Yeast studies indicate that deletion of thioredoxin reductase is not inhibiting p53 activity via an effect on the overall redox state of the cell, as defined by the redox state of glutathione (13
). Although the Δtrr1
mutation results in a 2.5-fold increase in the GSSG:GSH ratio, restoration of the normal GSSG:GSH ratio in Δtrr1
cells by overexpressing the GLR1
gene encoding glutathione reductase does not restore p53 activity (13
). Furthermore, deletion of the GLR1
gene, which results in more than a fivefold increase in the GSSG:GSH ratio (17
), does not inhibit p53 activity (13
). Thus, glutathione oxidation is neither sufficient nor necessary for p53 inhibition. Rather, p53 specifically depends on an intact thioredoxin system. We have previously shown that oxidized thioredoxin accumulates in yeast lacking thioredoxin reductase (13
). Given the current observation that thioredoxin deletion does not inhibit p53 activity and, in fact, suppresses the inhibitory effect of thioredoxin reductase deletion on p53 activity, our results suggest that it is the presence of oxidized thioredoxin rather than the absence of reduced thioredoxin that inhibits p53 activity. Oxidized thioredoxin may inhibit p53 activity by removing electrons from one or more cysteine residues on p53. Alternatively, it is possible that oxidized thioredoxin affects p53 activity by a mechanism that does not involve disulfide/dithiol exchange between the two proteins. For example, NADP+
and NADPH have been shown to have reciprocal allosteric effects on the DNA binding activity of circadium rhythm transcription factors (32
). Also, reduced, but not oxidized, thioredoxin binds and allosterically inhibits the apoptosis signaling kinase 1 (ASK1) (33
). Thus, it was possible that oxidized thioredoxin affected p53 activity by an allosteric mechanism rather than a mechanism involving disulfide-dithiol exchange. Our analyses using p53 cysteine substitution mutants were an attempt to obtain evidence for a mechanism involving disulfide-dithiol exchange between p53 and thioredoxin.
In mammalian cells, one mechanism for redox control of p53 is thought to be mediated by the dual function protein Ref1. Ref1 was originally purified and cloned by virtue of its ability to stimulate the DNA binding activity of the Fos/Jun transcription factor AP1 (34
) and was subsequently shown to possess apurinic/apyrimidinic endonuclease (APE) activity. The redox active domain and APE domain functionally map to the N-terminal and C-terminal halves of the protein, respectively (35
). Ref1 was purified a second time by virtue of its ability to stimulate the transcription-inducing activity of p53 (6
). Consistent with a role for Ref1 in controlling p53, p53 cysteine reduction and increased p53 reporter gene transactivation activity in the presence of selenomethionine are suppressed in cells co-transfected with a dominant-negative allele of the ref1
). However, it is also clear that the thioredoxin system can affect the activity of p53 in the absence of Ref1. Thioredoxin enhances p53 binding to DNA in vitro
in the absence of Ref1 (1
). Furthermore, thioredoxin reductase mutations affect p53 activity in yeast (7
), even though yeast contains no close homolog of Ref1, and the most similar yeast protein, the apurinic/apyrimidinic endonuclease APN2 (orf YBL019W), lacks the N-terminal domain and Cys65 residue that is essential for the redox activity of human Ref1 (35
). Finally, our current results showing that purified thioredoxin and p53 physically interact are consistent with a direct effect of thioredoxin on p53 activity. Although it remains possible that, in vivo
, oxidized thioredoxin affects p53 activity via a primary effect on an intermediary protein analogous to Ref1, the simplest explanation compatible with all observations is that thioredoxin directly interacts with p53 and thereby affects the ability of p53 to bind and transactivate target genes.
