From the
Department of Biochemistry, Paichai University, Taejon 302-735, Republic of Korea, the
Computer-aided Molecular Design Research Center, Soongsil University, Sangdo-5-dong, Dongjak Ku, Seoul 156-743, Republic of Korea, and the ¶Department of Bioengineering, Yonsei University, Seoul 120-749, Republic of Korea
Received for publication, March 14, 2003 , and in revised form, April 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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Although among the yeast TPx isoforms, the putative nuclear isoform of yeast TPx (nTPx) is a newly reported antioxidant protein localized in the nucleus, there is no evidence supporting in vivo function of nTPx as an antioxidant. In this report, nTPx, a putative yeast member of TSA/AhpC family, was characterized as a functional thiol peroxidase acting as alkyl-hydroperoxide reductase in nucleus during post-diauxic growth of Saccharomyces cerevisiae.
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EXPERIMENTAL PROCEDURES |
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Cloning and Mutagenesis of nTPxThe nTPx gene was amplified from yeast genomic DNA by PCR with two pairs of primers covering whole coding sequence. The forward primer has an NdeI site, and the reverse primer has a BamHI site for cloning. The following primers were used for the PCR of the nTPx gene, and enzyme sites are underlined: 5'-CGATCCATATG GGT GAA GCA CTA CGT AG (forward) and 5'-CGCGGATCC TTA TTC TTC TTT AAA CTT TTC AGC G (reverse). The resulting PCR products were cloned into Escherichia coli expression vector pT7-7. For substitution of two cysteine residues of nTPx, PCR-based strategy was employed to introduce nucleotide substitution at defined location (23). For replacement of putative functional cysteines of nTPx with serine, the respective cysteine codon was changed to a serine codon. Three mutant proteins (C106S, C111S, and C106S/C111S), in which Cys-106 and Cys-111 were individually replace with serine, were generated by the standard PCR-mediated site-directed mutagenesis with complementary primers containing a 1-base pair mismatch that converts the codon for cysteine to the codon for serine. The mutated PCR products were ligated into pT7-7 digested with NdeI and BamHI.
Expression and Purification of Recombinant TPx ProteinsTransformed cells were cultured at 37 °C overnight in LB medium supplemented with ampicillin (100 µg/ml) and then transferred to fresh medium to the ratio of 1:200. When the optical density of the culture at 600 nm reached 0.4, isopropyl-1-thio--D-galactopyranoside was added to final concentration of 0.5 mM. After induction for 3 h, the cells were harvested by centrifugation and stored at 70 °C until use. The frozen cells were suspended in 50 mM Tris-HCl (pH 8.0) containing 2 mM phenylmethylsulfonyl fluoride and 1 mM EDTA and disrupted. The supernatants after centrifugation at 18,000 rpm using a SS-34 rotor were loaded to a DEAE cellulose column that had been previously equilibrated with 50 mM Tris-HCl (pH 7.4). The proteins were eluted with a linear gradient of NaCl from 0 to 500 mM. The fractions corresponding to the peak of nTPx were pooled and precipitated with 80% ammonium sulfate. The dissolved sample was applied to Sephacryl-100 column that had been previously equilibrated with 50 mM Tris-HCl buffer (pH 7.4) containing 200 mM NaCl. The purified sample was stored at 70 °C.
Chemical Modification of nTPx with N-Ethylmaleimide The TPx was preincubated in the absence or presence of 1 mM DTT at 30 °C for 30 min, and then chemical modification was carried out in a 100-µl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 2.0 mg/ml nTPx, and 10 mM N-ethylmaleimide at 30 °C for 2 h as previously described. To remove unreacted reagents, the reaction mixture was extensively dialyzed against 10 mM Tris-HCl (pH 7.4) at 4 °C.
