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* Institute of Animal Physiology, University of Muenster, Germany;
Faculty of Science, An-Najah National University, Nablus, Palestine; and
Bernhard Nocht Institute, Hamburg, Germany
1Correspondence: Institute for Animal Physiology, University of Muenster, Hindenburgplatz 55, Muenster 48143, Germany. E-mail: liebaue{at}uni-muenster.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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300-nucleotide sequence upstream of the ATG start site necessary for GSTO-1 expression. Site-specific mutagenesis of a GATA transcription factor binding motif in the minimal promoter led to the loss of reporter expression. Similarly, RNA interference (RNAi) of Elt-2 indicated the involvement of this gut-specific transcription factor in GSTO-1 expression. Transcriptional up-regulation under stress conditions of GSTO-1 was confirmed by analyzing promoter-reporter constructs in transgenic C. elegans strains. To investigate the function of GSTO-1 in vivo, transgenic animals overexpressing GSTO-1 were generated exhibiting an increased resistance to juglone-, paraquat-, and cumene hydroperoxide-induced oxidative stress. Specific silencing of the GSTO-1 by RNAi created worms with an increased sensitivity to several prooxidants, arsenite, and heat shock. We conclude that the stress-responsive GSTO-1 plays a key role in counteracting environmental stress.—Burmeister, C., Lüersen, K., Heinick, A., Hussein, A., Domagalski, M., Walter, R. D., Liebau, E. Oxidative stress in Caenorhabditis elegans: protective effects of the Omega class glutathione transferase (GSTO-1).
Key Words: antioxidant • RNA interference • GST
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
GSTs probably evolved from thioredoxin and glutaredoxin enzymes, and a few GSTs that have a cysteine residue in proximity of the active site, which can form a mixed disulfide bond with GSH, distinct from the prototypical tyrosine or serine residues characteristic of other GST classes, still exist (2)
. It is therefore not surprising that the Omega class GSTs have a distinct substrate profile, most notably GSH-dependent oxidoreductase and dehydroascorbate reductase activity, reflecting their structural similarity to glutaredoxins (3)
. Recently their participation in the multistep biotransformation of inorganic arsenic to dimethylarsinate has been demonstrated (4
, 5)
. In addition to these enzymatic activities, variations in the human omega-1 genes that modify the age-at-onset of Alzheimer and Parkinson diseases have been described (6)
. Other proposed functions of the Omega class GSTs include a modulation of ryanodine receptor calcium release channels (7)
, participation in the posttranslational processing of interleukin-1β in monocytes (8)
, and a direct interaction with maspin, a novel serine protease inhibitor that suppresses tumor progression (9)
. The precise mechanisms by which the Omega class GSTs mediate all of these effects need further clarification. Recently the structural gene for the Drosophila eye color mutant sepia was identified, and a new catalytic activity for an Omega class GST (CG6781) was described. Here the enzyme is able to catalyze the synthesis of pyrimidodiazepine, an important intermediate in drosopterin biosynthesis (10)
.
A role of Omega class GSTs in the oxidative stress response has been invoked, with the reduction of glutathione-protein mixed disulfides proposed as the possible function of the enzyme (11
12
13)
. To elucidate the protective role of the Omega class GSTs in a multicellular organism, we investigated the GSTO-1 from Caenorhabditis elegans. This model system was chosen because genetic and transgenic techniques are well developed, and the system lends itself to performing of studies on an organismal level under normal and stress conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, were cultured on nematode growth medium plates [25 mM potassium phosphate, pH 6.0, 50 mM NaCl, 0.25% (w/v), 0.5% (w/v) cholesterol, 1 mM MgCl2, 1 mM CaCl2, and 1.7% agar] seeded with Escherichia coli OP50 (Caenorhabditis Genetics Center) as a food source. For bacteria-free culturing, adult worms were bleached in Clorox (40% sodium hypochlorite, 5% 10 N NaOH), and the remaining eggs were washed, incubated overnight in M9 buffer (22 mM KH2PO4, 42 mM Na2HPO4, 85 mM NaCl, and 0.1 mM MgSO4) and finally cultured in 25 ml of bacterial-free axenic medium [3% (w/v) yeast extract, 3% (w/v) soy peptone, 5 µg/ml cholesterol, and 50 mg/ml hemoglobin in 0.1 N KOH]. Animals were grown at 25°C (N2 Bristol) and 15°C [pha-1(e2123)]. Genomic DNA was prepared from homogenized worms by proteinase K digestion (Roche, Basel, Switzerland), followed by standard phenol/chloroform extraction and ethanol precipitation as described previously (15)
. Total RNA was prepared using TRIzol extraction according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA).
