Enrichment of Metabolite-Binding Proteins by Affinity Elution in

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Enrichment of metabolite-binding proteins by affinity elution in tandem hydrophobic interaction chromatography (AETHIC) reveals RKIP regulating ERK signaling in an ATP-dependent manner Wei-Chieh Huang, Der-Yen Lee, and Geen-Dong Chang J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00328 • Publication Date (Web): 16 Sep 2016 Downloaded from http://pubs.acs.org on September 19, 2016

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Journal of Proteome Research

Enrichment of metabolite-binding proteins by affinity elution in tandem hydrophobic interaction chromatography (AETHIC) reveals RKIP regulating ERK signaling in an ATP-dependent manner

Wei-Chieh Huang1, Der-Yen Lee2 and Geen-Dong Chang1* 1

Graduate Institute of Biochemical Sciences, National Taiwan University, No.1,

Section 4, Roosevelt Road, Taipei 106, Taiwan, 2Graduate Institute of Integrated Medicine, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan

Correspondence: Geen-Dong Chang, Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 106, Taiwan Tel: 886-2-3366-4071; Fax: 886-2-2363-5038; E-mail: [email protected].

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ACS Paragon Plus Environment


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Abstract To elucidate the molecular mechanisms underlying the action of bioactive compounds such as metabolites, identification of their binding targets is essential. However, available techniques for enriching metabolite-binding proteins are practically restrained by special equipment requirements and laborious efforts. Here, we have developed a novel method, affinity elution in tandem hydrophobic interaction chromatography (AETHIC), which enables enrichment of metabolite-binding proteins from a crude tissue extract. AETHIC constitutes two major steps, protein fractionation and affinity elution. The basic strategy of AETHIC uses a series of HIC matrices encompassing aliphatic chains of different length and thus provides a wide range of hydrophobicity for interactions with most proteins. Thereafter, target proteins are eluted selectively by a given ligand. As our first proof-of-principle, we demonstrated that AETHIC was able to enrich ATP-binding proteins from porcine brain extract. In addition, we have demonstrated that raf kinase inhibitory protein (RKIP) is an ATP-binding protein and ATP attenuates the interaction between RKIP and Raf-1. In parallel, short-term ATP depletion in cultured HEK293 cells augments interaction between RKIP and Raf-1 resulting in decreased activation of the downstream ERK signaling. Therefore, the ATP-binding function renders RKIP’s inhibition on Raf-1 modulated by cellular ATP concentrations. These data shed light on how energy levels affect the propagation of cellular signaling. Taken together, the enclosed results advocate the potential of AETHIC in the study of metabolite-protein interactions.

Keywords: hydrophobic interaction, metabolite, ATP, RKIP, Raf-1; glucose depletion

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Introduction The complicated molecular mechanisms of biological processes are not only mediated by nuclei acid-protein and protein-protein interactions, but also by protein-metabolite interactions 1. Recent studies have revealed a growing number of protein-metabolite interactions that regulate cellular activities, expanding our perception of molecular mechanisms underlying diverse cellular functions

2-5

. However, available tools and

techniques for the study of protein-metabolite or protein-small molecule interactions are relatively ineffective as compared to the existed methods for nuclei acid-protein and protein-protein interactions such as DNA microarray and yeast two hybrid, respectively 6. Traditionally, small molecule-binding proteins can be isolated from affinity chromatography where small molecules are immobilized to a solid matrix 7. However, the need of derivatization limits its usage because some small molecules do not possess the desired functional groups needed for the coupling reactions. In addition, introduction of a linker arm also causes non-specific interaction and steric hindrance for protein binding. Therefore whether the derivatized compounds perform biologically relevant functions remains questionable

8

. To circumscribe these

obstacles, we sought to develop a novel approach for enrichment of potential small molecule-binding proteins with lower levels of sophisticated designs and instrumental requirements.

Proteins are absorbed in hydrophobic interaction chromatography (HIC) through reversible interaction between the hydrophobic ligands of a chromatographic matrix and the hydrophobic surface patch on proteins, generally with the help of high concentrations of antichaotropic salt. Subsequently the absorbed proteins can be eluted by decreasing salt concentration

9-11

. In addition, calcium-dependent HIC has

been designed and used in the purification of calcium-binding proteins such as 3



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calmodulin 12, 13 due to the exposure of hydrophobic patches upon calcium binding 14. Calcium-binding proteins can then be eluted by calcium chelating agents, an example of affinity elution. In theory, ligand-binding induced conformational changes of proteins could result in varied surface hydrophobicity

9, 10

. Furthermore, small

compounds could possibly compete with HIC ligands for the substrate binding sites of target proteins. The rationale behind affinity elution in HIC is analogous to the practice of using substrates, co-factors or inhibitors in dye-ligand affinity chromatography resulting in elution of particular enzymes 15, 16.

