Characterization of the Translationally Controlled Tumor Protein

Sep 8, 2016 - (TCTP) Interactome Reveals Novel Binding Partners in Human ... analyzed the interactome of TCTP in HeLa cells by coimmunoprecipitation ...
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Characterization of the Translationally Controlled Tumor Protein (TCTP) Interactome Reveals Novel Binding Partners in Human Cancer Cells Siting Li,†,§ Minghai Chen,‡,§ Qian Xiong,† Jia Zhang,† Zongqiang Cui,*,‡ and Feng Ge*,† †

Key Laboratory of Algal Biology, Institute of Hydrobiology and ‡State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430072, China § Graduate University, Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: Translationally controlled tumor protein (TCTP) is a highly conserved housekeeping protein present in eukaryotic organisms. It is involved in regulating many fundamental processes and plays a critical role in tumor reversion and tumorigenesis. Increasing evidence suggests that TCTP plays a role in the regulation of cell fate determination and is a promising therapeutic target for cancer. To decipher the exact mechanisms by which TCTP functions and how all these functions are integrated, we analyzed the interactome of TCTP in HeLa cells by coimmunoprecipitation (IP) and mass spectrometry (MS). A total of 98 proteins were identified. We confirmed the in vitro and in vivo association of TCTP with six of the identified binding proteins using reciprocal IP and bimolecular fluorescence complementation (BiFC) analysis, respectively. Moreover, TCTP interacted with Y-box-binding protein 1 (YBX1), and their interaction was localized to the N-terminal region of TCTP and the 1−129 amino acid (aa) residues of YBX1. The YBX1 protein plays an important role in cell proliferation, RNA splicing, DNA repair, drug resistance, and stress response to extracellular signals. These data suggest that the interaction of TCTP with YBX1 might cooperate or coordinate their functions in the control of diverse regulatory pathways in cancer cells. Taken together, our results not only reveal a large number of TCTP-associated proteins that possess pleiotropic functions, but also provide novel insights into the molecular mechanisms of TCTP in tumorigenesis. KEYWORDS: translationally controlled tumor protein (TCTP), mass spectrometry (MS), coimmunoprecipitation (co-IP), bimolecular fluorescence complementation (BiFC), Peroxiredoxin 1 (PRDX1), Y-box-binding protein 1 (YBX1)



INTRODUCTION Translationally controlled tumor protein (TCTP), alternatively named tumor protein translationally controlled 1 (TPT1), histamine releasing factor (HRF), p23, or fortilin, was first identified in mouse erythroleukemia cells in the 1980s.1,2 It was subsequently found that TCTP is a highly conserved housekeeping protein present across diverse eukaryotic organisms.3−5 TCTP has been shown to play indispensable roles in many biological processes, including development,6,7 cell proliferation,8,9 cell death,10−12 immune responses,13,14 the cytoskeleton,15−17 protein synthesis,18,19 the cell cycle,20,21 malignant transformation,22 and induction of pluripotent stem cells or apoptosis.4,23−26 There is growing evidence in the literature to suggest that TCTP is a vital survival factor and a regulator of cell fate determination.11,27,28 A potential role of TCTP in tumor reversion was found in the pioneering work of Telerman et al., in which the process was defined as cancer cells that lose their malignant phenotype.29,30 We previously also demonstrated that down-regulation of TCTP can lead to tumor reversion in multiple myeloma cells.31 In recent years, TCTP has emerged as a promising therapeutic target for cancer prevention and intervention.3,32,33 © XXXX American Chemical Society

The diversity of the cellular functions of TCTP is the result of specific interactions with interacting proteins, including tubulin34 and actin,35 the translation elongation factors eEF1A and eEF1B-b,36 the myeloid cell leukemia 1 protein (Mcl-1), Bcl-xL,37−39 Hsp27,40 p53,41 Na, K-ATPase,42 and polo-like kinase (PLK1).17 Although the studies describing these interactions have provided possible explanations for the diverse functional roles of TCTP, the molecular mechanisms by which TCTP functions remain to be explored. Because of the involvement of TCTP in diverse regulatory mechanisms, the identification and characterization of its interacting proteins on a large scale is critical for the understanding of its diverse biological functions. To decipher the TCTP interactome and its cellular functions, we conducted a global proteomics analysis of TCTP protein complexes using coimmunoprecipitation (Co-IP) in HeLa cells followed by tandem mass spectrometry (MS/MS) analysis. As a result, 98 proteins were identified, the majority of which are novel TCTP interacting proteins. We confirmed the in vitro Received: June 19, 2016

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DOI: 10.1021/acs.jproteome.6b00556 J. Proteome Res. XXXX, XXX, XXX−XXX

