Abstract 14119: Oxidative Stress Inhibits mTOR Through Intermolecular Disulfide Bond Formation With Deptor in Cardiomyocytes.

Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth, metabolism and survival. In cardiomyocytes, mTOR is inhibited by oxidative stress partly through intermolecular disulfide bond formation at Cysteine 1483, which negatively regulates cell survival and mitochondrial function. However, the mechanism by which the disulfide bond formation inhibits mTOR remains unknown. Deptor is a component of the mTOR complex 1 (mTORC1) and an endogenous inhibitor of mTOR. Here we show that Deptor forms an intermolecular disulfide bond with mTOR. In the presence of hydrogen peroxide (H2O2, 100 nM), both Deptor and mTOR exhibited a band shift to a high molecular weight in Western blot analyses using non-reducing SDS-PAGE, suggesting that Deptor and mTOR are subjected to oxidative posttranslational modifications. The band shift was not observed in other components of mTORC1, such as Raptor, MLST8 and Pras40. H2O2 promoted the binding of Deptor to mTOR, which was inhibited by dithiothreitol, a reducing agent that cleaves disulfide bonds, or in the presence of mTOR-Cys1483Phe mutant. Knockdown of Deptor with short hairpin RNA (shDeptor) abolished the H2O2-induced band shift of mTOR. Thus, Deptor forms a disulfide bond with mTOR at Cys1483 in response to oxidative stress. In addition, shDeptor normalized H2O2-induced decreases in phosphorylation of mTORC1 substrates, such as S6 kinase and 4EBP1, suggesting that Deptor mediates H2O2-induced inhibition of mTORC1. shDeptor also inhibited H2O2-induced cardiomyocyte death, an effect which was diminished in the presence of rapamycin, an inhibitor of mTORC1 (relative cell viability: control: 1; control H2O2: 0.45; shDeptor H2O2: 0.81; Rapamycin/H2O2: 0.48; Rapamycin/shDeptor/H2O2: 0.48*, P<0.05 vs shDeptor H2O2). These results suggest that Deptor mediates oxidative stress-induced mTOR inhibition through formation of a disulfide bond with mTOR at Cys1483, which in turn negatively regulates cardiomyocyte survival. [ABSTRACT FROM AUTHOR]