Heme Transport and Detoxification in Nematodes: Subproteomics Evidence of Differential Role of Glutathione Transferases Samı¨rah Perally,*,† E. James LaCourse,‡ Alison M. Campbell,† and Peter M. Brophy† Institute of Biological, Environmental, and Rural Sciences, Aberystwyth University, Aberystwyth, Ceredigion SY23 3DA, United Kingdom, and Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, United Kingdom Received May 30, 2008
In contrast to their mammalian hosts, parasitic nematodes are heme auxotrophs and require pathways for the uptake and transport of exogenous heme for incorporation into hemoproteins. Phase II detoxification Nu-class glutathione transferase (GST) proteins have a proposed role as heme-binding ligandins in parasitic nematodes. The genome-verified free-living nematode Caenorhabditis elegans also cannot synthesize heme and is an ideal functional genomics model to delineate the role of individual nematode GSTs in heme trafficking and heme detoxification. In this study, C. elegans was exposed to externally controlled heme concentrations ranging from 20-fold suboptimal growth levels to 10-fold supra-optimal growth levels to mimic fluctuations in blood- and tissue-feeding parasitic cousins from the same nematode group. A new heme-responsive GST (GST-19) was identified by subproteomics approaches. Functional characterization of this and two other C. elegans GSTs revealed that they all have high affinity for heme compounds similar to mammalian soluble heme carrier proteins such as HBP23 (Kd ≈ 10-8 M). In the genomics-predicted absence of orthologous mammalian soluble hemebinding proteins in nematodes, we propose that Nu-class GSTs are candidates in the cellular processing of heme compounds. Toxic heme binding may be coupled to enzymatic protection from its breakdown as several GSTs possess glutathione peroxidase activity. Keywords: glutathione transferase • heme • transport • detoxification • Caenorhabditis elegans • nematode
Introduction Worldwide, parasitic nematodes are responsible for a multitude of debilitating diseases in man, his livestock and companion animals.1,2 Treatment of parasitic nematode infection relies on only a few classes of chemical anthelmintics. However, issues regarding drug toxicity, environmental impact of these anthelmintics, and global drug resistance in nematodes, especially of small ruminant livestock and human populations, are increasingly being reported.3,4 With the continued failure to produce anti-nematode vaccines, there is an urgent need for effective chemotherapeutics. Functional genomics-based target discovery and validation research may stimulate the industry to develop new anthelmintics.5 In the absence of completed working toolkits for parasitic nematodes, the closely related genome-verified model nematode Caenorhabditis elegans is fortuitously available now to fill the technical gap, enabling us to undertake functional genomics studies. C. elegans is of limited relevance for studying parasite targets related to derived adaptations to the host environment, such as living in low oxygen tension and avoiding immune initiated responses. However, both C. elegans, and its “luminal” * To whom correspondence should
[email protected]. † Aberystwyth University. ‡ Liverpool School of Tropical Medicine. 10.1021/pr800395x CCC: $40.75
be
addressed.
2008 American Chemical Society
E-mail:
parasitic nematode Strongylida cousins are morphologically similar, with shared microbe-feeding requirements in the soil environment of their early larval stages, and thus common metabolic and detoxification challenges. In addition, nematode free-living survival systems within a harsh soil environment containing plant and microbe secondary metabolites are likely retained for adult parasitic nematode survival challenges of feeding, uptake and metabolite detoxification of the host gut environment. Such generic nematode physiological mechanisms, not under host selection pressure, offer possibilities for broad spectrum target discovery. In contrast to most other free-living eukaryotes, all free-living and parasitic nematodes do not synthesize heme de novo and, in the absence of an extra-genome symbiont, rely totally on external intact heme capture. In parasites, heme is derived from feeding off host blood, tissue fluid and gut contents (or intestinal bacteria), while free-living nematodes can obtain heme from bacterial or yeast feeding in the environment. The common nematode problem is the transport of toxic heme to multiple tissues. Ingested heme is probably also used as an iron source when iron is limiting.6 A total reliance on external capture would appear to be an “Achilles heel” as heme is a key cofactor for a multitude of cellular processes.7 Nematodes and their human hosts have a common transmembrane protein family for heme uptake.8 However, once taken up, the heme Journal of Proteome Research 2008, 7, 4557–4565 4557 Published on Web 08/23/2008
research articles needs to be transported by a carrier within cells as it is insoluble, and also highly toxic on oxidization to the Fe3+ form, catalyzing the formation of lipid peroxides and damaging DNA via oxidative stress. In mammals, intracellular heme levels are tightly controlled by genes encoding enzymes not present in nematode genomes analyzed to date such as aminolaevulinic acid synthase (ALAS) and heme oxygenase (HO).9 Similarly, no evidence of a soluble heme-carrier protein could be detected in currently known nematode genomes, for example, HCP1 involved in heme absorption from the mammalian intestine.10 In mammals, heme taken up by the intestine is immediately degraded by HO to release metabolic iron. The apparent absence of HO and other heme-related enzymes in nematode genomes suggests either a lack of nematode/mammalian homology between potentially similarly functioning proteins, or as yet uncharacterized novel nematode heme activity and/ or pathways. Nematode proteins must be required to safely chaperone and transport heme from the intestine or possibly via the cuticle to the endoplasmic reticulum of tissues for its incorporation into known genome/EST predicted hemoproteins that are required for multicellular functions. In mammalian cells, suggested heme transporters from the site of synthesis in the mitochondria include Alpha-class glutathione transferases (GSTs), fatty acid binding proteins (FABP’s) and heme 23 kDa binding proteins.9 In nematodes, however, the delineation of heme transport and heme detoxification has yet to be fully resolved. The recently uncovered Nu-class and possibly nematodespecific GSTs from Haemonchus contortus and Ancylostoma caninum nematodes have a high-affinity binding site for heme, and may be key transport and/or detoxification proteins in nematode heme pathways. Importantly, there is, from a clinical perspective, a clear distinction between host and parasite GST classes in both structure and biochemical activity in vitro.11-13 Blood-feeding nematodes genome and Expressed Sequence Tag (EST) projects remain, at present, in early developmental stages, curtailing current systematic functional genomic studies. The close relationship between the genome-verified culture nematode C. elegans and parasitic nematodes (including H. contortus and Necator americanus) has been validated by numerous EST projects showing that up to 70% of the available 250 000-plus sequences from these parasitic nematodes have significant BLAST-based relationships to C. elegans proteins, including common classes of apparently nematode-specific GSTs.12-15 In this paper, we focus on delineating the transport and detoxification roles of one predicted component of the nematode heme cytosol-transport network, the Nu-class GSTs. Utilizing the free-living C. elegans nematode as a genomeverified experimental model, the response of nematode GSTs to varying heme exposures is investigated by subproteomic assays.
