Mammalian thioredoxin reductases: Functions and regulation

Selenium is an essential trace element and selenium-containing proteins (selenoproteins) play critical roles in various aspects of life. Mammalian thioredoxin reductases (TRs) are selenoproteins that play pivotal roles in redox regulation and signaling. Two of the three known TRs, TR1 and TR3, are essential proteins. We characterized the splicing pattern of the human TR1 gene and identified 12 different splicing patterns which result in 6 different ORFs. One of the splicing form results from an alternative promoter and encodes the glutaredoxin (Grx)-containing TR1 form. Genome comparison suggested this form occurs in human, chimpanzee and dog but not mouse or rat. Further studies using the recombinant form of this protein showed it was not active. The Grx domain itself exhibited no activity and also blocked the TR activity of the recombinant protein. A third member of the mammalian TR family, known as thioredoxin/glutathione reductase (TGR) in the TR family, is highly expressed in testes. We found that it has a close functional relationship with another selenoprotein, phospholipid hydroperoxide glutathione peroxidase (GPx4). GPx4 is known to play a critical role in sperm maturation and in addition, it is a structural component of sperm. We found that TGR is a disulfide isomerase that could help GPx4 to form correct disulfide bonds during sperm maturation. Further in vitro and in vivo studies showed that the Grx domain of TGR is responsible for the disulfide isomerase activity. We also identified protein targets of TGR, most of which are known to be involved in sperm development. We developed a new method for expression of mammalian selenoproteins in E. coli. We introduced a 3\u27UTR SECIS in the GPx4 gene with base-pairing capability to bring the SECIS close to the UGA codon that codes for selenocysteine. We found that Sec could be inserted by this 3\u27UTR SECIS, however, the major product had tryptophan inserted at the UGA codon. Changing the UGA codon to UAA, accompanied by coexpressing a mutant tRNA sec with the UAA anticodon, eliminated the opal suppression and achieved specific insertion of selenocysteine into polypeptide chain