Pamela Y. Gasdaska

Cloning and sequencing of a human thioredoxin reductase

The DNA sequence encoding human placental thioredoxin reductase has been determined. Of the 3826 base pairs sequenced, 1650 base pairs were in an open reading frame encoding a mature protein with 495 amino acids and a calculated molecular mass of 54,171. Sequence analysis showed strong similarity to glutathione reductases and other NADPH‐dependent reductases. Human thioredoxin reductase contains the redoxactive cysteines in the putative FAD binding domain and has a dimer interface domain not previously seen with prokaryote and lower eukaryote thioredoxin reductases.

The predicted amino acid sequence of human thioredoxin is identical to that of the autocrine growth factor human adult T-cell derived factor (ADF): Thioredoxin mRNA is elevated in some human tumors

The cDNA sequences of thioredoxin obtained by PCR cloning from human colon cancer cells, human lymphoblastoid cells, and human liver have been found to be identical with the cDNA sequence reported for the autocrine growth factor, human adult T-cell leukemia derived factor (ADF). Recombinant human thioredoxin was 95% reduced by dithiothreitol and was a substrate for reduction by human thioredoxin reductase. Human non-small cell primary lung tumors from subjects who were not cigarette smokers at the time of surgery showed significantly increased levels of thioredoxin mRNA compared to thioredoxin mRNA in paired normal human lung tissue. Subjects who were smokers did not show a significant increase in lung tumor thioredoxin mRNA. The results of the study show that human thioredoxin and ADF are identical species and suggest that there may be increased production of thioredoxin (ADF) by some human cancers.

Cloning, sequencing and functional expression of a novel human thioredoxin reductase

The DNA sequence encoding a novel human thioredoxin reductase has been determined. The protein is predicted to have 524 amino acids including a conserved ‐Cys‐Val‐Asn‐Val‐Gly‐Cys catalytic site and a selenocysteine containing C‐terminal ‐Gly‐Cys‐SeCys‐Gly. The predicted molecular mass is 56.5. The newly identified TR sequence exhibits 54% identity to a previously reported human thioredoxin reductase and 37% identity to human glutathione reductase. Transient transfection of human embryonal kidney cells results in a 5‐fold increase in thioredoxin reductase activity but no increase in glutathione reductase activity.

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.