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Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea
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Abstract |
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Introduction |
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Thioredoxin is well known for its role of intracellular redox regulator of gene expression (Okamoto et al. 1992; Makino et al. 1996; Akamatsu et al. 1997; Hirota et al. 1997) and cytosolic anti-oxidant (Hori et al. 1994; Sasada et al. 1996; Takagi et al. 1998). Many thioredoxins have been described as small (
12 kDa), ubiquitous disulfide reductases, being part of a system which also comprises NADPH (nicotinamide adenosine dinucleotide phosphate) and thioredoxin reductase. Their oxidoreductase activity is illustrated through a multitude of functions, which can be grouped into two major categories. First, they act as electron carriers, providing reducing equivalents for the catalytic cycles of the biosynthetic and anti-oxidant enzymes, such as ribonucleotide reductases, methionine sulfoxide reductases and peroxiredoxins; second, they protect the cytosolic proteins from aggregation and inactivation through intermolecular or intramolecular disulfides formation. Besides their anti-oxidative activity, thioredoxins have many other functions, some of them very specialized (subunit of T7 DNA polymerase, filamentous phage assembly), some others of high therapeutical significance—for example, their major regulatory effects on immune responses, including their ability to control the binding activity of immunologically active transcription factors (e.g. NF
B and AP-1) (Arner & Holmgren 2000).
It is noteworthy to mention that cells also have certain anti-oxidant molecules, like ascorbate (vitamin C), tocopherol (vitamin E) and the tripeptide glutathione, which are capable of reducing the reactive oxygen species in a nonenzymatical fashion (Di Mascio et al. 1991).
The active role that thioredoxin plays in many cellular processes is also revealed by the multitude of compartments where its activity has been found: outside the cell, where it is involved in cell growth stimulation and chemotaxis; in the cytoplasm, as an anti-oxidant and a reductant cofactor; in the nucleus, where it regulates the activity of several transcription factors, as well as in the mitochondria (Powis & Montfort 2001). Moreover, it is also known for its capacity of inhibiting apoptosis: thioredoxin binds to the ASK-1 (apoptosis signaling kinase 1) and thus inhibits its activation by TNF (tumor necrosis factor), hence being a major regulator of ASK-1 activity.
Here we characterize trx-1, a C. elegans thioredoxin homolog. CeTRX-1 displays oxidoreductase activity, being related to oxidative stress resistance and life span regulation.
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Results |
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Despite the fact that thioredoxines are known from several model species, none has so far been characterized from C. elegans. Besides the well-characterized 12-kDa thioredoxines, members of another subclass, of 16 kDa, have been described in other nematode species (Kunchithapautham et al. 2003). Along with the difference in size, these two subclasses display a slightly different sequence surrounding the active site; also, the 16 kDa proteins lack certain otherwise conserved cysteines, which seem to confer to these molecules specific biological properties (Gasdaska et al. 1996). A selenium-containing thioredoxin reductase has already been described in C. elegans, which implies the existence of the whole system (Buettner et al. 1999; Gladyshev et al. 1999).
By computer-assisted searching in the available C. elegans databases for human thioredoxin homologs, we discovered several candidate genes. These putative thioredoxins code for proteins that segregate into 12 and 16 kDa subclasses. The latter category is characterized by a slightly different active site sequence (WCGPCQ or WCPPCR) from human thioredoxin. Among 12 kDa candidate proteins, only one (B0228.5) had the active site motif fully conserved (WCGPCK). Therefore, based on such criteria as mass and active site sequence of the encoded proteins, we focused our attention on this gene (trx-1). Two thioredoxin isoforms are reported from ORFeom Project (Chen et al. 2004), B0228.5a (CB392184 [GenBank] ) and B0228.5b (CB389161 [GenBank] ). As shown in Fig. 1, these two isoforms, B0228.5a (CeTRX-1a) and B0228.5b (CeTRX-1b) present 36% and 33% identity with the human homolog, respectively. They are similar to each other, the mass difference being of just one amino acid (115 and 114 amino acids, respectively). B0228.5a has four exons, while B0228.5b has only three, the last two exons being common (Fig. 2). To obtain both isoforms, cDNA library PCR was conducted but only B0228.5b was cloned.
