Worm1:1,66–71;January/February/March2012;G2012LandesBioscience Quantitative proteomics by amino acid labeling identifies novel NHR-49 regulated proteins in C. elegans Julius Fredens and Nils J. Færgeman* DepartmentofBiochemistryandMolecularBiology;UniversityofSouthernDenmark;Odense,Denmark Stable isotope labeling by amino acids of the abundance of the sample protein.3 combined with mass spectrometry Such an approach does not require any is a widely used methodology to quanti- sample preparation prior to analysis, thus tatively examine metabolic and signaling this approach is applicable to all kinds of pathways in yeast, fruit flies, plants, cell samples and an indefinite number of cultures and mice. However, only meta- experiments can be compared. However, © 2012 Landes Bioscience. bolic labeling using 15N has been applied this methodology suffers from sensitivity to examine such events in the nematode to variations in sample composition that Caenorhabditis elegans. We have recently can easily affect ionization of the relevant shown that C. elegans can be completely peptide and hence alter its apparent labeled with heavy-labeled lysine by abundance.Consequently,assamplecom- feeding worms on prelabeled lysine plexity grows the utility of such strategy Do not distribute. auxotroph Escherichia coli for just one declines.Instead,severallabelingapproaches generation.Weappliedthismethodology have been developed that benefit from to examine the organismal response to stable isotopes having the same physico- functional loss or RNAi mediated knock chemical properties except from their down of the transcription factor NHR- masses, making distinguishable by mass 49, and found numerous proteins spectrometry. In vitro labeling includes involvedinlipidmetabolismtobedown- incorporation of 18O by enzymatic hydro- regulated, which is consistent with its lysisinheavywaterandchemicallabelingof previously proposed function as a tran- amino acids containing certain reactive scriptional regulator of fatty acid meta- groups.3 The latter features isotope-coded bolism.Thecombineduseofquantitative affinity tags to facilitate purification and proteomicsandselectivegeneknockdown isobaric tags for relative and absolute by RNAi provides a powerful tool with quantitation(iTRAQ).iTRAQareisobaric broad implicationsforC.elegansbiology. tags that, upon fragmentation, release reporter ions of unique masses. The advantage of these in vitro approaches is Quantitative proteomics is increasingly that any sample or tissue can be labeled. being applied to examine and understand However,thesamplesundergoanumberof how cells and organisms regulate their preparation steps before labeling and mix- Keywords: stable isotope labeling, metabolism in order to support growth, ing,whichincreasestheriskofintroducinga metabolic labeling, proteomics, SILAC, proliferation, differentiation, development quantitativebiasinthesamplesandthereby C. elegans, RNAi, lipid metabolism, and survival.1 Several different strategies decreasingthequantitativeaccuracy. nuclear hormone receptors for quantitative proteomics have been Particularly, metabolic labeling with Submitted: 11/07/11 developed including label-free quantifica- stable isotopes has become the prevailing tion, chemical labeling e.g., iTRAQ and strategy to quantitatively compare pro- Revised: 11/29/11 dimethyl labeling or metabolic labeling teomes of cells and organisms. C. elegans Accepted: 11/30/11 using heavy isotopes.2-5 The simplest has during the past decade proven to be a http://dx.doi.org/10.4161/worm.19044 strategy is label-free quantification, where powerful model in identification and signal intensities of a given peptide in a characterization of novel genes and signal- *Correspondenceto:NilsJ.Færgeman; Email:[email protected] number of spectra are used as an estimate ing pathways regulating cell division, 66 Worm Volume1Issue1 COMMENTARY development, aging, apoptosis and meta- applied on the