Experimental Methods Axenic Culture of C. elegans. Nematodes (C. elegans Bristol N2 strain) were cultured in Caenorhabditis elegans Habitation and Reproduction (CeHR) axenic liquid medium which was modified to control concentrations of exogenous heme with 25 µM being the growth optimum.6,16 Nematodes were grown in modified CeHR (mCeHR) medium containing six different concentrations of the heme-related and water-stable form of heme (hemin), from suboptimal to known growth inhibiting toxic levels (1, 4, 25, 50, 100 and 250 µM) under aerobic conditions at 20 °C for 5 days. Flasks containing 500 mL of 4558
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Perally et al. liquid medium were seeded with 2.5 × 10 nematodes. For initial culturing in axenic media, three generations of nematodes were consecutively bleached, with eggs harvested from each culture used to seed the next batch. This was carried out to remove any “carry-over” traces of bacterial heme as described.6 In all dose-response experiments, nematodes were growth synchronized by inoculating with newly hatched L1 larvae from eggs obtained after treatment of gravid hermaphrodite nematodes via the hypochlorite method.17 Harvesting of C. elegans. Nematodes were left to settle at 4 °C for 4 h. Excess medium was removed using a suction pump. Nematodes were collected by centrifugation at 1200 g at 4 °C, washed four times in M9 buffer (30 mM KH2PO4, 58 mM Na2HPO4, 178 mM NaCl, and 1 mM MgSO4), and then separated from bacteria by sucrose floatation by centrifugation in 30% w/v sucrose at 2500 g for 4 min at 4 °C. Worms were washed another four times in M9 buffer. One mL aliquots of worms were cryogenically frozen and stored at -80 °C. Samples of worms from each culture were observed under a light microscope to estimate life-cycle demographics in each culture. Individual Life-Stage Plate Culture. Individual life-stage nematode samples were obtained by synchronizing a culture of worms according to the hypochlorite bleaching method.17 The “egg pellet” was then spread over an uninoculated area of a large (20 cm × 30 cm) NGM-agar plate covered with an Escherichia coli OP50 lawn. Eggs were left to hatch at 20 °C and upon hatching, L1 larvae moved from the cell debris containing dead mixed-stages to the bacterial lawn spread over the remainder of the plate. Plates were incubated for differing times to obtain the appropriate life-stage. Incubation times were based upon body length and time elapsed since bleaching with harvests calculated to fall between moults.18 L1 stage worms were collected 9 h after hypochlorite treatment, L2s after 20 h, L3s after 29 h, L4s after 40 h, and pre-egglay gravid adults after 58 h. 6
Larvae of each life phase were washed from the plate with M9 buffer, taking care not to wash any of the contaminating cell debris/pellet into the collection. Washed worms were collected into 2 mL cryovials following centrifugation at 500 g for 1 min at 4 °C. One mL aliquots were cryogenically frozen and stored at -80 °C. Homogenization of C. elegans. Harvested nematodes were homogenized in 20 mM phosphate buffer, pH 7.4 containing 50 mM NaCl, 0.1% v/v Triton X-100 and protease inhibitors (mini-complete protease inhibitor cocktail tablets, Roche Applied Science) using 1 mm zircon beads and a mini bead-beater (Biospec Products). Cell debris was removed by centrifuging at 17 000 g for 15 min at 4 °C. The supernatant was ultracentrifuged at 100 000 g for 30 min at 4 °C in order to recover the cytosol. GSTs were purified from the cytosolic supernatants via glutathione (GSH)-affinity chromatography according to established protocols.19 Protein was determined using the BioRad Protein Assay kit. 2-D Electrophoresis (2-DE). GSH-affinity samples were subjected to 2-DE as previously described.20 A Protean IEF unit (Bio-Rad) was used for the isoelectric focusing step. Proteins eluted from a GSH-affinity column were resuspended into immobilized pH gradient (IPG) rehydration buffer to a final concentration of 6 M urea, 1.5 M Thiourea, 3% w/v CHAPS, 66 mM DTT, 0.5% v/v ampholytes pH 3-10 (Pharmalytes, Amersham Biosciences) at 300 µL for 17 cm IPG strips (Bio-Rad). In-gel passive rehydration of IPG gel strips with protein samples was performed at 20 °C for 16 h with each sample/strip overlaid
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Table 1. Identification of C. elegans Protein Spots from Heme-Dose Experiments and Single Life-Stage GSH-Affinity Sub-Proteome Gels (Figures 1 and 2) Following Trypsin Digestion, MALDI-TOF MS and PMF Analysisa spot no.