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We have examined the temporal and spatial expression pattern of trx-1 using the gfp (green fluorescent protein) reporter system. The gfp-fusion construct containing full-length trx-1 DNA including 5' promoter sequence (1.074 kb) was microinjected in order to obtain stable transgenic lines. We found that trx-1 expression was restricted to two pairs of head neurons (Fig. 3E,G) and to posterior intestine (Fig. 3A,B) and could be detected from the larval stages throughout the life span of the animals. The expression in hermaphrodites and males was identical (data not shown). Interestingly, this pattern is altered in certain stress conditions: following starvation, expression is enhanced and spreads toward the anterior intestine (data not shown). A similar modification of the gfp signal was seen in aged animals (Fig. 3C–D).
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We also examined the expression pattern of CeTRX-1 in situ, by staining whole-mount wild-type C. elegans with anti-TRX-1 antibodies. Staining was observed throughout the intestine of the animals, in a punctuated pattern (Fig. 3I–J).
Taking together the results of gfp expression and immunostaining, we conclude that CeTRX-1 is expressed in ASI and ASJ neurons as well as in the intestine and that protein expression is significantly enhanced as the worms are aging.
TRX-1 shows reductase activity
All thioredoxins reduce disulfide bonds, in this way exerting their major anti-oxidant function. In order to show that TRX-1 functions as an oxidoreductase, we cloned its cDNA from a C. elegans library and expressed it in E. coli. rCeTRX-1b (recombinant CeTRX-1b) was purified and subsequently used in the insulin reduction assay. As the thioredoxin is reducing insulin A and B chains, the B chain precipitates, and the level of turbidity is increasing. The precipitation is determined spectrophotometrically and constitutes a measure of reductase activity. We performed this insulin reducing assay at pH = 7, commercially available Spirullina thioredoxin being used as positive-control. In the absence of rCeTRX-1, no significant precipitation was observed. The rCeTRX-1b0-catalyzed reaction kinetics was similar to that of Spirullina thioredoxin (Fig. 4).
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To characterize in vivo functions of trx-1, we isolated and characterized a deletion mutant of this gene in C. elegans. trx-1(jh127) has a deletion that covers the promoter region and part of the coding region, knocking out the antepenultimate exon, which also comprises the active site of the protein. Null mutation was confirmed using anti-CeTRX-1 antibody (Fig. 2C). The deletion and the single homozygote worm were confirmed by PCR (Fig. 2A,B). This mutant was outcrossed 6 times with wild-type worms in order to segregate the trx-1 mutation from other accompanying mutations. The mutant worms lack any morphological defects.
The gfp expression being enhanced with aging suggested that trx-1 might be up-regulated in this condition as well; as the cells become older, it is more difficult to cope with the reactive oxygen species. Therefore, it was reasonable to assume that the functional nullity of thioredoxin might affect the life span. We measured the life span of deletion mutant at 20 °C and found that it is significantly reduced when compared to that of N2, and closer to that of daf-16, used as positive control (Fig. 5A). The mean life span of trx-1(jh127) is just 17.44 days, compared to 21.5 days of wild-type worms (Table 1).
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In order to prove that this phenotype was caused indeed by the lack of functional gene, we performed rescue experiments. The same gfp-fusion construct containing full-length trx-1 DNA was microinjected into trx-1(jh127) deletion mutant. We obtained a stable transgenic line, whose gfp expression pattern was indistinguishable from that of KJ560. Both the life span and the paraquat sensitivity of trx-1(jh127) were fully rescued (Fig. 5).
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Discussion |
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Thioredoxines have been described as ubiquitous molecules, with critical functions in redox regulation and signaling (Arner & Holmgren 2000). Therefore, we were expecting the same ubiquitous expression in the case of trx-1. Nevertheless, as our analysis reveals, the gene is restrictedly expressed in ASJ and ASI neurons and in intestine. Although different from our expectation, this expression pattern could be reasonably explained based on the initial observation that there are a number of putative thioredoxins in C. elegans, possibly with different tissue localizations. It is interesting to note that the expression pattern is altered in certain conditions, as previously described. We have previously reported another gene that is also over-expressed when aged (Kim et al. 2004). It was found that this gene ftn-1, which encodes a iron-binding protein, seemed to be regulated by iron concentration in the cell. Taking into account the anti-oxidant function of thioredoxin, these observations suggest that, as the animals become more and more exposed to oxidative stress, trx-1 might be up-regulated accordingly.
