Proteomic Analyses of Caenorhabditis elegans Dauer Larvae and Long-Lived daf-2 Mutants Implicates a Shared Detoxification System in Longevity Assurance Laura M. Jones,*,† Katharina Staffa,† Samı¨rah Perally,† E. James LaCourse,‡ Peter M. Brophy,† and Jo V. Hamilton† Aberystwyth University, Institute of Biological, Environmental and Rural Sciences, SY23 3DA, United Kingdom, and The University of Liverpool, School of Veterinary Science, L69 7ZJ, United Kingcom Received October 26, 2009
The insulin/insulin-like growth factor-1 (IGF-1) signaling system is a public regulator of aging in the model animals Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus. For the first time, proteomic analyses of the environmentally resistant and ‘nonaging’ C. elegans dauer stage and longlived daf-2 mutants has provided a unique insight into the protein changes which mediate survival against endogenously produced toxins. These changes support a diversion of energy consumption away from anabolic processes toward enhanced cellular maintenance and detoxification processes as previously described by the ‘Green Theory of Aging’. Important components of this enhanced longevity system identified in this proteomics study include the alpha-crystallin family of small heat shock proteins, anti-ROS defense systems and cellular phase II detoxification (in daf-2 only). Among those proteins involved in phase II cellular detoxification that were significantly upregulated was a Pi-class glutathione transferase (GST) CE00302. Targeting this GST with RNAi revealed compensatory regulation within the Pi-class GSTs. Furthermore, a recombinant form of the GST protein was found to detoxify and/or bind short-chain aldehydic natural toxic products of lipid peroxidation and long-chained fatty-acids at physiologically relevant concentrations, which may indicate a role in longevity. Keywords: C. elegans • dauer • daf-2 • longevity • Insulin/IGF-1 signaling • IIS • 2-DE • proteomics • RNAi • glutathione transferase • GST-1 • heat shock protein • HSP-12.6 • glutathione peroxidise • superoxide dismutase • SOD-1 • lipid binding protein • LBP-6 • ribosomal protein • RPS-12.
Introduction The biological process that causes aging is very complex and remains poorly understood at the mechanistic molecular level. Extensive genetic analyses have revealed the involvement of many genes and molecular pathways which determine lifespan. The insulin/insulin-like growth factor-1 (IGF-1) signaling system is a powerful regulator of aging in the metazoan model Caenorhabditis elegans: reduced insulin/IGF-1 signaling (IIS) can lead to more than a doubling of lifespan, the Age phenotype.1-3 A similar response has been observed in Drosophila melanogaster4,5 and also in Mus musculus.6,7 The IIS system is one of several molecular signal transduction pathways which regulate larval diapause in C. elegans.3,8 Under adverse environmental conditions (including high temperature, low food, and high population density), developing larvae may arrest at an environmentally resistant dauer stage and survive up to 8 times the normal 3-week lifespan of C. elegans.9-11 Dauer larvae exhibit a variety of morphological, behavioral and biochemical adaptations.10-13 Through misexpression of ISS * Corresponding author: Laura M. Jones, The University of Leeds, Faculty of Biological Sciences, LS2 9JT. E-mail: L.M.Jones@ Leeds.ac.uk. † Aberystwyth University. ‡ The University of Liverpool. 10.1021/pr9009639
2010 American Chemical Society
signaling and subsequent activation of daf-16, the daf-2 mutant also exhibits longevity and provides another suitable aging model, complementary to the dauer model.2 The accumulation of lipid peroxidation products including 4-hydroxy-2-nonenal (4-HNE) and reactive oxygen species (ROS) such as superoxide and hydrogen peroxide have long been regarded as potential major contributors to the molecular damage that underlies aging.14 Biochemical and transcriptional analyses in both dauer stage and daf-2 longevity systems support a diversion of energy consumption away from anabolic processes and toward enhanced cellular maintenance and detoxification processes15-23 as described in the ‘Green Theory of Aging’.22,24,25 Such a diversion of energy has also been supported in an extensive multilevel cross-species transcriptional analysis including long-lived mutant Drosophila and mice with reduced IIS.25 Important components of this enhanced longevity system include the alpha-crystallin family of small heat shock proteins, anti-ROS defense systems, cellular phase II detoxification (in daf-2 only) and possibly also enhanced cell maintenance, reduced growth and (in dauers only) reduced protein turnover. The use of proteomic analysis for studying developmental processes in C. elegans has gained recent recognition26 and shown high potential to complement transcriptional analyses as well as assess the gene product of Journal of Proteome Research 2010, 9, 2871–2881 2871 Published on Web 04/15/2010
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Jones et al. 27
RNAi-mediated knock-down. Protein expression profiles have now been described under a number of genetic, environmental, temporal, and 2-DE conditions.26 Recent proteomic investigations found an almost identical proteome between dauer and L3 stage, with only eight differently expressed proteins (represented by 23 spots), supporting a role in anaerobic fermentative metabolism in dauer larvae.28 A similar comparative proteomic study of 1-day old daf-2 and wild-type adults identified 86 proteins that were differentially expressed in daf-2 adults which suggested enhanced amino acid biosynthesis, oxygen and reactive oxygen species metabolism, and carbohydrate metabolism.29 However, no differences in the expression of enzymes involved in phase II cellular detoxification were uncovered in these proteomic analyses of dauer larvae and daf-2 adults, as predicted by transcriptional analyses.22 More recently, the use of a subproteomic approach has shown potential to investigate the role of glutathione transferases in C. elegans.30 For the first time, we have used combined global and subproteomic analyses of the environmentally resistant and ‘nonaging’ dauer stage and 6-day old daf-2 mutants to provide a unique insight into the regulation of these changes at the functionally relevant protein level, including proteins promoting survival against endogenously produced toxins. Among those proteins upregulated in the dauer stage and longlived daf-2 mutants was a Pi-class GST, CE00302. RNAi in 6-day old glp-4 C. elegans was also used to functionally characterize this GST. CE00302 was also cloned, produced as a recombinant, and found to conjugate and/or bind short-chain cytotoxic aldehydic natural products of lipid peroxidation and longchained fatty-acids at physiologically relevant concentrations, which may indicate a role in longevity.