DNA sequencing, Northern blotting analysis
The nucleotide sequence was determined either by the dideoxy chain termination procedure on double-stranded DNA using 35S-labeled dATP and Sequenase (Amersham Buchler GmbH & Co., Braunschweig, Germany) or by terminator cycle sequencing using Ampli TaqDNA polymerase (Applied Biosystems, Inc., Foster City, CA) on an Abi Prism automated sequencer (PerkinElmer, Wellsley, MA, USA). For Northern blotting, total RNA was separated on an agarose formaldehyde gel and transferred to a positively charged nylon membrane (Millipore Co., Bedford, MA, USA). The membrane was hybridized with a radiolabeled probe of GSTO-1 cDNA in 50% formamide, 5x standard saline citrate (SSC), 5x Denhardt’s solution, and yeast tRNA at 55°C overnight, followed by washing with 2x SSC and 0.1% sodium dodecyl sulfate (SDS) at 60°C.
Database search and identification of the C. elegans GSTO-1
The GSTO-1 from C. elegans (C29E4.7; accession number AAA27959) was identified by a Blast search of WormBase using the GSTO-1 from the human pathogenic nematode Onchocerca volvulus (OvGST3) (16)
. A study of the phylogeny was performed to clarify the homology of GSTO-1 with other GST classes and dehydroascorbate reductase-like GSTs. Sequences were aligned by the program CLUSTALW and manually adjusted. Phylogenetic analysis was performed using MEGA 2.1 and the PHYLIP 3.57c program package with default settings (17)
.
Cloning, expression, and purification of the recombinant protein
To obtain the GSTO-1 coding region for expression in E. coli, the sense primer EX-4660 and the antisense primer EX-4661AS (Table 1
) were used in a polymerase chain reaction (PCR) with the complete C. elegans cDNA as template. The amplified DNA was cloned into the prokaryotic expression plasmid pJC40 (18)
and transformed into the E. coli strain Rosetta-gami(DE3) (Novagen, Madison, WI, USA). After expression, recombinant GSTO-1 was purified from the supernatant by chelating chromatography on Ni2+-nitrilotriacetate (Ni-NTA) agarose according to the manufacturer’s instructions (Qiagen, Valencia, CA, USA). For further purification, the GSTO-1 was purified with an S-hexylglutathione affinity matrix (Sigma-Aldrich, St. Louis, MO, USA). Here, cell pellets were resuspended in 50 mM Tris-HCl, pH 7.8, containing 200 mM NaCl (buffer A). After sonification, the supernatant was applied to equilibrated S-hexylglutathione-Sepharose. After extensive washing with buffer A, freshly prepared 5 mM S-hexylglutathione diluted in buffer A, was used for elution of GSTO-1. Furthermore, the GSTO-1 was subjected to fast protein liquid chromatography on a Superdex 75 column (Amersham-Pharmacia Biotech, Piscataway, NJ, USA). The protein concentration was determined by the method of Bradford (19)
. The homogeneity of the enzyme preparation was analyzed with 12.5% SDS-PAGE. Proteins were revealed by Coomassie Blue staining or Western blot analysis using a polyclonal antibody against GSTO-1 at a dilution of 1:1000. Polyclonal antibodies were raised in mouse against the purified recombinant GSTO-1. Proteins were detected using ECL Plus Western Blotting Detection System (Amersham Biosciences Corp., Piscataway, NJ, USA).
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Site-directed mutagenesis
The oligonucleotides MUT-7360S and MUT-7361AS (Table 1)
were designed to replace Cys-33 with alanine. PCR-based mutagenesis was carried out according to the manufacturer’s recommendations (Stratagene, La Jolla, CA, USA). The mutation was verified by nucleotide sequencing, and the construct was transformed for expression in competent E. coli Rosetta gami(DE3) cells. The purification of the protein variant was carried out as described before.