Determination of Thiol-dependent Antioxidant ActivityThe antioxidant activity was determined by measuring the activity to protect the inactivation of E. coli glutamine synthetase (GS) by a thiol metal-catalyzed oxidation system (DTT/Fe3+/O2) (thiol metal-catalyzed oxidation system) as described previously (4). The 30-µl reaction mixture containing 100 mM Hepes-NaOH (pH 7.0), 1.0 µg of GS, 3 µM FeCl3, various concentrations of nTPx, and 10 mM DTT was incubated at 37 °C, and then 0.5 ml of -glutamyltransferase assay mixture was added. After incubation at 37 °C for 10 min, the remaining activity of GS was determined by measuring the absorbance at 540 nm.
Determination of Thioredoxin-linked Peroxidase Activity of nTPx Peroxidase reaction was performed in a 350-µl of a reaction mixture containing 50 mM Hepes-NaOH (pH 7.0), 0.8 µM thioredoxin, 0.3 µM thioredoxin reductase, 0.26 mM NADPH, varying concentrations of nTPx, and 300 µM of peroxide at room temperature. The peroxidase activity of TPx linked to NADPH oxidation was traced.
Construction of nTPx Promoter--Galactosidase (lacZ) FusionThe nTPx promoter-lacZ fusion plasmid was constructed using a PCR-amplified DNA fragment. A putative promoter sequence of nTPx was identified by the analysis with the SGD program (Stanford University). The promoter sequence (positions 345 to 1) was amplified using genomic DNA from a yeast strain (JD7-7C) and a pair of primers (5'-CGGGGTACCTTGTCACTTATTAATGATAAATTATAA and 5'-CGCGGATTCTCTATTAAGGAACTTTAATATTACC). These primers introduce KpnI (forward) and BamHI (reverse) sites (underlined) for in-frame directional cloning into plasmid digested with KpnI and BamHI. To make a
-galactosidase-fused promoter sequences, the lacZ gene was amplified with primers (5'-CGCGGATCCATGACCATGATTACGGATTCACT and 5'-GGTGAAGCTTATATTATTTTTGACACCAGACC), digested with BamHI and HindIII, and cloned into YEG
-HIR525 digested with same enzymes to produce pYLac. The PCR products of nTPx promoter was digested with a KpnI and BamHI and cloned into pYLac digested with KpnI and BamHI to give pYcTPxIIP-lacZ fusion vector. DNA sequencing confirmed that no mutation had been introduced in the promoter during PCR amplification.
Long Term Stationary Phase CultureTo test the viability of stationary phase yeast cells, after 72 h of culture in YPD medium, the cells were washed three times and resuspended in sterile distilled water, and incubation at 25 °C with shaking was continued. The cell viability was measured by plating serial dilutions of the yeast cultures on to YPD plates. The colonies formed were counted after 2 days of incubation at 30 °C. The experiments were repeated twice with 10 independent samples.
Other MethodsYeast cells were harvested and disrupted by vortexing with glass beads, and the -galatosidase activity was assayed using o-nitrophenyl-
-D-galactoside essentially as described previously (22). The
-galatosidase activity is expressed as unit (increase in A412 nm resulted from o-nitrophenyl-
-D-galactoside hydrolyzed by
-galatosidase/10 min/mg of protein). Patch assays were done as described below. Aliquots (10 µl) containing
103 or the indicated cell number of an overnight culture were spotted on YPD plates containing oxidants at the indicated concentration. The plates were monitored after 25 days of incubation at 30 °C. The protein concentration was determined using a Bradford protein assay kit (Bio-Rad). Yeast transformation, DNA, protein extraction from yeast, and other methods not mentioned were carried out according to a supplier manual or the standard protocol described (24). Immunoblot analysis was performed using rabbit polyclonal antibodies against nTPx. Transfer of proteins from 12% SDS-PAGE gels to nitrocellulose and processing of nitrocellulose blots were carried out according to a standard protocol. Northern blot analysis of nTPx mRNA was carried out according to a standard protocol. Yeast total RNA (20 µg) was fractionated in a 1.5% formaldehyde-agarose gel and transferred to a nylon membrane, and the resultant blot was hybridized with 32P-labeled nTPx structural gene.