Materials and Methods C. elegans Culture. C. elegans N2 (Bristol wild-type) strain and temperature-sensitive infertile glp-4(bn2ts) strain were provided by the Caenorhabditis Genetics Center. The temperature-sensitive infertile and long-lived glp-4(bn2ts); daf-2(e1370) strain was kindly provided by Professor David Gems at the University College of London, UK. Laboratory stock cultures were maintained at 16 °C on nematode growth media agar (NGM) as previously described.10 Culture in liquid media was performed as described31 with adaptations. Mixed-stage N2 (Bristol wild-type) worms were raised at 20 °C for 5 days prior to harvest and preparation of eggs via the hypochlorite method.32 Synchronous stage cultures were started from the egg preparation for collection of L3 and dauer larvae following 39 and 100 h, respectively. L3 stage were grown in culture containing 200 000 eggs and 25 mL of Escherichia coli HB101 resuspended in M9 buffer. Dauer larvae were grown from 12 000 000 eggs under conditions capable of inducing 100% dauer formation, that is, 470 mL of fresh medium, 30 mL of 7-day old exhausted culture medium, and 7.5 mL of E. coli HB101. Nematodes were separated from bacteria by floatation on 30% sucrose in M9 buffer and washed twice in M9.31 Live juvenile worms were separated from the dead carcasses, following sucrose floatation, by sieving through a 41 µm Nitex screen (Sefar, Bury, Lancashire, U.K.) stretched over a 9 cm embroidery hoop and supported in contact with M9 buffer contained in a 9 cm Petri dish.33 Three biological replicate cultures were grown for each life-cycle stage under each environmental condition. Mixed-stage glp-4 worms were raised at 16 °C for 7 days prior to harvest and bleaching to obtain eggs via the hypochlorite method.32 Further cultures containing 2872
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200 000 eggs and 25 mL of E. coli HB101 were then grown for 3 days at 16 °C to L4 stage. Upon further addition of nutrients and 25 mL of E. coli HB101, worms were grown for a further 6 days at 25 °C and fed 25 mL of E. coli HB101 every day until harvest. A typical yield was found to be approximately 4 g of wet-weight worm pellet. Following separation from bacteria as previously described,31 worms were cryogenically frozen and stored at -80 °C. C. elegans RNAi Culture. C. elegans glp-4(bn2ts) strain was provided by the Caenorhabditis Genetics Center. RNAi clones in vector pL4440 (ampicillin/carbenicillin resistant) of C. elegans CE00302 (GST-1) (WormBase codes www.wormbase.org) and control vector pL444034 were constructed in the Ahringer Laboratory at the The Wellcome CRC Institute, University of Cambridge, Cambridge, U.K.35,36 and obtained from Geneservice Ltd., U.K. (http://www.geneservice.co.uk/). The GST-1 construct does not have recognized secondary targets of RNAi within the C. elegans genome database (www.wormbase.org). Laboratory stock cultures were maintained at 16 °C on NGM as previously described.10 Culture in liquid media was performed as described31 with adaptations for RNAi suppression carried out according to bacterial feeding method protocols from the Fire Laboratory (http://firelab.stanford.edu/ accessed 20.05.07). Mixed-stage glp-4 worms were raised at 16 °C for 7 days prior to harvest and bleaching to obtain eggs via the hypochlorite method.32 Further cultures containing 200 000 eggs and 25 mL of tetracycline resistant, RNase III deficient strain E. coli HT115 (including appropriate RNAi vector construct) were then grown for 3 days at 16 °C to L4 stage.37 Upon further addition of nutrients and 25 mL of E. coli HT115, they were grown for a further 6 days at 25 °C and fed 25 mL of E. coli HB101 every day until harvest. As a control, a 1056 bp noncoding insert was ligated into the pl4440 vector and transformed into HT115 (DE3) as previously described.31 A typical yield was found to be approximately 4 g of wet-weight worm pellet. Cytosolic Protein Purification. Protein purification was carried out as previously described.27 Harvested nematodes were homogenized in 20 mM phosphate buffer, pH 7.4 containing 0.1% (v/v) Triton X-100 and protease inhibitors (mini-complete protease inhibitor cocktail tablets, Roche Applied Science, Burgess Hill, West Sussex, U.K.) using 1 mm zircon beads and a mini bead-beater (Biospec Products, Bartlesville, OK). Cell debris was removed by centrifuging at 17 000g for 15 min at 4 °C. The supernatant was ultracentrifuged at 100 000g for 30 min at 4 °C in order to recover the cytosol. Protein concentration was estimated using Bradford reagent (Sigma-Aldrich, Dorset, U.K.) according to manufacturer’s instructions based upon the method previously described.38 Protein extracts were purified for GSTs using a GSHaffinity wash-batch method according to the manufacturer’s instructions and as previously described.31 Briefly, 200 mg of GSH-agarose gel (Sigma-Aldrich) was equilibrated with 25 mL of 20 mM KHPO4 pH 7.0 buffer containing 1 M NaOH (equilibration buffer) for 15 min at 4 °C. A total of 400 µL of equilibrated gel was then incubated with 1 mg of global cytosolic protein extracts at 4 °C for 2 h prior to washing six times in equilibration buffer as previously described. Following each wash, the gel was centrifuged at low speed (100g) for 10 s and the supernatant removed and discarded. Proteins were eluted for 30 min at 4 °C in 1 mL of 50 mM Tris-HCl buffer pH 9.6 containing 5 mM glutathione (GSH, Sigma-Aldrich). Eluted proteins were pooled, washed in double-distilled H2O, and
