczepanowska
- Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw 02-093, Poland; E-Mails: (J.M.S.); (M.L.); (J.S.); (J.D.)
| | - Gyorgy Szabadkai
- University College London, Department of Cell and Developmental Biology, Consortium for Mitochondrial Research, London WC1E 6BT, UK; E-Mail:
| | - Jerzy Duszynski
- Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw 02-093, Poland; E-Mails: (J.M.S.); (M.L.); (J.S.); (J.D.)
| | - Maciej Pronicki
- Department of Pathology, The Children’s Memorial Health Institute, Warsaw 04-730, Poland; E-Mails: (A.K.-W.); (M.P.)
| | - Paolo Pinton
- Department of Experimental and Diagnostic Medicine, Section of General Pathology, Interdisciplinary Center for the Study of Inflammation (ICSI) and LTTA Center, University of Ferrara, Ferrara 9-44121, Italy; E-Mails: (C.G.); (P.P.)
| | - Mariusz R. Wieckowski
- Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw 02-093, Poland; E-Mails: (J.M.S.); (M.L.); (J.S.); (J.D.)
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Stanley BA, Sivakumaran V, Shi S, McDonald I, Lloyd D, Watson WH, Aon MA, Paolocci N. Thioredoxin reductase-2 is essential for keeping low levels of H(2)O(2) emission from isolated heart mitochondria. J Biol Chem 2011; 286:33669-77. [PMID: 21832082 DOI: 10.1074/jbc.m111.284612] [Citation(s) in RCA: 155] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Respiring mitochondria produce H(2)O(2) continuously. When production exceeds scavenging, H(2)O(2) emission occurs, endangering cell functions. The mitochondrial peroxidase peroxiredoxin-3 reduces H(2)O(2) to water using reducing equivalents from NADPH supplied by thioredoxin-2 (Trx2) and, ultimately, thioredoxin reductase-2 (TrxR2). Here, the contribution of this mitochondrial thioredoxin system to the control of H(2)O(2) emission was studied in isolated mitochondria and cardiomyocytes from mouse or guinea pig heart. Energization of mitochondria by the addition of glutamate/malate resulted in a 10-fold decrease in the ratio of oxidized to reduced Trx2. This shift in redox state was accompanied by an increase in NAD(P)H and was dependent on TrxR2 activity. Inhibition of TrxR2 in isolated mitochondria by auranofin resulted in increased H(2)O(2) emission, an effect that was seen under both forward and reverse electron transport. This effect was independent of changes in NAD(P)H or membrane potential. The effects of auranofin were reproduced in cardiomyocytes; superoxide and H(2)O(2) levels increased, but similarly, there was no effect on NAD(P)H or membrane potential. These data show that energization of mitochondria increases the antioxidant potential of the TrxR2/Trx2 system and that inhibition of TrxR2 results in increased H(2)O(2) emission through a mechanism that is independent of changes in other redox couples.
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Affiliation(s)
- Brian A Stanley
- Division of Cardiology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA
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29
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Iñarrea P, Casanova A, Alava MA, Iturralde M, Cadenas E. Melatonin and steroid hormones activate intermembrane Cu,Zn-superoxide dismutase by means of mitochondrial cytochrome P450. Free Radic Biol Med 2011; 50:1575-81. [PMID: 21397009 PMCID: PMC3144380 DOI: 10.1016/j.freeradbiomed.2011.03.003] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/02/2010] [Revised: 02/12/2011] [Accepted: 03/02/2011] [Indexed: 01/08/2023]
Abstract
Melatonin and steroid hormones are cytochrome P450 (CYP or P450; EC 1.14.14.1) substrates that have antioxidant properties and mitochondrial protective activities. The mitochondrial intermembrane space (IMS) Cu,Zn-superoxide dismutase (SOD1) is activated after oxidative modification of its critical thiol moieties by superoxide anion (O₂(•-)). This study was aimed at investigating the potential association between the hormonal protective antioxidant actions in mitochondria and the regulation of IMS SOD1 activity. Melatonin, testosterone, dihydrotestosterone, estradiol, and vitamin D induced a sustained activation over time of SOD1 in intact mitochondria, showing a bell-shaped enzyme activation dose response with a threshold at 50nM and a maximum effect at 1μM concentration. Enzyme activation was not affected by furafylline, but it was inhibited by omeprazole, ketoconazole, and tiron, thereby supporting the occurrence of a mitochondrial P450 activity and O₂(•-) requirements. Mitochondrial P450-dependent activation of IMS SOD1 prevented O₂(•-)-induced loss of aconitase activity in intact mitochondria respiring in State 3. Optimal protection of aconitase activity was observed at 0.1μM P450 substrate concentration, evidencing a likely oxidative effect on the mitochondrial matrix by higher substrate concentrations. Likewise, enzyme activation mediated by mitochondrial P450 activity delayed CaCl₂-induced loss of transmembrane potential and decreased cytochrome c release. Omeprazole and ketoconazole abrogated both protecting mitochondrial functions promoted by melatonin and steroid hormones.