MASCOT scoreb
expect value
queries matched
% coverage
Mr (Dalton)
1a 2a 3a 4a 5a 6a 7a 8a 9a 10a 11a 12a 13a 14a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
254 147 107 119 98 218 165 185 131 196 142 200 130 156 116 126 93 131 88 193 137 137 141 97 160 135 145 97 123 113 88
3.30E-20 1.60E-09 1.60E-05 1.00E-06 1.20E-04 1.30E-16 2.60E-11 2.60E-13 5.80E-08 2.10E-14 4.60E-09 8.20E-15 7.30E-08 2.10E-10 6.70E-08 6.70E-09 1.30E-05 2.20E-09 4.40E-05 1.30E-15 5.90E-08 5.30E-10 2.10E-10 4.90E-09 2.70E-12 8.40E-10 8.40E-11 5.30E-06 1.30E-08 1.30E-07 5.30E-05
15/16 8/10 6/7 8/10 5/6 11/11 9/10 10/11 12/12 11/13 14/16 10/10 11/13 9/10 8/15 9/15 8/10 10/11 8/11 11/13 9/15 9/15 10/15 7/15 10/15 9/15 9/15 6/8 8/18 7/9 9/10
59 46 35 42 33 57 49 49 53 54 58 47 60 45 46 43 43 50 40 59 45 45 39 33 54 45 49 36 46 37 44
24348 23588 23261 23876 23602 23277 23261 23943 23973 23128 23128 23295 23295 23317 24348 24894 23943 23943 23943 23270 23295 23295 23128 23128 23261 23876 23588 24348 23602 23918 23804
ID/Wormbase protein code
GST-13/ CE03737 GST-28/ CE22418 GST-27/ CE22417 GST-4/ CE06155 GST-6/ CE32622 GST-26/ CE22416 GST-27/ CE22417 GST-1/ CE00302 GST-1/ CE00302 GST-7/ CE07055 GST-7/ CE07055 GST-5/ CE01613 GST-5/ CE01613 GST-19/ CE09995 GST-13/ CE03737 GST-10/ CE21937 GST-1/ CE00302 GST-1/ CE00302 GST-1/ CE00302 GST-39/ CE22420 GST-5/ CE01613 GST-5/ CE01613 GST-7/ CE07055 GST-7/ CE07055 GST-27/ CE22417 GST-4/ CE06155 GST-28/ CE22418 GST-13/ CE03737 GST-6/ CE32622 GST-36/ CE30562 GST-38/ CE15958
predicted pI
5.14 5.37 5.86 5.40 5.70 5.78 5.86 6.31 6.31 6.80 6.80 7.58 7.58 8.38 5.14 5.41 6.31 6.31 6.31 6.98 7.58 7.58 6.80 6.80 5.86 5.40 5.37 5.14 5.70 6.25 6.14
a Spots 1a-14a are featured in Figure 1 and spots 1-17 are those depicted in Figure 2. b Mascot Score is -10*Log (p) where p is the probability that the observed match is a random event. Protein scores greater than 57 are significant (p < 0.05).
Figure 1. Representative gels showing response of GST to heme after growth of C. elegans in modified axenic medium (mCeHR) containing 1 µM (A), 4 µM (B), 25 µM (C), 50 µM (D), 100 µM (E) and 250 µM (F) hemin (silver-stained). Each IPG strip was rehydrated in buffer containing 10 µg of GSH-affinity proteins. Proteins were focused on a nonlinear pH 3-10 gradient IPG strip (Bio-Rad) and resolved in the second dimension via SDS-PAGE on 17 cm × 20 cm × 1 mm, 12.5% acrylamide gels. Spot identities are listed in Table 1. CE09995 (GST-19), present in worms exposed to higher concentrations of heme, is circled.
with 1 mL of mineral oil. Isoelectric focusing was performed at 20 °C according to IPG strip manufacturer’s instructions until 50 000 V hours were reached and proteins were focused. Focused proteins were reduced for 15 min in equilibration buffer (50 mM Tris-Cl pH 8.8, 6 M urea, 30% glycerol, 2%
sodium dodecyl sulfate (SDS)) containing 1% (w/v) dithiothreitol (DTT). This was followed by a 15 min alkylating step in equilibration buffer containing 2.5% (w/v) iodoacetamide. Each IPG focused sample was taken through second dimension SDSPAGE on vertical 17 cm × 20 cm × 1 mm, 12.5% polyacrylamide Journal of Proteome Research • Vol. 7, No. 10, 2008 4559
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Figure 2. GSH-affinity subproteomes of individual C. elegans lifecycle stages L1, L2, L3, L4 and adult (Coomassie Blue-stained). Each IPG strip was rehydrated in buffer containing 50 µg of GSHaffinity purified protein. Proteins were focused using a linear pH 3-10 gradient IPG strip (Bio-Rad) and resolved in the second dimension via SDS-PAGE on 17 cm × 20 cm × 1 mm. Spot identities are listed in Table 1.