fruitfly Drosophila melano- 330 proteins were differentially expressed bolism.1However,quantitativeproteomics gaster.12 SILAC based quantitative proteo- in response to disruption or knock down has only to a limited extent been applied mics is typically based on labeling with ofNHR-49function.15Amongthedown- to study signaling events governing meta- both arginine and lysine, which provides regulated proteins we identified proteins bolisminC.elegans.In2003Krijgsveldet one label per peptide after trypsin diges- involved in lipid metabolism to be sig- al. showed that C. elegans animals can be tion, and hence improved proteome nificantly overrepresented. In particular, metabolically labeled with 15N by feeding coverage. Although we only labeled C. wefoundthattheD9fattyaciddesaturases them on 15N-labeled E. coli for two elegans with lysine, and subsequently FAT-5 and FAT-6, which previously have generations,6 that, when combined with digested with lysyl endopeptidase (Lys-C) been shown to be controlled by NHR- an14N-labeledwormpopulation,couldbe resulting in longer peptides, we were able 49,18 were significantly downregulated in used to determine the relative protein to identify and quantify a vast number of response to functional loss or knockdown abundance among the two populations by proteins due to intensive peptide frac- of nhr-49. Moreover, we found FAT-1 mass spectrometry. Henceforth, they tionation prior to mass spectrometry and FAT-2, an v3 fatty acid desaturase applied this strategy to identify differenti- analysis. Moreover, arginine to proline and a D12 fatty acid desaturase, respect- ally expressed proteins in glp-4 animals conversion imposes a major challenge in ively, to be downregulated. This obser- compared with wild type animals.6 peptide identification and quantification. vation supports the notion that loss of Analogously, Yates and coworkers exam- Tothisend,Laranceetal.recentlyshowed NHR-49 function impedes on fatty acid ined how loss of functional insulin that up to 20% of the proline become desaturation leading to accumulation of receptors affects the proteome of C. labeled when C. elegans is labeled with saturated fatty acids.18 Besides fat-5, fat-6 elegans,andidentifiednovelkeyregulators arginine,whichcanbepreventedbyRNAi and fat-7, van Gilst et al. also found three © 2012 Landes Bioscience. of insulin regulated metabolic outputs.7 mediatedknockdownofornithinetransa- genes involved in mitochondrial β-oxida- Recently, Simonsen et al. used a similar minase, orn-1, required for the conver- tion of fatty acids (ech-1, cpt-5 and acs-2), approach to identify differentially sion.16 However, labeling with lysine three genes involved in peroxisomal β- expressedproteinsinC.elegansinresponse combined with extensive peptide frac- oxidation (two putative acyl-CoA oxidases to short- and long-term infection by a tionation may be advantageous as orn-1 andech-9),twogenesinvolvedinfattyacid pathogenic adherent-invasive strain of knock down may interfere with the binding/transport (lbp-7 and lbp-8) and Do not distribute. E. coli, that were isolated from patients metabolismofthenematode,andprevents two genes involved in the glyoxylate suffering from the inflammatory bowel that other genes are efficiently knocked pathway (gei-7 and sdha-1), arguing that disease Crohn disease.8 However, com- down by RNAi.17 NHR-49isrequiredforfattyaciddegrada- pared with metabolic labeling with 15N, One of the major advantages by C. tion in C. elegans.18 Accordingly, nhr-49 stable isotope labeling by amino acids in elegans as a model organism is the animals have enlarged lipid stores.18 cell culture9,10 (SILAC) provides a more unprecedentedapplicabilityofRNAinter- Consistently, we find an array of proteins precisemassspectrometry-basedquantitat- ference to systematically study gene func- (Fig.1 and Table1) to be downregulated ive strategy, as it provides a defined tions. We therefore rendered the lysine in nhr-49 animals that