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Affiliation(s)
- Pedro Iñarrea
- Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain.
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Zhang Q, Dai T, Zhang L, Zhang M, Xiao X, Hu H, Zou P, Liu X, Xiang Q, Su Z, Huang Y, He QY. Identification of potential biomarkers for predicting acute dermal irritation by proteomic analysis. J Appl Toxicol 2011; 31:762-72. [DOI: 10.1002/jat.1630] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2010] [Revised: 10/11/2010] [Accepted: 10/11/2010] [Indexed: 11/06/2022]
Affiliation(s)
- Qihao Zhang
- Institute of Life and Health Engineering, and National Engineering Research Center of Genetic Medicine; Jinan University; Guangzhou; 510632; China
| | - Taoli Dai
- Institute of Life and Health Engineering, and National Engineering Research Center of Genetic Medicine; Jinan University; Guangzhou; 510632; China
| | - Lei Zhang
- Institute of Life and Health Engineering, and National Engineering Research Center of Genetic Medicine; Jinan University; Guangzhou; 510632; China
| | - Minjing Zhang
- Institute of Life and Health Engineering, and National Engineering Research Center of Genetic Medicine; Jinan University; Guangzhou; 510632; China
| | - Xue Xiao
- Institute of Life and Health Engineering, and National Engineering Research Center of Genetic Medicine; Jinan University; Guangzhou; 510632; China
| | - Hao Hu
- Institute of Life and Health Engineering, and National Engineering Research Center of Genetic Medicine; Jinan University; Guangzhou; 510632; China
| | - Ping Zou
- Institute of Life and Health Engineering, and National Engineering Research Center of Genetic Medicine; Jinan University; Guangzhou; 510632; China
| | - Xia Liu
- Institute of Life and Health Engineering, and National Engineering Research Center of Genetic Medicine; Jinan University; Guangzhou; 510632; China
| | - Qi Xiang
- Institute of Life and Health Engineering, and National Engineering Research Center of Genetic Medicine; Jinan University; Guangzhou; 510632; China
| | - Zhijian Su
- Institute of Life and Health Engineering, and National Engineering Research Center of Genetic Medicine; Jinan University; Guangzhou; 510632; China
| | - Yadong Huang
- Institute of Life and Health Engineering, and National Engineering Research Center of Genetic Medicine; Jinan University; Guangzhou; 510632; China
| | - Qing-Yu He
- Institute of Life and Health Engineering, and National Engineering Research Center of Genetic Medicine; Jinan University; Guangzhou; 510632; China
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Wang X, Gong J, Liu X, Zhan R, Kong R, Zhao Y, Wan D, Leng X, Chen M, Qian L. Expression of uncoupling protein 3 in mitochondria protects against stress-induced myocardial injury: a proteomic study. Cell Stress Chaperones 2010; 15:771-9. [PMID: 20383618 PMCID: PMC3024060 DOI: 10.1007/s12192-010-0185-y] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2009] [Revised: 03/01/2010] [Accepted: 03/04/2010] [Indexed: 10/19/2022] Open
Abstract
It has been confirmed that stress plays an important role in the induction and development of cardiovascular diseases, but its mechanism and molecular basis remain unknown. In the present study, a myocardial injury model induced by restraint stress was established in rat. To screen for the related proteins involved in stress-induced myocardial injury, proteomic techniques based on 2-DE and mass spectrometry were used. In our results, ten proteins were found to be altered. The expression of eight of these proteins was increased after restraint stress, including cardiac myosin heavy chain, dihydrolipoamide succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial aldehyde dehydrogenase, H(+)-transporting ATP synthase, albumin, and apolipoprotein A-I precursor. The expression of uncoupling protein 3 (UCP3) and mitochondrial aconitase was decreased. Most of the proteins were related to energy metabolism. Further research indicated that UCP3 may mediate the myocardial cell response induced by restraint stress.