gels cast and run according to the discontinous system.21 Electrophoresis was performed in Tris/Glycine/SDS buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS). Gels were stained either with Coomassie Blue (Phastgel Blue R, Amersham Biosciences) or a MALDI-TOF compatible silver-staining procedure.22 Gel Image Analysis. 2-DE gels were scanned using a BioRad GS-800 densitometer. Image analysis was performed using the Progenesis PG200 v 2006 software package for proteomics (Nonlinear Dynamics). Gels of GSH-affinity samples from worms grown in the presence of 250 µM heme were omitted because there was not enough biological material to run replicate gels. Spots were automatically detected and manually edited. Background subtraction was performed according to mode of nonspot, accepting the default margin parameter of 45. Spot volumes were normalized by multiplying total spot volume by 100. Gels were warped where necessary and spots manually matched to the reference gel. The reference gel was automatically chosen by the software package and was the gel with the most spots, however virtual spots could be added later if absent from the base gel. An average gel was created from each set of 3 biological replicate gels representing the GSH-captured subproteome of C. elegans for each growth condition used. When creating an averaged gel, the maximum number of gels in which spots may be absent was set to 0. Expression levels of GSTs in the averaged subproteome gels were compared to the average gel of GSTs purified from C. elegans grown under optimal heme conditions 4560
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Perally et al. of 25 µM hemin chloride. Normalized spot volumes of GSTs present on each average gel were expressed as a percentage of total spot volume of all major spots represented on that gel and were deemed to be up- or down-regulated following >2.0fold changes in expression with statistical significance reported at p < 0.05. Tryptic Digestion of Protein Spots. Proteins were processed and identified in accordance with the basic principles and methods cited.23 Briefly, protein spots were cut from the gels and dehydrated in acetonitrile for 10 min before complete dehydration under vacuum. Gel plugs were rehydrated in 50 mM ammonium hydrogen carbonate buffer containing 10 ng trypsin/µL (porcine; Sigma T0134) at 4 °C for 45 min in 5 µL per mm2 of gel, before allowing proteins to digest overnight at 37 °C. Digested peptide fragments were eluted from the gel plugs through three changes of 5 µL of solution; 5% formic acid, 50% acetonitrile and 45% water (Sigma W3500). Supernatant was collected at each change after centrifugation at 7 000 g for 1 min, allowing 20 min at 20 °C between changes. Collected peptide supernatant was kept at 4 °C for the duration of the procedure, before vacuum dehydration, and resuspension of peptides in 3 µL of 0.1% trifluoroacetic acid (TFA). MALDI-TOF MS Analysis of Protein Spots. One microliter of trypsin-digested sample in 0.1% TFA was mixed with 1 µL of internal standard (human angiotensin I (1296.6853 Da; Sigma A9650) at 1 pmol/µL and 2 µL of alpha-matrix (R-cyano-4hydroxycinnamic acid) at 2 mg/mL in methanol. Two µL of this ‘sample/standard/matrix’ solution was dropped onto the metal target plate of a Micromass TOF-Spec 2E spectrometer (Micromass, Manchester, UK) and allowed to dry at room temperature. Samples were analyzed in reflectron mode set at 26 kV, source voltage 20 kV, pulse voltage 2700 V, detector voltage 1600 V, with laser energy of 20% coarse and 30-50% fine. External calibrants were; adrenocorticotropic hormone fragment 18-39 (2465.1989 Da; Sigma A0673), bradykinin fragment 1-7 (756.9 Da; Sigma B1651), human angiotensin I (1296.6853 Da; Sigma A9650), substance P (1347.6 Da; Sigma S6883), Glu-fibrinopeptide B (1570.6 Da; Sigma F3261), bovine insulin chain B (3495.9 Da; Sigma I6383). Spectra produced were analyzed using the MassLynx program (Micromass, UK) and calibrated using the human angiotensin I internal standard as lock mass. Recorded monoisotopic peptide masses were submitted to Mascot peptide mass fingerprint searches. Parameters set for all PMF searches were: Database: NCBInr.01 June 2005, Trypsin digest, maximum missed cleavages ) 1, cysteines modified by carbamidomethylation, oxidation of methionines, acetylation of peptide N-terminus, modification of peptide N-terminal Gln to pyroGlu, molecular weight limit ) 40 000 Da, mass-tolerance ) 50 ppm. All C. elegans proteins identified are referred to by their WormBase protein codes and their allocated gene names (http:///www.wormbase.org) (Table 1). Cloning and Recombinant Expression of GST. Recombinant forms of C. elegans GST-5 (CE01613), GST-7 (CE07055), and GST-19 (CE09995) were cloned into pET23d vector and expressed using the E. coli BL21/pET expression system (Novagen). Recombinant protein was purified by GSH-affinity chromatography.19 Biochemical Characterization. Conjugation of a range of model compounds by GSTs from the free-living and parasitic nematodes was determined spectrophotometrically at 25 °C.24,25 GSH-dependent peroxidase assays were measured in a coupled
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Table 2. Expression Changes of GST Subunits in Response to Heme Concentrations Expressed As Percentages of Total Spot Volumea relative proportion of GSTs as percentage of total volumes of spots spot
protein identity
1 µM heme
4 µM heme
25 µM heme
50 µM heme
100 µM heme
1a 2a 3a 4a 5a 6a 7a 8a 9a 10a 11a 12a 13a 14a
GST-13/ CE03737 GST-28/ CE22418 GST-27/ CE22417 GST-4/ CE06155 GST-6/ CE32622 GST-26/ CE22416 GST-27/ CE22417 GST-1/ CE00302 GST-1/ CE00302 GST-7/ CE07055 GST-7/ CE07055 GST-5/ CE01613 GST-5/ CE01613 GST-19/ CE09995
4.17 ( 0.98 2.29 ( 0.39 0.52 ( 0.15b,c 1.64 ( 0.31d 1.07 ( 0.22 3.77 ( 0.57b 8.75 ( 2.15 13.70 ( 2.70 3.77 ( 1.92c,d 5.61 ( 1.16 25.85 ( 2.98 7.02 ( 1.64b 19.44 ( 6.42d 2.39 ( 0.83 e
4.31 ( 1.36 3.32 ( 0.72 2.18 ( 0.70 2.90 ( 1.44 0.91 ( 0.57 6.56 ( 0.26 10.60 ( 0.73 13.25 ( 0.71 8.66 ( 0.97 7.54 ( 0.49d 28.16 ( 1.78 3.08 ( 0.21b 8.53 ( 1.44 0
7.05 ( 2.96 4.46 ( 1.93 1.60 ( 0.32 2.77 ( 0.55 1.14 ( 0.50 5.97 ( 0.65 11.43 ( 1.30 14.55 ( 3.95 7.55 ( 0.72 5.85 ( 0.99 23.86 ( 1.51 3.84 ( 0.24 9.93 ( 0.41 0
4.65 ( 2.41 3.37 ( 1.12 1.34 ( 0.49 2.27 ( 1.53 0.64 ( 0.40 5.66 ( 1.07 10.80 ( 0.66 12.66 ( 3.95 5.82 ( 1.70 5.37 ( 1.44 25.81 ( 2.40 3.52 ( 1.24 11.15 ( 3.09 6.94 ( 0.73 e
7.22 ( 0.32 3.06 ( 0.25 0.98 ( 0.26d 3.10 ( 0.53 0.89 ( 0.12 4.54 ( 0.26d 8.32 ( 0.37b 12.62 ( 1.53 6.30 ( 1.84 6.43 ( 0.45 23.84 ( 2.87 3.92 ( 0.70 12.82 ( 0.10 5.97 ( 0.76 e
a Significant fold changes compared with optimal growth conditions >1.5 fold are highlighted in bold. optimal growth conditions. d p < 0.05. e GST-19 not expressed under optimal conditions.