Enzymatic activity assays
Recombinantly expressed and Ni-NTA-purified GSTO-1 was dialyzed in 100 mM KH2PO4, pH 7.5, and used for enzyme assays. The assays were performed in triplicate from three independent enzyme preparations. The activities toward 1-chloro-2,4-dinitrobenzene (CDNB), cumene hydroperoxide, t-butylhydroperoxide, and trans-nonenal were determined as described previously (20)
. Thiol oxidoreductase activity was determined as described previously (21)
using 2-hydroxyethyl disulfide (HEDS).
Disc diffusion assay
The effect of heterologous expression of C. elegans GSTO-1 in E. coli Rosetta gami(DE3) cells on the susceptibility to different stressors was tested. Approximately 5 x 108 cells of the respective cultures were mixed with top agar (LB medium with 0.8% agar, 0.5 mM ampicillin, and 1 mM isopropyl β-D-thiogalactoside), plated on LB-ampicillin agar plates and incubated at 37°C for 1 h. Filter discs (8-mm diameter) soaked with different concentrations (10–400 mM) of juglone, cumene hydroperoxide, t-butylhydroperoxide, or paraquat were placed on the surface of the top agar. Cells were allowed to grow for 24 h at 37°C, before the inhibition zones around the paper discs were measured. P values were calculated in the Wilcoxon/Kruskal-Wallis one-way test using the program JMP 5.0.
Generation of transgenic C. elegans by microinjection
Germline transformation was performed using C. elegans pha-1(e2123) mutants by coinjecting vector constructs (80 µg ml–1) with the pBX plasmid (80 µg ml–1) containing the dominant marker gene, pha-1, into the germline of L4 pha-1 mutants (14)
. The selection of transgenic animals of the pha-1/pBX system (a kind gift from R. Schnabel, Braunschweig, Germany) is based on the temperature-sensitive embryonic lethal mutation pha-1. After microinjection, the animals were transferred to 25°C. Only transformed progeny survive and can be easily maintained by cultivation at 25°C.
Green fluorescent protein (GFP) reporter gene constructs
To investigate the cell-specific, developmentally regulated transcription of GSTO-1, lines of transgenic nematodes were created. The basic strategy involved the insertion of several fragments of the 5'-region of GSTO-1 into the multiple cloning site of pPD95.77 provided by A. Fire (Carnegie Institute, Baltimore, MD, USA). The putative promoter region of GSTO-1 was amplified using the Expand High Fidelity PCR System (Roche) with C. elegans genomic DNA as template and the gene-specific oligonucleotides GFP-6141S (sense, 1321 bp upstream of the translation start site) and GFP-6246AS (antisense, in exon II) (Table 1)
. For microinjection, the plasmid DNA was prepared using the Endo Free Plasmid Maxi Kit (Qiagen). Patterns of GFP expression were analyzed by fluorescence microscopy in multiple transgenic lines cultured in bacteria-free axenic medium.
Immunoelectron microscopy
Because the GFP signal obtained was weak, immunolocalization by electron microscopy was performed. For this, wild-type worms were fixated for 1 h at room temperature in a solution containing 1% paraformaldehyde and 0.025% glutaraldehyde in 0.2 M sodium cacodylate-HCl buffer (pH 7.2) and washed in cacodylate buffer. Fixated worms were dehydrated in ethanol and embedded in LR-White. Ultrathin sections were prepared on an Ultracut E (Reichert Analytical Instruments, Depew, NY, USA) and placed on 200-mesh nickel grids. Anti-GSTO-1 antibodies (1:1000) or preimmune antibodies (1:1000) were incubated with the grids for 1 h at 37°C and then overnight at 4°C. The sections were then treated for 1 h each with rabbit anti-mouse antibody (1:1000; Dako Diagnostics, Glostrup, Denmark) and with protein A-gold (10 nm, 1:100; Biocell Laboratories, Rancho Dominguez, CA, USA). Staining of subcellular structures was performed in 1% UO2 in water and Pb-citrate (Reynolds’ procedure). Electron micrographs were taken on a Philips CM-10 transmission electron microscope.