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Affiliation(s)
- Xinxing Wang
- Key Laboratory of Stress Medicine, Tianjing Institute of Hygiene and Environmental Medicine, 1 Dali Road, Heping District, Tianjing, 300050 China
- Department of Stress Medicine, Institute of Health & Environmental Medicine, No.1 DaLi Road, Tianjin, 300050 China
| | - Jingbo Gong
- Key Laboratory of Stress Medicine, Tianjing Institute of Hygiene and Environmental Medicine, 1 Dali Road, Heping District, Tianjing, 300050 China
- Department of Stress Medicine, Institute of Health & Environmental Medicine, No.1 DaLi Road, Tianjin, 300050 China
| | - Xiaohua Liu
- Key Laboratory of Stress Medicine, Tianjing Institute of Hygiene and Environmental Medicine, 1 Dali Road, Heping District, Tianjing, 300050 China
- Department of Stress Medicine, Institute of Health & Environmental Medicine, No.1 DaLi Road, Tianjin, 300050 China
| | - Rui Zhan
- Key Laboratory of Stress Medicine, Tianjing Institute of Hygiene and Environmental Medicine, 1 Dali Road, Heping District, Tianjing, 300050 China
- Department of Stress Medicine, Institute of Health & Environmental Medicine, No.1 DaLi Road, Tianjin, 300050 China
| | - Ruirui Kong
- Key Laboratory of Stress Medicine, Tianjing Institute of Hygiene and Environmental Medicine, 1 Dali Road, Heping District, Tianjing, 300050 China
- Department of Stress Medicine, Institute of Health & Environmental Medicine, No.1 DaLi Road, Tianjin, 300050 China
| | - Yun Zhao
- Key Laboratory of Stress Medicine, Tianjing Institute of Hygiene and Environmental Medicine, 1 Dali Road, Heping District, Tianjing, 300050 China
- Department of Stress Medicine, Institute of Health & Environmental Medicine, No.1 DaLi Road, Tianjin, 300050 China
| | - Di Wan
- Key Laboratory of Stress Medicine, Tianjing Institute of Hygiene and Environmental Medicine, 1 Dali Road, Heping District, Tianjing, 300050 China
- Department of Stress Medicine, Institute of Health & Environmental Medicine, No.1 DaLi Road, Tianjin, 300050 China
| | - Xue Leng
- Key Laboratory of Stress Medicine, Tianjing Institute of Hygiene and Environmental Medicine, 1 Dali Road, Heping District, Tianjing, 300050 China
- Department of Stress Medicine, Institute of Health & Environmental Medicine, No.1 DaLi Road, Tianjin, 300050 China
| | - Ming Chen
- Key Laboratory of Stress Medicine, Tianjing Institute of Hygiene and Environmental Medicine, 1 Dali Road, Heping District, Tianjing, 300050 China
- Department of Stress Medicine, Institute of Health & Environmental Medicine, No.1 DaLi Road, Tianjin, 300050 China
| | - Lingjia Qian
- Key Laboratory of Stress Medicine, Tianjing Institute of Hygiene and Environmental Medicine, 1 Dali Road, Heping District, Tianjing, 300050 China
- Department of Stress Medicine, Institute of Health & Environmental Medicine, No.1 DaLi Road, Tianjin, 300050 China
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Abstract
Mitochondria are primary loci for the intracellular formation and reactions of reactive oxygen and nitrogen species including superoxide (O₂•⁻), hydrogen peroxide (H₂O₂) and peroxynitrite (ONOO⁻). Depending on formation rates and steady-state levels, the mitochondrial-derived short-lived reactive species contribute to signalling events and/or mitochondrial dysfunction through oxidation reactions. Among relevant oxidative modifications in mitochondria, the nitration of the amino acid tyrosine to 3-nitrotyrosine has been recognized in vitro and in vivo. This post-translational modification in mitochondria is promoted by peroxynitrite and other nitrating species and can disturb organelle homeostasis. This study assesses the biochemical mechanisms of protein tyrosine nitration within mitochondria, the main nitration protein targets and the impact of 3-nitrotyrosine formation in the structure, function and fate of modified mitochondrial proteins. Finally, the inhibition of mitochondrial protein tyrosine nitration by endogenous and mitochondrial-targeted antioxidants and their physiological or pharmacological relevance to preserve mitochondrial functions is analysed.
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Affiliation(s)
- Laura Castro
- Department of Biochemistry and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
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Xu X, Chen CNJ, Arriaga EA, Thompson LV. Asymmetric superoxide release inside and outside the mitochondria in skeletal muscle under conditions of aging and disuse. J Appl Physiol (1985) 2010; 109:1133-9. [PMID: 20689097 DOI: 10.1152/japplphysiol.00174.2010] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Superoxide released from mitochondria forms reactive oxygen species that can cause severe oxidative damage and have been associated with aging- and disuse-induced muscle dysfunction. Superoxide is released to both the exterior and the matrix of mitochondria, where oxidative damage is not necessarily the same. This complicates determining the role of mitochondrial superoxide in eliciting oxidative stress in skeletal muscle. A newly developed capillary electrophoretic method analyzes hydroxytriphenylphosphonium ethidium, a superoxide-specific product of triphenylphosphonium hydroethidine, released to outside the mitochondria (supernatant) and retained in the matrix (pellet). In this study, we investigated the mitochondrial superoxide production of soleus (type I) and semimembranosus (type II) muscles of Fischer 344 rats affected by aging (13 vs. 26 mo) and disuse (hindlimb unloading). In agreement with previous studies, overall superoxide production increased with aging and disuse. On the other hand, the new experimental method revealed that superoxide production outside the mitochondria of the soleus does not show a significant age-related increase. Another observation was that the superoxide production increase in the matrix occurs earlier (7 days of disuse) compared with the outside mitochondria (14 days of disuse) in both muscle types. These findings indicate that superoxide release is complex as it occurs asymmetrically at both sides of the mitochondrial inner membrane, and that such release has muscle type and temporal specificity. These findings are important to refine current concepts on oxidative stress associated with muscle aging and disuse.
