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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.

ABSTRACT: 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.

SUBMITTER: Shu N

PROVIDER: S-EPMC6926358 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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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.

Shu Nan N Hägglund Per P Cai Huan H Hawkins Clare L CL Davies Michael J MJ

Redox biology 20191206

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 qu ...[more]

PMID: 31926625

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Project description:BackgroundCancer cells in the advanced stage show aberrant antioxidant capacity to detoxify excessive ROS resulting in the compensation for intrinsic oxidative stress and therapeutic resistance. PDAC is one of the most lethal cancers and often associated with a high accumulation of ROS. Recent studies identified gold(I) NHC complexes as potent TrxR inhibitors suppressing cell growth in a wide spectrum of human malignant cell lines at the low micromolar concentration. However, the mechanism of action is not completely elucidated yet.MethodsTo understand the biological function of gold(I) NHC complexes in PDAC, we used a recently published gold(I) NHC complex, MC3, and evaluated its anti-proliferative effect in four PDAC cell lines, determined by MTT and SRB assays. In further detailed analysis, we analyzed cellular ROS levels using the ROS indicator DHE and mitochondrial membrane potential indicated by the dye JC-1 in Panc1. We also analyzed cell cycle arrest and apoptosis by FACS. To elucidate the role of specific cell signaling pathways in MC3-induced cell death, co-incubation with ROS scavengers, a p38-MAPK inhibitor and siRNA mediated depletion of ASK1 were performed, and results were analyzed by immunoblotting, ELISA-microarrays, qRT-PCR and immunoprecipitation.ResultsOur data demonstrate that MC3 efficiently suppressed cell growth, and induced cell cycle arrest and apoptosis in pancreatic cancer cells, in particular in the gemcitabine-resistant cancer cells Panc1 and ASPC1. Treatment with MC3 resulted in a substantial alteration of the cellular redox homeostasis leading to increased ROS levels and a decrease in the mitochondrial membrane potential. ROS scavengers suppressed ROS formation and rescued cells from damage. On the molecular level, MC3 blocked the interaction of Trx with ASK1 and subsequently activated p38-associated signaling. Furthermore, inhibition of this pathway by using ASK1 siRNA or a p38 inhibitor clearly attenuated the effect of MC3 on cell proliferation in Panc1 and ASPC1.ConclusionsOur results confirm that MC3 is a TrxR inhibitor and show MC3 induced apoptosis in gemcitabine-resistant PDACs. MC3 mediated cell death could be blocked by using anti-oxidants, ASK1 siRNA or p38 inhibitor suggesting that the Trx-ASK1-p38 signal cascade played an important role in gold(I) NHC complexes-mediated cellular damage.

Dataset Information

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.

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.

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