Determination of the minimal promoter region of GSTO-1 and analyses of GATA elements
The promoter region was further analyzed by deletion mutants. Here, the sense primers DEL-6484S1, DEL-6486S2, DEL-6487S3, and DEL-6488S4 and the antisense primer DEL-6485AS were used in PCR. The fragments were digested with the enzyme HindIII and religated afterward (Table 1)
. The nomenclature of these constructs, GFP-397, GFP-325, GFP-254, and GFP-178, is based on the number of base pairs upstream of the initiator codon ATG
(Fig. 4)
. The effect of shortening the 5'-flanking regions on cell specific-expression in vivo was determined for all constructs in transgenic C. elegans pha-1(e2123) mutants. Successful introduction of constructs that did not mediate GFP expression was confirmed by single-worm PCR. Site-directed mutagenesis was performed to assess the contribution of a GATA element (–264 bp upstream of the initiation codon) on gene expression in vivo. Here, the GATA element was replaced by a BamHI restriction site using the primers GAT-7253S and GAT-7254AS (Table 1)
and the construct GFP-325 as template.
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Overexpression of GSTO-1 in transgenic worms
By using the oligonucleotides OE-6502S and OE-6503AS (Table 1)
, a PCR fragment was obtained, cloned into the vector pPD103.05, and microinjected into the worms as described above. The oligonucleotide OE-6503AS was designed to include a His-tag, making recognition of successful overexpression of GSTO-1 using an anti-His antibody in transgenic worms possible.
RNA-mediated interference (RNAi) of GSTO-1, Elt-2, and Elt-4
RNAi was performed as described in an established protocol (22
, 23)
. Briefly, double-stranded RNA was produced in the E. coli strain HT115 transformed with pPD129.36 containing cDNA fragments of GSTO-1 (RNA-6740S/RNA-6741AS), Elt-2 (ELT2–8038S/ELT2–8039AS), and Elt-4 (ELT4–8040S/ELT4–8041AS) (Table 1)
. IPTG (1 mM) was added to induce transcription of the double-stranded RNA, and L4-staged animals were added to plates and their progeny were evaluated. For Elt-2 the "reproductive defect" phenotype was used as an indicator for effective knock down of expression. For GSTO-1, the efficiency of knockdown was checked by Western blot using the anti-GSTO-1 polyclonal antibody
(Fig. 7)
.
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Stress resistance assays
For each survival assay 150 L4 larvae were cultivated on 10 small plates with the stressors cumene hydroperoxide, juglone, paraquat, or sodium arsenite mixed with the NGM agar. For heat shock, worms were incubated at 33°C for 1–3 h. As a food source E. coli OP50 was given on the plates. The survival rate of the worms was evaluated after 18 h at 25°C or in a time-dependent manner in liquid culture. A worm was scored as dead when it did not respond to a mechanical stimulus. Each experiment was performed three times, and the P value was calculated in a Kruskal-Wallis one-way test using the statistical analysis package JMP 5.0.
Phenotype analyses
To measure nematode life span, plates containing 10 worms were incubated at 20°C and scored daily for surviving animals. Worms were transferred to a new plate every 2–3 days. To analyze the rate of reproduction, each adult worm was moved on a fresh plate daily, and the remaining progeny were counted.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). The nucleotide sequence at the splice junctions is consistent with the canonical GT-AG rule. The cDNA sequence confirms the intron-exon boundaries predicted from the genomic sequence in the worm database. Interestingly, only two of the four introns are located in the same position as those described for the human and the O. volvulus Omega class GST (Fig. 1C
). No trans-splicing was observed at the 5' end of the transcripts obtained by "rapid amplification of cDNA ends" (data not shown). The 753-bp cDNA codes for 250 amino acids with a calculated molecular mass of 28.5 kDa. Alignment of the deduced amino acid sequence with representative sequences from different GST classes (data not shown) was used to generate a phylogenetic tree clearly grouping the GSTO-1 into the Omega class (Fig. 1B
). The GSTO-1 was aligned with the Omega class GST from the filarial nematode O. volvulus (OvGST3, AF203814) and the human Omega class GSTO1 (AF212303), demonstrating 26% and 34% identity. Residues that contribute to the binding of GSH, as described for the human Omega class GST, are either conserved or conservatively replaced. Another distinguishing feature is the active site cysteine (Cys-33) (3)
(Fig. 1C
).
Characterization of the recombinant GSTO-1
To investigate the enzymatic characteristics of GSTO-1, the enzyme was expressed recombinantly in E. coli (Fig. 2
A). About 1 mg of GSTO-1 was obtained from a 1-L E. coli culture after purification on Ni-NTA agarose. Western blot of the extract of E. coli overexpressing GSTO-1 demonstrates the quality of the anti-GSTO-1 polyclonal antibody (Fig. 3
D). Additional purification was performed by gel filtration. It was also possible to purify the enzyme using S-hexylglutathione-Sepharose (Fig. 2A
). However, release of GSTO-1 with S-hexylglutathione proved difficult and the resin-GSTO-1 complex had to be boiled in SDS-sample buffer to liberate the enzyme. Because the expression level of GSTO-1 in C. elegans is extremely low, affinity purification of 0.2 g of C. elegans followed by Western blotting had to be applied to visualize the native enzyme (Fig. 2B
). This visualization was necessary to demonstrate successful overexpression of GSTO-1 or gene silencing in transgenic C. elegans (Fig. 7)
.