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Affiliation(s)
- Xin Xu
- Department of Chemistry, Univ. of Minnesota, Minneapolis, MN, USA
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Abstract
The mitochondrion is the most important organelle in determining continued cell survival and cell death. Mitochondrial dysfunction leads to many human maladies, including cardiovascular diseases, neurodegenerative disease, and cancer. These mitochondria-related pathologies range from early infancy to senescence. The central premise of this review is that if mitochondrial abnormalities contribute to the pathological state, alleviating the mitochondrial dysfunction would contribute to attenuating the severity or progression of the disease. Therefore, this review will examine the role of mitochondria in the etiology and progression of several diseases and explore potential therapeutic benefits of targeting mitochondria in mitigating the disease processes. Indeed, recent advances in mitochondrial biology have led to selective targeting of drugs designed to modulate and manipulate mitochondrial function and genomics for therapeutic benefit. These approaches to treat mitochondrial dysfunction rationally could lead to selective protection of cells in different tissues and various disease states. However, most of these approaches are in their infancy.
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Malinska D, Kulawiak B, Kudin AP, Kovacs R, Huchzermeyer C, Kann O, Szewczyk A, Kunz WS. Complex III-dependent superoxide production of brain mitochondria contributes to seizure-related ROS formation. BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 2010; 1797:1163-70. [DOI: 10.1016/j.bbabio.2010.03.001] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/31/2009] [Revised: 02/19/2010] [Accepted: 03/01/2010] [Indexed: 01/16/2023]
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36
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Babich PS, Tsymbalenko NV, Klotchenko SA, Platonova NA, Masalova OO, Zatulovski EA, Shavlovskii MM, Sapronov NS, Puchkova LV. Effect of a Deficiency of Ceruloplasmin Copper in Blood Plasma on Copper Metabolism in the Brain. Bull Exp Biol Med 2010; 148:592-7. [DOI: 10.1007/s10517-010-0772-z] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
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Abstract
During aging, a significant loss of skeletal muscle mass and function occurs that can have a dramatic impact on the quality of life of older individuals. The processes underlying this loss of mass and function are unknown, but a chronic increase in cellular superoxide has been implicated in the contributory mechanisms. Mitochondria are a major cellular site for superoxide generation at complexes I or III of the electron transport chain. Within the mitochondrial matrix, superoxide is converted to hydrogen peroxide though activity of Mn-superoxide dismutase. A portion of the superoxide generated at complex III is also released into the mitochondrial intermembrane space, which contains a recently identified copper, zinc superoxide dismutase (Cu,ZnSOD). Deletion of Cu,ZnSOD has been shown to lead to a phenotype of accelerated age-related loss of skeletal muscle mass and function, although it is unclear whether loss of the enzyme in the intermembrane space or cytosol is important in this respect. It is hypothesized that the processes underlying loss of muscle mass and function in the Cu,ZnSOD knockout mice provide a model that can inform identification of the processes that occur during normal aging.
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Affiliation(s)
- Malcolm J Jackson
- School of Clinical Sciences, University of Liverpool, Liverpool, UK.
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Pardo M, Tirosh O. Protective signalling effect of manganese superoxide dismutase in hypoxia-reoxygenation of hepatocytes. Free Radic Res 2010; 43:1225-39. [PMID: 19905985 DOI: 10.3109/10715760903271256] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
This study investigates the mechanism by which MnSOD exerts its protective effect in hypoxia-reoxygenation (H/R) injury in hepatocytes. Following induction of H/R, MnSOD expression and activity levels increased and remained high for over 24 h. Hepatocytes silenced for MnSOD (siMnSOD) demonstrated increased susceptibility to H/R-induced apoptotic cell death and a lower capacity to generate mitochondrial reactive oxygen species. Microarray and real time PCR analysis of gene expression from siMnSOD cells revealed a number of down-regulated protective genes, including hemeoxygenase-1, glutamate-cysteine ligase and Nrf2, a master regulator of cellular adaptation to stress. Decreased Nrf2 protein expression and nuclear translocation were also confirmed in siMnSOD cells. siMnSOD cells showed low glutathione (GSH) content with no oxidation to GSSG, lower lipid peroxidation levels than their controls and lower mitochondrial membrane potential, which all were even more salient after H/R. Therefore, MnSOD appears to act as a signalling mediator for the activation of survival genes following H/R injury in hepatocytes.
