Mitochondrial Thioredoxin System

Patenaude, Alexandre; Murthy, M.R.V.; Mirault, Marc-Édouard

doi:10.1074/jbc.m402496200

Cited by 102 publications

(36 citation statements)

References 83 publications

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“…Earlier reports (51,58,62) have suggested an important function of the thioredoxin system in redox regulation of cell growth. In our study, we found a significantly slower growth of the TrxR1-and TrxR2-overexpressing cells as compared with their controls.…”

Section: Discussionmentioning

confidence: 99%

Overexpression of Enzymatically Active Human Cytosolic and Mitochondrial Thioredoxin Reductase in HEK-293 Cells

Nalvarte

Damdimopoulos

Nystöm

et al. 2004

Journal of Biological Chemistry

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The mammalian thioredoxin reductases (TrxR) are selenoproteins containing a catalytically active selenocysteine residue (Sec) and are important enzymes in cellular redox control. The cotranslational incorporation of Sec, necessary for activity, is governed by a stem-loop structure in the 3-untranslated region of the mRNA and demands adequate selenium availability. The complicated translation machinery required for Sec incorporation is a major obstacle in isolating mammalian cell lines stably overexpressing selenoproteins. In this work we report on the development and characterization of stably transfected human embryonic kidney 293 cells that overexpress enzymatically active selenocysteine-containing cytosolic TrxR1 or mitochondrial TrxR2. We demonstrate that the overexpression of selenium-containing TrxR1 results in lower expression and activity of the endogenous selenoprotein glutathione peroxidase and that the activity of overexpressed TrxRs, rather than the protein amount, can be increased by selenium supplementation in the cell growth media. We also found that the TrxRoverexpressing cells grew slower over a wide range of selenium concentrations, which was an effect apparently not related to increased apoptosis nor to fatally altered intracellular levels of reactive oxygen species. Most surprisingly, the TrxR1-or TrxR2-overexpressing cells also induced novel expression of the epithelial markers CK18, CK-Cam5.2, and BerEP4, suggestive of a stimulation of cellular differentiation.Thioredoxin reductases (TrxRs) 1 are members of the nucleotide-disulfide oxidoreductase family and are ubiquitously found in mammalian tissues. Two main isoforms of thioredoxin reductases exist in mammals: the classical cytosolic form (TrxR1, the TXNRD1 gene product) (1, 2), and the mitochondrial form (TrxR2) (3, 4). A third form, thioredoxin and glutathione reductase, is mainly expressed in testis (5). All mammalian TrxRs are homodimeric selenocysteine-containing enzymes (6) that share high sequence homology between each other and contain an NADPH binding domain and a FAD binding domain per subunit (7). TrxRs reduce and thereby activate thioredoxins (Trxs), which are small ubiquitous proteins with a conserved active site sequence (-Trp-Cys-Gly-ProCys-) that catalyze many redox reactions through the reversible oxidation of the active site dithiol to a disulfide. Trxs are involved in a variety of reactions, such as redox regulation of transcription factors (8, 9), reduction of hydroperoxides (4, 10), and modulation of cell growth (11) and apoptosis (12, 13). The selenocysteine residue (Sec) that is found at the C terminus in TrxRs (-Gly-Cys-Sec-Gly-COOH) is encoded by a UGA codon that is normally identified as a stop codon, but which can be transcribed as Sec in the presence of a selenocysteine insertion sequence (SECIS) element in the 3Ј-UTR of the corresponding mRNA (14). The C-terminal Cys-Sec motif is located on a presumably flexible arm of the enzyme that is kept reduced by the N-terminal redox active motif (-Cys-Val-Asn-Val-Gly...