To evaluate the ability of GSTO-1 to protect against oxidative stress, E. coli expressing GSTO-1 were exposed to various stressors. Here, the redox quinones juglone and paraquat that are able to cross cell boundaries were used as internal inducers of reactive oxygen species, generating superoxide anion from molecular oxygen during metabolism. As external environmental stressors t-butylhydroperoxide and cumene hydroperoxide were used. After overnight exposure, the killing zones around the drug-soaked filters were smaller on plates with E. coli overexpressing GSTO-1 than in control bacteria with a 77% (t-butylhydroperoxide), 42% (cumene hydroperoxide), 25% (juglone), and 21% (paraquat) halo reduction (Fig. 3A
). Interestingly, the replacement of the active site Cys-33 with alanine resulted in about half of the halo size reduction compared with control bacteria and not complete loss of protection (Fig. 3B-D
).
To identify catalytic activities that may reveal the biological function of the GSTO-1, substrate specificity of the recombinant enzyme with a broad range of substrates was determined. Elimination of the His-tag by factor Xa (Fig. 2A
) did not influence enzyme activity (data not shown). The purified enzyme showed detectable GSH conjugating activity toward CDNB (Vmax=106±10 nmol mg–1 min–1; Km=51.52±6.92 µM) and measurable GSH peroxidase activity toward cumene hydroperoxide (Vmax=32±4 nmol mg–1 min–1). There was no detectable activity with 1,2-dichloro-4-nitrobenzene, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol, ethacrynic acid, 1,2-epoxy-3-(4-nitrophenoxy) propane, p-nitrophenyl acetate, and trans-nonenal (data not shown). In contrast, the GSTO-1 was able to use GSH as an electron donor to reduce dehydroascorbate (Vmax=186±37 nmol mg–1 min–1; Km=184.1±25.45 µM) and HEDS (Vmax=192±14 nmol mg–1 min–1; Km=70.77±7.71 µM). The specific activities for the two reactions were similar to each other.
Localization of GSTO-1 in C. elegans
To analyze the expression pattern of GSTO-1, transgenic C. elegans were created expressing a GSTO-1::GFP fusion protein under the control of the GSTO-1 promoter. Under the fluorescence microscope multiple transgenic lines were analyzed. Low GFP signals were detected exclusively in the intestinal cells of late embryos, L1–L4, and adult hermaphrodites (Fig. 4
A). Immunolocalization by electron microscopy of GSTO-1 using wild-type worms confirmed expression of GSTO-1 in intestinal cells (Fig. 4C
).
Deletion analysis of the GSTO-1 promoter, mutagenesis of the GATA box, and knock down of Elt-2
To analyze conserved cis-regulatory elements in the promoter region, deletion constructs were generated, and the minimal promoter region that mediates cell-specific transcription of GSTO-1 was determined. Worms were microinjected with the constructs GFP-397, GFP-325, GFP-254, and GFP-178 and the fluorescence of transgenic worms was analyzed (Fig. 4B
). Although the constructs GFP-397 and GFP-325 still promote GFP expression, no signal was obtained for the shorter constructs GFP-254 and GFP-178. These observations suggest a minimal promoter of 300 bp, with essential information for proper transcription present in the short region between –325 and –254 bp upstream of the initiation codon. Within this region is a motif fitting the consensus sequence T/A(GATA)A/G, the binding site for the GATA family transcription factors, shown to be necessary for expression of other intestinal cell-specific genes. To investigate the participation of the GATA element 259–265 bp upstream of the initiation codon in regulating transcription, site-directed mutagenesis was used. Transgenic worms expressing this construct were then generated and examined for GFP expression. Mutation resulted in complete loss of GFP expression. This result suggests that this GATA element within the minimal promoter region is required for GSTO-1 transcription. There are seven GATA-type transcription factors expressed in the C. elegans endoderm. Whereas four of these factors are expressed early and transiently during the specification phase of the endoderm, only three are expressed in the intestine after specification, with Elt-2 likely to be the dominant transcription factor in the C. elegans intestine (24)
. To investigate the involvement of Elt-2 or Elt-4 in GSTO-1 expression, RNAi was used to knock down expression of the gut-specific transcription factors in GFP-325 worms. GFP signals were unchanged in Elt-4-RNAi-treated worms and, as expected (25)
, no obvious phenotype was detected. RNAi of Elt-2 led to the distinctive gut-obstructed phenotype, with growth arrest at the L1 larval stage (24)
. Additionally, knockdown led to loss of reporter expression, suggesting that Elt-2 is necessary for GSTO-1 expression in the developing gut.