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Affiliation(s)
- Michal Pardo
- Department of Biochemistry and Nutrition, Institute of Biochemistry, Food Science and Nutrition, Robert H Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
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Tikunov A, Johnson CB, Pediaditakis P, Markevich N, Macdonald JM, Lemasters JJ, Holmuhamedov E. Closure of VDAC causes oxidative stress and accelerates the Ca(2+)-induced mitochondrial permeability transition in rat liver mitochondria. Arch Biochem Biophys 2010; 495:174-81. [PMID: 20097153 DOI: 10.1016/j.abb.2010.01.008] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2009] [Accepted: 01/16/2010] [Indexed: 02/07/2023]
Abstract
The electron transport chain of mitochondria is a major source of reactive oxygen species (ROS), which play a critical role in augmenting the Ca(2+)-induced mitochondrial permeability transition (MPT). Mitochondrial release of superoxide anions (O(2)(-)) from the intermembrane space (IMS) to the cytosol is mediated by voltage dependent anion channels (VDAC) in the outer membrane. Here, we examined whether closure of VDAC increases intramitochondrial oxidative stress by blocking efflux of O(2)(-) from the IMS and sensitizing to the Ca(2+)-induced MPT. Treatment of isolated rat liver mitochondria with 5microM G3139, an 18-mer phosphorothioate blocker of VDAC, accelerated onset of the MPT by 6.8+/-1.4min within a range of 100-250microM Ca(2+). G3139-mediated acceleration of the MPT was reversed by 20microM butylated hydroxytoluene, a water soluble antioxidant. Pre-treatment of mitochondria with G3139 also increased accumulation of O(2)(-) in mitochondria, as monitored by dihydroethidium fluorescence, and permeabilization of the mitochondrial outer membrane with digitonin reversed the effect of G3139 on O(2)(-) accumulation. Mathematical modeling of generation and turnover of O(2)(-) within the IMS indicated that closure of VDAC produces a 1.55-fold increase in the steady-state level of mitochondrial O(2)(-). In conclusion, closure of VDAC appears to impede the efflux of superoxide anions from the IMS, resulting in an increased steady-state level of O(2)(-), which causes an internal oxidative stress and sensitizes mitochondria toward the Ca(2+)-induced MPT.
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Affiliation(s)
- Andrey Tikunov
- Department of Cell & Developmental Biology, University of North Carolina at Chapel Hill, 27599, USA
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Abstract
With the aging of the population, we are seeing a global increase in the prevalence of age-related disorders, especially in developed countries. Chronic diseases disproportionately affect the older segment of the population, contributing to disability, a diminished quality of life and an increase in healthcare costs. Increased life expectancy reflects the success of contemporary medicine, which must now respond to the challenges created by this achievement, including the growing burden of chronic illnesses, injuries and disabilities. A well-developed theoretical framework is required to understand the molecular basis of aging. Such a framework is a prerequisite for the development of clinical interventions that will constitute an efficient response to the challenge of age-related health issues. This review critically analyzes the experimental evidence that supports and refutes the Free Radical/Mitochondrial Theory of Aging, which has dominated the field of aging research for almost half a century.
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Affiliation(s)
- Mikhail F Alexeyev
- Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, AL 36688, USA.
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41
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Crane MS, Howie AF, Arthur JR, Nicol F, Crosley LK, Beckett GJ. Modulation of thioredoxin reductase-2 expression in EAhy926 cells: implications for endothelial selenoprotein hierarchy. Biochim Biophys Acta Gen Subj 2009; 1790:1191-7. [PMID: 19595745 DOI: 10.1016/j.bbagen.2009.07.001] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2008] [Revised: 06/10/2009] [Accepted: 07/01/2009] [Indexed: 02/07/2023]
Abstract
BACKGROUND We examined the expression of the mitochondrial selenoenzyme TrxR2 in the endothelial cell line EAhy926 under conditions known to modify its cytoplasmic counterpart TrxR1. METHODS Cells were cultured with varying concentrations of selenite, sulforaphane or the Ca2+ ionophore A23187 for 72-h, prior to assay of TrxR concentration and activity. Further cultures underwent prolonged (7-day) Se-depletion before selenoprotein measurement. RESULTS In Se-deficient cultures, neither Se, A23187 or sulforaphane affected TrxR2 concentration, while these treatments induced TrxR1 concentration (p<0.05). When co-incubated, optimal concentrations of Se (40 nM) and sulforaphane (4 microM) only modestly increased TrxR2 protein (approximately 1.3-fold), compared with TrxR1 (approximately 4-fold). In Se-deficient cells, TrxR activity was unaffected by sulforaphane or A23187. Prolonged Se-depletion caused a comparatively small reduction in TrxR2 (66% TrxR2 retained) against TrxR1 and glutathione peroxidase-1 activity (38% and 17% retained, respectively). CONCLUSIONS The relative resistance of TrxR2 to Se-deprivation and induction by sulforaphane and A23187 suggests TrxR2 lies near the top of the selenoprotein hierarchy in EAhy926 cells and exhibits near maximum expression under a range of culture conditions. In Se deficiency an inactive (possibly truncated) TrxR1 is produced in response to stimulus by sulforaphane and A23187. GENERAL SIGNIFICANCE These observations underpin a likely critical antioxidant role for TrxR2 and TrxR1 in the endothelium.
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Affiliation(s)
- Michael S Crane
- Department of Clinical Biochemistry, Royal Infirmary of Edinburgh, Little France, EH16 4SA Edinburgh, UK.
