1 Molecular and Cellular Biology Program, Oregon State University, Corvallis, Oregon 97331,
2 Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331,
3 Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA
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Abstract |
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Abbreviations: DIT, dithiothreitol; IAA, iodoacetate; IAM, iodoacetamide; PDTC, pyrrolidine dithiocarbamate; PEMSA, protein electrophoretic mobility shift assay
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Introduction |
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In budding yeast, genetic and biochemical evidence indicates that the thioredoxin and glutathione systems carry out partially overlapping functions (30). Cells lacking the TRX1 and TRX2 genes encoding thioredoxin or the GLR1 gene encoding glutathione reductase are viable, but triple mutants lacking all three genes are not. Furthermore, deletion of the TRX1 and TRX2 genes results in a significant increase in the percentage of glutathione that is in the oxidized (GSSG) form (30). Given that glutathione is present at high, c.a. 5 mM, concentrations in the cell, the redox state of glutathione is often considered to be representative of the general redox state of the cell particularly with respect to cellular thiols. If the oxidation of glutathione known to occur in trx1
trx2 yeast also occurs in
trr1 yeast, the inhibitory effect of deleting thioredoxin reductase on p53 activity could be secondary to a primary effect on the redox state of glutathione. To investigate this possibility, the redox state of thioredoxin and glutathione in wild-type and
trr1 yeast was determined, and the effects of overexpressing glutathione reductase on p53 activity and glutathione redox state in
trr1 cells was assayed. The results showed that, although glutathione indeed does become more oxidized in
trr1 yeast, the oxidation of glutathione is neither sufficient nor necessary for p53 inhibition.
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Materials and methods |
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The pRS314PGKp53 effector plasmid expressing human p53 from the yeast PGK1 promoter and the pRS315-p53RE-Z reporter plasmid containing a p53 response element cluster fused upstream from the basal CYC1 promoter and LacZ coding region were described previously (41), where they were referred to as pRS314-PGK-SN and pRS315-PG-ß-gal, respectively. The pRS316-TRR1 plasmid containing the complete TRR1 gene was described previously (34), as was the pRS316 control plasmid (39). The pRS416GPDp53 effector plasmid was made by subcloning a p53-encoding EcoRI/SalI fragment of pBTM-p53 (29) into the yeast expression vector pRS416GPD (32). The highcopy YEp195-GLR1 plasmid was constructed by subcloning a 2072 bp XbaI/HindIII DNA fragment containing the GLR1 gene from pDA21 (1) into the 2 µ vector YEp195 (13).
Yeast were grown with vigorous shaking (120 r.p.m.) at 25°C in flasks that were 10-fold greater than the medium volume. Growth medium was yeast nitrogen broth (YNB) containing 2% glucose and required supplements (38). Cells were harvested at 107 cells/ml, which was determined by A600.
ß-Galactosidase activity was assayed by the rapid-freeze/sarkosyl method (23) and is expressed as nmol ONP min 107 cells (1 A420 = 222 nmol ONP/ml).
Thioredoxin and glutathione redox state assays
Exponentially growing yeast (50 ml cultures) were harvested by centrifugation (5 min at 1000 g), washed once with water, and flash frozen in liquid nitrogen. Frozen pellets were pulverized by grinding under liquid nitrogen using a mortar and pestle. For GSH and GSSG determinations, ground pellets were processed as described by Fariss and Reed (12). Data were analyzed by ANOVA followed by pairwise t-tests, with P < 0.01 taken as significant. For thioredoxin redox state determinations, ground pellets were processed as described by Bersani et al. (3). Briefly, lysates were alkylated with 30 mM iodoacetate (IAA), washed with cold acetone, reduced with 3 mM dithiothreitol (DTT), and alkylated with 10 mM iodoacetamide (IAM), as described by Takahashi and Hirose (40). All reactions were done in 8 M urea, to denature proteins and expose all thiols to alkylating and reducing agents. Protein concentration was determined (5) and equal amounts of protein (515 µg) were separated by ureaPAGE and blotted to Hybond ECL membrane (Amersham Pharmacia Biotech, Piscataway, NJ). Blots were immunostained with a 1:10 000 dilution of rabbit anti-yeast thioredoxin primary antibody (31), 1:10 000 dilution of HRP-conjugated goat anti-rabbit IgG secondary antibody (Promega, Madison, WI) and ECL chemoluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ). All incubations were in TBS-T (15 mM Tris pH 7.6, 140 mM NaCl, 0.1% Tween). The intensity of each thioredoxin band was quantitated using a Molecular Dynamics densitometer and ImageQuant software. Variation in the total thioredoxin signal between lanes was sometimes observed. When the source of this variation was investigated by analyzing aliquots of the alkylation reactions by standard SDSPAGE and immunoblotting, we found that the samples contained different levels of total thioredoxin. We concluded that the recovery of thioredoxin during the extraction or alkylation reactions was non-uniform. Variation in total thioredoxin signal between lanes did not affect our analysis of the fraction of thioredoxin in each isoform within a lane.
