Stimulation of transcription by the human tumor suppressor protein p53 is compromised in yeast lacking the enzyme thioredoxin reductase (Trr1). The result suggests that p53 is prone to oxidative inactivation. This hypothesis and the mechanism of inactivation will be investigated using genetic tools unique to the yeast system. Unlike deletion of the TRR1 gene, deletion of both yeast thioredoxin genes (TRX1 and 2) does not affect p53 activity. Before concluding that Trr1 affects p53 independently of thioredoxin, the possibility that loss of p53 activity is due to accumulation of oxidized thioredoxin will be investigated by determining whether deleting TRX1 and 2 suppresses the effect of deleting TRR1 (Aim 1). Deletion of TRR1 results in accumulation of oxidized glutathione. To test whether reduced p53 activity in deltatrr1 yeast is an indirect consequence of increased glutathione oxidation, the effect of deleting and overexpressing the glutathione reductase gene GLR1 on p53 activity and glutathione redox state will be determined (Aim 2). If deleting GLR1 results in glutathione oxidation but does not affect p53 activity, and if high copy GLR1 restores glutathione to the reduced state in deltatrr1 yeast but does not restore p53 activity, it would indicate that the thioredoxin system and not the glutathione system is critical for p53 activity. A LexA/full length p53 fusion protein stimulates a Lex operator/LacZ reporter gene in wildtype but not deltatrr1 yeast. A fusion protein lacking the last 256 p53 residues is active in both yeast. To identify the domain that causes p53 to be Trr1- dependent, a finer set of C-terminally-truncated LexA/p53 fusion proteins will be analyzed (Aim 3). To identify cysteines that cause p53 to be Trr-dependent, the effect of Cys-to-Ser mutations on p53 activity in wildtype and deltatrr1 yeast will be determined (Aim 4). To complement the genetic approaches, biochemical evidence of p53 oxidation will be sought (Aim 5). Immunoblots done in the presence or absence of reducing agent will investigate whether p53 is disulfide-bonded to other proteins in deltatrr1 yeast. Differential alkylation assays will investigate whether p53 has fewer free thiols in deltatrr1 yeast. Band shift assays will determine whether p53 modifications that result in Trr1-independent transcriptional activity in vivo result in dithiothreitol-independent DNA binding activity in vitro. Understanding the basis for Trr1-dependent p53 activity in yeast will provide a framework for testing whether p53 is subject to oxidative inactivation in higher eucaryotes, and may provide a molecular explanation for the effects of antioxidants and hypoxia on tumor incidence and progression. Equally important, the experiments test a general approach for deriving oxidation-resistant proteins using yeast.