In this communication, we exploited affinity elution in HIC as a strategy for enrichment of a group of proteins that exhibit affinities for a given ligand. Instead of using single hydrophobic matrix, we assembled an array of HIC comprising five tandem columns packed with butyl (C4), hexyl (C6), octyl (C8), decyl (C10) and dodecyl (C12)-Sepharose, respectively. The protein samples are applied to the series of columns connected with increasing hydrophobicity. This design intends to fractionate proteins into five major fractions as the most hydrophobic proteins are absorbed by C4 column and the least hydrophobic proteins are absorbed by C12 column, prior to affinity elution with small molecules. Importantly, this step gradually reduces the complexity of protein samples. Most proteins are absorbed by columns by a mild hydrophobic interaction so that they are not tightly bound, resulting in better elution. Therefore, these modifications improve the recovery of ligand-binding proteins from the HIC columns to support a better coverage of surveyed proteome. Herein we name the method affinity elution in tandem hydrophobic interaction chromatography (AETHIC). Using this approach, we investigated the ATP-binding proteins in porcine brain extract and identified raf kinase inhibitor protein (RKIP) as an ATP-binding protein. Our subsequent studies uncover distinct roles of RKIP in ERK signaling 4


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responding to cellular ATP concentrations.

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Materials and Methods Cell culture HEK293 cells were cultured in DMEM high glucose (HyClone Laboratories, Inc., South Logan, UT) supplemented with 10% fetal bovine serum (FBS, HyClone Laboratories, Inc.), 1% penicillin and streptomycin within 5% CO2 atmosphere at 37 ℃. Transfection was conducted with Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. For the ATP depletion experiments, culture medium was replaced with DMEM without glucose (Thermo Fisher Scientific) plus 10 mM 2-deoxylglucose (Sigma-Aldrich Corporation, St. Louis, MO) and incubated for 40 min. Sodium azide was added to the medium at a final concentration of 10 mM for 5 min before cell harvest. For the study of ERK signaling, cells were serum-starved in DMEM containing 0.2% FBS for 16 h prior to treatments of ATP depletion and then stimulated with the indicated concentrations of epidermal growth factor (EGF, Cell Signaling Technology, Boston, MA) for an additional 5 min. In the experiments of glucose-deprivation, cells were washed once with phosphate-buffered saline (PBS) then the medium was replaced with DMEM no glucose containing 10% dialyzed FBS (Thermo Fisher Scientific).

Affinity elution in tandem hydrophobic interaction chromatography 12 g porcine or mouse brain tissues were homogenized in 100 ml Tris-buffered saline (TBS: 20 mM Tris-HCl, pH 7.6, 150 mM NaCl) supplemented with 4 mM MgCl2 by a polytron homogenizer (Kinematica AG, Luzern, Switzerland). The tissue extract was centrifuged at 20,000Xg, at 4 ℃ for 30 min. The supernatant was filtrated through filter paper to remove small aggregates. The clarified lysate was applied to the series of columns (5 ml packed gel) with the assist of a peristaltic pump (Gilson, 6


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Inc., Middleton, WI). After sample entirely entered the gel bed of the first column, an additional 4X column volume (CV) of TBS was applied to prompt proteins to flow through the five columns sequentially. The connecting loops between the tandem columns were subsequently disassembled. Each column was washed with 10X CV TBS followed by elution with 5X CV elution buffer (TBS containing 4 mM MgCl2 and 3 mM ATP). For recovery of total proteins, each column was eluted with 0.5 M arginine in 10% ethanol as previously described