assay system with glutathione reductase and NADPH.26 Activity toward the natural compound 4-hydroxynonenal (4-HNE) was tested at 30 °C.27 Intrinsic fluorescent measurements were performed at 20 °C in a Shimadzu RF-5301PC spectrofluorimeter (Shimadzu, Milton Keynes, UK) as described.13,28 The inhibitory concentration of hematin leading to a 50% reduction in GST activity (IC50) was also determined.12,29
Results Modulation of C. elegans GSTs in Response to Heme. At least three biological replicates of six growth synchronized C. elegans populations (approximately 2.5 × 106 nematodes per replicate) were exposed to a different fixed hemin concentration from 20-fold suboptimal growth levels to 10-fold supra-optimal growth levels. Soluble C. elegans GST proteins were isolated from each heme exposure by GSH-affinity chromatography. Initial inspection of the GSH-captured subproteome revealed plasticity in C. elegans GST response to heme, with the production of GST-19 (CE09995) occurring at higher heme doses (Figure 1 and Table 1). While we observed expression of GST-19 following exposure to 1 µM heme, the protein was not always present in worms cultured under these conditions (results not shown). In addition, GST-19 expression was significantly higher following dosage with 50-100 µM heme and was always present compared to the lowest heme exposure (>2-fold with p < 0.01). GST-19 has not been identified previously in GST subproteomes of mixed-stage C. elegans under standard growth conditions where worms were fed on E. coli.30,31 2-DE analysis of cytosolic extracts from worms grown under varying concentrations of heme failed to reveal any significant changes in over 500 soluble proteins analyzed (results not shown) and may emphasize the relative importance of the GST-19 response to heme. Life-cycle stages were isolated from monoxenically cultured worm populations in order to verify that potential hemeresponse changes are not simply a result of changes in lifecycle demographics. GSH-affinity/ 2-DE analysis (Figure 2, Table 1) suggested changes in the GST subproteomes of the larval stages, most notably GST-5 (CE01613) which completely disappears in L3 larvae. The life-stage analysis suggested that the relatively high levels of GST-5 present at low heme levels
b
p < 0.01.
c
Fold changes >2.0 compared with
could reflect a higher percentage of L1 and L2 larvae present in the culture population due to slower development at 1 µM heme (∼45% L1/L2’s cf. 10% at >25 µM). GST-19 was not detected in any of the GSH-affinity subproteomes of the individual larval stages promoting argument for the specific role of GST-19 in a toxic heme level response or metabolic imbalance. Quantitative assessment of changes in GST subunit expression in response to external heme was made by image analysis software. Spot volumes after background subtraction and normalization were expressed as a percentage of the total volume of all spots (Table 2). Comparisons of GSH-captured subproteomes of worms grown under observed optimal growth conditions (25 µM heme) show that several GSTs are relatively under-synthesized at low heme levels (1 µM) including GST-1 (CE00302), GST-4 (CE06155), GST-26 (CE22416) and GST-27 (CE22417). There are at least two forms of GST-1, each with a different pI, one of which is also reduced at low heme levels. GST-5 (CE01613) accounted for nearly 25% of the GST subproteome at low levels of heme, and approximately 11-15% in worms exposed to 4-100 µM heme. An increase in cytosolic GST activity toward the model substrate 1-chloro, 2, 4-dinitrobenzene (CDNB) was observed in worm extracts from cultures exposed to >50 µM hemin and high somatic GST activity was also observed at low heme levels (1 µM) (results not shown), probably reflecting changes in the GST superfamily composition. Characterization of Nematode GSTs. Recombinant forms of three C. elegans GSTs were produced in order to investigate heme interaction. GST-19 was selected because it is upregulated in supra-optimal external heme levels while GST-5 may be involved in early larval development given its relative abundance in L1 larvae. GST-5 (CE01613) and GST-7 (CE07055) share the highest amino acid similarity to already characterized and structurally resolved parasite nematode Nu-class GSTs from the human hookworm N. americanus (60% identity/ 75% similarity) while GST-19, according to primary sequence analysis, shows more similarity (43% identity/ 60% similarity) to the pig roundworm, Ascaris suum GST, AsGST-1 (Accession no. CAA53218).11-13,32 Phylogenetic analysis of C. elegans GSTs captured by GSH-affinity following heme exposure, together with representative sequences GST classes from various species Journal of Proteome Research • Vol. 7, No. 10, 2008 4561