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42
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Kurosawa T, Nakamura H, Yamaura E, Fujino H, Matsuzawa Y, Kawashima T, Murayama T. Cytotoxicity induced by inhibition of thioredoxin reductases via multiple signaling pathways: Role of cytosolic phospholipase A2α-dependent and -independent release of arachidonic acid. J Cell Physiol 2009; 219:606-16. [DOI: 10.1002/jcp.21703] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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Stowe DF, Camara AKS. Mitochondrial reactive oxygen species production in excitable cells: modulators of mitochondrial and cell function. Antioxid Redox Signal 2009; 11:1373-414. [PMID: 19187004 PMCID: PMC2842133 DOI: 10.1089/ars.2008.2331] [Citation(s) in RCA: 351] [Impact Index Per Article: 21.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/10/2008] [Revised: 01/12/2009] [Accepted: 01/13/2009] [Indexed: 12/14/2022]
Abstract
The mitochondrion is a major source of reactive oxygen species (ROS). Superoxide (O(2)(*-)) is generated under specific bioenergetic conditions at several sites within the electron-transport system; most is converted to H(2)O(2) inside and outside the mitochondrial matrix by superoxide dismutases. H(2)O(2) is a major chemical messenger that, in low amounts and with its products, physiologically modulates cell function. The redox state and ROS scavengers largely control the emission (generation scavenging) of O(2)(*-). Cell ischemia, hypoxia, or toxins can result in excess O(2)(*-) production when the redox state is altered and the ROS scavenger systems are overwhelmed. Too much H(2)O(2) can combine with Fe(2+) complexes to form reactive ferryl species (e.g., Fe(IV) = O(*)). In the presence of nitric oxide (NO(*)), O(2)(*-) forms the reactant peroxynitrite (ONOO(-)), and ONOOH-induced nitrosylation of proteins, DNA, and lipids can modify their structure and function. An initial increase in ROS can cause an even greater increase in ROS and allow excess mitochondrial Ca(2+) entry, both of which are factors that induce cell apoptosis and necrosis. Approaches to reduce excess O(2)(*-) emission include selectively boosting the antioxidant capacity, uncoupling of oxidative phosphorylation to reduce generation of O(2)(*-) by inducing proton leak, and reversibly inhibiting electron transport. Mitochondrial cation channels and exchangers function to maintain matrix homeostasis and likely play a role in modulating mitochondrial function, in part by regulating O(2)(*-) generation. Cell-signaling pathways induced physiologically by ROS include effects on thiol groups and disulfide linkages to modify posttranslationally protein structure to activate/inactivate specific kinase/phosphatase pathways. Hypoxia-inducible factors that stimulate a cascade of gene transcription may be mediated physiologically by ROS. Our knowledge of the role played by ROS and their scavenging systems in modulation of cell function and cell death has grown exponentially over the past few years, but we are still limited in how to apply this knowledge to develop its full therapeutic potential.
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Affiliation(s)
- David F Stowe
- Anesthesiology Research Laboratories, Department of Anesthesiology, The Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
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44
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Brigelius-Flohé R, Kipp A. Glutathione peroxidases in different stages of carcinogenesis. Biochim Biophys Acta Gen Subj 2009; 1790:1555-68. [PMID: 19289149 DOI: 10.1016/j.bbagen.2009.03.006] [Citation(s) in RCA: 218] [Impact Index Per Article: 13.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2009] [Revised: 03/05/2009] [Accepted: 03/08/2009] [Indexed: 01/12/2023]
Abstract
Cancer cells produce high amounts of reactive oxygen species (ROS) and evade apoptosis. Hydroperoxides support proliferation, invasion, migration and angiogenesis, but at higher levels induce apoptosis, thus being pro- and anti-carcinogenic. Accordingly, glutathione peroxidases (GPxs) regulating hydroperoxide levels might have dual roles too. GPx1, clearly an antioxidant enzyme, is down-regulated in many cancer cells. Its main role would be prevention of cancer initiation by ROS-mediated DNA damage. GPx2 is up-regulated in cancer cells. GPx1/GPx2 double knockout mice develop colitis and intestinal cancer. However, GPx2 knockdown cancer cells grow better in vitro and in vivo probably reflecting the physiological role of GPx2 in intestinal mucosa homeostasis. GPx2 counteracts COX-2 expression and PGE(2) production, which explains its potential to inhibit migration and invasion of cultured cancer cells. Overexpression of GPx3 inhibits tumor growth and metastasis. GPx4 is decreased in cancer tissues. GPx4-overexpressing cancer cells have low COX-2 activity and tumors derived therefrom are smaller than from control cells and do not metastasize. Collectively, GPxs prevent cancer initiation by removing hydroperoxides. GPx4 inhibits but GPx2 supports growth of established tumors. Metastasis, but also apoptosis, is inhibited by all GPxs. GPx-mediated regulation of COX/LOX activities may be relevant to early stages of inflammation-mediated carcinogenesis.
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Affiliation(s)
- Regina Brigelius-Flohé
- Department Biochemistry of Micronutrients, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, D-14558 Nuthetal, Germany.