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. The fractions containing eluted

proteins were combined and concentrated by centrifugal filters with 10,000 Da cutoff (Merck Millipore Corporation, Darmstadt, Germany). The concentrated samples were separated on SDS-PAGE followed by Coomassie brilliant blue staining for visualization. In-gel digestion and mass spectrometry analysis The protein samples were loaded to 8% SDS-PAGE and separated for a distance of 1 cm. Each lane of gel was cut into 2 mm slices. These gel slices were soaked in 100 mM dithiothreitol (DTT) /25 mM ammonium bicarbonate followed by alkylation with 65 mM iodoacetamide. The gel slices were washed with 50% acetonitrile in 25 mM ammonium bicarbonate. The dehydrated gels were moisturized with trypsin solution (10 ng/µl, Promega, Madison, WI)) and incubated at 37 ℃ for 16 h. The tryptic peptides were extracted with 50% and 100% acetonitrile containing 0.1% trifluoroacetic acid sequentially. The peptide mixtures were desalted by C18 Zip-tip and subjected to proteomics analysis using an LTQ-Orbitrap Velos hybrid mass spectrometer (Thermo Fisher Scientific). Peptide mixtures were loaded onto a 75-µm × 250-mm nanoACQUITY UPLC BEH130 column packed with C18 resin (Waters Corp., Milford, CT) and were separated at a flow rate of 300 nl/min using a linear gradient of 5 to 40% solvent B (95% acetonitrile with 0.1% formic acid) in 30 min, 7



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followed by a sharp increase to 85% B in 1 min and held at 85% B for another 10 min. Solvent A was 0.1% formic acid in water. The effluent from the HPLC column was directly electrosprayed into the mass spectrometer. The LTQ Orbitrap Velos instrument was operated in data-dependent mode to automatically switch between full scan MS and MS/MS acquisition. Instrument control was through Tune 2.6.0 and Xcalibur 2.1. For the CID-MS/MS top20 method, full scan MS spectra (m/z 350– 1600) were acquired in the Orbitrap analyzer after accumulation to a target value of 106 ions in the linear ion trap. Resolution in the Orbitrap system was set to R = 60,000 (all Orbitrap system resolution values are given at m/z 400). The 20 most intense peptide ions with charge states ≥2 were sequentially isolated to a target value of 5,000 and fragmented in the high-pressure linear ion trap by low-energy CID with normalized collision energy of 35%. The resulting fragment ions were scanned out in the low-pressure ion trap at the normal scan rate and recorded with the secondary electron multipliers. Ion selection threshold was 500 counts for MS/MS, and the maximum allowed ion accumulation times were 500 ms for full scans and 100 ms for CID-MS/MS measurements in the LTQ. An activation q = 0.25 and activation time of 10 ms were used. The peptides were identified from the MS/MS data searched against the Swiss-Prot database using the Mascot search engine 2.3.02 (Matrix Science Inc., Boston, MA). Search criteria used were as follows: trypsin digestion; variable modifications set as carbamidomethyl (Cys) and oxidation (Met); up to two missed cleavages allowed; and mass accuracy of 10 ppm for the parent ion and 0.60 Da for the fragment ions.

Gene Ontology (GO) analysis The protein identification from ATP eluate of AETHIC was converted to UniProt

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accessions and uploaded to DAVID (Database for Annotation, Visualization and Integrated Discovery) version 6.7 for functional annotation clustering. The analysis was performed using the default setting provided by the DAVID system. The overrepresented annotation terms were defined by SP_PIR_KEYWORDS database. The enriched pathways were determined by KEGG database.

Pull-down assays For the RKIP pull-down assay, 2 µg of His-RKIP was immobilized to 10 µl of TALON Co2+-affinity resin (Clontech Laboratories, Incorporation) and then incubated with Raf-1 protein in PBS containing 3% bovine serum albumin (BSA) supplemented with the indicated concentrations of ATP at 4 ℃for 45 min. The pull-down proteins were detected by SDS-PAGE and immunoblotting. Experimental procedures for purification of recombinant RKIP and Raf-1 protein are given in supplemental information. For ATP-agarose pull-down assay, equal amount of RKIP or BSA were incubated with ATP-agarose (hydroxyl-linked) (Sigma-Aldrich Corporation) at 4 ℃ for 30 min. The bound proteins were eluted with 3 mM ATP or 0.5 M arginine in 10% ethanol.

ATP probe labeling assay Desthiobiotin-ATP probe (Thermo Fisher Scientific) was reconstituted in ultrapure water on ice. The recombinant RKIP was incubated with 0-100 μM of probes in the reaction buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT and 0.1% BSA) at room temperature for 10 min. For competition assays, nucleotides were pre-incubated with RKIP at room temperature for 10 min before the probe labeling. The reactions were stopped by adding an equal volume of 2X SDS sample buffer and 9



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separation on SDS-PAGE. Proteins were transferred to PVDF membrane. Biotinylated RKIP was detected by peroxidase-conjugated streptavidin overlay assay. Total RKIP was detected by Coomassie blue G-250 staining or by an anti-RKIP immunoblotting.