Shared Detoxification System in C. elegans concentrated using 10 kDa Molecular Weight Cut Off (MWCO) spin filter tubes (Amicos Ultra, Houston, TX). Samples of cytosolic extract nonbinding flow-through and purified GST were retained to check for purification efficiency when assayed for activity with the universal model substrate 1-chloro-2, 4-dinitrobenzene (CDNB) at 25 °C under the conditions described in Table 5.39 Two-Dimensional Gel Electrophoresis (2-DE). Cytosolic global protein and GST samples were subjected to 2-DE as previously described.40 A Protean IEF unit (Bio-Rad, Hemel Hempsted, Herts, U.K.) was used for the isoelectric focusing step. A total of 200 µg of global cytosolic protein or 10 µg of eluted GST proteins was 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, and 0.5% (v/v) ampholytes pH 3-10 (Pharmalytes, Amersham BioSciences, Little Chalfont, Buckinghamshire, U.K.) at a final volume of 300 µL for 17 cm pH 3-10 nonlinear IPG strips (BioRad, U.K.). In-gel passive rehydration of IPG gel strips with protein samples was performed at 20 °C for 16 h with each sample/strip overlaid with 1 mL of mineral oil (Bio-Rad). Isoelectric focusing was performed at 20 °C according to IPG strip manufacturer’s instructions until 50 000 Vh were reached and proteins 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 global cytosolic sample was separated in the second-dimension by a 5-20% T acrylamide/ bisacrylamide 29:1 (3.3% C) gradient gel using the automated 2-DE electrophoresis Optimiser system (NextGen Sciences, Huntingdon, Cambridgeshire, U.K.). Each IPG focused GST sample was taken through second dimension SDS-PAGE by a 12.5% T acrylamide/bisacrylamide 29:1 (3.3% C) gel, using the PROTEAN II xi Cell (Bio-Rad, U.K.), cast and run according to the discontinous system.41 Electrophoresis was performed in Tris/Glycine/SDS buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS). Gels were stained with a MALDI-TOF compatible silver-staining procedure42 for image analysis. For mass spectrometric analysis of protein spots 2-DE was performed with 1 mg of global cytosolic proteins and 50 µg GST proteins, prior to staining with Coomassie Blue (Phastgel Blue R, Amersham Biosciences). Gels were also completed for samples obtained from the replicate set of cultures in the same manner. At least three biological replicates were incorporated into the experimental design. 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, Newcastle, U.K.). Gel images (for three biological replicates) were matched to a composite 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. Spots were automatically detected and were manually edited. Background subtraction was then performed according to mode of nonspot, accepting the default margin parameter of 45. Spot volumes were normalized to accurately compare spot measurements across different images with the following parameters: Total spot volume multiplied by 100. Gels were then warped and spots were manually matched to the reference gel. An averaged gel was created from each set of three biological
research articles replicate gels representing the global cytosolic proteome and GST 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. Normalized spot volumes of GSTs were calculated and ANOVA was performed. Tryptic Digestion of Protein Spots. Proteins were processed and identified in accordance with the basic principles and methods cited.43 Briefly, protein spots were excised 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 mL-1 trypsin (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 7000g for 1 min, allowing 20 min at 20 °C between changes. Collected peptide supernatant was maintained 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-1 and 2 µL of alpha-matrix (R-cyano-4hydroxycinnamic acid) at 2 mg mL-1 in methanol. Two microliters of this ‘sample/standard/matrix’ solution was dropped onto the metal target plate of a Micromass TOF-Spec 2E spectrometer (Micromass, Manchester, U.K.) 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, U.K.) 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 2007; 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). Recombinant CE0302 GST Production. A recombinant form of C. elegans GST-1 (CE00302) was cloned into pET23d and expressed using the BL21/pET expression system (Novagen, Merk KGaA, Darmstadt, Germany). Overexpression of the protein was induced by adding 1 mM final concentration isopropyl β-D-1-thio-galactopyranoside (IPTG), at 28 °C for 6 h and at 200 rpm before harvesting. Overexpression of recombinant protein was verified by SDS-PAGE. Recombinant GST was purified by GSH-affinity chromatography and purity was assessed by 2-DE and Coomassie stain (Phastgel Blue R, Amersham Biosciences). Expected molecular mass of the recombinant CE00302 GST protein was assessed by Electrospray Ionization (ESI) QToF-MS. A 20 mg mL-1 stock concenJournal of Proteome Research • Vol. 9, No. 6, 2010 2873
