Article pubs.acs.org/jpr
Intestinal Amino Acid Availability via PEPT‑1 Affects TORC1/2 Signaling and the Unfolded Protein Response Kerstin E. Geillinger,† Katja Kuhlmann,‡ Martin Eisenacher,‡ Pieter Giesbertz,† Helmut E. Meyer,‡,§ Hannelore Daniel,† and Britta Spanier*,† †
ZIEL Research Center of Nutrition and Food Sciences, Molecular Nutrition and Biochemistry Unit, Technische Universität München, Gregor-Mendel-Str. 2, 85350 Freising, Germany ‡ Medizinisches Proteom-Center, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Germany § Leibniz-Institut für Analytische Wissenschaften − ISAS − e.V., 44139 Dortmund, Germany S Supporting Information *
ABSTRACT: The intestinal peptide transporter PEPT-1 plays an important role in development, growth, reproduction, and stress tolerance in Caenorhabditis elegans, as revealed by the severe phenotype of the pept-1-deficient strain. The reduced number of offspring and increased stress resistance were shown to result from changes in the insulin/IGF-signaling cascade. To further elucidate the regulatory network behind the phenotypic alterations in PEPT1-deficient animals, a quantitative proteome analysis combined with transcriptome profiling was applied. Various target genes of XBP-1, the major mediator of the unfolded protein response, were found to be downregulated at the mRNA and protein levels, accompanied by a reduction of spliced xbp-1 mRNA. Proteome analysis also revealed a markedly reduced content of numerous ribosomal proteins. This was associated with a reduction in the protein synthesis rate in pept-1 C. elegans, a process that is strictly regulated by the TOR (target of rapamycine) complex, the cellular sensor for free amino acids. These data argue for a central role of PEPT-1 in cellular amino acid homeostasis. In PEPT-1 deficiency, amino acid levels dropped systematically, leading to alterations in protein synthesis and in the IRE-1/XBP-1 pathway. KEYWORDS: Caenorhabditis elegans, unfolded protein response, XBP-1, PEPT-1, TOR
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development,4 and increased fat accumulation.7 The phenotype of daf-2 mutants largely depends on the increased activity of the forkhead transcription factor DAF-16/FOXO, which regulates the expression of genes involved in phase II detoxification (e.g., sod-3, ctl-1), metabolism (e.g., fat-7, gei-7), and aging (e.g., ins-7). A deletion of daf-16 in the daf-2 background reverses the effects on life span, development, brood size, and thermotolerance, even though levels are not returned to those of the wild type, and daf-16;daf-2;pept-1 triple mutants are phenotypically like pept-1 animals.4 Thus, pept-1 deletion affects the IIS pathway and other signaling pathways that appear to act in parallel, regulating stress tolerance, brood size, and development. Therefore, we aimed to explore the regulatory mechanisms that are affected by loss of pept-1 and performed quantitative proteomics in combination with a transcriptomic approach. Here, we demonstrate that loss of pept-1 affects the TOR signaling pathway and the XBP-1/IRE-1 axis as part of the unfolded protein response (UPR). Low amino acid concentrations in pept-1 C. elegans affect TOR-mediated signaling pathways comprising ribosomal protein translation and de novo protein synthesis. Thus, our analysis shows a complex interplay between TOR, UPR signaling processes, and the provision of amino acids.
