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 (Kd = 0.9 microM 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 Deltatrr1 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.
Stoner
Christopher S
CS
Department of Biochemistry and Biophysics, Oregon State University, 2011 ALS Building, Corvallis, Oregon 97331-7305, USA.
Pearson
George D
GD
Koç
Ahmet
A
Merwin
Jason R
JR
Lopez
Nathan I
NI
Merrill
Gary F
GF
D019943:Amino Acid Substitution; D001483:Base Sequence; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D017353:Gene Deletion; D005800:Genes, Fungal; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; D011994:Recombinant Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine
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