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Figure 3. Neighbor-joining tree representing phylogenetic relationship of C. elegans GSTs purified via GSH-affinity and representative GSTs from Alpha, Mu, Pi, Sigma, Omega, Zeta and Theta classes in different species. Bootstrap values are indicated at the nodes (1000 replicates). (a) C. elegans GST sequences are referred to by their WormBase protein codes (b) LrubGSTA (LRP02027_1) from Lumbricus rubellus is present in LumbriBASE (http://www.nematodes.org/Lumbribase/lumbribase.php) (c) Other sequences represented in the tree are listed with their GenBank or Swiss-Prot accession numbers below: Human Alpha GST (homoGSTA1), NP_66583; mouse Alpha GST (musGSTA1), NP_032207; Bombyx mori Sigma GST (BmorGSTS), BAD911071; Ascaris suum Sigma GST (AsGST-1), CAA53218; Haemonchus contortus GST (HcGST-1), AAF81283; Ancylostoma caninum GST (AcGST-1), AAT37718; human Sigma GST (homoGSTPGD2), NP_055300; mouse Sigma GST (musGSTPGD2), NP_062328; Dermatophagoides pteronyssinus Mu GST (DerGSTM1), AAB32224.1; Fasciola hepatica Mu GST (FhepGSTM27), P31670; human Mu GST (homoGSTM1) CAG46666; mouse Mu GST (musGSTM1), AAH91763; Unio tumidus Pi GST (UtumGSTP), AAX20373.1; human Pi GST (homoGSTP1), AAH10915; mouse Pi GST (musGSTP1), P19157; Onchocerca volvulus Pi GST (OvolGSTP), AAA53575; Drosophila melanogaster Theta GST (DmelGSTT1), P20432; Strongylocentrotus purpuratus Theta GST (SpurGSTT1), XP_790223.1; human Theta GST (homoGSTT1), NP_000844; mouse Theta GST (musGSTT1), NP_032211; Schistosoma mansoni Omega GST (SmanGSTO), AA049385; human Zeta GST (homoGSTZ1), AAH31777; mouse Zeta GST (musGSTZ1), NP_034493; Drosophila melanogaster Zeta GST (DmelGSTZ1), AAC28280.2; Bombyx mori Zeta GST (BmorGSTZ4), ABC79691; Bombyx mori Omega GST (BmorGSTO2), NP_001037406; human Omega GST (homoGSTO1), NP_004823; mouse Omega GST (musGSTO1), AAH85165; XP_790223.1.
confirms the presence of the novel Nu-class GST family in nematodes, although there may be at least two subfamilies of GST within this class (Figure 3). Most of the GSH-captured GSTs belong to the Nu-class with the exception of Pi-class GST-1 (CE00302) and GST-10 (CE21937). Substrate specificities of the three recombinant GSTs were tested with a range of potential natural and model substrates (Table 3). All three GSTs have activity toward the universal GST model substrate CDNB, however only GST-7 and GST-19 have detectable GSH-dependent lipid peroxidase activity. GST-7 also 4562
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shows GSH-conjugation activity toward the major lipid peroxidation product 4-hydroxynonenal (4-HNE) within the same range observed for C. elegans Pi-class GST-10. Binding of the heme-related compound, hematin to GSTs was determined by intrinsic fluorescence quenching of GST protein following titrations with ligand (Figure 4) and enzyme inhibition assays. The GSTs have dissociation constants (Kd) for hematin similar to values observed for major GSTs of parasitic blood-feeding nematodes, with Kd ) 6.47 ( 1.24, 5.89 ( 0.40, and 3.46 ( 0.20 µM for GST-5, GST-7, and GST-19,
research articles
Heme Trafficking and Detoxification in Nematodes Table 3. Substrate Specificities of Nu-Class Recombinant GSTs from C. elegans
specific activity/nmol min-1 mg protein-1 substrate
1-chloro-2, 4-dinitrobenzene Trans-2-nonenal Trans,trans- 2,4-decadienal Cumene hydroperoxide Ethacrynic acid 1,2-Dichloro-4-nitrobenzene Trans-4,phenyl-3-buten-2-one 4-Hydroxy-2-nonenal a
[substrate] [GSH] (mM) (mM) pH λmax (nm)
1 0.023 0.023 1.2 0.08 1 0.05 0.1
1 1 1 1 1 5 0.25 0.5
6.5 6.5 6.5 7 6.5 7 6.5 6.5
340 225 280 340 270 345 290 224
GST-5/CE01613a GST-7/CE07055a GST-19/CE09995a
∆ε -1
9.6 × 10 cm .mol 19.2 mM-1.cm-1 29.7 mM-1.cm-1 6.22 × 106 cm2.mol-1 5.0 mM-1.cm-1 9.6 × 106 cm2.mol-1 -24.8 mM-1.cm-1 13.75 mM-1.cm-1 6
2
10002 ( 90 96 ( 2 ND 515 ( 6 63 ( 5 ND 68 ( 2 391 ( 89
23932 ( 748 656 ( 46 73 ( 12 2639 ( 97 114 ( 15 30 ( 3 ND 4210 ( 370
12152 ( 1249 ND ND 3617 ( 96 ND ND ND 389 ( 27
ND - No activity detected under conditions used. All values are listed ( standard deviation of the mean of 4 replicates.
response to heme stress and life-stage development, and report the presence of the genome-predicted Nu-class GST-19 protein (CE09995) within a postgrowth-optimal heme environment. This study further demonstrates the plasticity of the nematode proteome in response to changing external environments.33 Nu-class GSTs are presumed to be major detoxification enzymes in C. elegans developing larval stages and adult worms given that they comprise 36 of the 44 annotated GSTs in the model organism.