INTRODUCTION Dietary proteins deliver essential and nonessential amino acids for growth, development, and body maintenance. Proteins are hydrolyzed in the intestinal lumen by various proteases and membrane-bound peptidases with the release of small peptides and free amino acids. Whereas free amino acids are taken up via different transport systems,1 there is only one transporter that mediates the uptake of di- and tripeptides into enterocytes. PEPT-1 acts as a low-affinity/high-capacity proton-coupled symporter, utilizing the transmembrane electrochemical proton gradient as its driving force. Although the mechanism of transport is conserved across species,2 PEPT-1 loss-of-function induces species-specific phenotypic outcomes. In mice lacking the intestinal peptide transporter, no apparent phenotypic changes were observed,3 whereas the disruption of the pept-1 gene in Caenorhabditis elegans leads to decreased body length and brood size, retarded embryonic development, and excessive fat accumulation.4,5 However, the lack of pept-1 also increases the tolerance toward thermal and oxidative stress. Additionally, the knockout of pept-1 in animals harboring a defect in the daf-2 gene, encoding the insulin-like receptor, shows additive effects on the phenotypic outcome,4 with a pronounced extension of life span and a superior stress tolerance. Interruption of the insulin/IGF-like signaling (IIS) pathway by loss-of-function of daf-2 alone already leads to an extended life span, increased thermotolerance, a reduction of brood size,6 retarded © 2014 American Chemical Society
Received: March 14, 2014 Published: July 7, 2014 3685
dx.doi.org/10.1021/pr5002669 | J. Proteome Res. 2014, 13, 3685−3692
Journal of Proteome Research
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Article
Measurement of pept-1 Promoter Activity and Detection of GFP Fusion Proteins
MATERIAL AND METHODS
C. elegans Strains and Nematode Culture
Synchronized worms expressing GFP under the control of the hsp-16.2 promoter4 as well as HSP-4::GFP fusion protein or HSP-60::GFP fusion protein-expressing C. elegans were cultured under the described conditions. After microscopic determination of the body length, 50 worms/well were placed into a black 384-well plate. Each sample was performed in technical triplicate. GFP expression was measured using a fluorometer (Thermo Fisher Scientific, Ulm, Germany) with excitation at 395 nm and emission at 513 nm. The obtained fluorescence signal was normalized to the mean body length. For determination of GFP-synthesis rate, hsp-16.2p::GFP was induced by incubation at 34 °C for 1 h, fluorescence was detected every 60 min for a total of 4 h, and signals from noninduced worms were subtracted.
Wild-type N2 (var. Bristol), pept-1(lg601) (BR2742), hsp16.2p::GFP (CL2070), HSP-4::GFP (SJ4005), and HSP60::GFP (SJ4058) C. elegans were used in this study. Maintenance of worms was carried out on NGM agar plates as previously described8 unless otherwise stated. Synchronization of worm culture was conducted using hypochloride treatment. RNAi was performed on NGM agar plates containing IPTG (1 mM) and carbenicillin (25 μg/mL) following the protocol of Kamath et al.9 All RNAi clones in Escherichia coli HT115, including vector control (VC, containing empty vector pPD129.36 (L4440)) were obtained from the Ahringer C. elegans RNAi library (GeneService, Cambridge, UK). Protein Extraction
Transcriptomics Analysis
After nematodes were harvested and extensively washed with M9 buffer (42 mM Na2HPO4, 22 mM KH2PO4, 86 mM NaCl, 1 mM MgSO4), the resulting pellet was disrupted using glass beats in combination with mechanical force (3 × 30 s, level 5, FastPrep, MP Biomedicals, Germany). Lysis buffer (100 mM Tris/HCl pH7.4, 200 nM NaCl, 2 μM EDTA, 8% Glycerol, 1.25 mM DTT) containing 1 mM PMSF was used, and all steps were performed at 4 °C. The whole worm lysate was centrifuged in order to remove cell debris (1 min, 10 000g at 4 °C), and the supernatant was used for further protein analysis.
Synchronized L4-larvae cultures of N2 wild-type and pept-1 C. elegans (n = 5) were subjected to transcriptome analysis using C. elegans whole-genome DNA microarrays (Affymetrix Inc., Santa Clara, CA, USA). Parts of the data sets have already been published.12,5,13 The nematode culture, RNA preparation, microarray analysis, data analysis, and statistics were performed according to described protocols.12 Gene ontology clustering was undertaken using DAVID software.14 Quantitative Proteome Analysis
Western Blot