either have been number of labels per identified peptide auxotroph E. coli strain RNAi compatible shown,orbasedonsequencesimilaritiesto and therefore enables easier and more by modifying it to express the T7 RNA functionally characterized gene products comprehensive peptide identification polymerase from an IPTG-inducible pro- from other model organisms like and data analysis. Such methodologies moter and by eliminating RNaseIII to Saccharomyces cerevisiae and mice, are have proven to be a highly valuable prevent degradation of dsRNA in E. coli. predicted to be involved in β-oxidation tool for studies in in vivo systems Subsequently, to validate the use of lysine offattyacidsormetabolismofacetyl-CoA. like the yeast Saccharomyces cerevisiae,9,11 labeling of C. elegans in quantitative Since these proteins contain a C-terminal the fruit fly Drosophila melanogaster,12 proteomics studies we aimed to identify peroxisomal targeting signal or that their the plant Arabidopsis thaliana13 and differentially expressed proteins in mammalian counterpart previously has mice.14 Recently, our laboratory and response to functional loss or RNAi been identified to the peroxisomes, our others added C. elegans to the SILAC zoo mediated knock down of the nuclear observations suggest that NHR-49 prim- (see below).15,16 hormone receptor NHR-49. The expres- arily regulates peroxisomal β-oxidation We have shown that C. elegans can be sionofgenesinvolvedinlipidmetabolism rather than mitochondrial β-oxidation, as completely labeled by stable isotope inC.elegansiscoordinatelycontrolledbya suggestedbyvanGilstetal.18Theinability labeled lysine by feeding animals on a number of transcription factors including to degrade long-chain fatty acids would lysine auxotroph E. coli strain for a single the NHR-49, which is a hepatocyte consequently result in increased intracel- generation.15 Moreover, following protein nuclear factor (HNF)-4a ortholog and lular levels of unbound fatty acids and extraction from light and heavy labeled C. has a function analogous to that of fatty acyl-CoA esters. Consistent with this elegans we showed that peptides can be peroxisome proliferator-activated receptor notion, we find the predicted fatty acid identified and quantified with high accu- a, PPARa, in mammals. By quantitative binding protein LBP-3 and acyl-CoA racy (standard deviation of log = 0.22), proteomics we identified 3949 and 4627 bindingproteinACBP-1tobeupregulated 2 which is comparable to similar approaches proteins, respectively, of which 143 and in response to loss of NHR-49 function. www.landesbioscience.com Worm 67 © 2012 Landes Bioscience. Do not distribute. Figure1.Downregulationofnhr-49byRNAiaffectstheabundanceofproteinsinvolvedinfattyacidmetabolism.Stableaminoacidlabelingandquantitative proteomicswasusetoidentifythedifferentiallyexpressedproteinsinL4stagenematodestreatedwithnhr-49RNAicomparedwithemptyvectorcontrols. Amongtheregulatedproteins,enzymesinvolvedinfattyacidmetabolism,especiallyperoxisomalb-oxidation,aresignificantlyoverrepresented.The indicatedproteinisknownorpredicted,basedonsequencehomologytoyeastormouseorthologs,tobeinvolvedintheindicatedbiochemicalpathway. Greenandredindicateproteinsthatbecomelessandmoreabundant,respectively,inresponsetoRNAimediatedknockdownofnhr-49. These binding proteins may bind, seques- level.15ThismaybeduetoalternativeRNA Incontrasttometaboliclabelingwith15N, terandhenceprotectcellsfromdetrimental splicing, differences in the half-lives of stable isotope labeling with amino acids effects of large increases in free fatty acid mRNAs and proteins, as well as rates of providesaninvivostrategytolabelproteins and acyl-CoA levels, respectively. The fact transcriptionandtranslation.21 with different stable isotopic forms of the thatwealsofindglutathioneandxenobiotic amino acids (e.g., lys0, lys4, lys8, Arg4 or metabolism to be upregulated may also Conclusion Arg10), making it possible to monitor reflect an