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Transactivational activity of the tumor suppressor protein p53 is dependent on thioredoxin reductase activity in mammalian cells
Reporter gene transactivation by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. Thioredoxin reductase specifically catalyzes the NADPH-dependent reduction of thioredoxin. Thioredoxin provides a source of electrons for disulfide reduction in various cellular processes. Reduction of disulfides within the cell can be accomplished by the separate but partially overlapping glutathione reductase - glutathione - glutaredoxin pathway. The basis for p53 inhibition was investigated by measuring the redox state of thioredoxin and glutathione in wild-type and Δtrr1 yeast lacking the gene encoding thioredoxin reductase. The Δtrr1 mutation caused an increased in oxidation in both molecules. Highcopy expression of the GLR1 gene encoding glutathione reductase in Δtrr1 yeast restored the redox state of glutathione to wild-type levels, but did not restore p53 activity. Also, p53 activity was unaffected by be a Δglr1 mutation, even though the mutation was known to result in glutathione oxidation. These results indicate that p53 activity has a specific requirement for an intact thioredoxin system, rather than a general dependence on the intracellular reducing environment. In order to test if p53 activity requires an intact thioredoxin system in mammalian cells, dominant-negative and RNAi approaches designed to suppress thioredoxin reductase activity were used in a breast adenocarcinoma cell which contains an endogenous wild-type p53. In cells stably transformed with a plasmid encoding a dominant-negative form of thioredoxin reductase, thioredoxin reductase activity was inhibited 4.3-fold and p53 reporter gene expression was inhibited by 2-fold. In cells stably transformed with a RNAi plasmid designed to target thioredoxin reductase mRNA, thioredoxin reductase activity was inhibited by 1.7-fold and p53 reporter gene expression was inhibited by 1.6-fold. A decrease in the protein levels of the p53 endogenous target genes p21 and Bax was also observed in both dominant-negative and RNAi transformants. Additionally, thioredoxin was shown to bind p53 in vitro (Kd=0.9 μM), and a LexA-thioredoxin fusion protein was shown to bind p53 in vivo. These results suggest that p53 activity is regulated by thioredoxin reductase in mammalian cells through a direct interaction with thioredoxin
Effect of thioredoxin deletion and p53 cysteine replacement on human p53 activity in wild-type and thioredoxin reductase null yeast
Reporter gene transactivation by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. To investigate the role of thioredoxin in controlling p53 activity, the level of reporter gene transactivation by p53 was determined in yeast lacking the TRX1 and TRX2 genes encoding cytosolic thioredoxin. Surprisingly, p53 activity was unimpaired in yeast lacking thioredoxin. Subsequent analyses showed that thioredoxin deletion suppressed the inhibitory effect of thioredoxin reductase deletion, suggesting that accumulation of oxidized thioredoxin in mutant yeast was necessary for p53 inhibition. Purified human thioredoxin and p53 interacted in vitro (K d = 0.9 μM thioredoxin). To test the idea that dithio-disulfide exchange reactions between p53 and thioredoxin were responsible for p53 inhibition in mutant yeast, each p53 cysteine was changed to serine, and the effect of the substitution on p53 activity in TRR1 and Δtrr1 yeast was determined. Substitutions at Zn-coordinating cysteines C176, C238, or C242 resulted in p53 inactivation. Unexpectedly, substitution at cysteine C275 also inactivated p53, which was the first evidence for a non-zinc-coordinating cysteine being essential for p53 function. Cysteine substitutions at six positions (C124, C135, C141, C182, C229, and C277) neither inactivated p53 nor relieved the requirement for thioredoxin reductase. Furthermore, no tested combination of these six cysteine substitutions relieved thioredoxin reductase dependence. The results suggested that p53 dependence on thioredoxin reductase either was indirect, perhaps mediated by an upstream activator of p53, or was due to oxidation of one or more of the four essential cysteines