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Results |
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Table I shows the range of values obtained when the ratio of fully-oxidized, half-oxidized and fully-reduced thioredoxin was determined in several independently-grown yeast cultures. The 70:12:18 ratio obtained for
trr1 yeast differed significantly from the 34:13:54 ratio obtained for wild-type yeast. The
trr1 mutation resulted in a 2.1-fold increase in the fraction of thioredoxin that was in the disulfide form. As an alternative means of quantitating and summarizing the extent of thioredoxin oxidation under each condition, the percent of thioredoxin cysteines oxidized in each individual sample was calculated by multiplying the fraction of thioredoxin in each isoform by the fraction of oxidized cysteine in that isoform (1 for the disulfide, 0.5 for the intermediate, 0 for the dithiol) and summing the products. Mean values for the percent of thioredoxin cysteines oxidized in wild-type cells versus
trr1 cells are shown in Table I
. The
trr1 mutation resulted in a 1.9-fold increase in the percent of thioredoxin cysteines oxidized.
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In summary, although some fully reduced thioredoxin persisted in trr1 cells, the
trr1 mutation resulted in a significant shift of thioredoxin to the disulfide form. The observation that efficient reduction of thioredoxin in vivo was dependent on the product of the TRR1 gene, confirmed the physiological relationship between thioredoxin reductase and thioredoxin long suggested from in vitro enzyme assays.
It was previously shown that S.cerevisiae lacking thioredoxin have increased total glutathione levels and a higher GSSG:GSH ratio (30). To determine if deletion of thioredoxin reductase had similar effects, the amounts of GSH and GSSG in wild-type and trr1 yeast were determined. As shown in Figure 2A
, the
trr1 mutation resulted in significantly higher total glutathione levels and a disproportionate increase in the levels of GSSG. Wild-type yeast contained 51 nmol of glutathione per mg protein, 18% of which was in the GSSG form. In contrast,
trr1 yeast contained 146 nmol of glutathione per mg protein, 32% of which was in the GSSG form. When
trr1 yeast were transformed with a TRR1-containing plasmid (pRS316-TRR1), total glutathione and the percent of total glutathione in the GSSG form dropped to levels near that of wild-type cells. In contrast, when
trr1 cells were transformed with an empty control plasmid (pRS316), total glutathione and the percent of total glutathione in the GSSG form remained significantly elevated. In our experiments, wild-type and
trr1 yeast yielded 2.5 and 3.8 µg of extract protein per 107 cells, respectively. Thus, wild-type and
trr1 yeast contained 0.13 nmole and 0.60 nmol of total glutathione per 107 cells, respectively. Our values for total glutathione in wild-type yeast agree closely with previous reports (30,46). Elevated total glutathione levels in
trr1 yeast may be the result of induced expression of the GSH1 and GSH2 genes encoding
-glutamylcysteine synthetase and glutathione synthetase, respectively. Oxidative stress in general, and deletion of TRR1 in particular, has been shown to induce expression of several genes encoding proteins with antioxidant roles, including GSH1 and GSH2 (6). The results in Figure 2A
suggest that, in the absence of thioredoxin reductase, greater demands were put on the glutathione system, resulting in a higher GSSG:GSH ratio and higher total glutathione levels.
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Glutathione reductase overexpression in trr1 yeast restores the GSSG:GSH ratio but not p53 activity
Given that oxidized glutathione accumulates in trr1 yeast, inhibition of p53 activity may be a consequence of glutathione oxidation, rather than thioredoxin oxidation. Therefore, to distinguish the possible role of glutathione redox status from that of thioredoxin redox status in p53 inhibition, we introduced a highcopy plasmid containing the yeast glutathione reductase gene GLR1 (orf YPL091W) into
trr1 cells, and determined the effect of GLR1 overexpression on both glutathione redox status and p53 reporter gene transactivation. As shown in Figure 3
, in
trr1 yeast transformed with the highcopy GLR1 plasmid (YEp195-GLR1), 15% of total glutathione was in the GSSG form, which was not significantly different from the 18% level present in wild-type cells (Figure 2A
) and was significantly less than the 32% level present in
trr1 cells transformed with an empty highcopy control plasmid (YEp195).
