Mammalian thioredoxin reductases: roles in redox homeostasis and analysis of cellular targets

Abstract

Thioredoxin reductase (TR) and thioredoxin (Trx) constitute a major cellular redox system. In the present study, we characterized subcellular localization of mouse TR3 forms which resided in mitochondria and cytosol. We generated recombinant selenoprotein forms of TR1 and TR3 and found that these enzymes were inhibited by zinc. A proteomic method for identification of targets of TRs in mammalian cells and tissues was developed. We found that Trx1 was the major target of TR3 and TR1 in rat and mouse liver cytosol and that Trx1 was the major target of TR1 in rat brain cytosol and could be enriched on the TR affinity columns from mouse serum. TR3 knockdown cells had increased sensitivity to hydrogen peroxide, whereas TR1 knockdown cells were more sensitive to diamide. To further examine the roles of TRs in redox homeostasis, we used knockout mice, in which selenocysteine tRNA gene was disrupted specifically in the liver. We found that the levels of cytosolic TR1 and mitochondrial TR3 were significantly lower in the knockout samples compared to wild type, whereas the levels of cytosolic Trx1 and mitochondrial Trx2 were elevated. Trx1 was mostly in the oxidized state in the knockout samples. Overall, these data provide important insights into the roles of mammalian TRs in redox homeostasis.