The significance of this neuronal expression could be several fold. Recently, it was shown that a peroxiredoxin, which may act as a terminal reductase on the thioredoxin system, is restrictedly expressed in two interneurons in C. elegans (Isermann et al. 2004). It is possible that trx-1 plays a neuromodulation role in ASJ and ASI, which are well known as secretor neurons. One of the well-known functions of ASI and ASJ is to control dauer formation (Bargmann & Horvitz 1991). So we have tested whether these trx-1 mutants are defective in dauer formation or not by using purified pheromon (Jeong et al. 2005). Results showed that trx-1 mutants were normal in dauer formation when induced by pheromone just like wild-type worms (P. Y. Jeong and Y. K. Paik, personal communication). Nevertheless, the function(s) of trx-1 in ASI and ASJ neurons is not obvious at this point and it will require further studies to elucidate its neuron specific function.
In C. elegans, the intestine is the most highly metabolically active organ; consequently, it is more exposed to oxidative stress than other organs. In the intestine, the level of expression is dynamic, being directly correlated with external (food availability) as well as internal (aging) factors. It is interesting that the trx-1 expression is readily induced throughout the intestine in the aging animals. This suggests that thioredoxin is indeed needed for its anti-oxidant functions, especially in the cells of C. elegans, the aging of whom equals that of its cells.
We also isolated a deletion mutant of trx-1. The deleted region covers both the promoter and the active site and null mutation was confirmed using anti-CeTRX-1 antibody (Fig. 2C). The mutant worms do not present any defects with respect to morphology, egg production or brood size, but they do display a specific phenotype, characterized by reduced life span and sensitivity toward oxidative stress, which can be rescued by the re-introduction of trx-1 into trx-1(jh127) deletion mutant. These data confirm that trx-1 knockout is responsible for the mutant's phenotype.
The life span of C. elegans is regulated by an insulin-like signaling pathway (Guarente & Kenyon 2000). Therefore, we have generated a double mutant between trx-1(jh105) and daf-2 (e1370) and then tested paraquat sensitivity of this double mutant. The trx-1(jh127) mutant did not significantly suppress the resistance of daf-2(e1370) suggesting that trx-1(jh127) could be independent from the daf-2/daf-16 pathway. However, this does not exclude a possibility of cross-talk between the daf-2/daf-16 pathway and the trx-1 gene. Currently we are looking at the link between trx-1 and the daf-2/daf-16 pathway in lifespan regulation.
The anti-oxidative functions of thioredoxins are important both through their direct activity and through the action of their substrate molecules, of which peroxiredoxines have prominent scavenging activities. C. elegans has three genes coding for peroxiredoxines; nevertheless, it was shown that at least one of them is not able to confer any resistance against immediate oxidative stress (Isermann et al. 2004). Characterization of trx-1(jh127) reveals that trx-1 is likely to have important functions in both the rapid response to ROS (reactive oxygen species)—generating stress as well as the chronic oxidative stress, which would explain the reduced life span. Further studies are needed in order to elucidate the possibly complex implications of thioredoxin in oxidative stress response and redox signaling in C. elegans.
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Experimental procedures |
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Bristol N2, DR26 daf-16(m26)I, were obtained from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota. trx-1(jh127)II was isolated by TMP/UV mutagenesis as described (Barstead 1999). Worm breeding and handling were conducted as described (Brenner 1974).
Plasmid construction and expression analysis
The gfp reporter gene construct was obtained by PCR amplifying a 1725 bp fragment from cosmid B0228 (comprising full coding region of trx-1 including 1074 bp of 5' promoter region) and cloning it into pPD 95.75 vector (a kind gift from A. Fire) within PstI and BamHI restriction enzyme sites. Microinjection of the gfp expression constructs was performed as described (Mello & Fire 1995). After the stable transgenic lines were obtained, worms were treated with 2.5% (w/v) levamisole to immobilize them and gfp signals were observed by fluorescent microscopy (Olympus BX50). To obtain a full-length cDNA clone of trx-1, nested PCR with an embryonic C. elegans phagemid cDNA library was performed with the following primers: outer upstream and downstream primers, 5'- TTT CAT CAT GTC TCT CAC CAA GGA GCC-3'; and 5'-TCG AAC ATC AAA AAT TTG GCC GTT G-3', respectively; inner upstream and downstream primers, 5'- GAA GAG CTC GAG GAG CAG ATG ATT GGT-3'; and 5'- TAT GGT GGA TCC ATG TTG AAC GAT GC-3', respectively. The amplified product was subcloned into a pGEM-T Easy vector (Promega) as pLV001 at XhoI and BamHI restriction sites, and subsequently sequenced using phage T7 and SP6 primers.