Figure 4. Binding of hematin to recombinant CE07055 (GST-7). (A) Quenching of intrinsic fluorescence in CE07055 upon each addition of hematin (0.33-10.67 µM). Lines A and B indicate fluorescence measurements with and without CE07055 respectively. (B) Plot of quenching of intrinsic fluorescence against concentration of free hematin fitted by nonlinear regression with double-reciprocal plot (inset).
respectively.12,13 Enzyme inhibition assays using CDNB as substrate confirmed that all three C. elegans GSTs are strongly inhibited by the iron tetrapyrrole with IC50 between 0.4 to 1.5 µM as also observed for parasitic Nu-class GSTs.12,13 Dissociation constants were much higher than calculated IC50s, and may be attributed to the detection of different heme-binding sites in GST.12 Conserved high-affinity for heme-related compounds in both C. elegans and parasitic Nu-class GSTs suggests that in contrast to previous proposals, heme-binding is not an adaptation to parasitism.12
Discussion Using C. elegans as an experimental nematode model, we have identified changes within a GSH-affinity subproteome in
There are a number of Nu-class GSTs present in EST databases of several parasitic nematodes including H. contortus, A. caninum and Ancylostoma ceylanicum, although the number of GSTs found to date in each species is fewer than in C. elegans. It remains to be seen whether complete annotation of a parasitic Clade V nematode genome such as the H. contortus sequencing project currently underway will reveal numbers of Nu-class GSTs similar to those in C. elegans. It has previously been shown by global RNAi analysis that individual Nu-class GSTs are not essential for C. elegans survival (albeit at normal external heme levels).34 This study suggests that GST plasticity (genetic buffering) and functional redundancy may disguise phenotype outcomes of global-based analysis, and in vitro characterization suggests differences in biological activity profiles, at least for the three GSTs in this study. A robust, targeted RNAi analysis is required to reveal the role of individual superfamily proteins in C. elegans response to environmental challenge. Ayyadevara and colleagues35 primed C. elegans with reactive oxygen species (ROS) to confirm that several GSTs were important for protection against oxidative stress. Therefore, if validated by robust RNAi, nematode Nuclass GST(s) could present themselves as potential drug targets against parasitic worms of veterinary and medical importance since interruption, possibly by a heme-derivative, would deprive worms of hemoproteins. If functional redundancy of GST is reduced in parasitic nematodes, given the relative fewer number of phase II detoxification genes predicted in their genomes compared with C. elegans, this offers additional advantages for the use of Nu-class GSTs as targets for therapy. Since GST-5 has high affinity for hematin and is relatively more abundant in L1 and L2 larvae, we suggest that this GST could be more important in intracellular movement of heme in larval development, while heme-induced GST-19 may be involved in detoxification at higher lethal concentrations of heme, possibly in more heme-exposed tissues. GST-5 appears to have a limited enzymatic activity profile toward most compounds tested, supporting a more focused role. In contrast, GST-19 shows high GSH-dependent lipid peroxidase activity, consistent with high levels of lipid peroxides produced as a Journal of Proteome Research • Vol. 7, No. 10, 2008 4563
research articles 36
result of heme stress. High GSH-dependent lipid peroxidase activity has previously been detected in GSH-affinity purified fractions from N. americanus, therefore a functional ortholog of GST-19 is predicted in blood-feeding parasitic nematodes.37,38 Although, GST-19 is produced under high heme levels, its ability to bind heme as assessed by our spectrofluorimetric assays does not appear to be different from other Nu-class GSTs. This suggests that either GST-19 is part of a tissuespecific pattern to heme stress or it indirectly contributes to heme response pathways. Global microarray analysis suggests that GST-19 is expressed in reproductive hermaphrodite adult stages and embryos where vitally important heme may be accumulated for embryo and early larval development.39,40 Interestingly, the C. elegans interactome indicates that the heme-responsive GST-19 interacts with a nuclear hormone receptor (nhr-111 (CE35538)) which may form part of a protein network “hub” by interacting with numerous proteins including CE05663, a putative ABC transporter with a heme exporter domain.41 Therefore, as well as detoxifying lipid peroxides potentially induced by heme, GST-19 may indirectly influence heme trafficking at high intracellular heme concentrations. Previous investigations have identified a GST-19 response following exposure to cadmium, with RNAi suppression of the GST leading to cadmium hypersensitivity.42 These findings may support a generic GST-19 response to metal toxicants including iron. However Nu-class GSTs in this study and others interact strongly with hematin and other heme-related compounds such as protoporphyrin IX which lacks an iron core at the center of its tetrapyrrole ring structure,13 thus supporting the phase II detoxification enzymes’ proposed heme ligandin role. Heme-binding may rely upon hydrophobic interactions between GST and the porphyrin ring structure since we were unable to identify a conserved heme-binding domain in Nuclass GSTs in our previous studies12 or in subsequently resolved protein crystal structures.11,32 C. elegans GST-7 appears to have the broadest substrate specificity, with high GSH-dependent lipid peroxidase and 4-HNE activity. However, its GSH-dependent lipid peroxidase activity is 60-fold lower than observed for mammalian Alphaclass GSTs which are generally known for their catalytic efficiency in the degradation of lipid peroxides.43,44 Unlike GST19, expression of GST-7 appears to be unaffected by heme (Figure 1, Table 2) implying that this GST detoxifies 4-HNE and lipid peroxides under “normal” growth conditions as part of its general house-keeping. An SKN-1 binding site, similar to ARE-type response elements has been located upstream of GST-7, confirming the general role for GST-7 in protecting C. elegans against oxidative stress.45 Apart from the newly uncovered role of hrg-4 protein in heme uptake,8 a number of membrane-bound heme transporter orthologs are present in the genome of C. elegans such as FLVCR, involved in heme efflux46 and ABC-type transporters present in both plasma and mitochondrial membranes.47 No soluble mammalian heme-binding protein orthologs could be detected in nematodes following searches within WormBase and nematode EST databases. The Nu-class GSTs have a lower affinity for heme compounds compared to previously isolated mammalian soluble heme carrier proteins such as the extracellular heme carrier hemopexin (Kd ≈ 10-12 M).48 However, the rat cytosolic 23 kDa heme-binding protein (HBP23) (Kd ≈ 10-8 M) and serum albumin (Kd ≈ 10-6-10-8 M) also have a similar affinity for heme compared to nematode GSTs in this study.49 Therefore, in contrast to mammals, nematodes may 4564
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Perally et al. have a simpler heme transport system, with more reliance on hydrophobic ligand binding proteins, such as the Nu-class GSTs and possibly as yet uncharacterized with respect to hemebinding, FABPs. Heme capture may be functionally coupled to detoxification of heme-induced ROS given the relatively high lipid peroxidase activity detected in Nu-class GSTs.