Immunoblotting and immunoprecipitation After the indicated treatments, cells were rinsed with ice-cold PBS twice and then lysed with an IP-lysis buffer consisting 20 mM HEPES, 150 mM NaCl, 5% glycerol, 0.5% Triton X-100, 10 mM beta-glycerolphosphate, 1 mM sodium orthovanadate and protease inhibitor cocktails (Roche, Basel, Switzerland). The cell lysate was spun at 12,000Xg, 4 ℃ for 15 min to obtain clarified lysate. For immunoblotting analysis, cell lysates were mixed with an equal volume of 2X SDS sample buffer and boiled for 5 min followed by SDS-PAGE. Proteins transferred to PVDF membrane were then probed with antibodies with a 1:2,000 dilution in TBS with 0.05% Tween (TBST) containing 3% non-fat milk for overnight. For immunoprecipitation, clarified lysates were incubated with 20 µl of anti-Flag M2 beads (Sigma-Aldrich Corporation) for overnight at 4 ℃. After wash with IP-lysis buffer for three times, the beads were boiled with SDS sample buffer. The proteins were resolved on SDS-PAGE for immunoblotting. To address the effect of ATP to the RKIP-Raf-1 complex, cell lysate was passed through a Sephadex G25 (Sigma-Aldrich Corporation) spin column equilibrated with IP-lysis buffer to remove endogenous nucleotides. Subsequent immunoprecipitation was performed as described above with the exception of adding 2 mM nucleotides to experimental groups. The association of Raf-1 with RKIP was determined by immunoblotting.

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Results Experimental design of AETHIC To set up AETHIC method, we derivatized Sepharose beads with five aliphatic chains and examined their performance. As expected, the hydrophobicity of the HIC matrix increases with increasing number of carbon atoms in the alkyl chains (Figure 1A). To determine whether ligand-induced thermodynamic changes of proteins could lead to target protein desorption from HIC, we applied glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to a C6-Sepharose column and subsequently eluted the column with the known ligand, nicotinamide adenine dinucleotide (NAD+). Indeed, GAPDH was eluted from HIC columns with NAD+ (Figure 1B). We also noticed that the most efficient elution was achieved from the C6-Sepharose column, despite C8-Sepharose and C10-Sepharose columns had higher binding capacities suggesting the optimized elution is achieved only with an appropriate degree of hydrophobic interaction (Figure S1). To expand the applications of AETHIC, we scale up and implement the HIC array to a framework for enrichment of metabolites-binding proteins. A schematic presentation of the workflow of AETHIC approach is illustrated in Figure 1C. Following protein binding, individual column is disassembled, washed and eluted with an excess of ligand. The eluate form each column is collected, concentrated, analyzed and subjected to mass spectrometry analysis.

Using AETHIC to identify ATP-binding proteins ATP as the energy currency of living organisms regulates various cellular processes by serving as a substrate or allosteric regulator

18

. The fact that many drugs are

designed as ATP competitors targeting the ATP-binding site of protein kinases dictates the importance of ATP-binding proteins in therapeutic interventions

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.

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However, due to the diversity of their ligand-binding motif, the whole picture of ATP-binding proteins has not been well characterized

20

. To investigate

ATP-regulated processes, we attempted to identify novel ATP-binding targets as our first trial of AETHIC approach. The clarified lysate from porcine brain tissue was applied to the five tandem columns. The proteins eluted with ATP from each column were collected and digested with trypsin for subsequent mass spectrometry analysis.

The Profiling of ATP-binding proteins To validate whether AETHIC specifically enriches the ATP-binding targets, the resulting mass spectrometric data were analyzed by bioinformatics tools: the Database for Annotation, Visualization and Integrated Discovery (DAVID), for unbiased statistical analysis.21 The gene ontology (GO) terms derived from protein identification were categorized into several groups according to their functional networks. The top-ranked annotations belong to phosphoprotein, cytoplasm, acetylation, nucleotide-binding, cytoskeleton, disease mutation, ATP-binding, actin-binding, oxidoreductase and kinase (Figure 2A). Proteins involved in ATP-consuming processes including glycolysis, cytoskeleton remodeling, endocytosis and purine metabolism were enriched in the inquired protein entries (Figure 2B). Taken together, 52 out of the 354 proteins identified in the practice are assigned ATP binding proteins, while the remaining parts consist of novel ATP-binding proteins, associated proteins of ATP-binding proteins and possible some non-specific proteins. A summarized list of protein identifications from ATP eluate is in (Table S1). We also verified the presence of ATP-binding proteins in the ATP eluate from mouse brain extract by immunoblotting against several known markers including valosin-containing protein (VCP) and heat-shock protein 90 (Hsp90) (Figure 2C) 22, 23.