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Jezek P, Plecitá-Hlavatá L. Mitochondrial reticulum network dynamics in relation to oxidative stress, redox regulation, and hypoxia. Int J Biochem Cell Biol 2009; 41:1790-804. [PMID: 19703650 DOI: 10.1016/j.biocel.2009.02.014] [Citation(s) in RCA: 76] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2008] [Revised: 02/17/2009] [Accepted: 02/19/2009] [Indexed: 02/03/2023]
Abstract
A single mitochondrial network in the cell undergoes constant fission and fusion primarily depending on the local GTP gradients and the mitochondrial energetics. Here we overview the main properties and regulation of pro-fusion and pro-fission mitodynamins, i.e. dynamins-related GTPases responsible for mitochondrial shape-forming, such as pro-fusion mitofusins MFN1, MFN2, and the inner membrane-residing long OPA1 isoforms, and pro-fission mitodynamins FIS1, MFF, and DRP1 multimers required for scission. Notably, the OPA1 cleavage into non-functional short isoforms at a diminished ATP level (collapsed membrane potential) and the DRP1 recruitment upon phosphorylation by various kinases are overviewed. Possible responses of mitodynamins to the oxidative stress, hypoxia, and concomitant mtDNA mutations are also discussed. We hypothesize that the increased GTP formation within the Krebs cycle followed by the GTP export via the ADP/ATP carrier shift the balance between fission and fusion towards fusion by activating the GTPase domain of OPA1 located in the peripheral intermembrane space (PIMS). Since the protein milieu of PIMS is kept at the prevailing oxidized redox potential by the TOM, MIA40 and ALR/Erv1 import-redox trapping system, redox regulations shift the protein environment of PIMS to a more reduced state due to the higher substrate load and increased respiration. A higher cytochrome c turnover rate may prevent electron transfer from ALR/Erv1 to cytochrome c. Nevertheless, the putative links between the mitodynamin responses, mitochondrial morphology and the changes in the mitochondrial bioenergetics, superoxide production, and hypoxia are yet to be elucidated, including the precise basis for signaling by the mitochondrion-derived vesicles.
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Affiliation(s)
- Petr Jezek
- Department of Membrane Transport Biophysics, No. 75, Institute of Physiology, v.v.i., Academy of Sciences of the Czech Republic, Vídenská 1083, CZ 14220 Prague, Czech Republic.
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Arnér ESJ. Focus on mammalian thioredoxin reductases--important selenoproteins with versatile functions. Biochim Biophys Acta Gen Subj 2009; 1790:495-526. [PMID: 19364476 DOI: 10.1016/j.bbagen.2009.01.014] [Citation(s) in RCA: 504] [Impact Index Per Article: 31.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2008] [Accepted: 01/30/2009] [Indexed: 02/07/2023]
Abstract
Thioredoxin systems, involving redox active thioredoxins and thioredoxin reductases, sustain a number of important thioredoxin-dependent pathways. These redox active proteins support several processes crucial for cell function, cell proliferation, antioxidant defense and redox-regulated signaling cascades. Mammalian thioredoxin reductases are selenium-containing flavoprotein oxidoreductases, dependent upon a selenocysteine residue for reduction of the active site disulfide in thioredoxins. Their activity is required for normal thioredoxin function. The mammalian thioredoxin reductases also display surprisingly multifaceted properties and functions beyond thioredoxin reduction. Expressed from three separate genes (in human named TXNRD1, TXNRD2 and TXNRD3), the thioredoxin reductases can each reduce a number of different types of substrates in different cellular compartments. Their expression patterns involve intriguingly complex transcriptional mechanisms resulting in several splice variants, encoding a number of protein variants likely to have specialized functions in a cell- and tissue-type restricted manner. The thioredoxin reductases are also targeted by a number of drugs and compounds having an impact on cell function and promoting oxidative stress, some of which are used in treatment of rheumatoid arthritis, cancer or other diseases. However, potential specific or essential roles for different forms of human or mouse thioredoxin reductases in health or disease are still rather unclear, although it is known that at least the murine Txnrd1 and Txnrd2 genes are essential for normal development during embryogenesis. This review is a survey of current knowledge of mammalian thioredoxin reductase function and expression, with a focus on human and mouse and a discussion of the striking complexity of these proteins. Several yet open questions regarding their regulation and roles in different cells or tissues are emphasized. It is concluded that the intriguingly complex regulation and function of mammalian thioredoxin reductases within the cellular context and in intact mammals strongly suggests that their functions are highly fi ne-tuned with the many pathways involving thioredoxins and thioredoxin-related proteins. These selenoproteins furthermore propagate many functions beyond a reduction of thioredoxins. Aberrant regulation of thioredoxin reductases, or a particular dependence upon these enzymes in diseased cells, may underlie their presumed therapeutic importance as enzymatic targets using electrophilic drugs. These reductases are also likely to mediate several of the effects on health and disease that are linked to different levels of nutritional selenium intake. The thioredoxin reductases and their splice variants may be pivotal components of diverse cellular signaling pathways, having importance in several redox-related aspects of health and disease. Clearly, a detailed understanding of mammalian thioredoxin reductases is necessary for a full comprehension of the thioredoxin system and of selenium dependent processes in mammals.