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Section: Discussionmentioning

confidence: 99%

Overexpression of Enzymatically Active Human Cytosolic and Mitochondrial Thioredoxin Reductase in HEK-293 Cells

Nalvarte

Damdimopoulos

Nystöm

et al. 2004

Journal of Biological Chemistry

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“…The mitochondrial thioredoxin sys tem may be essential for mammalian development because disruption of Trx2 gene in the mouse resulted in massive apoptosis during early embryogenesis and embry onic lethality [166]. However, overexpression of Trx2 or TrxR2, or both, does not necessarily improve cell survival or resistance to ROS promoting factors, indicating that perhaps an unidentified variable controls the effect of these proteins [167].…”

Section: Mitochondrial Ros Detoxifying Systemsmentioning

confidence: 98%

Mitochondrial metabolism of reactive oxygen species

Andreyev

Kushnareva

Starkov

2005

Biochemistry (Moscow)

1,135

957

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Oxidative stress is considered a major contributor to etiology of both "normal" senescence and severe pathologies with serious public health implications. Mitochondria generate reactive oxygen species (ROS) that are thought to augment intracellular oxidative stress. Mitochondria possess at least nine known sites that are capable of generating superoxide anion, a progenitor ROS. Mitochondria also possess numerous ROS defense systems that are much less studied. Studies of the last three decades shed light on many important mechanistic details of mitochondrial ROS production, but the bigger picture remains obscure. This review summarizes the current knowledge about major components involved in mitochondrial ROS metabolism and factors that regulate ROS generation and removal. An integrative, systemic approach is applied to analysis of mitochondrial ROS metabolism, which is now dissected into mitochondrial ROS production, mitochondrial ROS removal, and mitochondrial ROS emission. It is suggested that mitochondria augment intracellular oxidative stress due primarily to failure of their ROS removal systems, whereas the role of mitochondrial ROS emission is yet to be determined and a net increase in mitochondrial ROS production in situ remains to be demonstrated.

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“…After removal of the solvent, the crude product was purified over silica gel using ethyl CH 2 Cl 2 /MeOH (v/v, 9:1) as the eluent to yield Mito-Naph as a yellow solid (0. 13 Synthetic Materials and Methods. All reactions were carried out under nitrogen atmosphere.…”

Section: ■ Conclusionmentioning

confidence: 99%

Mitochondrial Thioredoxin-Responding Off–On Fluorescent Probe

et al. 2012

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We synthesized a new probe, Mito-Naph, to visualize mitochondrial thioredoxin (Trx) activity in cells. A fluorescence off-on change is induced by disulfide cleavage of the probe, resulting from a reaction with Trx and subsequent intramolecular cyclization by the released thiolate to give a fluorescent product. By measuring the fluorescence at 540 nm, Trx activity can be detected at nanomolar concentrations (down to 50 nM) well below its physiological levels. The in vitro and in vivo Trx preference of Mito-Naph was demonstrated by fluorometric and confocal microscopic experiments. In vitro kinetic analysis of the disulfide bond cleavage revealed that the second-order rate constant for Trx is (4.04 ± 0.26) × 10(3) (M s)(-1), approximately 5000 times faster than that for GSH. The inhibition experiments involving PX-12, a selective inhibitor of Trx, also revealed that the emission from Mito-Naph significantly decreased in PX-12 dose-dependent manners, both in living cells and in cellular protein extracts. The Trx preference was further supported by an observation that the fluorescence intensity of rat liver extract was decreased according to the Trx depletion by immunoprecipitation. On the basis of these results, it is concluded that Mito-Naph preferentially reacts with Trx, compared with other biological thiols containing amino acids in vitro and in vivo.

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Mitochondrial Thioredoxin System

Cited by 102 publications

References 83 publications

Overexpression of Enzymatically Active Human Cytosolic and Mitochondrial Thioredoxin Reductase in HEK-293 Cells

Overexpression of Enzymatically Active Human Cytosolic and Mitochondrial Thioredoxin Reductase in HEK-293 Cells

Mitochondrial metabolism of reactive oxygen species

Mitochondrial Thioredoxin-Responding Off–On Fluorescent Probe

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