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.

The Benzene Hematotoxic and Reactive Metabolite 1,4-Benzoquinone Impairs the Activity of the Histone Methyltransferase SET Domain Containing 2 (SETD2) and Causes Aberrant Histone H3 Lysine 36 Trimethylation (H3K36me3)

“…We also found that BQ led to the formation of non-reducible SETD2 protein cross-links/oligomers as previously observed for other enzymes inhibited by BQ (Fig. 2A and 2C) (Shu, Cheng, et al, 2020;Shu, Hägglund, et al, 2020). Interestingly, SETD2 has been shown to be inherently prone to protein crosslink/aggregation which can be detected by SDS-PAGE/western blot or immunofluorescence (Bhattacharya and Workman, 2020).…”

“…As shown in Fig. 4.A, exposure of transfected cells to BQ led to the formation of inactive SETD2 cross-links/oligomers as previously observed for other enzymes inhibited by BQ (Shu, Cheng, et al, 2020;Shu, Hägglund, et al, 2020). SETD2 is inherently prone to protein crosslink/aggregation and SETD2 aggregates/oligomers have been observed by immunofluorescence as puncta in cells (Bhattacharya and Workman, 2020).…”

“…BQ is considered as the major hematotoxic metabolite of BZ (Smith, 2010;North et al, 2020). The leukemogenic properties of BQ are thought to rely, at least in part, on the formation of covalent Michael adducts with cysteine residues that may affect the structure and the function of proteins involved in the regulation of hematopoietic cells (Smith et al, 2011;Bolton and Dunlap, 2017;North et al, 2020;Shu, Hägglund, et al, 2020). So far, inhibition of topoisomerase II enzymes by BQ is considered as one of the key mechanisms contributing to BZ hematotoxicity (Bender et al, 2007;Eastmond et al, 2014;Holmes and Winn, 2019).…”

“…Cysteine residues are chemically and kinetically favored sites for BQ adduction and, as such, certain enzymes can be impaired by formation of BQ adducts on reactive cysteines (Bender et al, 2007;Mbiya et al, 2013;Duval et al, 2019;Shu, Hägglund, et al, 2020). SETD2 catalytic domain possesses two Zn-finger regions (AWS and SET/post-SET) containing three Zn atoms chelated by cysteines that contribute to the folding of the domain and insure enzyme function (Yang et al, 2016;Zhang et al, 2017) (supplementary Fig.…”

Elevated serum 4HNE plus decreased serum thioredoxin: Unique feature and implications for acute exacerbation of chronic obstructive pulmonary disease

“…The substrates of Trx1 includes hydroperoxide [ 21 ], glutathione peroxidases that terminate lipids peroxidation cascade, and ribonucleotide reductase that is a key enzyme in DNA replication [ 17 ]. Moreover, the reduced form of Trx1 interacts with the N-terminal portion of apoptosis stimulating kinase 1 (ASK1) in vitro and in vivo , acting as a physiological inhibitor of ASK1, while the oxidation and release of Trx1 from ASK1 links cytotoxic stresses, such as TNF-α and H 2 O 2 , leading to activation of the p38 MAPK and SAPK/JNK stress response pathways [ 22 , 23 ]. Thus, Trx1 system is important for cell growth/survival as well as oxidative stress-induced signal transduction [ 24 ].…”