research articles tration of rCE00302GST was prepared in 1% acetic acid, 50% of methanol, and 48.5% of H2O. The sample mix was pumped at 5 µL min-1 in a Harvard syringe pump 11. Biochemical Characterization. GST activity of rCE00302 toward the model substrates CDNB (ClC6H3(NO2)2), Ethacrynic acid (C13H12Cl2O4) and Trans-4,phenyl-3-buten-2-one (C10H10O) was determined by spectrophotometric assay at 25 °C under the conditions described in Table 5.39,44 GST activity of rCE00302 was also determined toward a range of natural lipid associated substrates at 25 °C and under conditions described in Table 5. These substrates included Stearic acid (C18H36O2),45 Oleic acid (C18H34O2)45 Arachidonic acid (C20H32O2),45 Linoleic acid (C18H32O2),45 Dauemone (C13H24O6, KDR Biotech Co., Ltd., Korea), 4-Hydroxy-2-nonenal (C9H16O2),46 Trans-2-nonenal (C9H16O)47 and Trans,trans-2,4 decadienal (C10H16O).47 GSHdependent peroxidise activity toward Cumene hydroperoxide (C9H12O2) was measured in a coupled assay system with glutathione reductase and NADPH.48 CE00302 binding of ligands was also assessed by inhibition of GSH conjugation, using CDNB as a second substrate.39,44 GSH-dependent conjugation was estimated using the free thiol colorimetric assay, measured at 412 nm at 20 °C as previously described.49 Briefly, a standard GST assay was set up in 1 mL of 100 mM KHPO4 buffer pH 6.5 containing 50 mM GSH, 50 mM CDNB, and 0.25 µM GST. Then, 50 µL aliquots were removed at regular intervals over 80 min and tested for thiol content in an assay containing 0.2 mM DTNB and 300 mM NaHPO4 buffer, pH 8.0.
Results Global Proteome Analyses. Silver-stained gels from three biological replicates were analyzed by Progenesis 220 Software, version 2006 (Nonlinear Dynamics, U.K.). A mean of 863 spots were represented on each global L3 and dauer stage gel with a mean of 75% spots matched to the reference gel. One-way ANOVA (calculated in Progenesis, Nonlinear Dynamics Ltd., U.K.) revealed that seven proteins were significantly upregulated and five were significantly down-regulated (at p < 0.05, N ) 3) during the dauer stage in comparison to the L3 control (see Figure 1). However, due to low abundance, only 10 of these spots were visible on a Coomassie-stained gel and available for excision and preparation for identification by mass spectrometry. Among those identified were Heat Shock Proteins (HSP)-12.6 (CE03294) and -12.3 (CE03293), which were uniquely expressed in dauers in comparison to the L3 control. Glutathione peroxidase (CE18107) was also significantly higher in dauers (at p ) 0.057). Among those down-regulated were a Small Ribosomal Protein (RPS)-12 (CE26896) and a Nucleoside diphosphate kinase (CE09650) (see Table 1). A mean average of 761 spots were represented on each glp-4 and glp-4; daf-2 mutant global gel with a mean average of 79% spots matched to the reference gel. One-way ANOVA (calculated in Progenesis, Nonlinear Dynamics Ltd., U.K.) revealed that 10 different spots were up-regulated and three were downregulated in glp-4; daf-2 adults in comparison to glp-4 adults (p < 0.05, N ) 3 see Figure 2). Only five of these spots were visible on a Coomassie gel and available for excision and preparation for mass spectrometry. Similar to the dauer stage, the Heat-Shock Protein (HSP)-12.6 (CE03294) was uniquely expressed in glp-4; daf-2 adults in comparison to glp-4 adults. Superoxide Dismutase (SOD)-1 (CE23550) was also significantly up-regulated (at p < 0.01). Unlike the dauer stage, the Small Ribosomal Protein (RPS)-12 (CE26896) and Lipid Binding Protein (LBP)-6 (CE14426) were also significantly up-regulated 2874
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Figure 1. A total of 200 µg of global cytosolic proteome (average gel) from three biological culture replicates of (A) L3 stage and (B) dauer larvae with (C) reference gel from the comparative analysis of these two proteomes, including labeled spots with significantly different regulation. Proteins were resolved using a 17 cm pH 3-10 nonlinear IPG IEF strip and 5-20% T acrylamide/ bisacrylamide 29:1 (3.3% C). Gels were stained with a MALDITOF compatible silver-staining procedure.