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37 °C, alkylated for 10 min in 50 mM iodacetamide at room temperature (RT) in the dark, and digested with 20 μg/mL sequencing grade modified trypsin in 50 mM NH4HCO3. The gel pieces were then swelled using adequate trypsin solution, kept on ice for 45 min, and then incubated at 37 °C for 16−18 h. After that, the supernatants were transferred into eppendorf tubes, and the gel pieces were sonicated twice in extraction buffer (67% ACN and 2.5% trifluoroacetic acid). Finally, the peptide extraction and the supernatant were mixed together and then dried using a SpeedVac.

and in vivo association of TCTP with beta-actin (ACTB), tubulin, alpha 1c (TUBA1C), X-ray repair complementing defective repair in chinese hamster cells 6 (XRCC6), peroxiredoxin 1 (PRDX1), Y-box-binding protein 1 (YBX1), and heat shock 70 kDa protein 9 (HSPA9) using immunoblotting and bimolecular fluorescence complementation (BiFC) analyses, respectively. Moreover, the specific interaction region of TCTP and YBX1 was localized to TCTP42−83 and YBX11−129. Taken together, our results not only reveal the diverse functions of TCTP, but also provide new insights into the role of TCTP in cancer cells.



LC−MS/MS Analysis

We performed a nanoelectrospray tandem mass analysis using the Ultimate 3000 nano-LC system (Dionex, Sunnyvale, California, USA) combined with the electrospray ion-trap mass spectrometer HCT ultra (Bruker Daltonics, Bremen, Germany). The samples were injected into a C18 precolumn (Acclaim PepMap, Dionex, Sunnyvale, USA) of 0.300 mm in diameter and 5 mm in length, and conducted on a C18 reversed phase analytical column (Acclaim, Dionex) of 0.75 mm in diameter and 150 mm in length. Reversed phase chromatograph separation was performed during a gradient (Buffer A, 0.1% formic acid; Buffer B, 100% CAN and 0.1% formic acid) for 120 min at an estimated flow rate of 300 nL/min. The mass spectrometer was equipped in the positive ion mode at 2 kV at 180 °C. Mass spectra were recorded in stand-enhanced mode (speed, 8100 m/z/s). Tandem mass spectra were acquired in ultra scan operating mode (speed, 26000 m/z/s). We selected the four most intense peptides for CID MS/MS (m/z range 100−2000) after a survey scan (enhanced mode, 300−2000 m/ z) that consisted of four scans. The MS/MS spectra parameters were set at a normalized collision energy of 0.5 V and a precursor selection threshold of 100 000 Abs. Selected ions were dynamically excluded for 0.25 min.

EXPERIMENTAL PROCEDURES

Cell Cultures and Protein Extraction

HeLa cells (a human cervical cancer cell line) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% v/v fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 100 U/mL streptomycin, and 100 U/mL penicillin, cultured at 37 °C in a humidified incubator with 5% CO2. When plated at about 90−95% confluence in tissue culture dishes, the cells were harvested by trypsinization and were washed with phosphatebuffered saline (PBS) following centrifugation (4 °C, 500 × g, 5 min). To extract cellular protein, the cells were lysed in moderate Western and IP lysis buffer (Beyotime Biotechnology, Beijing, China; supplemented with phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA) on a rotator at 4 °C for 0.5 h. The cell debris were removed by centrifugation (4 °C, 16 000 × g, 10 min). Protein concentration was measured using a BCA Protein Assay Kit (TANGEN, Beijing, China). Co-IP and Reciprocal Co-IP

Protein Identification

Co-IP was used to isolate immune complexes binding to TCTP in HeLa cells. The experiments were performed according to the instructions of Dynabeads Protein G (Invitrogen) with the following modifications. (1) The beads were incubated with 5 μg of antibody (anti-TCTP, XRCC6, ACTB, TUBA1C, HSPA9, PRDX1, and YBX1 antibodies or IgG (nonimmune, control; Abcam, Cambridge, UK)) diluted in PBS for 4−6 h at 4 °C on a rotator. Rabbit or mouse IgG was used as a control for nonspecific antibody interaction. (2) After the supernatant was removed, the bead−antibody complexes were incubated with total cell lysate (containing 2 mg of protein in each sample) for 10−12 h at 4 °C on a rotator. (3) The bead− antibody−protein complexes were eluted and denatured with 20 μL of loading buffer and boiled for 5 min. For the identification of TCTP binding proteins, three biological replicates were analyzed.

Raw files were processed using Data Analysis 4.0 (Bruker Compass Software) and converted into mgf files. Protein identification was performed by searching the MS/MS data on a local MASCOT 2.3 (Matrix Science, London, UK) against the IPI human 3.87 database (including 91 491 entries) concatenated with a reverse decoy database and protein sequences of common contaminants. The search parameters were enzyme specificity, trypsin/with no proline restriction; maximum missed cleavages, 2; fixed modification, carbamidomethyl (+57.0215 Da, Cys); variable modification, oxidation (+15.9949 Da, Met); precursor ion mass tolerance, 0.4 Da (monoisotopic); and MS/MS mass tolerance, 0.6 Da (monoisotopic). Our final data set only included proteins with a minimum of two identified peptides. The false discovery rate (FDR) was set to 0.01. Additionally, we only retained proteins with a minimum of two identified peptides and an identification coverage over 5%.

