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. / Graduation date: 2004

Graduation date: 1999

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 199-212).

Thesis (doctoral)--Umeå Universitet, Sweden, 1988. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Umeå Universitet, Sweden, 1988. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1962. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph.D.)--Montana State University--Bozeman, 2007. / Typescript. Chairperson, Graduate Committee: John W. Peters. Includes bibliographical references (leaves 164-187).