Control of extracellular cysteine/cystine redox state by HT-29 cells is independent of cellular glutathione

Anderson, Corinna L.; Iyer, Smita S.; Ziegler, Thomas R.; Jones, Dean P.

doi:10.1152/ajpregu.00195.2007

Cited by 55 publications

(60 citation statements)

References 33 publications

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“…Concentrations of GSH and GSSG obtained from HPLC analysis were normalized to the protein concentration of each sample prepared using cultured HT29 cells. Because of the proliferating nature of HT29 cells under normal conditions, GSH (3.8 -4.6 mM) and GSSG (41.7-113.8 M) concentrations and E h GSSG level (-254.0 ϳ Ϫ241.2 mV) measured in HT29 cells at control condition varied slightly by experiments, consistent with previous reports (20,28,36).…”

Section: Measurements Of Trx1 and Gsh/gssg Redoxsupporting

confidence: 90%

“…In the current study, we selected human colon carcinoma HT29 cells because the Trx and GSH systems have been extensively characterized and have different steady states of oxidation under defined cell culture conditions (20,28,29). We performed dose-response studies to obtain concentration of ARF that oxidized Trx1 without oxidation of GSH/GSSG and to obtain concentration of BSO that oxidized GSH/GSSG without oxidation of Trx1.…”

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confidence: 99%

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Selective Targeting of the Cysteine Proteome by Thioredoxin and Glutathione Redox Systems

Roede

Walker

et al. 2013

Molecular & Cellular Proteomics

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Thioredoxin (Trx) and GSH are the major thiol antioxidants protecting cells from oxidative stress-induced cytotoxicity. Redox states of Trx and GSH have been used as indicators of oxidative stress. Accumulating studies suggest that Trx and GSH redox systems regulate cell signaling and metabolic pathways differently and independently during diverse stressful conditions. In the current study, we used a mass spectrometry-based redox proteomics approach to test responses of the cysteine (Cys) proteome to selective disruption of the Trx-and GSH-dependent systems. Auranofin (ARF) was used to inhibit Trx reductase without detectable oxidation of the GSH/GSSG couple, and buthionine sulfoximine (BSO) was used to deplete GSH without detectable oxidation of Trx1. Results for 606 Cys-containing peptides (peptidyl Cys) showed that 36% were oxidized more than 1.3-fold by ARF, whereas BSO-induced oxidation of peptidyl Cys was only 10%. Mean fold oxidation of these peptides was also higher by ARF than BSO treatment. Analysis of potential functional pathways showed that ARF oxidized peptides associated with glycolysis, cytoskeleton remodeling, translation and cell adhesion. Of 60 peptidyl Cys oxidized due to depletion of GSH, 41 were also oxidized by ARF and included proteins of translation and cell adhesion but not glycolysis or cytoskeletal remodeling. Studies to test functional correlates showed that pyruvate kinase activity and lactate levels were decreased with ARF but not BSO, confirming the effects on glycolysis-associated proteins are sensitive to oxidation by ARF. These data show that the Trx system regulates a broader range of proteins than the GSH system, support distinct function of Oxidative stress is associated with numerous human diseases and results from alteration in cellular redox systems (1-3). Cellular redox mechanisms and signaling are controlled by two major thiol antioxidants, thioredoxin (Trx) 1 and glutathione (GSH). The Trx system, composed of Trx, Trx reductase (TrxR), Trx peroxidase/peroxiredoxin (Prx), and NADPH, has a wide range of cellular activities in redox control, cell proliferation, growth and survival, regulation of transcription, and cell morphology and structure (4 -6). The Trx family includes evolutionary conserved proteins that contain two catalytically active cysteine (Cys) residues that can reduce disulfide bonds of numerous target proteins in the Cys proteome, e.g. apoptosis signaling kinase-1 (ASK-1), Trx1-interacting protein (Txnip), transcription factors, and actin. In addition to catalytic activity, Trx binds to target proteins and further controls protein activity and biological function. For example, ASK-1 binds to Trx in physiologic conditions; however, under stressful conditions, it is dissociated from Trx, which then stimulates apoptotic signaling leading to cell death (7). The diverse functions of Trx suggest a nonspecific nature to its function, yet kinetic studies also show differences in activities with different substrates (8 -10), implying that an underlying redox organi...

