Balanced Regulation of Redox Status of Intracellular Thioredoxin Revealed by in-Cell NMR

Mochizuki, Ayano; Saso, Arata; Zhao, Qingci; Kubo, Shugo; Nishida, N.; Shimada, Ichio

doi:10.1021/jacs.8b00426

Cited by 41 publications

(64 citation statements)

References 30 publications

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“…This competition between GSH and Trx1 for BQ will depend, at least in part, on the concentrations of these compounds and their relative rates of reaction with BQ. Whilst the GSH concentration in cells is typically 1–5 mM [42], the cellular concentration of Trx appears to be more variable and cell-type dependent [43,44]. Recent (minimally-invasive) NMR analyses indicate that normal physiological levels of Trx are 10–70 μM, with this dependent on external factors/stimuli [43], and much lower than for GSH.…”

Section: Discussionmentioning

confidence: 99%

“…Whilst the GSH concentration in cells is typically 1–5 mM [42], the cellular concentration of Trx appears to be more variable and cell-type dependent [43,44]. Recent (minimally-invasive) NMR analyses indicate that normal physiological levels of Trx are 10–70 μM, with this dependent on external factors/stimuli [43], and much lower than for GSH. We have reported kinetic data for the reaction of GSH with BQ, with k 2 ~ 6 × 10 5 M −1 s −1 at 10 °C and pH 7.0 [9].…”

Section: Discussionmentioning

confidence: 99%

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Modification of Cys residues in human thioredoxin-1 by p-benzoquinone causes inhibition of its catalytic activity and activation of the ASK1/p38-MAPK signalling pathway

et al. 2020

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Quinones can modify biological molecules through both redox-cycling reactions that yield radicals (semiquinone, superoxide and hydroxyl) and via covalent adduction to nucleophiles (e.g. thiols and amines). Kinetic data indicate that Cys residues in GSH and proteins are major targets. In the studies reported here, the interactions of a prototypic quinone compound, p-benzoquinone (BQ), with the key redox protein, thioredoxin-1 (Trx1) were examined. BQ binds covalently with isolated Trx1 forming quinoprotein adducts, resulting in a concentration-dependent loss of enzyme activity and crosslink formation. Mass spectrometry peptide mass mapping data indicate that BQ forms adducts with all of the Trx1 Cys residues. Glutathione (GSH) reacts competitively with BQ, and thereby modulates the loss of activity and crosslink formation. Exposure of macrophage-like (J774A.1) cells to BQ results in a dose-dependent loss of Trx and thioredoxin reductase (TrxR) activities, quinoprotein formation, and a decrease in GSH levels without a concomitant increase in oxidized glutathione. GSH depletion aggravates the loss of Trx and TrxR activity. These data are consistent with adduction of GSH to BQ being a primary protective pathway. Reaction of BQ with Trx in cells resulted in the activation of apoptosis signal-regulating kinase 1 (ASK1), and p38 mitogen-activated protein kinase (MAPK) leading to apoptotic cell death. These data suggest that BQ reacts covalently with Cys residues in Trx, including at the active site, leading to enzyme inactivation and protein cross-linking. Modification of the Cys residues in Trx also results in activation of the ASK1/p38-MAPK signalling pathway and promotion of apoptotic cell death.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Modification of Cys residues in human thioredoxin-1 by p-benzoquinone causes inhibition of its catalytic activity and activation of the ASK1/p38-MAPK signalling pathway

et al. 2020

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“…where EDTT, pH7 is the redox mid-potential of DTT (-332 mV [45,46]), R is the gas constant (1.987 10 -3 kcal.K −1 .mol −1 ) and T is the temperature (283 K), n is the number of electrons (2), and F is the Faraday constant (95484.6 J.V -1 .mol -1 ). 1 H- 15 N fast HSQC were recorded at 283K for all samples, the data were transformed in nmrPipe [40], and the signal intensity at frequency of reduced C23 (δ 15 [41].…”

Section: Thermodynamic Of the Redox Transitionmentioning

confidence: 99%

In-Situ Flexibility of Both Redox-States of the Chloroplast Regulatory Protein CP12

Launay¹,

Bornet²,

Cantrelle³

et al. 2021

Preprint

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In the chloroplast, Calvin-Benson-Bassham enzymes are active in the reducing environment imposed in the light via the electrons from the photosystems. In the dark these enzymes are inhibited, and this regulation is mainly mediated via oxidation of key regulatory cysteine residues. CP12 is a small protein that plays a role in this regulation with four cysteine residues that undergo a redox transition. Using amide-proton exchange with solvent measured by nuclear magnetic resonance (NMR) and mass-spectrometry, we confirmed that reduced CP12 is intrinsically disordered. Using real-time NMR, we showed that the oxidation of the two disulfide bridges are simultaneous. In oxidized CP12, the C23-C31 pair is in a region that undergoes a conformational exchange in the NMR-intermediate timescale. The C66-C75 pair is in the C-terminus that folds into a stable helical turn. We confirmed that these structural states exist in a physiologically relevant environment that is, in cell extract from Chlamydomonas reinhardtii. Consistent with these structural equilibria, the reduction is slower for the C66-C75 pair compared to the C23-C31 pair. Finally, the redox mid-potentials for the two cysteine pairs differ and are similar to those found for phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase, that we relate to the regulatory role of CP12.

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“…The versatility of NMR stems from the significant freedom allowed in experimental conditions. One such highlight is in-cell NMR, which allows the analysis of the dynamic behavior of proteins and nucleic acids inside living cells [113][114][115][116][117][118][119]. The application of NMR dynamics information to drug development has also not been limited to conventional modalities.…”

Section: Future Perspectivesmentioning

confidence: 99%

Spotlight on the Ballet of Proteins: The Structural Dynamic Properties of Proteins Illuminated by Solution NMR

Tokunaga

Viennet

Arthanari

et al. 2020

IJMS

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Solution NMR spectroscopy is a unique and powerful technique that has the ability to directly connect the structural dynamics of proteins in physiological conditions to their activity and function. Here, we summarize recent studies in which solution NMR contributed to the discovery of relationships between key dynamic properties of proteins and functional mechanisms in important biological systems. The capacity of NMR to quantify the dynamics of proteins over a range of time scales and to detect lowly populated protein conformations plays a critical role in its power to unveil functional protein dynamics. This analysis of dynamics is not only important for the understanding of biological function, but also in the design of specific ligands for pharmacologically important proteins. Thus, the dynamic view of structure provided by NMR is of importance in both basic and applied biology.

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Balanced Regulation of Redox Status of Intracellular Thioredoxin Revealed by in-Cell NMR

Cited by 41 publications

References 30 publications

Modification of Cys residues in human thioredoxin-1 by p-benzoquinone causes inhibition of its catalytic activity and activation of the ASK1/p38-MAPK signalling pathway

Modification of Cys residues in human thioredoxin-1 by p-benzoquinone causes inhibition of its catalytic activity and activation of the ASK1/p38-MAPK signalling pathway

In-Situ Flexibility of Both Redox-States of the Chloroplast Regulatory Protein CP12

Spotlight on the Ballet of Proteins: The Structural Dynamic Properties of Proteins Illuminated by Solution NMR

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