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Contrary to the ATP elution, a non-specific control elution with regeneration buffer (0.5M arginine in 10% ethanol) shows no significant enrichment of ATP-binding protein as determined by gene enrichment analysis (Figure S2). These results indicated the capability of AETHIC for enrichment of metabolite-binding proteins from tissue extracts.

RKIP is an ATP-binding protein To gain new insights into ATP-regulated processes, we then focus on finding new ATP-binding proteins that are able to modulate cell signaling. Among the protein identification list, we found RKIP, top scorer in ATP eluate from C10 column, was an interesting target to study. RKIP, also named phosphatidylethanolamine binding protein (PEBP1) due to its ability in phospholipid binding 24, is involved in a variety of cellular functions by regulating Raf-1 NFκB

27

25

, G protein-coupled receptor kinase 2

and glycogen synthase kinase 3 signaling

26

,

28

. RKIP belongs to an

evolutionary conserved protein family whose structure features a surface pocket accommodating its ligands 29. However, the physiologically relevant ligands of RKIP in cells are still under debate 24, 30-34. Given the pivotal roles of RKIP in cell signaling and oncogenic diseases, the characterization of its ligand may provide valuable insights into the molecular mechanisms underlying its functional diversity

35, 36

. To

validate the affinity of RKIP for ATP, we applied recombinant RKIP to five individual HIC columns. RKIP was mainly eluted from C10 column by ATP although C12 column bound most abundant amount of RKIP (Figure 3A). The absence of RKIP in C12 eluate suggests the strong hydrophobicity of the C12 column tightly retained RKIP hence resulting in poor elution. By contrast, RKIP did not bind to an ATP-conjugated agarose, possible due to the steric hindrance of linker arm (Figure

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3B). We also determined the binding of other nucleotides: GTP, ADP and AMP to RKIP by affinity elution from C10 column. RKIP exhibited a stronger affinity for ATP albeit it also had relatively weak affinity for ADP and GTP, but no binding for AMP (Figure 3C).

The ATP binding of RKIP was further confirmed by labeling with an acyl nucleotide probe, desthiobiotin-ATP (dtb-ATP), which is a nucleotide analogue allowing covalent labeling of ATP-binding proteins with a biotin tag

37

. As revealed by

streptavidin-peroxidase overlay assay, RKIP was labeled by dtb-ATP in a dose dependent manner (Figure 4A). Also, we introduced the probe to the lysates from cells expressing FLAG-tagged RKIP. In agreement with the in vitro labeling experiments using purified RKIP, cellular RKIP was biotinylated by the dtb-ATP probe (Figure 4B) while GAPDH was not. Collectively, these results confirm the ATP binding activity of RKIP and underpin the potential of AETHIC in the study of ligand-protein interactions.

ATP attenuates the interaction between RKIP and Raf-1 The Ras-ERK signaling is crucial for governing cell proliferation and death

38

. The

ERK activity is tightly regulated by the upstream kinase, MEK and Raf-1 39. As one of the most characterized binding partners of RKIP, the binding of RKIP to Raf-1 impairs the Raf-1 dependent activation of MEK and thereby inhibits ERK signaling 40. These clues led us to study the effects of ATP on RKIP binding to Raf-1. We conducted pull-down assays using recombinant RKIP and Raf-1. RKIP physically interacted with HA-Raf-1 as expected (Figure 5A, left panel) whereas the addition of ATP reduced the extent of the interaction dose-dependently (Figure 5A, right panel).

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Accordingly, the replenishment of ATP to desalted cell lysate decreased the RKIP-Raf-1 interaction whereas other nucleotides had little effect (Figure 5B and Figure S3). We next addressed whether the interaction was responsive to the changes of ATP concentration in cells. The bound fraction of Raf-1 was only slightly increased in the RKIP immunoprecipitates when cellular ATP was decreased to near 80% of the control by glycolysis inhibitor, 2-deoxyglucose (2-DG) (Figure 6A and Figure 6B). However, the interaction was further enhanced when ATP level was reduced to around 10% by combining the treatment of 2-DG with the mitochondria complex IV inhibitor, sodium azide (NaN3).