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RESULTS |
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Members of the TSA/AhpC family can be divided into two subgroups, such as one-cysteine and two-cysteine groups according to the number of conserved cysteines within the protein (3). The two Cys-containing protein exists as a homodimer via an intermolecular disulfide bond. Previously, we suggested that except for nTPx, all of the yeast TPx isoforms exist as the dimer (22). Nuclear TPx was detected at the molecular mass corresponding to that of monomer regardless of the presence or absence of DTT (22). Analysis of nTPx mutant proteins on a nonreducing and reducing SDS-PAGE gel showed that in contrast to other mutant and wild type proteins, most of the C111S proteins exist in the dimer form (Fig. 1). The existence of C111S in the dimer form can be taken as an evidence supporting the intramolecular disulfide linkage between two cysteines of nTPx. To confirm the intramolecular disulfide bond, we reacted nTPx with an excessive amount of a thiol-specific modification reagent, N-ethylmaleimide, in the absence or presence of DTT. The treatment of nTPx with N-ethylmaleimide only in the presence of DTT resulted in a considerable loss of antioxidant activity (data not shown), which also indicates that the SH group of the functional Cys-106 forms the disulfide bond with the SH group of Cys-111 during the catalytic cycle of the reduction reaction. Previously, mammalian Prx V (a mammalian TPx) and yeast cTPx III were shown to form an intermediate with an intramolecular disulfide (22, 25). These results, therefore, indicate that nTPx is an atypical two-Cys subgroup member, which forms an intramolecular disulfide as an intermediate (25).
To investigate the substrate specificity of nTPx, we measured the Trx-linked thiol peroxidase activity toward H2O2, t-butyl hydroperoxide, and cumene-hydroperoxide. Fig. 2C showed that among the peroxides as the substrate, cumenehydroperoxide was most rapidly reduced, but H2O2 was not significantly reduced (cumene-hydroperoxide > t-butyl-hydroperoxide » H2O2). The preference of nTPx to alkyl-hydroperoxide as a substrate suggests the in vivo function of nTPx as an alkyl-hydroperoxide reductase. In addition, it is noteworthy that in contrast to other yeast TPx isoforms (22), nTPx more easily reduced bulky hydroperoxide such as cumene-hydroperoxide containing benzyl group as the alkyl moiety. The preference for bulky substrate suggests that nTPx may be designed to remove the hydroperoxide linked to bulky groups such as the base groups of DNA. Collectively, these data demonstrate that nTPx is a functional thiol peroxidase exerting alkyl-hydroperoxide reductase activity.
Physiology of nTPx Null Mutant StrainTo investigate the in vivo antioxidant function of nTPx, we imposed various oxidants on nTPx null mutant (nTPx) growing aerobically on LB medium containing varying concentrations of 4-NQO. The growth of nTPx
was considerably retarded by 4-NQO, a DNA oxidation agent, when compared with the isogenic strain (Fig. 3). To test the viability of nTPx mutant toward oxidative stress, we exposed various oxidants to the mutant. As expected, nTPx
was much more sensitive to paraquat, diamide, and 4-NQO than the isogenic strain (Fig. 4). In addition, the sensitivity of nTPx
to various peroxides was examined. As shown in Fig. 5, nTPx
showed higher sensitivity toward alkyl-hydroperoxide such as t-butyl-hydroperoxide and cumene-hydroperoxide than the wild strain but did not toward H2O2, indicating the in vivo function of nTPx as an alkyl-hydroperoxide reductase. The alkyl-hydroperoxide-specific sensitivity of nTPx
can be explained in terms of its alkyl hydroperoxideselective kinetic properties shown in Fig. 2C. Furthermore, the reduction of the viability of nTPx
against various oxidants supports an in vivo antioxidant role for nTPx.
