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 through their substrate, thioredoxin, in the proper folding of enzymes and redox regulation of transcription factor activity. These enzymes are overexpressed in certain tumors and cancer cells and down-regulated in apoptosis and may play a role in regulating cell growth. Mammalian thioredoxin reductases contain a selenocysteine residue, encoded by a UGA codon, as the penultimate carboxyl-terminal amino acid. This amino acid has been proposed to carry reducing equivalents from the active site to substrates. We report expression of a wild-type thioredoxin reductase selenoenzyme, a cysteine mutant enzyme, and the UGA-terminated protein in mammalian cells and overexpression of the cysteine mutant and UGA-terminated proteins in the baculovirus insect 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 selenocysteine 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 AU-rich instability elements. These sequences are found in a number of cytokine and proto-oncogene mRNAs and have been shown to confer rapid mRNA turnover.
Gasdaska
J R
JR
Arizona Cancer Center, Tucson, Arizona 85724, USA.
Harney
J W
JW
Gasdaska
P Y
PY
Powis
G
G
Berry
M J
MJ
D020413:3' Untranslated Regions; D002460:Cell Line; D004251:DNA Transposable Elements; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger / Q000235:genetics*; D017279:Selenocysteine / Q000235:genetics; D013880:Thioredoxin-Disulfide Reductase / Q000096:biosynthesis / Q000235:genetics*
D020413:3' Untranslated Regions; D004251:DNA Transposable Elements; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger; D017279:Selenocysteine; D013880:Thioredoxin-Disulfide Reductase