To identify proteins that are differentially expressed during the development of WT and pept-1 C. elegans, synchronized cultures of N2 wild-type and pept-1 C. elegans were allowed to grow for 20, 40, or 60 h, respectively, before whole protein extract was subjected to quantitative proteome analysis. Protein extraction, 15N labeling, LC−MS/MS, and statistical and bioinformatical analysis are described in Geillinger et al.15 Briefly, a standard for indirect quantification was generated by including fully 15N-labeled WT and pept-1 at every developmental stage to ensure the integrity of the standard. After each sample was combined with this standard, they were processed by a short acrylamide gel run, trypsin digestion, and peptide extraction, and peptides were analyzed by nanoHPLC−ESI−MS/MS (UltiMateTM 3000 RSLCnano system coupled to LTQ Orbitrap Velos). Data analysis was accomplished using Mascot Distiller version 2.4.0.0. Mascot (version 2.3) search engine with the UniProt/Swiss-Prot database (UniProt/Swiss-Prot release 2011_06) and restriction for taxonomy (C. elegans, 3332 sequences) was used for identification. Search parameters were as follows: tryptic specificity, one missed cleavage site, methionine oxidation as variable modification, 4 ppm as precursor mass tolerance, and 0.5 Da for fragment mass tolerance. A decoy version of the database was used that was complemented with a duplicate of itself in which the amino acid sequence of each protein entry was randomly shuffled in order to enable the calculation of a false discovery rate. Resulting lists were truncated at a false discovery rate of 1%. Quantification was achieved using Mascot Distiller. Impurity of labeling was set to 92%. Protein ratios were calculated on the basis of a minimum of two peptides meeting the selection criteria of a correlation threshold above 0.9 and standard deviation below 0.2, whereby at least one peptide had to be unique to the protein. The list of identified proteins was merged for the two biological replicates and three time points to yield a combined list of identified proteins. Proteins that were not detected in all samples were
Detection of proteins of interest was achieved with minor changes as described earlier.10 A solution of 1% BSA in PBST was used as a blocking solution. The antibody against PEPT-1 was used as described earlier.10 The antibody against heat shock protein HSP-60 (P50140) was purchased at Enzo Life Science and used at a dilution of 1:1000. The antibody against ATP synthase subunit alpha H28016.1 (Q9XXK1) was purchased from Abcam and used at a dilution of 1:1000. All primary antibodies were incubated overnight at 4 °C followed by incubation with IR-dye-coupled secondary antibodies from Li-COR Biosciences for 2 h. Bound antibodies were analyzed and quantified using an Odyssey scanner (Li-COR Biosciences, USA). RNA Isolation
Ice-cold RNA lysis reagent (750 μL; 5Prime, Hilden, Germany) and glass beads were added to a 50 μL worm pellet. Disruption was achieved as described in the Protein Extraction section. Glass beads were washed with 500 μL of RNA lysis reagent, and the resulting mixture was incubated with chloroform (20% of sample volume). After centrifugation for 15 min at 20 000g at 4 °C, the aqueous phase was mixed with the same volume of cold ethanol and further processed using RNAeasy (Qiagen, Hilden, Germany) following the manufacturer’s instructions. RT-PCR
Isolated RNA was diluted to 10 ng/μL; primers were used at a concentration of 20 μM. Mastermix was prepared as recommended by the manufacturer (Quantitect Probe RT-PCR, Qiagen, Hilden, Germany). Measurement was conducted in a 96-well plate format using a Realplex system (Eppendorf, Hamburg, Germany). Primer sequences for pept-1, rsks-2, daf-15, and the large subunit of RNA polymerase II ama-1 (for normalization) are reported in the Supporting Information. Primer sequences for total xbp-1 as well as the spliced variant were previously published.11 3686
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excluded from the list. Statistical analysis was accomplished using R software package applying a two-way ANOVA test. Quantification of Amino Acids in C. elegans
Synchronized L4-larvae cultures of N2 wild-type and pept-1 C. elegans were harvested, washed in M9 buffer, and frozen in liquid N2 as 200 μL samples. The nematodes were disrupted in a liquid N2-cooled mortar and pestle, and the cytosolic fraction was collected by centrifugation. The total content of free amino acids in the samples was determined by amino acid analysis using the amino acid analyzer Biochrom 30 based on the ninhydrin detection/ion exchange chromatography method (Biochrom, Cambridge, UK).