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Transcription of nTPx Gene Is Turned On at the Diauxic ShiftWhen the cells are cultured in a liquid-rich medium in which the major carbon source is a fermentable carbohydrate (e.g. YPD), they exhibited two distinct growth phases, followed by a stationary phase in which cells cease to divide. During the first phase, cells meet their energy requirements primarily by fermentation. The second growth phase is initiated when cells exhaust most of the fermentable carbon source, undergo major physiological change, and begin to grow at a much slower rate. The shift between these two phases is called the diauxic shift (a switch from fermentative to oxidative metabolism) (26). Several genes such as HSP26 are transcriptionally induced during the diauxic shift, in which dramatic changes in gene expression occur (27). Therefore, it is necessary to monitor the transcriptional activity of nTPx during growth.
To follow a growth-dependent transcription of nTPx gene, we monitored the transcriptional activity of nTPx as function of the growth. Comparison of growth curve before the diauxic shift with their corresponding transcriptional activity (Fig. 6A) indicates that the transcriptional activity of nTPx is not activated in the log phase cells. Therefore, this result suggests the possibility that nTPx gene could be transcriptionally induced during the diauxic shift. To test the possibility, we measured the possible transcriptional activation of nTPx gene upon changing the carbon source from fermentable carbohydrate (i.e. glucose) to nonfermentable carbohydrate (glycerol plus ethanol). As shown in Fig. 6B, the transcriptional activity in the yeast growing on a nonfermentable carbon was about four times higher than that of the yeast on a fermentable carbon when the activities were measured at the mid-log phase growth. Furthermore, the transcriptional activities of Tor1 were much higher when compared with those of the isogenic strain (Fig. 6B). Rapamycin forms a complex with FKBP12 that inhibits components of signal transduction pathways, named TOR (target of rapamycin) pathway. The target of the complex was first identified in yeast as the Tor1p and Tor2p (28). The loss of TOR function at the diauxic shift induces several other physiological changes characteristic of starved cells entering the stationary phase. Inhibition of the TOR signaling pathway by a lack of fermentable carbon induces a set of proteins necessary for the stationary phase yeasts (29, 30). Taken together with the function of the TOR signaling pathway described above, these results suggest that the transcription of nTPx is turned on at the diauxic shift.
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The increase of the nTPx transcript upon deletion of Tor1 suggests that the transcription of nTPx is under down-regulation of TOR pathway. To demonstrate negative control on transactivation of the nTPx promoter by TOR pathway, we monitored the transcriptional activity of the nTPx promoter in tor1 mutants throughout the yeast growth cycles. Figs. 6B and 7A show that disruption of Tor1 resulted in a dramatic increase, which is consistent with the Northern blot analysis (Fig. 7A, inset). Taken together, these results demonstrate that Tor1p suppresses the transactivation of nTPx via activation of the TOR pathway.
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In S. cerevisiae, Ras proteins, which encode GTP-binding proteins, are activated by both growth signals (e.g. glucose) (31) and stress signals (e.g. UV radiation and starvation) (3234). The unregulated Ras/cAMP pathway suppresses the activation of a large number of stress-related genes (3234). Ras proteins activate the cAMP-dependent protein kinase A activity, which in turn turns on TOR pathway before the diauxic growth. We have demonstrated the negative control of the TOR signaling pathway on the expression of nTPx in response to glucose starvation (Fig. 8). To elucidate the transactivation of nTPx under negative control of Ras/cAMP pathway, we determined the transactivational ability of nTPx in Ras1 and Ras2 mutants (Fig. 7B). The induction pattern of the transcriptional activities in Ras mutants shows that in contrast to the wild type cell (YPK9), the transactivation of nTPx in Ras1 and Ras2
occurs during early log phase ras1
and ras2
cells (Fig. 7B). Each disruption of Ras1 and Ras2 resulted in an increase in the transcriptional activity with
3- and
10-fold inductions, respectively, even in the mid-log phase cells (cells cultured for 12 h), which is consistent with the Northern blot analysis (Fig. 7B, inset). Taken together, these results indicate that the transactivation of nTPx gene at the diauxic shift resulted from the derepression of the negative control of the Ras/cAMP-TOR pathways.