(p ) 0.040 and p ) 0.091, respectively) in glp-4; daf-2 adults in comparison to glp-4 adults (see Table 1). GST Subproteome Analyses. A one-way ANOVA (N ) 3) revealed a significantly higher (p ) 0.001) GST activity in the total cytosolic proteins of dauer stage in comparison to L3 stage. A higher mean amount of GST was also extracted from 1 mg of cytosolic protein, but this was not statistically significant at p < 0.05. GST activity remained significantly higher (p ) 0.017) in the cytosol of dauers following GSH affinity purification compared to L3 (see Table 2 and Supplementary Figure 2). A one-way ANOVA (N ) 3) revealed a significantly higher (p ) 0.002) GST activity of the total cytosolic proteins in glp-4; daf-2 prior to GST-extraction (see Table 2 and Supplementary Figure 2) and a significantly higher (p ) 0.005) amount of GST extracted from 1 mg of cytosolic protein compared to glp-4 (see Table 2 and Supplementary Figure 2). GST activity of cytosolic protein of glp-4; daf-2 after GST extraction and of purified GSTs showed no significant difference at p < 0.05 compared to glp-4 (see Table 2). An average of 22 spots was detected on each GSH-affinity gel. Seven spots showed a significant change in normalized spot volume (ANOVA p < 0.05, N ) 3) calculated in Progenesis (Nonlinear Dynamics Ltd., U.K.) between dauer and L3 stage (see Figure 3 and Table 3). Five spots showed a significant (ANOVA p < 0.05, N ) 3) change in normalized spot volume, calculated in Progenesis (Nonlinear Dynamics Ltd., U.K.) between glp-4; daf-2 adults and glp-4 adults (see Figure 3 and Table 3). Three isoforms of CE01613 (GST-5) and two isoforms of CE07055 (GST-7) had a significant higher expression in dauers compared to L3 (p ) 0.05, 0.002, 0.004 and p ) 0.01,
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Table 1. Summary of Significant Changes in Normalized Spot Volumes between Global Proteomes of Dauer and L3 Stage and between 6-Day Old glp-4 and glp-4; daf-2 Adults, with Spot Identifications following Tryptic Digestion, MALDI-TOF MS and PMF Analyses normalized spot vol
spot no. L3/con L3d/Daf-2 12
293
679
13
739 815 1,319
22
1,429 328 302 228 1,319
23
-
14 16 17 18 21
norm spot vol ANOVA P-value
pred E-value sequence no. matched MW Wormbase (MOWSE) coverage (%) peptides (kDa) pred pI entry
description
v0.020 L3d
0.008 (81)
36
6/11
18.1
7.34
CE18107
1,107 1330 614
v0.091 L3d v0.007 daf-2 V0.017 L3d
0.033 (59)
39
5/14
18.7
6.63
CE23550
0.056 (72)
10
8/9
17.1
7.63
CE09650
392 684 557 372 1,028 654 614
V0.050 L3d v0.040 daf-2 v0.071 L3d v0.091 daf-2 Unique to L3d Unique to daf-2 v0.013 L3d
0.002 (89)
63
8/22
15.1
6.65
CE26896
0.038 (75) 0.083 (71) 0.003 (87)
58 55 79
9/11 6/12 7/11
15.6 15.6 12.6
6.82 7.68 5.85
CE24993 CE14426 CE03294
Serine/threonine kinase Lipid binding protein-6 (LBP-6) Heat Shock Protein 12.6 (HSP-12.6)
0.007 (76)
43
7/15
12.6
5.85
CE03294
Unique to L3d
0.013 (63)
32
5/13
12.3
6.80
CE03293
Heat Shock Protein 12.6 (HSP-12.6) Heat Shock Protein 12.3 (HSP-12.3)
173
Glutathione peroxidase Superoxide Dismutase-1 (SOD-1) Nucleoside diphosphate kinase Ribosomal binding protein-12 (RPS-12)
Table 2. Mean Specific GST Activity and GST Amount Extracted from 1 mg of Total Cytosolic Protein from L3 Stage, Dauer Larvae, glp-4 Adults, and glp-4;daf-2 Adultsa specific activity (nmol/min/mg)
strain and stage N2 L3 stage
Mean SD N2 dauer stage Mean SD D.f. F-value P-value glp-4 adults
Mean SD glp-4; daf-2 adults
Figure 2. A total of 200 µg of global cytosolic proteome (average gel) from three biological culture replicates of (A) glp-4 and (B) glp-4; daf-2 6-day adults with (C) reference gel from the comparative analysis of these two proteomes, including labeled spots with significantly different regulation. Proteins were resolved using a 17 pH 3-10 nonlinear IPG IEF strip and 5-20% T acrylamide/bisacrylamide 29:1 (3.3% C). Gels were stained with a MALDI-TOF compatible silver-staining procedure.