results suggest that p53 is extremely prone to oxidation. Binding of p53 to target DNA in vitro
requires the presence of reductant in the binding buffer (2
), and incubation of p53 in the absence of reductant leads to rapid changes in immunoreactivity with conformation-specific antibodies (2
). Alkylation studies with [14
C]-iodoacetamide indicate that p53 rapidly becomes refractive to labeling when the protein is incubated or stored in the absence of reductant (2
). Other studies suggest that p53 is prone to oxidation in vivo
. Treatment of human cells with the copper chelator pyrrolidine dithiocarbamate inhibits p53 activity, and the pattern of p53 reactivity with alkylating reagents suggests that an undetermined number of p53 cysteines become oxidized (36
). Furthermore, essentially no p53 cysteines are accessible to thiol-directed alkylating reagents unless the cells are pre-treated with the micronutrient selenomethionine (27
Recent biochemical and pharmacological evidence suggests that the link between thioredoxin reductase and p53 activity suggested by our yeast results also exists in mammalian cells. Using an approach involving sequential treatment of cell lysates with N-ethylmaleimide, DTT and 3-(maleimidopropioryl)-biocytin, it was demonstrated that transiently-expressed p53 in human H1299 cells contain at least one and possibly several oxidized cysteines (27
). Significantly, if cells are pre-incubated with selenomethionine, all of the p53 cysteines that were previously oxidized become reduced. Similarly, in cells expressing a p53 fragment containing only Cys275 and Cys277, no cysteines are reactive with an alkylating reagent unless the cells are pre-incubated with selenomethionine (27
). In addition to affecting the redox state of p53, pre-incubation with selenomethionine also modestly induces a p53-dependent reporter gene in transfected H1299 cells (27
). Thioredoxin reductase is one of twenty-five selenoproteins identified in the mammalian proteome (38
). Its penultimate selenocysteine residue is essential for catalytic activity (39
), and incubation with selenium has been shown to boost thioredoxin reductase activity levels in several cells lines (40
). Although the evidence is indirect, the effects of selenomethionine on the redox state and activity of p53 (27
) are consistent with the idea that p53 is prone to oxidation in certain mammalian cell lines and that thioredoxin reductase helps to maintain p53 in the reduced and active state.
Further support for the control of p53 activity through redox communication with the thioredoxin reductase system comes from a recent study that shows high concentrations of prostaglandins PGA1
, and certain other electrophillic lipids inhibit both thioredoxin reductase activity and p53-dependent reporter gene transactivation in transfected mammalian cells (41
). In addition, a biotinylated PGA1
-derivative forms covalent adducts with several cellular proteins, including thioredoxin reductase and thioredoxin, but does not form adducts with p53. Furthermore, pre-incubation of cells with selenite partially suppresses the inhibitory effect of PGA2
on p53-dependent reporter gene transactivation (41
). Although prostaglandin and selenite treatment may affect the activity of other proteins in the cell, the results are consistent with the conclusion that efficient target gene transactivation by p53 in mammalian cells requires thioredoxin reductase. Proof of a linkage between thioredoxin reductase activity and p53 activity in mammalian cells will require the development of methods to specifically inhibit thioredoxin reductase in higher eukaryotes.
We proposed and tested a model for regulation of p53 transactivation activity by direct cysteine redox communication between p53 and thioredoxin in yeast. No single replacement of cysteine with serine relieved the dependence of p53 on thioredoxin reductase, nor did any combination of replacements involving the six nonessential residues (C124S, C135S, C141S, C182, C229S and C277S). We could not assess thioredoxin reductase dependence of the p53 C135/229S allele due to extremely low levels of reporter gene transactivation in both TRR1 and Δtrr1 cells. However, the C135/141A-HEX mutant, which contains substitutions at codon 135 and 229, was both active and regulated by thioredoxin reductase. For these reasons, we consider it unlikely that oxidation of any of the six nonessential cysteine residues of p53 was responsible for thioredoxin reductase dependence.
Since bound zinc is required for p53 DNA binding activity and zinc is lost from p53 under oxidizing conditions in vitro
), it is possible that p53 activity is controlled in vivo
through redox regulation of the zinc-coordinating cysteine residues C176, C238 and C242. X-ray crystallography of murine p53 shows zinc-free protein containing a disulfide between C173 and C239 (homologs of human p53 C176 and C242, respectively) (42
). The structure is superimposable on the structure of zinc-containing reduced p53, with the exception of the L3 loop containing C235, C239 and R245 (homologs of human C238, C242 and R248). This loop is rotated nearly ninety degrees from the normal position in the oxidized structure, thereby moving R245 away from the minor groove of DNA. We speculate that disulfide formation between C176 and C242 may serve as a mechanism for preventing gross unfolding and degradation of of the apoprotein when zinc is lost due to either stochastic or regulated events.
The possibility of C275 serving as a redox control point is intriguing. The residue lies in the major groove when p53 binds DNA. Mutation of the residue to either serine or alanine — shape and dipole mimics, respectively — uniformly inactivated p53 transactivation in the yeast model system. The incompatibility of both shape and dipole analogs indicated that a cysteine thiolate anion may either be required for DNA contact or for maintaining local structure such that an essential DNA contact by a nearby residue is made. For example, the essential DNA-contacting residue R273 is in the same sheet-loop structure (24
), and may be dependent on a C275 thiolate for proper orientation. In addition, mutation of C275S strongly impaired the ability of p53 to transactivate a panel of response elements. Finally, it was previously shown that at least one unidentified p53 cysteine is oxidized under standard cell culture conditions, that the residue is fully reduced in response to selenomethionine treatment, and that a truncated p53 peptide containing only cysteines 275 and 277 exhibits the same pattern (27
). Our result, showing that mutation of C277 has no effect on thioredoxin reductase dependence, leaves C275 as the remaining available site for redox chemistry.