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nTPx Is Inducible in Response to Oxidative StressIn yeast, Yap1p and Msn2p/4p have been known to be a transcriptional factor in response to oxidative stress. Yap1-mediated transcription can be activated by oxidative stress (35), and the activation is attributed to oxidative stress-induced nuclear localization of the Yap1 involving the nuclear export receptor Crm1 (Xpo 1) (36, 37). Another important transcriptional factor, Msn2p, and the partially redundant factor Msn4p are key regulators of oxidative stress-responsive genes expression (38). In addition to their involvement in the oxidative stress response, they are also implicated in the control of the multiple stress responses to carbon source starvation, osmotic stress, and heat stress (38). We performed semi-quantitative studies of nTPx transcripts in yeast mutants lacking Yap1p and Msn2p/4p (i.e. yap1 and msn2p/4p
, respectively). To test whether Msn2p/4p and Yap1p are involved in the induction of nTPx, the transcriptional activity of nTPx in msn2p/4p
and yap1
strains were examined. Fig. 8 shows that the transcriptional activity in the msn2p/4p
strain is
50% of that of the isogenic strain, but the activity in the Yap1
strain is not changed when compared with the parent type strain, which indicates that Msn2p/4p acts as a transcriptional factor, but Yap1p does not.
nTPx (YIL010W/DOT5) was suggested to be involved in the derepression of telemelic silencing (39), and its mRNA was 3-fold induced after exposure to alkylating agent, methylmethanesulfonate (40). However, the transcriptional response of nTPx upon oxidative stress as an antioxidant was not investigated. To examine the transcriptional response of nTPx to oxidative stress, Northern blots were performed using total RNA from exponentially growing yeast (W303-1a) in the presence of varying concentrations of H2O2 and diamide (Fig. 9A). The nTPx RNA level was not greatly but significantly induced upon H2O2 and diamide. The induction was considerably reduced in the Msn2/4
strain (Fig. 9B) compared with its wild type strain, which is consistent with the transcriptional activity data shown in Fig. 8B. Taken together, these data suggest that nTPx is an inducible protein in response to oxidative stress and that Msn2p/4p acts as one of the transcriptional factors to induce the expression of nTPx in response to oxidative stress.
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Nuclear TPx Acts as an Antioxidant Necessary for Stationary Phase Survival of Yeast CellsWe have demonstrated that the transcription of nTPx is activated at the diauxic shift, which is strictly negatively regulated by Ras-TOR signaling pathway (Fig. 7). The transcriptional activation at the diauxic shift suggests that nTPx takes an important antioxidant role in stationary phase growth, and thus nTPx is one of the antioxidants necessary for stationary phase growth. To address the antioxidant function of nTPx in stationary phase yeast cells, we explored the effect of nTPx mutation on the survival in the stationary phase. Nuclear TPx mutant and its parent strain as a control were grown to the stationary phase under the condition of high aeration for 3 days and then were washed and resuspended in water, as described under "Experimental Procedures." The viability of the stationary phase cells was then determined as a function of time (Fig. 10A). The wild type strain maintained viability of 50% for 16 days, whereas the nTPx mutant died much sooner. Only
10% of the nTPx
strain survived for the same time. The 5-fold decrease in the viability of nTPx
indicates that nTPx acts an important antioxidant necessary for stationary phase survival of yeast cells
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To demonstrate further the antioxidant function of nTPx in the stationary phase growth rather than in exponential phase growth, 4-NQO as an oxidant was subjected to stationary phase and log phase nTPx mutant cells, and the cell viabilities were investigated (Fig. 10B). Exponential and stationary growth phase cells were plated on the YPD plate containing 0.5 µM 4-NQO, and the cell viability was measured in terms of the number of the survival colonies. For the wild type strain, the stationary phase cells survived longer than the log phase cells, which is consistent with the fact that stationary phase cells are more resistant to oxidative stress. However, as seen in the experiment with stationary phase cells (Fig. 10B), the cell viabilities of the nTPx mutant cells reduced by 10%, when compared with the viability of the log phase nTPx mutant, and the viability of the mutant cells were 3-fold decreased compared with the viability of the stationary phase parent strain, which suggests the antioxidant function of nTPx in the stationary phase growth rather than in exponential phase growth. The model carcinogen 4-NQO can exert genotoxic potential via the generation of reactive oxygen species (ROS). Therefore, the high susceptibility of nTPx mutant toward a DNA-damaging agent such as 4-NQO can elucidate the antioxidant function of nTPx to protect against DNA oxidation by ROS.