0.002. respectively, see Table 3). Three isoforms of Nu-class CE01613 (GST-5) and two isoforms of Nu-class CE07055 (GST7) had a significantly higher expression in compared to L3 (p ) 0.05, 0.002, 0.004 and p ) 0.01, 0.002. respectively, see Table 3). Two (out of three) isoforms of GST-5 (CE01613) (p ) 0.0004 and p ) 0.005. respectively) and one (out of two) isoform of GST-7 (CE07055) also had a significantly higher relative expression in daf-2 adults (p ) 0.05, see Table 3). One isoform of Nu-class CE30562 (GST-36) had a significantly lower relative expression in dauers (p ) 0.003) but not in daf-2 adults. One isoform of Pi-class CE21937 (GST-10) was unique in dauers.
Mean SD d.f. F-value P-value a
cytosolic protein cytosolic protein GST prior to GST post GST purified amount extraction extraction GST (µg) 26.3 34.3 28.8 29.8 4.06 110.3 100.5 84.2 98.3 13.2 8 48.7 0.001 170.3 146.7 121.6 146.2 24.3 371.2 296.0 317.7 328.4 38.7 5 8.21 0.002
0.00 2.56 2.46 1.67 1.45 26.1 57.1 32.7 38.7 16.3 8 11.2 0.017 84.2 62.1 73.7 81.8 11.1 94.8 127.2 66.4 96.1 30.4 5 1.44 0.289
2776 3709 4034 3507 652 3045 4029 3989 3688 557 8 0.40 0.733 4387 5081 4501 4656 2489 7326 4541 3165 5011 2120 5 0.62 0.790
2.2 4.4 5.3 4.0 1.6 5.5 3.2 5.7 4.8 1.4 8 0.44 0.533 9.0 8.5 11.7 9.7 4.2 15.0 16.7 16.6 16.2 1.0 5 14.5 0.005
See Supplementary Fig. 2a-c for graphical representation of results.
One isoform of Pi-class CE00302 (GST-1) and CE06155 (GST4) showed a significantly higher expression in the daf-2 model only (p ) 0.03 and p ) 0.0002, respectively). RNAi and Biochemical Analyses. During RNAi, normalized spot volumes for CE00302 (GST-1) were reduced by 79.5% and 100% (see Table 4, Figure 4 and Supplementary Figure 4). However, no visible phenotypic traits, such as changes in morphology or survival, were observed during the time course of any of the RNAi-mediated knock-down experiments. There were no significant differences in global cytosolic proteomes, GST activity, or GST amount for cytosolic or glutathione affinity purified samples. However, both Pi-class GST-26 (CE22416) and Journal of Proteome Research • Vol. 9, No. 6, 2010 2875
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Jones et al. 25
Figure 3. Ten micrograms of GST average subproteome from three biological culture replicates of wild-type (A) L3 stage and (B) dauer larvae and 6-day (C) glp-4 and (D) glp-4;daf-2 adults, including labeled spots with significantly different regulation. Proteins were resolved by a 17 cm pH 3-10 nonlinear IPG IEF strip and 12.5% T acrylamide/bisacrylamide 29:1 (3.3% C). Gels were stained with a MALDI-TOF compatible silver-staining procedure.