Acknowledgment. S.P. and E.J.L. acknowledge Aberystwyth University for PhD funding. P.M.B. acknowledges support of Natural Environment Research Council UK (NER/T/S/2002/00021). We thank Kevin Bailey and James Heald for assistance with mass spectrometry analysis. References (1) Jasmer, D. P.; Goverse, A.; Smant, G. Parasitic nematode interactions with mammals and plants. Ann. Rev. Phytopathol. 2003, 41, 245–270. (2) Chan, M. S.; Medley, G. F.; Jamison, D.; Bundy, D. A. P. The evaluation of potential global morbidity attributable to intestinal nematode infections. Parasitology 1994, 109, 373–87. (3) Sangster, N. C. Anthelmintic resistance: past, present and future. Int. J. Parasitol. 1999, 29, 115–24. (4) Kaminsky, R. Drug resistance in nematodes: a paper tiger or a real problem. Curr. Opin. Infect. Dis. 2003, 16, 559–64. (5) Geary, T. G.; Thompson, D. P.; Klein, R. D. Mechanism-based screening: discovery of the next generation of anthelmintics depends upon more basic research. Int. J. Parasitol. 1999, 29, 105– 12. (6) Rao, A. U.; Carta, L. K.; Lesuisse, E.; Hamza, I. Lack of heme synthesis in a free-living eukaryote. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4270–5. (7) Rodgers, K. R. Heme-based sensors in biological systems. Curr. Opin. Chem. Biol. 1999, 3, 158–167. (8) Rajagopal, A.; Rao, A. U.; Amigo, J.; Tian, M.; Upadhyay, S. K.; Hall, C.; Uhm, S.; Mathew, M. K.; Fleming, M. D.; Paw, B. H.; Krause, M.; Hamza, I. Haem homeostasis is regulated by the conserved and concerted functions of HRG-1 proteins. Nature 2008, 453, 1127–31. (9) Latunde-Dada, G. O.; Simpson, R. J.; McKie, A. T. Recent advances in mammalian haem transport. Trends. Biochem. Sci. 2006, 31, 182–188. (10) Shayeghi, M.; Latunde-Dada, G. O.; Oakhill, J. S.; Laftah, A. H.; Takeuchi, K.; Halliday, N.; Khan, Y.; Warley, A.; McCann, F. E.; Hider, R. C.; Frazer, D. M.; Anderson, G. J.; Vulpe, C. D.; Simpson, R. J.; McKie, A. T. Identification of an intestinal heme transporter. Cell 2005, 122, 789–801. (11) Schu ¨ ller, D. J.; Liu, Q.; Kriksunov, I. A.; Campbell, A. M.; Barrett, J.; Brophy, P. M.; Hao, Q. Crystal structure of a new class of glutathione transferase from the model human hookworm nematode Heligmosomoides polygyrus. Proteins 2005, 61, 1024–1031. (12) van Rossum, A. J.; Jefferies, J. R.; Rijsewijk, F. A.; LaCourse, E. J.; Teesdale-Spittle, P.; Barrett, J.; Tait, A.; Brophy, P. M. Binding of hematin by a new class of glutathione transferase from the bloodfeeding parasitic nematode Haemonchus contortus. Infect. Immun. 2004, 72, 2780–2790. (13) Zhan, B.; Liu, S.; Perally, S.; Xue, J.; Brophy, P. M.; Xiao, S.; Liu, Y.; Feng, J.; Williamson, A.; Wang, Y.; Bueno, L. L.; Mendez, S.; Goud, G.; Bethony, J. M.; Hawdon, J. M.; Loukas, A.; Jones, K.; Hotez, P. J. Biochemical characterization and vaccine potential of a hemebinding glutathione transferase from the adult hookworm Ancylostoma caninum. Infect. Immun. 2005, 73, 6903–6911. (14) Parkinson, J.; Mitreva, M.; Whitton, C.; Thomson, M.; Daub, J.; Martin, J.; Schmid, R.; Hall, N.; Barrell, B.; Waterston, R. H.; McCarter, J. P.; Blaxter, M. L. A transcriptomic analysis of the phylum Nematoda. Nat. Genet. 2004, 36, 1259–1267. (15) Britton, L.; Murray, C. Using Caenorhabditis elegans for functional analysis of genes of parasitic nematodes. Int. J. Parasitol. 2006, 36, 651–659. (16) Clegg, E. D.; LaPe´notie`re, H. F.; French, D. Y.; Szilagyi, M. Use of CeHR Axenic Medium for exposure and gene expression studies; East Coast Worm Meeting, 2002, Abstract 91. (17) Lewis, J. A.; Flemming, J. T. Basic culture methods in Caenorhabditis elegans: Modern Biological Analysis of an Organism; Epstein, H. F., Shakes, D. C., Eds.; Academic Press: San Diego, 1995; Vol. 48, pp 3-29.
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