The inhibitory effect of RKIP to ERK signaling upon stimulation with mitogen is proposed to act like a modulator shaping the oscillatory activation of kinase activity 41. To understand the effects of ATP-dependent regulation for Raf-1-RKIP interaction, we compared the amplitude of ERK signaling activated by EGF under short-term energy depletion with the control. The phosphorylated ERK was significantly upregulated in the serum-starved cells after stimulation of EGF. Interestingly, EGF-elicited ERK activation was severely attenuated under both serum- and ATP-depleted conditions (Figure 6C). Similar effects were observed when 12-O-Tetradecanoylphorbol-13-acetate (TPA) was used to activate ERK signaling (Figure S4). We speculate that the increased binding of RKIP to Raf-1 under low ATP concentrations could contribute to the decreased activation of ERK signaling. To examine the possibility, we used locostatin, a RKIP inhibitor, to impair the inhibition 15



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of RKIP to Raf-1

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42

. As shown in (Figure 7A), locostatin indeed hindered the

association of RKIP and Raf-1 in vitro as previously reported

43

. Under normal

conditions, EGF-provoked ERK activation levels were about the same between control and locostatin-treated cells. However, the pre-treatment of locostatin restored the sensitivity of ERK signaling to EGF in ATP-depleted cells (Figure 7B). Therefore, the augmented RKIP-Raf-1 interaction dampens the activation of ERK signaling under low concentrations of ATP. To further elucidate whether the inhibition of RKIP on Raf-1 responding to energy levels was dependent on its ligand-binding function, we constructed a mutant RKIP lacking the ATP-binding ability. Based on sequence prediction, the Arg119 of RKIP is one of possible residues mediating ATP-binding (Figure S5)

44, 45

. As the dtb-ATP labeling assay revealed, the R119A was labelled to

a lower degree (Figure 7C). In line with this result, only wild-type RKIP, but not R119A mutant was eluted from C10 column by ATP, which indicates R119A mutant does not bind ATP (Figure 7D). Interestingly, R119A exhibited stronger binding affinity to Raf-1 under normal conditions as compared with the wild-type RKIP (Figure 7E). Furthermore, the depletion of ATP did not enhance R119A mutant binding to Raf-1. Thus, the ATP-binding function is essential for RKIP to fine-tune its inhibition on Raf-1 according to the cellular ATP concentrations, which can be affected by nutrient supply and metabolic activities. To know if the observed

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ATP-dependent regulation was physiologically relevant, we changed culture medium to a low-glucose medium containing 2.5 mM glucose, a concentration observed in hypoglycemia 46. In agreement with the treatment of ATP depletion, the RKIP-Raf-1 interaction was enhanced when cells were cultured under the low-glucose condition (Figure 7F).

The roles of RKIP in glucose deprivation-induced metabolic stress Recent studies have uncovered the important roles of ERK signaling in triggering death signaling when cells are deprived of glucose for a prolonged period

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.

Especially, the central components of the kinase cascades are isoform specific, comprising Raf-1, MEK1 and ERK2 isoforms. We asked whether RKIP is involved in metabolic stress induced by glucose depletion where cellular ATP levels are low. HEK293 cells underwent morphology changes featuring detachment from culture dish, round-up shape in most cells and membrane blebbing in some cells after deprivation of glucose for 24 h (Figure 8A). At this time point, an elevated ERK activity was coincided with the downregulation of anti-apoptotic protein Bcl-2 and phosphorylated Akt in glucose-starved cells (Figure 8B). In addition, co-treatment of locostatin exaggerated the changes in Bcl-2, phosphorylated Akt and phosphorylated ERK as well as the alterations in cell morphology. Conversely, the blockade of ERK activation by U0126 mitigated the alterations in morphology and the downregulated Akt activity. The data indicate hyper-activation of ERK might be detrimental when cells are confronted with metabolic stress induced by long-term glucose depletion. The enhanced inhibition of Raf-1 by RKIP under low ATP levels might provide a protective effect in short-term glucose depletion by delaying the onset of 17



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ERK-dependent death program

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. However, the prolonged withdrawal of glucose

eventually led to massive cell death at around 48 h (Figure 8C). The results indicate that an unknown mechanism sustains the hyperactive ERK in cells suffering long-term glucose deprivation, even RKIP may exert more potent inhibition on Raf-1 under low ATP conditions.