Induction of the GSTO-1 promoter by prooxidants
Transgenic worms expressing a GSTO-1::GFP fusion protein under the control of the GSTO-1 promoter were incubated with t-butylhydroperoxide and a significant increase of GFP expression was observed (Fig. 5
A). To confirm this result, conventional Northern blot analysis was performed. However, the amount of GSTO-1 mRNA is not detectable under standard Northern blot conditions of total RNA prepared from unstressed/stressed worms. Therefore, transcriptional up-regulation was confirmed by Northern blotting using RNA of the GFP-397 transgenic worms and a radiolabeled GFP probe. Thereby a 2- to 3-fold increase of hybridization signal intensity was observed (Fig. 5B
).
Overexpression of GSTO-1 leads to higher stress resistance
Transgenic worms were generated to examine in vivo whether the stress-responsive GSTO-1 is capable of protecting C. elegans under stress conditions using cumene hydroperoxide, juglone, paraquat, arsenite, and heat shock treatment (Fig. 6
). The survival rate of the GSTO-1 worms was compared with the survival rate of pha-1(e2123) mutants rescued with pBX under the same stress conditions. The GSTO-1 overexpressing worms showed a significantly higher resistance to the stressors than the control strain did (Fig. 6)
. The increase in stress resistance was robust across independent transgenic lines and experiments.
Gene silencing of GSTO-1 by RNAi results in higher stress sensitivity
The gene silencing of GSTO-1 resulted in almost complete loss of the corresponding protein, as evidenced by Western blot analysis (Fig. 7
A); furthermore, feeding of double-stranded GSTO-1 RNA to transgenic C. elegans expressing GSTO-1::GFP fusion protein resulted in a strong inhibition of GFP fluorescence (data not shown). In the absence of stress, the phenotype of RNAi worms was similar to that of control nematodes, except for an observed 17% reduced reproduction rate. No differences in life span, morphology, pharyngeal pumping, defecation rate, and movement were detected (data not shown). RNAi-mediated inhibition of GSTO-1, however, led to an increased susceptibility to cumene hydroperoxide, juglone, paraquat, arsenite, and heat shock treatment, with a reduction in survival rates of 95.6% (P<0.001), 82.2% (P<0.001), 68.6% (P<0.001), 25.9% (P<0.05), and 44% (P<0.001), respectively (Fig. 7C, D
). Incubation of RNAi worms with cumene hydroperoxide was also performed in liquid culture at a lower concentration and analyzed in a time-dependent manner for 5 h. The survival rate of the RNAi worms was significantly lower than that of the control worms (Fig. 7B
). To investigate whether depletion of the GSTO-1 transcript leads to unfolded stress response induction, hsp-4::GFP animals (CGC strain SJ4005, zcls4[hsp-4::GFP]V) were used as markers. In comparison with treatment with tunicamycin, a drug that induces endoplasmic reticulum stress, RNAi inactivation of GSTO-1 did not activate the endoplasmic reticulum stress marker (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Because the mutation of Cys-33 to Ala eliminates the GSH-dependent oxidoreductase activity, it is clear that Cys-33 plays an important catalytic role in the oxidoreductase activity of the enzyme. However, the disc diffusion assay clearly shows that the mutation of Cys-33 to Ala does not entirely abolish the protective effect against the stressor insult of E. coli overexpressing GSTO-1, perhaps reflecting the low GSH-dependent peroxidase activity by reducing organic hydroperoxides to the less toxic monohydroxy alcohols. In the human enzyme GSTO1-1, mutation of Cys-32 to Ala elevated CDNB activity, presumably by allowing the bound GSH to form a stable thiolate that readily participates in conjugation reactions (26)
. Whereas most Omega class GSTs described so far have no detectable peroxidase activity, a recent study by Garcera et al. (27)
demonstrates that the Omega class enzymes Gto2 and Gto3 from yeast also exhibit very low but reproducible activity against cumene hydroperoxide.