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Figure 3 also shows the effect of GLR1 overexpression on p53 activity. If inhibition of p53 in
trr1 yeast was due to an increased GSSG:GSH ratio, reduction of that ratio, through overexpression of GLR1, should alleviate the inhibition of p53 activity. Contrary to this prediction, overexpression of GLR1 in
trr1 cells had no restorative effect on p53 activity. As it is the GSSG:GSH ratio that governs the equilibrium position of glutathione-dependent protein disulfide reduction reactions, the failure of highcopy GLR1 to restore p53 activity, even though it restored the GSSG:GSH ratio to normal levels, indicates that p53 is not responsive to the glutathione system. Highcopy GLR1 expression in
trr1 cells did not affect the redox state of thioredoxin, which remained primarily oxidized (data not shown). The data in Figure 3
show that glutathione oxidation is not necessary for p53 inhibition in
trr1 yeast, and support the idea that p53 activity is specifically sensitive to the redox state of thioredoxin.
Deletion of the glutathione reductase gene does not affect p53 activity
As an additional approach to test glutathione involvement in controlling p53 activity and to test the specificity of thioredoxin involvement, the effect of deleting the GLR1 gene on p53-dependent reporter gene expression was determined. The GLR1 gene is the only glutathione reductase gene present in the yeast genome. Two previous studies showed that deletion of the yeast GLR1 gene results in more than a four-fold increase in the GSSG:GSH ratio (17,30). As shown in Table II, deletion of GLR1 did not affect p53 activity. Identical levels of p53-dependent reporter gene transactivation were observed in congenic yeast strains CY4 and CY7, even though the latter strain bears a
glr1 null mutation and has a 4.6-higher GSSG:GSH ratio (17). The
glr1 mutation did not affect the redox state of thioredoxin (data not shown). The data in Table II
support the conclusion drawn from Figure 3
that p53 activity is not responsive to the glutathione system.
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Discussion |
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In both fission yeast and budding yeast, efficient reporter gene transactivation by p53 requires thioredoxin reductase (7,29,34). Immunoblotting experiments established that reduced reporter gene expression is not due to an effect of thioredoxin reductase deletion on p53 protein levels (7,34). Sequence-specific DNA binding proteins such as p53 stimulate transcription by recruiting coactivators with histone acetylase activity to promoter regions (18,25). It is unlikely that reduced reporter gene transactivation by p53 in trr1 yeast is due to an effect on coactivator activity, as reporter gene transactivation occurs equally well in wild-type and
trr1 yeast, if the N-terminal activation domain of p53 is fused to the heterologous DNA binding protein LexA (29). Additional LexA/p53 fusion protein experiments indicate that loss of thioredoxin reductase inhibits both the transactivation activity of p53, by a mechanism that requires the C-terminal negative regulatory domain, and the DNA binding activity of p53, by a mechanism that is independent of the negative regulatory domain (29). The
trr1 mutation specifically inhibits transcription of p53-dependent reporter genes. Expression of reporter genes containing the same basal CYC1 promoter used in our studies, but possessing either a native CYC1 upstream element or a heterologous MCB cell cycle box element rather than a p53 binding site, is stimulated, rather than inhibited, by a
trr1 mutation (26). In addition, northern blot analyses showed that the
trr1 mutation does not affect the level of several endogenous gene mRNAs (26).
Deletion of the two yeast thioredoxin genes does not phenocopy the effect that deleting the yeast thioredoxin reductase gene has on p53 activity. In fact, the inhibitory effect of the trr1 mutation on p53-dependent reporter gene transactivation is suppressed if the yeast thioredoxin genes are also deleted (unpublished data). This suggests that it is the increase in oxidized thioredoxin, rather than the decrease in reduced thioredoxin (Figure 1
and Table I
), that is responsible for p53 inhibition in
trr1 cells. Thus far, we have not been able to determine the redox state of p53 in yeast or mammalian cells. When the PEMSA method used to measure the redox state of thioredoxin (Figure 1
) was applied to p53, the protein formed an aggregate that failed to enter the resolving gel during ureaPAGE. Other strategies, based on alkylation reactions that add bulky, fluorescent, or immunoreactive adducts have thus far also been unsuccessful.
It is not clear whether thioredoxin directly affects p53 activity in vivo, or acts through intermediates. Deletion of thioredoxin reductase probably has many pleiotrophic effects, any of which could modulate p53 activity. However, several observations support the idea of a direct interaction. In a screen for mutations that bypassed a p53-induced cell cycle arrest in Sc. pombe, only the TRR1 gene was identified (7). Furthermore, purified thioredoxin has been demonstrated to promote p53 binding to target DNA in vitro (42). Finally, it was reported in a recent review that the two proteins can be co-immunoprecipitated from mammalian cell extracts (19).
