Metallothionein Disulfides Are Present in Metallothionein-overexpressing Transgenic Mouse Heart and Increase under Conditions of Oxidative Stress

Abstract

Metallothionein (MT) releases zinc under oxidative stress conditions in cultured cells. The change in the MT molecule after zinc release in vivo is unknown although in vitro studies have identified MT disulfide bond formation. The present study was undertaken to test the hypothesis that MT disulfide bond formation occurs in vivo. A cardiac-specific MT-overexpressing transgenic mouse model was used. Mice were administered saline as a control or doxorubicin (20 mg/kg), which is an effective anticancer drug but with severe cardiac toxicity at least partially because of the generation of reactive oxygen species. A differential alkylation of cysteine residues in MT of the heart extracts was performed. Free and metal-bound cysteines were first trapped by N-ethylmaleimide and the disulfide bonds were reduced by dithiothreitol followed by alkylation with radiolabeled iodoacetamide. Analyses of the differentially alkylated MTs in the heart extract by high preformance liquid chromatography, SDS-PAGE, Western blot, and mass spectrometry revealed that disulfide bonds were present in MT in vivo under both physiological and oxidative stress conditions. More disulfide bonds were found in MT under the oxidative stress conditions. The MT disulfide bonds were likely intramolecular and both ␣-and ␤-domains were involved in the disulfide bond formation, although the ␣-domain appeared to be more easily oxidized than the ␤-domain. The results suggest that under physiological conditions, the formation of MT disulfide bonds is involved in the regulation of zinc homeostasis. Additional zinc release from MT under oxidative stress conditions is accompanied by more MT disulfide bond formation. Previous studies have shown that metallothionein (MT)2 functions in cardiac protection against oxidative damage (1-4). The mechanism of the antioxidant action of MT remains elusive. It has been shown in vitro that MT directly interacts with reactive oxygen and nitrogen species to prevent oxidative and nitrosative damage to macromolecules such as lipids, proteins, and DNA (5-8). However, this interaction has never been demonstrated in vivo and would unlikely be a major mechanism for the antioxidant action of MT in vivo because the reactivity of reactive oxygen and nitrogen species would only allow their reaction with molecules that have a close proximity to their generation sites. Alternatively, in vitro studies have demonstrated that the multiple cysteine residues of MT can be oxidized. The sulfur clusters that bind zinc in MT create an oxidoreductive environment for zinc at a redox potential so low that MT can be readily oxidized by mild cellular oxidants, such as glutathione disulfide, with the release of zinc (9). This MT oxidation and zinc release process would play a critical role in the cellular response to oxidative and nitrosative stress.The high content of cysteine residues enables MT to avidly bind zinc and other metals (10). The binding of divalent metals to MT occurs in two dumbbell-shaped domains, of which the N-terminal ␤-domain usually...

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