Mammalian thioredoxin reductase 1: roles in redox homoeostasis and characterization of cellular targets

Abstract

The classical Trx (thioredoxin) system, composed of TR (Trx reductase), Trx and NADPH, defines a major pathway of cellular thiol-based redox regulation. Three TRs have been identified in mammals: (i) cytosolic TR1, (ii) mitochondrial TR3 and (iii) testes-specific TGR (Trx-glutathione reductase). All three are selenocysteine-containing enzymes with broad substrate specificity in in vitro assays, but which protein substrates are targeted by TRs in vivo is not well understood. In the present study, we used a mechanism-based approach to characterize the molecular targets of TR1. Cytosolic Trx1 was the major target identified in rat and mouse liver, as well as in rat brain and mouse serum. The results suggest that the main function of TR1 is to reduce Trx1. We also found that TR1-based affinity resins provide a convenient tool for specific isolation of Trxs from a variety of biological samples. To better assess the role of TRs in redox homoeostasis, we comparatively analysed TR1- and TR3-knockdown cells. Although cells deficient in TR1 were particularly sensitive to diamide, TR3-knockdown cells were more sensitive to hydrogen peroxide. To further examine the TR1–Trx1 redox pair, we used mice with a liver-specific knockout of selenocysteine tRNA. In this model, selenocysteine insertion into TR1 was blocked, but the truncated form of this protein was not detected. Instead, TR1 and TR3 levels were decreased in the knockout samples. Diminished hepatic TR1 function was associated with elevated Trx1 levels, but this protein was mostly in the oxidized state. Overall, this study provides evidence for the key role of the TR1–Trx1 pair in redox homoeostasis.

The Role of Selenium in Inflammation and Immunity: From Molecular Mechanisms to Therapeutic Opportunities

“…Thioredoxin reductases. The thioredoxin reductase (TXNRD) enzymes are another well-characterized subfamily of selenoproteins that perform an essential redox role by regenerating reduced thioredoxin (TXN or TRX) within cells (147,251). TXN is a small redox active protein distributed ubiquitously in various mammalian tissues and cells that serves to reduce oxidized moities (e.g., Cys-Cys disulfide bonds), and the TXN/TXNRD system is one of the most important mechanisms for regulating cellular redox balance (108).…”

Mechanism-based Proteomic Screening Identifies Targets of Thioredoxin-like Proteins

“…Anti-Trx1 and anti-TR1 were obtained from Covance. CXXC and CXXS recombinant human Trx1 and CXXU TR1 were prepared as previously described (34). Recombinant human AIF (AIF(⌬1-120)) (35) and human Mia40, with a His tag at their C terminus, were prepared after cloning AIFM1 and CHCHD4 (Mia40) from the HEK 293T cDNA library into pET28a vector (Novagen), both between EcoRI and SalI restriction sites.…”

Crystal structure of the human thioredoxin reductase–thioredoxin complex

“…Trx1 is a cytosolic and nuclear protein, whereas Trx2 is targeted to mitochondria 1,8 . Cytosolic Trx1 was the major substrate captured by immobilized TrxR1, indicating that the main function of TrxR1 is to reduce Trx1 9 . TrxRs belong to a family of homodimeric pyridine nucleotide disulphide oxidoreductases with high homology to GRs.…”