In summary, the yeast results reported herein indicate that p53 is not sensitive to the overall redox state of the cell, as reflected in the GSSG:GSH ratio. Deletion of the glutathione reductase gene did not inhibit p53 activity (Table II), even though it is known to increase the GSSG:GSH ratio more than four-fold (17,30). Furthermore, overexpression of glutathione reductase in
trr1 null yeast did not restore p53 activity, even though it restored the GSSG:GSH ratio to near that of wild-type cells (Figure 3
). Thus, glutathione oxidation is neither sufficient nor necessary for p53 inhibition. The results indicate that p53 inhibition in
trr1 yeast is not due to a non-specific, generalized increase in the oxidation state of the cell. Rather, p53, or a protein controlling p53, is specifically sensitive to the redox state of thioredoxin.
The yeast results raise interesting questions concerning redox control of p53 activity and cancer. First, it suggests a potential mechanism for the cancer-preventive activity of selenium in clinical trials (10). Mammalian thioredoxin reductase is one of the few selenoproteins in the proteome (14) and its C-terminal selenocysteine is critical for enzyme activity (15). Selenium has been shown to increase the level and specific activity of thioredoxin reductase in several cultured cell lines (2). If dietary selenium similarly induces thioredoxin reductase activity in vivo, it may help prevent or reverse oxidative inactivation of p53 by insuring that the disulfide form of thioredoxin is quickly regenerated to the dithiol form under oxidative conditions. Secondly, p53 dependence on the redox state of thioredoxin may explain why inactivating p53 mutations are prevalent in non-vascularized large solid tumors. Perhaps, the hypoxic environment of non-vascularized tumors conserves reduced thioredoxin and favors p53-induced cell cycle arrest or apoptosis, thus limiting tumor volume and selecting for clonal expansion of cells with inactivating p53 mutations (16). Hypoxia has been shown to induce p53 protein levels in several cell lines and to enhance p53 target gene induction in response to DNA damage in MCF-7 cells (24). Why would cells evolve a p53 protein that is prone to oxidative inactivation or dependent on the redox state of thioredoxin? Oxidative inactivation may be an important mechanism for limiting p53 activity. For example, the oxygen-rich conditions resulting from tissue vascularization may help insure against inappropriate p53 activation during normal development. Similarly, following a DNA damage-induced cell cycle arrest, a mechanism for inactivating p53 once the damaged DNA has been repaired would be important. Oxidative inactivation of p53 may be a passive process. Alternatively, p53 dependence on thioredoxin may constitute a regulatory mechanism for redox communication between p53 and other protein thiols. For example, increased flux through ribonucleotide reductase during a normal S phase or as replication resumes following DNA repair might oxidize thioredoxin and thereby help inactivate p53.
Regulation of p53 activity is complex. The protein interacts with multiple proteins and is subject to several covalent modifications such as phosphorylation, acetylation and sumoylation. Dependence on an intact thioredoxin system suggests that p53 is subject to yet another type of covalent modification cysteine oxidation. Mouse p53 protein contains 12 cysteines, three of which are essential for DNA binding in vitro and reporter gene transactivation in transfected mammalian cells (37). The homologs of these three cysteines, as well as a fourth cysteine (Cys275), are similarly essential for reporter gene transactivation by human p53 in yeast (unpublished data). Mutation of the six non-essential human p53 cysteines to serine, singly or in combination, does not allow human p53 to escape thioredoxin reductase-dependence in yeast (unpublished data). Thus, either the cysteines causing p53 to be prone to oxidative inactivation are essential for protein function, or the dependence of p53 on an intact thioredoxin system is more complex than the simple reduction of p53 disulfides.
Yeast offers several advantages as a system for studying fundamental aspects of p53 function. In non-transformed mammalian cells, p53 induction of target gene expression triggers feedback inhibition of p53 through Mdm2 and results in complex physiological responses such as cell cycle arrest and apoptosis. These complicating sequelae do not occur in yeast. Furthermore, in yeast, genetic tools can be used to identify core processes required for p53 transactivation of target gene expression. On the other hand, the mammalian cell relevance of yeast results is open to question. For example, an important unresolved question raised by our yeast work is whether p53 requires an intact thioredoxin system in mammalian cells. Several strategies aimed at reducing thioredoxin reductase levels in mammalian cells in culture such as selenium deprivation, dominant negative transgenes and morpholino antisense compounds have thus far failed to give sufficient thioredoxin reductase inhibition to affect the redox state of thioredoxin (unpublished data). Thus, to investigate the relationship between thioredoxin reductase and p53 activity in mammalian cells, gene ablation may be necessary. As previous studies have shown that deletion of a mouse thioredoxin gene or a selenocysteine tRNA gene results in early embryonic lethality (4,27), it may be necessary to develop conditional null alleles in order to investigate the role of the thioredoxin system in mammalian growth, development, oncogenesis and tumor suppression.
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Notes |
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5 To whom correspondence should be addressed Email: merrillg{at}ucs.orst.edu
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Acknowledgments |
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References |
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