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Section: Measurements Of Trx1 and Gsh/gssg Redoxsupporting

confidence: 90%

mentioning

confidence: 99%

Selective Targeting of the Cysteine Proteome by Thioredoxin and Glutathione Redox Systems

Roede

Walker

et al. 2013

Molecular & Cellular Proteomics

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“…This is 10-to 1000-fold less than the metabolic rate of ATP synthesis, yet maximal rates are orders of magnitude greater than GSH synthesis. The synthesis of GSH is low (~ 0.024 mM/min in HT-29 cells [26], 0.1 mM/min for liver [111]) such that the distribution among cellular compartments can happen on a much faster time scale. From this, one can deduce that other factors are driving the fluxes of GSH and GSSG between compartments and not the synthesis rate of GSH.…”

Section: Non-equilibrium Thermodynamicsmentioning

confidence: 99%

“…For high import rates across a membrane such as in the mitochondria, this forced balance means that either the mitochondria must utilize GSH at a high rate, or bidirectional transport exists to reduce the net rate into the mitochondria. From the diagram, one can observe that with a low synthesis rate (24 µM/min) [26,108], the transport balance must be very fast because a high utilization/degradation rate would rapidly deplete cytoplasmic GSH. The discrimination between the two options may be compartment-specific.…”

Section: Linear Equations For Modeling Non-equilibrium Systemsmentioning

confidence: 99%

Nonequilibrium thermodynamics of thiol/disulfide redox systems: A perspective on redox systems biology

Kemp

Jones

2008

Free Radical Biology and Medicine

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Understanding the dynamics of redox elements in biologic systems remains a major challenge for redox signaling and oxidative stress research. Central redox elements include evolutionarily conserved subsets of cysteines and methionines of proteins which function as sulfur switches and labile reactive oxygen species (ROS) and reactive nitrogen species (RNS) which function in redox signaling. The sulfur switches depend upon redox environments in which rates of oxidation are balanced with rates of reduction through the thioredoxins, glutathione/glutathione disulfide and cysteine/cystine redox couples. These central couples, which we term redox control nodes, are maintained at stable but non-equilibrium steady states, are largely independently regulated in different subcellular compartments and are quasi-independent from each other within compartments. Disruption of the redox control nodes can differentially affect sulfur switches, thereby creating a diversity of oxidative stress responses. Systems biology provides approaches to address the complexity of these responses. In the present review, we summarize thiol/disulfide pathway, redox potential and rate information as a basis for kinetic modeling of sulfur switches. The summary identifies gaps in knowledge especially related to redox communication between compartments, definition of redox pathways and discrimination between types of sulfur switches. A formulation for kinetic modeling of GSH/GSSG redox control indicates that systems biology could encourage novel therapeutic approaches to protect against oxidative stress by identifying specific redox-sensitive sites which could be targeted for intervention.

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“…The efflux of GSH from astrocytes and its subsequent metabolism by ␥-glutamyltranspeptidase and aminopeptidase N provides an extracellular source of cysteine that is transported into neurons to support glutathione production in these cells (Yudkoff et al, 1990;Dringen et al, 1999Dringen et al, , 2001Wang and Cynader, 2000). Additional studies indicate that antioxidant support provided by these astrocytes is not limited to the efflux of glutathione, but should also be expanded to include the export of cysteine (Anderson et al, 2007;Banjac et al, 2008;Conrad and Sato, 2012). Thus, accumulating evidence suggests that extracellular .…”

Section: System X Cmentioning

confidence: 99%

Thinking Outside the Cleft to Understand Synaptic Activity: Contribution of the Cystine-Glutamate Antiporter (System xc−) to Normal and Pathological Glutamatergic Signaling

Bridges

Lutgen

Lobner

et al. 2012

Pharmacological Reviews

183

143

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Control of extracellular cysteine/cystine redox state by HT-29 cells is independent of cellular glutathione

Cited by 55 publications

References 33 publications

Selective Targeting of the Cysteine Proteome by Thioredoxin and Glutathione Redox Systems

Selective Targeting of the Cysteine Proteome by Thioredoxin and Glutathione Redox Systems

Nonequilibrium thermodynamics of thiol/disulfide redox systems: A perspective on redox systems biology

Thinking Outside the Cleft to Understand Synaptic Activity: Contribution of the Cystine-Glutamate Antiporter (System xc−) to Normal and Pathological Glutamatergic Signaling

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