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RESULTS
Proteome Analysis of pept-1 C. elegans Reveals Major Alterations in Expression of Ribosomal Proteins
The loss of pept-1 leads to major changes in postembryonal development as well as during adulthood.4 Therefore, we profiled the proteome of pept-1 and wild-type C. elegans at different time points during larval development as well as at the young adult stage to gain insight into the underlying mechanisms. A quantitative shotgun proteomics approach using 15N-based metabolic labeling was conducted at 20, 40, and 60 h during development starting with L1-larvae of both strains. In total, 166 proteins were identified and quantified in all samples (Table S1). In contrast to large changes in the proteome during the ontogenesis of wild-type animals, as described in our previous publication,15 the proteome of pept-1 remained rather static. A direct comparison of the developmental changes using a two-way ANOVA with “genotype” and “time” as variables revealed 35 proteins with a distinct strain−time interaction, 49 proteins were defined by strain, and 39 showed time-dependent changes (Table S2). Thereof, 16 proteins overlap between all groups. To avoid redundancy, proteins overlapping between groups were assigned to only one group. Hence, 35 proteins were assigned to the group of strain−time interaction, leaving 20 proteins in the strain-related group. The remaining 11 proteins, showing only time-dependent changes (Figure 1A), were not further considered. Protein quantification for proteins of the strain−time interaction group was validated using western blot analysis with appropriate antibodies (Figure 1B). While ATP synthase subunit alpha H28016.1 (Q9XXK1) did not show any significant regulation in the proteomics data set or the western blot analysis, heat shock protein HSP-60 (P50140) was significantly downregulated in pept-1, as was also observed in western blot analysis. Furthermore, the low expression of two proteins involved in C1 metabolism, S-adenosyl methionine synthetase SAMS-1 (O17680) and S-adenosyl homocysteine hydrolase AHCY-1 (P27604), confirmed earlier data from our group.13 Agglomerative hierarchical cluster analysis of proteins of the strain−time category revealed three main groups with an agglomerative coefficient of 0.88 (Figure S1) and thus a robust clustering structure. Although clusters 1 and 3 contained only two and three proteins, respectively, 30 proteins were assigned to cluster 2. Therefore, gene ontology and functional annotation clustering was performed only for proteins in cluster 2 using the annotation tool DAVID (Database for Annotation, Visualization and Integrated Discovery).14 This revealed a strong over-representation of proteins involved in “translation”, “larval development”, and “postembryonic development” (Figure 1C). Functional annotation clustering revealed an approximate 16-fold
Figure 1. Quantitative analysis of changes in the pept-1 proteome: (A) Venn diagram of significantly regulated proteins selected by two-way ANOVA. Proteins were classified into three groups showing straindependent regulation, time-dependent regulation, or an interaction of both parameters. (B) Synchronized wild-type and pept-1 C. elegans were harvested at indicated time points, starting with starved L1-larvae at time point 0 h. After total protein extraction, western blot analysis was conducted, and resulting bands for Q9XXK1/H28016.1 and P50140/HSP-60 were quantified using Odyssey V3.0 software and compared to values retrieved by the quantitative proteomics approach. PEPT-1 was detected as a genotypic control, and actin was used as a loading control. Ratios of protein abundances between pept-1 and WT were made for each experiment, and the mean and SD were subsequently calculated. (C) Top five terms of gene ontology clustering of proteins showing strain−time interaction. X axis shows number of proteins associated with the term; terms are sorted by p value (depicted at the top of the bars).
enrichment of “ribosomal components” followed by “larval development” (5.4-fold) and “mitochondrial” (2.2-fold). Using the KEGG pathway mapper,16 15 out of the 30 proteins were assigned as ribosomal components (Figure S1), all of them showing a reduced protein abundance in pept-1 when compared to that in wild-type. The diminished protein synthesis capacity might explain the observed decline in protein expression between 20 and 40 h of postembryonal development in pept-1 but not in wild-type animals. Proteome and Transcriptome of pept-1 Indicates Decreased Activity of the XBP-1/IRE-1 Axis
To enlighten the strain-specific changes in protein expression, the 20 proteins in the strain-related group were analyzed. 75% of these 20 proteins (Figure 2A; Table S2) revealed decreased 3687
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Figure 2. Differentially expressed proteins showing genotype-specific regulation are associated with protein folding: (A) 20 proteins showing genotype-dependent regulation after two-way ANOVA are depicted in rows; time points 20, 40, and 60 h after hatching in columns. (B) Top five terms of gene ontology clustering of proteins showing strain-dependent regulation. X axis shows number of proteins associated with the term; terms are sorted by p value (depicted at the top of the bars). (C) Gene ontology clustering of genes downregulated in pept-1 compared to that in wild-type with cluster terms (p value depicted at the top of the bars).
levels in pept-1 as compared to levels in wild-type animals, whereas only 5 proteins displayed increased levels. Gene ontology search of downregulated proteins in pept-1 classified enrichment in the categories of “nematode larval development”, “larval development”, and “postembryonic development”. Each category had 11 proteins assigned with p values of ∼3 × 10−4. With a comparable p value, 4 proteins were grouped in the category of “protein folding” (Figure 2B). Further categories retrieved were “growth”, “regulation of growth rate”, and “translation” (Table S3), reflecting parameters impaired in pept-1. To determine if these changes at the protein level are caused by modifications in mRNA expression, we analyzed transcriptome data of wild-type and pept-1 nematodes at the L4stage (Table S4)12,5 using gene ontology clustering. Genes that showed either changes in transcript levels >2 or