Isolation of a trx-1 deletion mutant
TMP (Trimethylpsoralen)/UV method was used to generate C. elegans deletion mutants. Screening of mutants from the mutagenized library was carried out by a nested PCR-based method and subsequent sib selections as described (Barstead 1999). Primers were designed based on the predicted sequences spanning the full genomic DNA of trx-1 (B0228.5): outer upstream primer (5'-GAA ATT CCG GAT CAA TAC AAA GCC ATC-3') and downstream primer (5'-CAT TAG GCA TGT CAT GTC TCT TAA TGG C-3'), inner upstream primer (5'-CCC AAT TCT TTC GCG ATT TTT CAT ACG-3'), and downstream primer (5'-AAC GTA CAG GAC GAG ACA AAA GAC CAA GC-3'). We designed an inside primer to confirm homozygote deletion mutants. Inside downstream primer (5'-CAA AGT TGA TGT CGA TGA AGC GGT TAG TG-3') was paired with inner upstream primer. A homozygous line of animals bearing a deletion of 1.2 kb was isolated. This animal was outcrossed 6 times to wild-type animals to establish the strain KJ562 trx-1(jh127) and was used in subsequent analysis. Deletion region for the trx-1 hermaphrodites was determined by nested PCR followed by sequencing of the PCR products.
DiI dye filling
KJ560 dpy-20Ex[trx-1::gfp, dpy-20] transgenic worms were allowed to take up the DiI lipophilic dye for 3 h, and then transferred to NGM (nematode growth medium) plate with OP50 and allowed to crawl for 1 h in order to distain. The identification was accomplished by comparing the positions of DiI-filled and gfp-expressing neurons.
Protein purification and analysis
The trx-1 cDNAb was subcloned into pGEX4T-1 vector (Pharmacia Biotech) using XhoI and BamHI restriction sites; the protein expression was induced in BL21 (DE3) strain of Escherichia coli with 1 mM IPTG (isopropylthiogalactopyranoside) for 2.5 h at 30 °C. The cells were subjected to sonication and the soluble proteins were separated by mass to produce a preparation highly enriched for rCeTRX-1-GST; the fused protein was purified on column, using Gluthation Sepharose 4B (Pharmacia Biotech). The recombinant thioredoxin was separated from the GST moiety by cutting with thrombin (10 units in PBS—phosphate buffer saline) and subsequently eluted. Western blot analyses were carried out by use of the semidry blotting technique. Specific rabbit antisera against CeTrx-1 and as secondary antibody an alkaline phosphatase conjugated anti-rabbit IgM were used. The proteins were detected using the 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/nitro blue tetrazolium (NBT) color developmental substrate (Promega).
Insulin reduction assay
Thioredoxin-mediated catalysis of insulin reduction was measured spectrophotometrically at 650 nm and 25 °C as an increase in turbidity resulting from precipitation of the free insulin-chain (Holmgren 1979). The assay mixture contained 100 mM potassium phosphate, 2 mM EDTA (ethylene diamine tetraacetic acid) (pH 7.0), 0.13 mM insulin (0.75 mg/mL), and different concentrations (139 µM or 277.8 µM per reaction) of rCe-TRX-1 or 0.033 units of Spirullina thioredoxin (Sigma). The reaction was initiated by the addition of 0.33 mM dithiothreitol (DTT). Total volume reaction was 600 µL.
Life span assays and paraquat sensitivity
The life span was assayed at 20 °C and it was initiated on the first day of adulthood of all animals. Animals were transferred away from progeny to new plates every day or every other day until they stopped laying eggs. If worms failed to move when provoked or lacked pharyngeal pumping, they were scored as dead. The worms which crawled off the plate were excluded from the total number of calculations of life span. To assay sensitivity to paraquat (methyl viologen, Sigma) young adults were transferred from NGM agar plates into 24-well plates (6 per well) containing 150 µL of M9 that either did or did not contain 50 mM paraquat. Worms were incubated at 20 °C, and the number of dead animals was counted by the continuous absence of swimming movements and pharyngeal pumping.
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Acknowledgements |
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Footnotes |
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These authors equally contributed to this work. 
* Correspondence: E-mail: joohong{at}gist.ac.kr |
References |
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Received: 25 June 2005
Accepted: 25 September 2005
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