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Affiliation(s)
- Elias S J Arnér
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
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Ahtoniemi T, Jaronen M, Keksa-Goldsteine V, Goldsteins G, Koistinaho J. Mutant SOD1 from spinal cord of G93A rats is destabilized and binds to inner mitochondrial membrane. Neurobiol Dis 2008; 32:479-85. [PMID: 18817872 DOI: 10.1016/j.nbd.2008.08.010] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2008] [Revised: 08/20/2008] [Accepted: 08/22/2008] [Indexed: 11/17/2022] Open
Abstract
Mutations in Cu/Zn superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS). Mechanisms of mutant SOD1 toxicity are unknown, but increased SOD1 activity can boost production of reactive oxygen species (ROS) in the mitochondrial intermembrane space (IMS). Using non-reducing SDS-PAGE we found that in G93A-SOD1 rats the mutant SOD1 was prominently destabilized only in the diseased spinal cord, where this mutant enzyme was also up regulated in the IMS with increased ability to bind the inner membrane of isolated non-transgenic mitoplasts. These mitoplasts increased ROS production when exposed to mutant SOD1 from the spinal cord at the presymptomatic stage. The levels of disulfide-reduced SOD1 peaked at the end stage of the disease, whereas protein disulfide isomerase (PDI), a chaperone capable of rearranging disulfide bonds between cysteine residues of SOD1, was increased prior to the end stage. IMS binding and increased ROS production by destabilized SOD1 may contribute to mitochondrial damage in G93A-SOD1 rats.
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Affiliation(s)
- Toni Ahtoniemi
- Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland
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Cox AG, Brown KK, Arner ESJ, Hampton MB. The thioredoxin reductase inhibitor auranofin triggers apoptosis through a Bax/Bak-dependent process that involves peroxiredoxin 3 oxidation. Biochem Pharmacol 2008; 76:1097-109. [PMID: 18789312 DOI: 10.1016/j.bcp.2008.08.021] [Citation(s) in RCA: 124] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2008] [Revised: 08/10/2008] [Accepted: 08/14/2008] [Indexed: 01/14/2023]
Abstract
Thioredoxin reductase (TrxR) is a key selenoprotein antioxidant enzyme and a potential target for anti-cancer drugs. One potent inhibitor of TrxR is the gold (I) compound auranofin, which can trigger mitochondrial-dependent apoptosis pathways. The exact mechanism of apoptosis induction by auranofin is not yet clear, but there are indications that mitochondrial oxidative stress is a central event. We assessed the redox state of the peroxiredoxins (Prxs) in Jurkat T-lymphoma cells treated with auranofin, and found that mitochondrial Prx3 was considerably more sensitive to oxidation than the cytosolic Prx1 and 2, indicating selective mitochondrial stress. Prx3 oxidation was detected at apoptotic doses of auranofin in several cell types, and occurred before other mitochondrial events including cytochrome c release and mitochondrial depolarisation. Auranofin was also able to sensitise U937 cells to TNF-alpha-mediated apoptosis. Auranofin-induced apoptosis was effectively blocked by the overexpression of Bcl-2, and Bax/Bak deficient mouse embryonic fibroblasts were also resistant to apoptosis, indicating a central role for the pro-apoptotic proteins of this family in auranofin-triggered apoptosis. Auranofin exposure inhibited the proliferation of apoptosis-resistant cells, and at higher doses of auranofin could cause cell death through necrosis. We conclude that auranofin induces apoptosis in cells through a Bax/Bak-dependent mechanism associated with selective disruption of mitochondrial redox homeostasis in conjunction with oxidation of Prx3.
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Affiliation(s)
- Andrew G Cox
- Free Radical Research Group, Department of Pathology, University of Otago, Christchurch, New Zealand
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Gertz M, Fischer F, Wolters D, Steegborn C. Activation of the lifespan regulator p66Shc through reversible disulfide bond formation. Proc Natl Acad Sci U S A 2008; 105:5705-9. [PMID: 18413607 PMCID: PMC2311372 DOI: 10.1073/pnas.0800691105] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2007] [Indexed: 11/18/2022] Open
Abstract
Cell fate and organismal lifespan are controlled by a complex signaling network whose dysfunction can cause a variety of aging-related diseases. An important protection against these failures is cellular apoptosis, which can be induced by p66(Shc) in response to cellular stress. The precise mechanisms of p66(Shc) action and regulation and the function of the p66(Shc)-specific N terminus remain to be identified. Here, we show that the p66(Shc) N terminus forms a redox module responsible for apoptosis initiation, and that this module can be activated through reversible tetramerization by forming two disulfide bonds. Glutathione and thioredoxins can reduce and inactivate p66(Shc), resulting in a thiol-based redox sensor system that initiates apoptosis once cellular protection systems cannot cope anymore with cellular stress.
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Affiliation(s)
| | - Frank Fischer
- Analytical Chemistry, Ruhr-University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany
| | - Dirk Wolters
- Analytical Chemistry, Ruhr-University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany
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