-27 (CE22417) were significantly up-regulated at p < 0.01 (see Table 4, Figure 4 and Supplementary Figure 4). Only one main isoform of recombinant CE00302 was present on a 12.5% 7 cm 2-DE gel in contrast to the several isoforms present in native GST complements (see Supplementary Figure 5a). One main peak representing the loss of a methionine at the N-terminal, along with a peak corresponding to that predicted were observed in ESI spectra for recombinant CE00302 (rGST-1, see Supplementary Figure 5b). The loss of a methionine is a common post-translational modification in both native and recombinant proteins which is unlikely to affect the function of the protein. From the range of model and natural substrates tested, recombinant CE00302 (GST-1) displays relatively high activity toward ethacrynic acid, 4-hydroxy-2-nonenal (4-HNE), trans-2-nonenal, and some activity toward trans,trans-2,4decadienal and trans-4,phenyl-3-buten-2-one (see Table 5). A high affinity for ethacrynic acid, 4-hydroxy-2-nonenal (4-HNE) and trans-2-nonenal has also been implicated by a relatively low IC50 (0.080, 0.015, and 0.008, respectively). A relatively low IC50 of less than 0.1 mM was also found for the polyunsaturated linoleic acid (C18:2) and arachidonic acid (C20:1) with CE00302 (GST-1) and no glutathione conjugation activity was detectable by thiol depletion assays (results not shown).
Discussion Daf-16 regulates a wide variety of genes involved in longevity, stress responses, metabolism, and development.20 It is, therefore, unsurprising that the global and GST subproteome analyses of this study have found a common protein expression pattern in the dauer and daf-2 longevity systems. These differences support a diversion of energy consumption away from anabolic processes and toward enhanced cellular maintenance and detoxification processes in both longevity systems as previously described in the ‘Green Theory of Aging’.22,24,25 This observation has also been reported in other transcriptional and also biochemical analyses of the dauer stage and daf-2 mutants.15-23 This diversion of energy has also been supported in an extensive multilevel cross-species transcriptional analysis including long-lived mutant Drosophila and mice with reduced 2876
Journal of Proteome Research • Vol. 9, No. 6, 2010
IIS. Important components of this enhanced longevity system include the alpha-crystallin family of small heat shock proteins, anti-ROS defense systems, cellular phase II detoxification (in daf-2 only), and possibly also enhanced cell maintenance, reduced growth, and (in dauers only) reduced protein turnover. Increased Regulation of Proteins Involved in Cellular Conservation in Dauer Larvae and daf-2. The increased expression of HSP-12.3 (CE03293) and HSP-12.6 (CE03294) in the dauer stage and daf-2 adults (either uniquely expressed or at p e 0.05, see Table 1) has also been observed on the level of the transcript.18,20-23 These HSP-12s have been reported as the most abundant transcripts in dauer larvae.18 However, an upregulation of these specific heat-shock proteins has not been reported in previous proteomic experiments,28,29 which may reflect differences in 2-DE conditions and subsequent underrepresentation. Also complementary to enhanced cellular conservation is the down-regulation (at p e 0.05) of a Small Ribosomal Protein (RPS)-12 (CE26896) (see Table 1) in the dauer stage (in comparison to the L3) which supports the down-regulation of transcript encoding proteins localized to the mitochondria, nucleus, and endoplasmic reticulum involved in protein synthesis, RNA binding, processing, and modification reported in dauers.21 However, RPS-12 was significantly up-regulated in daf-2 adults, which supports an up-regulation reported for transcript associated with protein synthesis in daf-2 adults.22,23 The significantly up-regulated (at p e 0.05) nucleoside diphosphate kinase (CE09650) in the dauer stage may be involved in several processes necessary for survival, growth, and reproduction, whereas increased expression of serine/threonine kinase family member, FKB-1 Cyclin T-dependent kinase (CE24993) in the dauer stage may be involved in signal transduction directing arrested growth and development. The nonfeeding dauer is dependent on internal energy reserves, which accumulate during dauer formation in the intestine and hypodermis, predominantly in the form of triglycerides.50,51 These reserves can then be metabolized prior to the citric acid cycle and oxidative phosphorylation via fatty acid β-oxidation, the glyoxylate cycle, and gluconeogenesis to acetyl coA. An increase in transcript for all three pathways has previously been reported in dauer larvae,21,22,52 while daf-2 adults appear to have normal oxidative metabolism.22,23 Previous proteomic studies of dauer larvae and 1-day old daf-2 adults have also reported a similar increase in proteins involved in these anaerobic fermentative pathways.28,29 Transcriptomic data have also suggested a down-regulation of the citric acid cycle and mitochondrial respiratory chain in dauers, but not in daf-2 adults.22 The increased expression in the daf-2 model of a lipid binding protein-9, belonging to the fatty-acid binding protein (FABP) family (p ) 0.051, see Table 1) found in this study also supports this. An increased expression of fatty-acid and retinol-binding proteins was also found by previous proteomic studies of 1-day old daf-2 adults.29 The overall enhanced conversion of fat to carbohydrate and conservation of ATP stocks in daf-2 adults implies a state of increased energy availability.22,51 This has been suggested by the disposable soma theory to fuel enhanced somatic maintenance activity.25 Increased Regulation of Anti-ROS in Dauer Larvae and daf-2. An up-regulation of Cu/Zn-SOD (CE23550), which converts O2•- and H2O to H2O2, dependent on a glutathione pathway rather than a copper chaperone, was observed in daf-2 mutants (p < 0.01) and in dauer larvae. However, although clearly visible by eye in dauers, this was not statistically
research articles
Shared Detoxification System in C. elegans
Table 3. Summary of Significant Changes in Normalized Spot Volumes between GST Subproteomes of Dauer (L3d) and L3 Stages and between 6-day old glp-4 and glp-4; daf-2 Adults, with Spot Identifications following Tryptic Digestion, MALDI-TOF MS, and PMF Analyses normalized spot volume spot no.
