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
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/30498 |
| Date | 11 September 2003 |
| Creators | Merwin, Jason R. |
| Contributors | Merrill, Gary F. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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