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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Antibody-drug conjugates (ADCs) have achieved great success in cancer therapy in recent years. Some peptidyl microtubule inhibitors consisting of natural and unnatural amino acids, such as monomethyl auristatin E (MMAE) and F (MMAF), are extremely cytotoxic and have been used as a payload in ADCs.However, their precise molecular interaction with tubulin and microtubules remains unclear. We determined the crystal structures of tubulin in complex with three ultra-potent peptidyl microtubule inhibitors [MMAE,, and tubulysin M] at 2.5 Å. Our data showed that the three peptides bound to the vinca domain and shared a common and key pharmacophore containing two consecutive hydrophobic groups (Val, Ile-like side chain). These groups protruded in opposite directions into hydrophobic pockets on the tubulin b and a subunits. Nitrogen and oxygen atoms from the same backbone formed hydrogen bonds with Asn329 from the a subunit and Asp179 from the b subunit in a direction normal to the surface formed by the aforementioned hydrophobic groups. In addition, our crystal structure data indicated that tubulysin M bound to the b subunit alone, providing a structural explanation for its higher affinity. We also compared the conformations of two representative structurally different vinca domain compounds, ustiloxin D and vinblastine, with those of the aforementioned peptidyl ligands, and found that they shared a similar pharmacophore. Our findings lay a foundation for the rational design of novel vinca domain ligands and may facilitate the development of microtubule inhibitors with high specificity, affinity, and efficiency as payloads for ADCs in cancer therapy.

Acrylonitrile (AN) is an industrial vinyl monomer that is acutely toxic. When administered to rats, AN covalently binds to tissue proteins in a dose-dependent but nonlinear manner [Benz, F. W., Nerland, D. E., Li, J., and Corbett, D. (1997) Fundam. Appl. Toxicol. 36, 149-156]. The nonlinearity in covalent binding stems from the fact that AN rapidly depletes liver glutathione after which the covalent binding to tissue proteins increases disproportionately. The identity of the tissue proteins to which AN covalently binds is unknown. The experiments described here were conducted to begin to answer this question. Male Sprague-Dawley rats were injected subcutaneously with 115 mg/kg (2.2 mmol/kg) [2,3-(14)C]AN. Two hours later, the livers were removed, homogenized, and fractionated into subcellular components, and the radioactively labeled proteins were separated on SDS-PAGE. One set of labeled proteins was found to be glutathione S-transferase (GST). Specific labeling of the mu over the alpha class was observed. Separation of the GST subunits by HPLC followed by scintillation counting showed that AN was selective for subunit rGSTM1. Mass spectral analysis of tryptic digests of the GST subunits indicated that the site of labeling was cysteine 86. The reason for the high reactivity of cysteine 86 in rGSTM1 was hypothesized to be due to its potential interaction with histidine 84, which is unique in this subunit.

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Covalent binding of reactive chemical species to tissue proteins is a common, but poorly understood, mechanism of toxicity. Identification of the proteins and the specific amino acid residues within the proteins that are chemically modified will aid our understanding of the toxification/detoxification mechanisms involved in covalent binding. Acrylonitrile (AN) is a commercial vinyl monomer that is acutely toxic and readily binds to tissue proteins. Total covalent binding of AN to tissue proteins is highly correlated with acute toxicity. Two-dimensional PAGE and autoradiography were used to locate proteins in male rat liver cytosol that are radiolabeled following administration of [2,3-(14)C]AN in vivo. Four intensely labeled spots were prominent in the autoradiogram and formed an apparent "charge-train" at approximately 30 kDa. Tryptic peptide mapping by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS was used to identify all of the spots as carbonic anhydrase III (CAIII). HPLC of the tryptic digests combined with MALDI-TOF MS was used to localize the radiolabel to tryptic fragment T22 containing amino acids 171-187. This tryptic fragment contains two Cys residues (Cys181 and Cys186) in the rat CAIII sequence. Electrospray ionization ion-trap MS was used to sequence the peptide and establish that only Cys186 was labeled. Thus, although AN is considered to be highly reactive, our data indicate that it does not react indiscriminately with rat CAIII but rather is selective for one out of five Cys residues. Rat liver CAIII has previously been shown to protect cells against oxidative stress. Our data suggest that CAIII is also capable of scavenging reactive xenobiotics and may help prevent covalent binding to more critical macromolecules.

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