Abstract 5211: Identification of a Direct Substrate of Thioredoxin 1 in Cardiac Myocytes
Thioredoxin 1 (Trx1), a 12 kD anti-oxidant, reduces oxidized proteins by transferring electrons from its conserved cysteine residues (Cys-32 and Cys-35) to its targets, a reaction termed the thiol-disulfide exchange reaction. Trx1 inhibits pathological hypertrophy in the heart, which is in part mediated by reduction of DnaJb5 and class II histone deacetylases. Thus, the goal in this study was to identify additional redox-sensitive targets of Trx1 in cardiac myocytes. When Trx1 reduces its target, Cys-32 in the catalytic center transiently forms an intermolecular disulfide bond with substrate proteins, which is in turn reduced by Cys-35. Thus, Trx1C35S, a Trx1 mutant, is expected to act as a substrate trapping mutant due to an incomplete dithiol-disulfide exchange reaction. To identify direct substrate proteins of Trx1 in cardiac myocytes, Flag-Trx1C35S-HA was expressed in cultured cardiac myocytes with adenovirus transduction. After myocytes were treated with H2O2, proteins trapped by Flag-Trx1C35S-HA were immunoprecipitated with anti-HA antibody. No protein was trapped when Flag-Trx1CC32/35SS-HA was used, suggesting that the trapping is Cys-32-dependent. Trapped substrates were separated by non-reducing SDS-PAGE and then subjected to mass spectrometry. We identified rat peroxiredoxin 1 (Prdx1), a Trx1-dependent peroxidase, as a major protein trapped by Flag-Trx1C35S-HA in hydrogen peroxide treated cardiac myocytes. Overexpression of Trx1 inhibited H2O2-induced oxidation of Prdx1 in cells in vitro (Oxidized/Reduced form of Prdx1: control 1.58, Trx1 overexpression 0.7), confirming that Prdx1 is reduced by Trx1. In order to identify in vivo targets of Trx1, transgenic mice (Tg) with cardiac specific expression of Flag-Trx1C35S-HA were generated. Heart homogenates obtained from Tg-Flag-Trx1C35S-HA mice were subjected to immunoprecipitation with anti-HA antibody and separated by non-reducing SDS-PAGE. Subsequent immunoblot analyses showed that Prdx1 was among the proteins trapped by Flag-Trx1C35S-HA in vivo. Thus, using a new Trx1 substrate trapping assay, we identified Prdx1 as a major direct substrate of Trx1 in cardiac myocytes both in vitro and in vivo.