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DISCUSSION |
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Nuclear localization of nTPx indicates the antioxidant role in the nucleus. Despite the higher sensitivity of nTPx toward various oxidants including alkyl-hydroperoxide such as t-butylhydroperoxide and cumene-hydroperoxide, the insensitivity of nTPx
toward H2O2 suggests its in vivo physiological function as an alkyl-hydroperoxide peroxidase. These results imply the antioxidant function of nTPx as an alkyl-hydroperoxide peroxidase to prevent from DNA oxidation against alkyl-hydroperoxide. It has been known that as yeast grows older, oxidative stress is gradually increased, and then stationary phase yeast cells suffer from the high level of ROS. In the stationary phase in which glucose as a carbon source is depleted, yeast begin to use ethanol and other secondary metabolites such as lipid as energy sources. In that situation, ROS including alkyl hydroperoxide such as fatty acid hydroperoxide should be highly accumulated in the cells. Therefore, during the post-diauxic shift, protection of DNA oxidation against ROS is essential for cell survival. The induction of nTPx at the diauxic shift suggests that nTPx acts as an important alkyl hydroperoxide peroxidase to maintain the yeast cells during stationary phase growth. The significant decrease in the viability of nTPx
compared with the isogenic strain supports the idea that nTPx acts an antioxidant necessary for stationary phase survival in the aerobic life of yeast.
In addition to the severe growth retardation of nTPx in the presence of 4-NQO, a DNA-damaging oxidant, it is noteworthy that in contrast to other yeast isoenzymes (cTPx I, cTPx II, cTPx III, and mTPx III) (22), nTPx is more active toward bulky hydroperoxide (cumene-hydroperoxide > t-butyl-hydroperoxide » H2O2). Cumene-hydroperoxide is a benzene-substituted alkyl-hydroperoxide (C6H5C(CH3)2OOH). The 4-NQO effect described above and the preference of nTPx for the cumenehydroperoxide as a substrate give us a hint that nTPx might be designed for the peroxidase, which can reduce the bulky form of hydroperoxide such as DNA base-anchored hydroperoxide, an alkyl-hydroperoxide.
Five isoenzymes of TPx are well compartmentalized in yeast cells. The peroxide selectivity of each TPx toward H2O2 and alkyl hydroperoxides is different from each other (22). Previously, based on their peroxidase activities toward H2O2 and alkyl hydroperoxides, we divided them into two groups such as hydrogen-peroxide peroxidase (cytoplasmic TPx I, cytoplasmic TPx II, and mitochondrial TPx) and alkyl hydroperoxidase (cytoplasmic TPx III) (22). In this report, we have demonstrated that as one of yeast TPx isoforms, nTPx acts as an in vivo alkyl-hydroperoxide reductase in the nucleus.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Dept. of Biochemistry, Paichai University, 439-6 Doma-2-Dong Seo-Gu Taejon 302-735, Republic of Korea. Tel.: 82-42-520-5379; E-mail: ihkim{at}mail.paichai.ac.kr.
1 The abbreviations used are: TPx, thioredoxin peroxidase; ROS; reactive oxygen species, nTPx; nuclear thiol peroxidase; DTT, dithiothreitol: Trx, thioredoxin; GS, glutamine synthetase, 4-NQO; 4-nitroquinoline N-oxide; TSA, thiol-specific antioxidant protein.
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REFERENCES |
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