increased cellular stress response differences at the protein level between inresponsetolossofNHR-49function. Stable isotope labeling of C. elegans with multiple conditions or over time in a Gene expression levels are often inter- lysine and/or arginine provides a simple quantitative manner. Thus, stable isotope preted based on mRNA levels, yet, it is and straightforward approach for in vivo labeling with amino acids can be used to increasinglyrecognizedthatthemRNAand incorporation of a label into proteins for monitor how genetic, chemical or envir- protein levels maynot correlate.19,20While mass spectrometry-based quantitative pro- onmentalperturbationsaffecttheproteome the abundance of the majority of the teomics. We anticipate that the recently of C. elegans over time. Combined with proteins,weidentifiedtoberegulatedupon described labeling methodologies15,16 enrichment of posttranslational modified impairedNHR-49function,correlatedwell greatly will facilitate characterization of peptides, e.g., phosphopeptides, this with the mRNA levels previously reported gene functions in the multicellular organ- approach can also delineate how various by van Gilst et al.,18 the level of some ism C. elegans and become a widely used signaling cascades are affected in response proteins did not correlate with the mRNA techniqueinallareasofC.elegansbiology. toaspecificperturbation. 68 Worm Volume1Issue1 Table1.NHR-49affectsabundanceofmetabolicenzymes.QuantitativeproteomicswasusetoidentifythedifferentiallyexpressedproteinsinL4stage nematodestreatedwithnhr-49RNAicomparedtoemptyvectorcontrols.Amongthetotalnumberofidentifiedregulatedproteinsasubsetisshown.The log ratiosindicatelessormoreabundantproteinsafterRNAiagainstNHR-49.SeeFredensetal.fordetails.15 2 Yeast Mouse BiochemicalProcess WormProtein Log Function homolog homolog 2 Aminoacidmetabolism K10H10.2 -1,28 Cysteinesynthase YGR012W CBS F26H9.5 -0,59 Phosphoserineaminotransferase SER1 PSAT1 C31C9.2 -0,35 3-phosphoglyceratedehydrogenase SER33 3-PGDH R102.4 0,35 Threoninealdolase GLY1 THA1 M02D8.4 -0,91 Asparaginesynthetase ASN2 ASNS Y51H4A.7 -0,62 Urocanatehydratase UROC1 CTH-1 -1,37 Cystathioninegamma-lyase CYS3 CTH CTH-2 -0,69 Cystathioninegamma-lyase CYS3 CTH R12C12.1 0,19 Glycinedecarboxylase GCV2 GLDC DDO-2 -0,89 D-aspartateoxidase DDO Carbohydratemetabolism W02H5.8 -0,52 Dihydroxyacetonekinase DAK1 DAK F53B1.4 0,35 UDP-glucose-4-epimerase GAL10 TGDS R11A5.4 -0,34 Phosphoenolpyruvatcarboxykinase PEPCK1 © 2012 Landes Bioscience. FBP-1 -0,34 Fructose1,6-bisphosphatase FBP1 FBP2 Mitochondrialenergymetabolism ANT-1.2 -0,70 ADP/ATPtranslocator AAC1 SLC25A31 C44B7.10 -0,35 Acetyl-CoAhydrolase ACH1 MAI-2 -0,41 ATPaseinhibitor ATPIF1 SUR-5 0,47 Acetoacetyl-CoAsynthetase ACS2 AACS1 Do not distribute. W10C8.5 -0,44 Creatinekinase CKM ZC434.8 -0,41 Creatinekinase CKM FAtransport ACS-22 -0,46 Fattyacidtransportprotein(FATP) FAT1 SLC27A4 ACBP-1 0,27 Acyl-CoA-bindingprotein ACB1 L-ACBP LBP-3 0,45 Fattyacidbindingprotein(FABP) FABP4 FAdesaturation FAT-1 -0,51 v3-desaturase FAT-2 -0,50 D12-desaturase FAT-5 -2,31 D-9desaturase OLE1 SCD1 FAT-6 -1,31 D-9desaturase OLE1 SCD1 MitochondrialFAmetabolism T20B3.1 -1,33 CarnitineO-acyltransferase CAT2 CROT ACS-2 -1,17 Acyl-CoAsynthetase FAA2 ACSF2 MCE-1 -0,44 MethylmalonylCoAepimerase MCEE PYC-1 -0,52 Pyruvatecarboxylase PYC1 PCX D1005.1 0,31 ATP-citratesynthase:succinyl-CoAtosuccinate LSC1 ACLY GEI-7 -0,84 Malatesynthase MLS1 ECH-4 -0,40 Enoyl-CoAhydratase/Acyl-CoAbindingprotein ECI1 ECI2 K09H11.1 -0,93 Acyl-CoAdehydrogenase ACAD12 ECH-7 0,28 EnoylCoAhydratase EHD3 ECHS1 PeroxisomalFAmetabolism T20B3.1 -1,33 CarnitineO-acyltransferase CAT2 CROT ACS-1 -0,69 Acyl-CoAsynthetase FAT2 ACSF2 ACS-7 -0,89 Acyl-CoAsynthetase FAT2 ACSF2 ZK550.6 -1,49 Convertsphytanoyl-CoAto2-hydroxyphytanoyl-CoA PHYH B0334.3 -0,78 2-hydroxyacyl-CoAlyase YEL020C HACL1 B0272.4 -1,18 Enoyl-CoAhydratase/isomerase ECI1 PECI F53C11.3 -0,69 2,4-dienoyl-CoAreductase SPS19 