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Discussion Here, our work has envisaged AETHIC as an alternative approach allowing enrichment of metabolite-binding proteins in biological samples. AETHIC relies on the thermodynamic changes of proteins induced by a given ligand resulting in specific elution from HIC matrix. Operation of AETHIC accomplishes substantial enrichment of target proteins rendering subsequent analysis more feasible. Without the needs of probe labeling and chemical derivatization generally used in affinity chromatography and chemical proteomics

49

, this method does not modify small molecules and thus

retains their native features. As a complementary technology to other systems, AETHIC approach is advantageous when derivatization of small ligand is not possible or target protein binds only natural ligand. For example, RKIP as a weak ATP-binder binds free form of ATP but not ATP-conjugated resin (Figure 3B).

Using the newly developed AETHIC approach, we have characterized RKIP as an ATP-binding protein. Little is known about how RKIP changes its affinity for various binding partners, except that PKC-dependent phosphorylation of RKIP at S153 leads to blockade of RKIP-Raf-1 binding and activation of MEK

26, 50

. However, under

short-term energy depletion conditions, EGF-induced activation of ERK became largely attenuated albeit RKIP phosphorylation at S153 levels persisted (Figure 6C). Given the cellular ATP concentrations are in a narrow range far above the typical disassociation constant of kinases for ATP, ATP levels are doubtful determinant for kinase activities

51, 52

. Recent studies have uncovered that acetoacetate could regulate

ERK signaling by modulating physical interaction between B-Raf and MEK1

53, 54

.

Our data suggest cellular energy levels, partially represented by ATP concentrations,

19



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play an additional role in MEK activation by modulation of RKIP and Raf-1 binding. This might be one important mechanism for cells to adapt to energetic stress, as the interference of RKIP-Raf-1 interaction by locostatin led HEK293 cells susceptible to the metabolic stress induced by glucose depletion (Figure 8C).

Nevertheless, several potential limitations in AETHIC need to be emphasized. First, to maintain the hydrophobic interaction, the use of detergents is avoided. Therefore, proteins with poor solubility such as integral membrane proteins and extracellular matrix components are inevitably omitted during tissue extraction. In addition, proteins tightly or poorly bound to columns may also be underestimated. In the case of proteins that exert increased surface hydrophobicity when binding to ligand, a reverse operation may be used: that is to supply excess ligand in sample applied to the column followed by ligand removal to elute target proteins as previously referred purification of calmodulin

14

. Second, the AETHIC system does not discriminate

between the direct and indirect ligand-binding targets, since some of the associated proteins may be co-eluted with the direct targets. Finally, one common challenge in binding assays is that the kinetic features of each metabolite-protein pair could be distinct so that one experimental condition cannot possibly meet all determinants of binding events, and AETHIC is no exception

55

. The thermodynamic features of

proteins in HIC and affinity elution cannot be predicted, and detailed experimental conditions such as pH, buffer type, salt requirement, ionic strength of buffer should be determined empirically.

20


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Conclusion AETHIC is generally applicable in the early stage of investigation of ligand-protein interactions. The mass spectrometry analysis of enriched fractions provides initial “hits” of potential ligand-binding proteins increasing the possibility of successful target identification. The gentle elution conditions used in AETHIC also allows the framework highly compatible with other analytic platforms. In conjunction with other techniques such as quantitative proteomics, the efficiency of AETHIC can be further improved in the future. In conclusion, we demonstrated the utilization of AETHIC in facilitating the study of metabolite-protein interactions. The use of AETHIC may accelerate the progress of the identification of ligand-protein interactions, which is the bottleneck in understanding the mechanisms of small ligand-mediated effects.

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ACKNOWLEDGMENTS This work was supported by grants from National Science Council and National Taiwan University; NSC 102-2311-B-002-046-MY3, NTU-CESRP-102R7602B1, NTU-CESRP-103R7602B1,

NTU-CESRP-103R8600.

Metabolomic

mass

spectrometry analyses were performed by the Metabolomics Core located at the TechComm, College of Life Science, National Taiwan University. Proteomic mass spectrometry analyses were performed by the Core Facilities for Protein Structural Analysis located at the Institute of Biological Chemistry, Academia Sinica, supported by a National Science Council grant (NSC100-2325-B-001-029) and the Academia Sinica.

