No abstract

No abstract

Quinones are common in nature, and often cytotoxic. Their proposed toxicity mechanisms involve redox cycling with radical generation, and/or reactions with nucleophiles, such as protein cysteine (Cys) residues, forming adducts via Michael addition reactions. The selenenyl anion of selenocysteine (Sec) is a stronger nucleophile, more prevalent at physiological pH, and more reactive than the corresponding thiolate anion of Cys. We therefore hypothesized that Sec residues should be readily modified by quinones and with potential consequences for the structure and function of selenoproteins. Here, we report data on the interaction of p-benzoquinone (BQ) with the selenoprotein thioredoxin reductase-1 (TrxR1), which exposes an accessible Sec residue upon physiological reduction by NADPH. Our results reveal that BQ targets NADPH-reduced TrxR1 and inhibits its activity using 5,5′-dithiobis(2-nitrobenzoic acid) or juglone as model substrates, consistent with the targeting of both the Cys and Sec residues of TrxR1. In the absence of NADPH, BQ modified the non-catalytic Cys residues, leading to subunit crosslinking, mainly through disulfides, which also resulted in some loss of activity. This crosslinking was time-dependent and independent of the Sec residue. Addition of NADPH after BQ pre-treatment could resolve the disulfide-linked crosslinking. TrxR activity loss was also observed upon incubation of J774A.1 cells or cell lysates with BQ. These data suggest that BQ readily targets TrxR1, albeit in a rather complex manner, which results in structural changes and loss of enzyme activity. We suggest that TrxR1 targeting can explain some of the cytotoxicity of BQ, and potentially also that of other quinone compounds.

No 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.