L3/Con
L3d/Daf-2
norm spot volume ANOVA P-value
1 2 3 4 5
1,012 36.6 73.6 176 40.8 663 145 555 663 1,155 302
183 1,323 71.5 249 534 75.4 1,138 190 147 1,318 1,969 391
Unique to L3d v0.03 daf-2 v2.0 × 10-4 daf-2 v0.054 L3d v0.002 L3d v0.005 daf-2 v0.004 L3d v0.0004 daf-2 V0.003 L3d v0.010 L3d v0.002 L3d v0.05 daf-2
6 7 8 9
E-value (MOWSE)
sequence coverage (%)
no. matched peptides
pred MW (kDa)
pred pI
Wormbase entry
description
1.0 × 10-7 (131) 0.0063 (83) 2.6 × 10-8 (137) 1.3 × 10-6 (120) 2.4 × 10-4 (97)
66 47 53 45 33
17/29 9/26 11/17 10/20 8/16
24.9 23.9 23.9 23.3 23.3
5.50 5.91 5.40 6.84 6.84
CE21937 CE00302 CE06155 CE01613 CE01613
GST-10 (π) GST-1 (π) GST-4 (η) GST-5 (η) GST-5 (η)
6.4 × 10-6 (113)
45
10/20
23.3
6.84
CE01613
GST-5 (η)
0.0067 (83) 1.6 × 10-4 (99) 3.2 × 10-5 (106)
47 38 46
7/21 8/15 11/28
23.9 23.1 23.1
5.88 6.30 6.30
CE30562 CE07055 CE07055
GST-36 (η) GST-7 (η) GST-7 (η)
Table 4. Summary of Significant Changes in Normalized Spot Volumes between GST Subproteomes from RNAi and Control Cultures, with Spot Identifications following Tryptic Digestion, MALDI-TOF MS, and PMF Analyses
spot
Con
RNAi
norm spot volume ANOVA P-value
1 2 3 4
799 82.0 262 326
164 491 596
V0.004 Unique v0.010 v0.003
normalized spot volume
E-value (MOWSE)
sequence coverage (%)
no. matched peptides
pred MW (kDa)
pred pI
Wormbase entry
description
0.0063 (83) 0.028 (77) 2.6 × 10-7 (157) 4.1 × 10-8 (135)
47 42 62 61
9/26 8/22 12/18 11/18
23.2 23.2 23.2 23.3
5.91 5.91 5.53 5.79
CE00302 CE00302 CE22416 CE22417
GST-1 (π) GST-1 (π) GST-26 (π) GST-27 (π)
Table 5. IC50 Values and Specific Activities of Recombinant Pi-Class GST-1 (CE00302) towards Model and Natural Substrates
substrate
conc. (mM)
pH
Λmax (nm)
1-chloro-2,4-dinitobenzene Ethacrynic acid Stearic acid Oleic acid Arachidonic acid Linoleic acid Dauemone 4-Hydroxy-2-nonenal Trans-2-nonenal Trans,trans-2,4-decadienal Trans-4,phenyl-3-buten-2-one Cumene hydroperoxide
1.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5 6.5 7.0
340 270 195 235 205 233 200 224 225 280 290 340
significant (p ) 0.091) and was most likely to be due to problems with spot saturation. Previous enzymatic activity assays have revealed an age-dependent increase in SOD activity in daf-2 and age-1 mutant adult worms as well as in dauer
Figure 4. Ten micrograms of GST average subproteome from three biological culture replicates of (A) Control, (B) CE00302 (GST-1) targeted RNAi, including labeled spots with significantly different regulation. Proteins were resolved by a 17 cm pH 3-10 nonlinear IPG IEF strip and 12.5% T acrylamide/bisacrylamide 29:1 (3.3% C). Gels were stained with a MALDI-TOF compatible silver-staining procedure.
extinction coefficient (mM-1 cm-1)
IC50 (mM)
specific activity (nmol min-1 mg-1)
thiol depletion
9.6 5.0 6.2 6.2 6.2 6.2 Unknown 13.75 -19.2 -29.7 -24.8 6.2
0.080 ND 18% at 0.1 mM 0.025 0.045 ND 0.015 0.008 0.025 >0.100(∼0.6) >0.100 (∼1.2)
315 ( 24 1610 ( 230 ND ND ND ND ND 1778 ( 338 1026 ( 85