Competing financial interests

No

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Legends to figures Figure 1. Establishment of AETHIC approach (A) Characterization of the binding capacities of five HIC columns. An equal amount of bovine serum albumin (BSA) was loaded to the five columns equilibrated with Tris-buffered saline (TBS), respectively. The bound BSA was eluted with 0.5 M arginine in 10% ethanol from each column and analyzed by SDS-PAGE. IN: input, FT: flowthrough. C4: butyl, C6: hexyl, C8: octyl, C10: decyl C12: dodecyl-Sepharose. (B) Affinity elution of glyeraldehyde-3-phosphate dehydrogenase (GAPDH) from C6-Sepharose by NAD+. Purified GAPDH was loaded to a C6-Sepharose column. After wash with TBS, the column was eluted with 10 mM NAD+ in TBS and then regenerated with 0.5 M arginine in 10% ethanol. F2~F11 represent the collected fraction numbers. The changes of buffer are indicated by arrows on the bottom of the gel staining. (C) Schematic representation of AETHIC workflow.

Figure 2. Using AETHIC approach to survey potential ATP-binding proteins in porcine and mouse brain tissues (A) Gene Ontology (GO) profiling of protein identification obtained from ATP eluate in AETHIC. The protein counts on top 10 annotation terms defined by SP_PIR_KEYWORDS in the DAVID sever were shown as bar graph. (B) Top 10 enriched pathways identified by KEGG (Kyoto Encyclopedia of Genes and Genomes) database. (C) The SDS-PAGE analysis of ATP-eluted proteins from five AETHIC columns. Mouse brain extract was loaded to AETHIC columns. After TBS wash, each column was eluted with 3 mM ATP. The five fractions were probed with antibodies against several known ATP-binding proteins, valosin-containing protein (VCP) and heat-shock protein 90 (Hsp90) to demonstrate elution profiles. 28


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Figure 3. Verification of RKIP as an ATP-binding protein (A) Binding and affinity elution of recombinant RKIP from C10 column. Recombinant RKIP was applied to a C10 column. The column was either eluted with 3 mM ATP in TBS containing 4 mM MgCl2 (upper panel) or 0.5 M arginine in 10% ethanol (lower panel). (B) ATP-agarose chromatography for recombinant RKIP. Recombinant RKIP in TBS containing 4 mM MgCl2 was loaded to an ATP-agarose column. The column was either eluted with 3 mM ATP in TBS containing 4 mM MgCl2 or 0.5 M arginine in 10% ethanol. BSA was also used as a negative control. (C) Elution of His-tagged RKIP from C10 column by different nucleotides. The experiments were the same as described above except that different nucleotide were used for elution.

Figure 4. Specific labeling of RKIP by nucleotide acyl phosphates (A) Dose-dependent labeling of desthiobiotin-ATP (dtb-ATP) to RKIP. Recombinant RKIP was incubated with the indicated concentrations of probes at room temperature for 10 min. An equal volume of SDS sample buffer was added to stop the reactions and the samples were subjected to SDS-PAGE. Total RKIP was stained by Coomassie brilliant blue and biotinylated RKIP was detected by peroxidase-conjugated streptavidin (SA-HRP). (B) In vitro affinity labeling of RKIP in cell lysate. HEK293 cells transfected with Flag-RKIP were lysed and then either treated with dtb-ATP or left untreated for the control group. Labeled proteins were pull-downed by streptavidin beads and followed by anti-Flag immunoblotting analysis. GAPDH was used as a negative control.

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Figure 5. Effects of ATP on RKIP-Raf-1 interaction (A) Interaction of RKIP and Raf-1 by the in vitro pull-down assay. Left panel shows HA-Raf-1 specifically bound to his-tagged RKIP immobilized on Ni2+-NTA beads but not the plain control. Right panel: The Co2+-affinity beads immobilized with His-RKIP were used to pull down HA-tagged Raf-1 at various concentrations of ATP. (B) RKIP-Raf-1 complex formation in desalted cell lysate supplemented with ATP. Left panel, the experimental procedures for removal of endogenous small molecules in cell lysate prepared from cells expressing Flag-RKIP and HA-Raf-1. Right panel, the comparison of IP efficiency between desalted and ATP-replenished cell extract. Flag-RKIP was immunoprecipitated with anti-Flag antibody following the addition of ATP and MgCl2, and the immunoprecipitate was examined by immunoblotting. The arrowhead (

Enrichment of Metabolite-Binding Proteins by Affinity Elution in

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