Abstract
Thioredoxin 1 (Trx1) is a 12-kDa oxidoreductase that catalyzes thiol-disulfide exchange reactions to reduce proteins with disulfide bonds. As such, Trx1 helps protect the heart against stresses, such as ischemia and pressure overload. Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth, metabolism, and survival. We have shown previously that mTOR activity is increased in response to myocardial ischemia-reperfusion injury. However, whether Trx1 interacts with mTOR to preserve heart function remains unknown. Using a substrate-trapping mutant of Trx1 (Trx1C35S), we show here that mTOR is a direct interacting partner of Trx1 in the heart. In response to H2O2 treatment in cardiomyocytes, mTOR exhibited a high molecular weight shift in non-reducing SDS-PAGE in a 2-mercaptoethanol-sensitive manner, suggesting that mTOR is oxidized and forms disulfide bonds with itself or other proteins. The mTOR oxidation was accompanied by reduced phosphorylation of endogenous substrates, such as S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1) in cardiomyocytes. Immune complex kinase assays disclosed that H2O2 treatment diminished mTOR kinase activity, indicating that mTOR is inhibited by oxidation. Of note, Trx1 overexpression attenuated both H2O2-mediated mTOR oxidation and inhibition, whereas Trx1 knockdown increased mTOR oxidation and inhibition. Moreover, Trx1 normalized H2O2-induced down-regulation of metabolic genes and stimulation of cell death, and an mTOR inhibitor abolished Trx1-mediated rescue of gene expression. H2O2-induced oxidation and inhibition of mTOR were attenuated when Cys-1483 of mTOR was mutated to phenylalanine. These results suggest that Trx1 protects cardiomyocytes against stress by reducing mTOR at Cys-1483, thereby preserving the activity of mTOR and inhibiting cell death.
Highlights
Thioredoxin 1 (Trx1) is a 12-kDa oxidoreductase that catalyzes thiol-disulfide exchange reactions to reduce proteins with disulfide bonds
We show that Mechanistic target of rapamycin (mTOR) was subjected to redox modification. mTOR formed intermolecular disulfides in response to oxidation that were reduced by Trx1. mTOR oxidation in cardiomyocytes was linked to mTOR inhibition
Interaction between mTOR and FLAG-TrxC35S-HA was observed in primary cultured cardiomyocytes, and the interaction was abolished with a lysis buffer containing DTT, a reducing agent (Fig. 1C), suggesting that the interaction between Trx1 and mTOR is mediated through an intermolecular disulfide bond
Summary
Thioredoxin 1 (Trx1) is a 12-kDa oxidoreductase that catalyzes thiol-disulfide exchange reactions to reduce proteins with disulfide bonds. The mTOR oxidation was accompanied by reduced phosphorylation of endogenous substrates, such as S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1) in cardiomyocytes. H2O2-induced oxidation and inhibition of mTOR were attenuated when Cys-1483 of mTOR was mutated to phenylalanine These results suggest that Trx protects cardiomyocytes against. Stress by reducing mTOR at Cys-1483, thereby preserving the activity of mTOR and inhibiting cell death. Increased ROS are frequently observed under various pathological and stressed conditions and are generally considered to promote the pathology because of oxidative damage of proteins, lipids, and DNAs. To protect against ROS, cells express antioxidant systems, including thioredoxins and glutathione, and reducing enzymes, such as superoxide dismutase, and catalase. Trx directly reduces specific substrates, including NFB, class II histone deacetylases, caspase, and AMP-activated protein kinase (AMPK), through thiol-disulfide exchange reactions [7]. Identification and characterization of Trx substrates may allow discovery of novel biological reactions that serve to counteract oxidative stress
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