Regulation of human thioredoxin reductase expression and activity by 3 '-untranslated region selenocysteine insertion sequence and mRNA instability elements

Thioredoxin reductases function in regulating cellular redox and function t hrough their substrate, thioredoxin, in the proper folding of enzymes and r edox regulation of transcription factor activity. These enzymes are overexp ressed in certain tumors and cancer cells and down-regulated in apoptosis a nd may play a role in regulating cell growth. Mammalian thioredoxin reducta ses contain a selenocysteine residue, encoded by a UGA codon, as the penult imate carboxyl-terminal amino acid. This amino acid has been proposed to ca rry reducing equivalents from the active site to substrates. We report; exp ression of a wild-type thioredoxin reductase selenoenzyme, a cysteine mutan t enzyme, and the UGA-terminated protein in mammalian cells and overexpress ion of the cysteine mutant and UGA-terminated proteins in the baculovirus i nsect cell system. We show that substitution of cysteine for selenocysteine decreases enzyme activity for thioredoxin by 2 orders magnitude, and that termination at the UGA codon abolishes activity. We further demonstrate the presence of a functional selenacysteine insertion sequence element that is highly active but only moderately responsive to selenium supplementation. Finally, we show that thioredoxin reductase mRNA levels are down-regulated by other sequences in the 3'-untranslated region, which contains multiple A U-rich instability elements. These sequences are found in a number of cytok ine and proto-oncogene mRNAs and have been shown to confer rapid mRNA turno ver.