By reporter gene analysis and immunolocalization, the spacial expression pattern of GSTO-1 was determined, and low GFP signals were detected exclusively in the intestinal cells of late embryos, L1–L4, and adult hermaphrodites. The intestine is at the interface between the organism and its luminal environment and represents a defense barrier against toxic agents such as gut-derived oxidants or endogenously generated reactive oxygen species. In C. elegans the intestine is a highly metabolically active organ and is therefore more exposed to oxidative stress than other organs. However, the generally low abundance of the GSTO-1—even under stress conditions—and the low thiol oxidoreductase activity perhaps point to a participation in specialized functions, such as the maintenance of the adequate redox state of specific protein targets. Unlike for the GSTO-1, immunohistochemical analysis of the human enzyme GSTO1-1 shows relatively high expression of GSTO1-1 in a wide range of human tissues. Specific expression of GSTO1-1 was further localized in nuclear membranes and nuclei of many cell types (28)
. The mouse Omega class GST p28 was shown to be overexpressed in a lymphoma cell line with resistance to radiation and chemotherapeutics. Interestingly, Omega class p28 was shown to move from a cytosolic location to the nucleus in response to heat shock, and the authors speculate that p28 may be involved in the cellular defense against or in the adaptive response to altered cellular redox conditions (13)
.
Deletion analysis and generation of transgenic worms combined with fluorescence microscopy identified a compact promoter region of 300 nucleotides necessary for intestine-specific expression of GSTO-1. Transgenic analysis and site-specific mutagenesis show that gut expression is critically dependent on a single GATA transcription factor binding site. GATA factors are a family of transcription factors that have characteristic zinc-finger motifs and bind to a consensus DNA sequence of target genes. Their essential role, not only in the development of endoderm but also for gene expression in terminally differentiated endodermal tissues, has been reported for various animals (29)
. C. elegans has 11 GATA factor genes, with three genes, Elt-2, Elt-4 and Elt-7, expressed in the specified gut lineage. The predominant Elt-2 appears at the two-cell stage of the gut and expression continues in every cell of the gut throughout the life of the worm. Because early gut markers such as the gut-specific esterase gene ges-1 are still expressed in the Elt-2 knockout, the gut transcription factor network appears to be partially redundant in the early embryo, with Elt-7 acting in parallel (30
, 31)
. However, this redundancy does not apply for GSTO-1 expression. By performing Elt-2 RNAi in transgenic worms carrying the GFP-325 construct, we demonstrate that Elt-2 is necessary for in vivo expression of GSTO-1. Here, Elt-2 RNAi leads to the arrest of L1 larvae, the previously observed gut-obstructed phenotype, and to the loss of reporter expression. The complete dependence on Elt-2 gene expression has also been described for other genes with expression beginning late in embryogenesis [acid phosphatase Pho-1 (31)
; sphingosine-1-phosphate lyase (25)
]. RNAi of Elt-4 had no obvious effect on expression of the reporter gene.
Transcriptional up-regulation of GSTO-1 under prooxidant stress was shown using transgenic worms expressing a GSTO-1::GFP fusion protein under the control of the GSTO-1 promoter. This 2- to 3-fold up-regulation was confirmed by Northern blotting. In the human parasitic nematode O. volvulus an Omega-class GST (OvGST3) has been identified by differential display RT-PCR that is dramatically up-regulated at the steady-state transcription level in response to oxidative stress (16)
. Because O. volvulus is not accessible to genetic manipulations, OvGST3 was overexpressed in C. elegans, and an increased resistance to oxidative stress was observed in transgenic worm lines. The authors suggest that OvGST3 plays an important role in the protection of the parasite against reactive oxygen species derived from the host’s immune system (12)
. Our own observation that OvGST3 is an extracellular protein supports a possible modification or manipulation of the host environment by the protein (N. Brattig, J. Hoeppner, and E. Liebau, unpublished data).