DECR1 www.landesbioscience.com Worm 69 Table1.NHR-49affectsabundanceofmetabolicenzymes.QuantitativeproteomicswasusetoidentifythedifferentiallyexpressedproteinsinL4stage nematodestreatedwithnhr-49RNAicomparedtoemptyvectorcontrols.Amongthetotalnumberofidentifiedregulatedproteinsasubsetisshown.The log ratiosindicatelessormoreabundantproteinsafterRNAiagainstNHR-49.SeeFredensetal.fordetails.15(continued) 2 Yeast Mouse BiochemicalProcess WormProtein Log Function homolog homolog 2 ACOX-1 -1,06 Acyl-CoAoxidase POX1 ACOX1 F08A8.2 -0,86 Acyl-CoAoxidase POX1 ACOX1 F58F9.7 -1,30 Acyl-CoAoxidase POX1 ACOXL Enoyl-CoAhydratase/3-hydroxyacyl-CoA MAOC-1 -1,49 dehydrogenase FOX2 MFE2 Enoyl-CoAhydratase/3-hydroxyacyl-CoA DHS-18 -1,97 dehydrogenase FOX2 HSDL2 Enoyl-CoAhydratase/3-hydroxyacyl-CoA DHS-28 -0,62 dehydrogenase FOX2 HSD17B4 Enoyl-CoAhydratase/3-hydroxyacyl-CoA DAF-22 -1,25 dehydrogenase FOX2 SCP2 F53A2.7 -0,39 Acetyl-CoAacyltransferase ERG10 ACAA2 Lipidsynthesis Y71H10A.2 0,36 FattyacylCoAreductase FAR1 MBOA-3 0,52 Lysophospholipidacyltransferase ALE1 MBOAT1 © 2012 Landes Bioscience. SPTL-1 0,26 Serinepalmitoyltransferase LCB1 SPTLC1 SPTL-2 0,49 Serinepalmitoyltransferase LCB2 SPTLC3 TAG-38 3,45 Sphingosinephosphatelyase DPL1 SGPL1 PMT-1 -0,52 PhosphoethanolamineN-methyltransferase PMT-2 -0,45 PhosphoethanolamineN-methyltransferase ISE1 Do nR06oC1.2 t 0,3d2 isFartnesyrldipihospbhatesuynthetatse e. ERG20 FDPS Cholesteroltransport VIT-6 -2,38 Cholesteroltransport VIT-2 -2,09 Cholesteroltransport VIT-4 -1,36 Cholesteroltransport Othergroups,dehydrogenases F54F3.4 -1,24 short-chaindehydrogenases/reductasesfamily SPS19 DHRS4 DHS-9 -0,72 short-chaindehydrogenases/reductasesfamily YMR226C DHRS1 DHS-15 0,38 short-chaindehydrogenases/reductasesfamily YMR226C DHRS4 DHS-20 -0,96 Mitochondrialshort-chaindehydrogenase YMR226C HSD16B6 DHS-22 0,30 Mitochondrialshort-chaindehydrogenase ENV9 RDH12 CytochromeP450 CYP-25A2 -1,02 CytochromeP450 ERG11 CYP3A11 CYP-29A2 -0,63 CytochromeP450 ERG11 CT033759.1 CYP-33A1 0,48 CytochromeP450 ERG5 CYP17A1 CYP-35C1 0,54 CytochromeP450 ERG11 CYP17A1 CYP-33C7 0,73 CytochromeP450 ERG5 CYP17A1 CYP-13A5 0,99 CytochromeP450 DIT2 CYP46A1 CYP-13A4 1,72 CytochromeP450 DIT2 CYP46A1 Proteases F21F8.4 -0,53 Vacuolaraspartylprotease(proteinaseA) PEP4 BACE2 Y16B4A.2 0,24 Putativeserinetypecarboxypeptidase YBR139W CPVL Y40D12A.2 0,32 Putativeserinetypecarboxypeptidase YBR139W CTSA ASP-2 0,33 Vacuolaraspartylprotease(proteinaseA) PEP4 BACE2 ASP-1 0,34 Vacuolaraspartylprotease(proteinaseA) PEP4 BACE2 K12H4.7 0,35 Serineprotease PRCP ASP-3 0,40 Vacuolaraspartylprotease(proteinaseA) PEP4 CTSD ASP-6 0,41 Vacuolaraspartylprotease(proteinaseA) PEP4 CTSD F13D12.6 0,43 Putativeserinetypecarboxypeptidase YBR139W CTSA 70 Worm Volume1Issue1 Table1.NHR-49affectsabundanceofmetabolicenzymes.QuantitativeproteomicswasusetoidentifythedifferentiallyexpressedproteinsinL4stage nematodestreatedwithnhr-49RNAicomparedtoemptyvectorcontrols.Amongthetotalnumberofidentifiedregulatedproteinsasubsetisshown.The log ratiosindicatelessormoreabundantproteinsafterRNAiagainstNHR-49.SeeFredensetal.fordetails.15(continued) 2 Yeast Mouse BiochemicalProcess WormProtein Log Function homolog homolog 2 C15C8.3 1,18 Vacuolaraspartylprotease(proteinaseA) PEP4 BACE2 K10B2.2 1,27 Putativeserinetypecarboxypeptidase YBR139W CTSA Lonprotease -0,60 Serineprotease PIM1 LONP2 ATP-bindingcassette(ABC)transporter HAF-4 0,23 ATP-bindingcassettetransporter MDL1 TAP2 ABT-4 0,36 ATP-bindingcassettetransporter YOL075C EP300 MRP-2 0,38 ATP-bindingcassettetransporter YCF1 ABCC1 PGP-6 0,56 ATP-bindingcassettetransporter STE6 ABCB11 MRP-5 0,59 ATP-bindingcassettetransporter YOR1 ABCC12 PGP-9 0,82 ATP-bindingcassettetransporter STE6 ABCB11 GlutathioneS-transferase GST-6 0,73 GlutathioneS-transferase HPGDS GST-7 0,38 GlutathioneS-transferase HPGDS GST-38 1,01 Glutathione-S-transferase HPGDS © 2012 Landes Bioscience. 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