Structure-functional analysis of mammalian thioredoxin reductases

Mammalian thioredoxin reductases (TRs) are central enzymes in the thioredoxin (Trx) redox pathway. These proteins are FAD-containing pyridine nucleotide disulfide oxidoreductases that utilize NADPH for reduction of active site disulfide of Trx. In addition to a previously known isozyme TR1, we identified and characterized two new thioredoxin reductase isozymes, TR2 and TR3. All three TR isozymes contain a selenocysteine residue that is located in the C-terminal penultimate position and is encoded by the UGA codon. TR1 is cytosolic, TR2 is microsomal, and TR3 is a mitochondrial protein. TR1 and TR3 are expressed in various organs, whereas TR2 is specifically expressed in adult testes. We also demonstrated a remarkable heterogeneity within TRs, which, at least in part, results from evolutionary conserved genetic mechanisms employing alternative first exon splicing. Multiple transcription start sites within TR genes may be relevant to complex regulation of expression and/or organelle- and cell type-specific location of animal thioredoxin reductases. We identified selenocysteine of TR1 as a cellular redox sensor and demonstrated an essential role for mammalian TR isozymes in redox-regulated cell signaling. The generation of reactive oxygen species in a human carcinoma cell line was shown to result in both the oxidation of the selenocysteine in TR1 and a subsequent increase in the expression of this enzyme. TR2 exhibited specificity for both thioredoxin and glutathione systems, and so we designated it as thioredoxin and glutathione reductase (TGR). This enzyme can reduce Trx, GSSG and a GSH-linked disulfide in in vitro assays. This unusual substrate specificity is achieved by an evolutionarily conserved fusion of the TR and glutaredoxin domains. These observations, together with the biochemical probing and molecular modeling of the TGR structure, suggested a reaction mechanism for mammalian TRs whereby the C-terminal selenotetrapeptide serves a role of a protein-linked GSSG and shuttles electrons from the disulfide center within the TR domain to either the glutaredoxin domain or Trx