Transgenic C. elegans overexpressing GSTO-1 were generated to examine resistance to artificially generated oxidative stress. The results obtained here and by posttranscriptional gene silencing by RNAi conclusively demonstrate that GSTO-1 protects the worms under prooxidant-induced stress. Overexpression of CeGSTP2-2, the gene product of the gst-10 gene, in transgenic worms not only led to stress resistance but also to an increase in median life span. In addition to this cause-effect relationship, correlative evidence was obtained in which life span paralleled the conjugating activity of the lipid peroxidation product 4-hydroxynonenal, strongly suggesting that 
,β-unsaturated carbonyl compounds affect life span (32)
. Whereas overexpression of GSTO-1 in transgenic worms led to an increase in stress resistance, no correlation between increased life span and overexpression of the GSTO-1 was observed. This observation has also been described for C. elegans strains overexpressing the stress-responsive gst-4 gene product. Whereas overexpression enhances stress resistance, their median life span did not differ from that of wild-type controls (33)
.
The specific silencing of GSTO-1 confirmed that the higher stress resistance of the above-mentioned transgenic animals depends on overexpression of this GST. The knockdown worms are hypersensitive to cumene hydroperoxide, juglone, paraquat, arsenite, and heat shock exposure. Here, the model prooxidant cumene hydroperoxide was used because it is more stable than H2O2 under the incubation conditions applied. Because the redox-active juglone and paraquat are able to cross cell boundaries, they were used as intracellular inducers of reactive oxygen species. The Omega-class GSTs are able to catalyze the reduction of monomethylarsonate (MMAV), an intermediate in the methylation pathway of arsenic biotransformation, making it feasible that RNAi worms are more sensitive to the environmental contaminant due to accumulation of MMAV (5)
. Toxic mechanisms of arsenic include mutation, inhibition of DNA repair, uncoupling of oxidative phosphorylation, or combination with thiol groups on the active site of proteins. Furthermore, reactive oxygen species are assumed to be involved in the stress response induced by arsenic. By using 
-glutamylcysteine synthase (gcs-1) mutant worms, Liao and Yu (34)
demonstrated the protective role of cellular GSH against arsenic-induced oxidative stress in C. elegans. In accordance with the above, induction of a variety of genes related to oxidative stress and cellular redox control have been described after arsenic exposure (35)
.
S-Glutathionylation has long been known to occur on oxidative stress, with protein glutathionylation serving the dual purpose of oxidative signal transduction and protection of key regulatory molecules from oxidative insult (36
, 37)
. The crystal structure of the human GSTO1-1 indicates that other proteins might be substrates for the enzyme, with the potential role of modulating the mixed disulfide status of critical cysteine residues on these proteins. However, no protein substrate has been found so far (26)
.
In a recent study, C. elegans was chosen as an ideal organism to study how protein-protein interaction networks relate to multicellular functions, and the interactome network was analyzed with high-throughput yeast two-hybrid screens providing functional hypotheses for thousands of uncharacterized proteins (38)
. Here an interaction was observed between GSTO-1 and the E1-like enzyme Uba5, the activating enzyme of a novel protein-conjugating system for a unique ubiquitin-fold modifier (Ufm1). Similar to protein ubiquitylation, this system consists of sequential reactions of multiple enzymes consisting of activating (E1), conjugating (E2), and ligation (E3) enzymes with high-energy thioester linkages between components. Because the target proteins have not been identified, the biological role of the Ufm1-modifying system still needs to be assessed (39)
. Of interest, retina cells show an increase in the glutathione disulfide/GSH ratio on oxidative stress, with a concomitant decrease in activity of the ubiquitination pathway, suggestive of the cellular redox status modulating protein ubiquitination via reversible S-thiolation of E1 and E2 (40)
. Furthermore, inhibition of the E1 protein is associated with the formation of mixed disulfides in response to oxidative insult (41)
. Recently, the regulation of SUMOylation (small ubiquitin-related modifiers) by reversible oxidation of respective conjugation enzymes has been described (42)
. Because the E1-like enzyme Uba5, involved in the attachment of Ufm1 to substrate proteins, contains an active site sulfhydryl that might be covalently modified, we further investigated the specific interaction of GSTO-1 with Uba5. By using coaffinity purification assays we were able to confirm this interaction (unpublished data) and the functional role of GSTO-1 in the regulation of this novel ubiquitin-like pathway is currently under investigation.
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ACKNOWLEDGMENTS |
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Received for publication January 15, 2007. Accepted for publication August 30, 2007.
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, intron-exon borders, gaps are indicated by –.