Protein Separation and In-Gel Digestion

The samples from Co-IP were separated on 12% SDS-PAGE gels, and the proteins were visualized by silver staining. The entire gel lane was divided into five slices, then further diced into ∼1 mm3 cubes. During the process of in-gel digestion, the gel pieces were first destained completely using destaining solution (30 mM potassium hexacyanoferrate (III) (K3Fe(CN)6) and 100 mM sodium thiosulfate (Na2S2O3)) and were washed thoroughly by deionized water. Then the gel pieces were dehydrated by washing several times in acetonitrile(ACN) until the gel pieces shrank and looked completely white. The proteins were disulfide reduced for 30 min in 25 mM DTT at

Bioinformatics Analysis

To investigate the biological relevance of the identified TCTP binding partners, we carried out bioinformatics analysis. We used the protein analysis through evolutionary relationships (PANTHER) ontology,43 a highly controlled vocabulary made up of ontology terms, to classify the identified TCTP binding proteins into several subfamilies and families with common functions by biological process, molecular function, and cellular component. Furthermore, a gene ontology (GO) enrichment distribution of the identified TCTP binding proteins was B

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Figure 1. Systematic identification of TCTP binding proteins. (A) Overview of the experimental workflow used in this study. Cell lysates were immunoprecipitated using magnetic beads and anti-TCTP antibody or IgG (control). The complexes eluted from Co-IP were separated by a 12% SDS-PAGE gel and processed with silver staining. The entire gel lanes were excised from the gels, and in-gel digestion was performed for LC−MS/ MS analysis. MS data were processed, and a protein list of TCTP interacting proteins was generated. Bioinformatics analyses were performed, and critical TCTP interacting proteins were selected for further validation. (B) Bar chart of the GO enrichment of identified TCTP binding proteins using Cytoscape. The top eight GO terms enriched in the TCTP interacting proteins are shown.

generated using a Cytoscape plugin, BINGO.44 In addition, the protein−protein interaction (PPI) networks of the identified TCTP binding proteins were generated by the search tool for the retrieval of interacting genes/proteins (STRING)45 with default settings, except that “organism” was set to “human”. The proteins in the network were classified into several functional groups on the basis of protein class in PANTHER.

TCTP, pXRCC6-MC156, pACTB-MC156, pTUBA1CMC156, pHSPA9-MC156, pPRDX1-MC156, and pYBX1MC156 protein expression plasmids. DNA sequencing verified all the inserted sequences. To map the specific binding region of YBX1 in TCTP, we constructed three deletion mutants of TCTP according to the protein’s crystal structure:12 one encoding the first 83 amino acids (aa) of TCTP (TCTP1−83), one encoding aa 84−132 (TCTP84−132), and a final mutant encoding aa 133−172 (TCTP133−172). The three split region fragments were inserted into pMN155 plasmids to obtain pMN155TCTP1−83, pMN155-TCTP84−132, and pMN155-TCTP133−172. On the basis of our experimental results, we further divided the TCTP1−83 aa fragment into two regions, a TCTP1−41 aa mutant and a TCTP42−83 aa mutant, and generated the pMN155TCTP1−41 and pMN155-TCTP42−83 plasmids in the same way. To map the TCTP-interacting region with YBX1, we divided the coding gene of YBX1 into three regions according to its structure47 and constructed three YBX1 deletion mutants encoding the N-terminal domain (YBX11−50), the central cold shock domain (CSD; YBX151−129), and the carboxyl-tail domain (CTD; YBX1130‑C‑terminus). The three split fragments were inserted into pMC156 plasmids to obtain the pMC156YBX11−50, pMC156-YBX151−129, and pMC156-YBX151−324 vectors. All sequences were verified by DNA sequencing.

Immunoblot Analysis

Each complex from Co-IP or reverse Co-IP was resolved by SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Blots were blocked with 5% nonfat dry milk in Tris-buffered saline supplemented with 0.1% Tween-20 for 2 h at RT and were then incubated with primary anti-TCTP, XRCC6, ACTB, TUBA1C, HSPA9, PRDX1, or YBX1 antibody (Abcam, Cambridge, UK) at 1:1000 dilution for 10−12 h at 4 °C. After washing, the blots were incubated for 2 h in peroxidaseconjugated antirabbit or antimouse IgG (KPL, Gaithersburg, MD, USA) at 1:5000 dilution for 30 min at RT. The signals were detected using ECL detection reagent (GE Healthcare Biosciences, Uppsala, Sweden) and autoradiography films. The images were scanned using a CCD camera and Graphic Converter software. Plasmids Construction

Transfection and Fluorescence Microscopy

The human genes TCTP, XRCC6, ACTB, TUBA1C, HSPA9, PRDX1, and YBX1 were cloned by PCR from the cDNA of HeLa cells. The sequences of these primers are listed in Table S1. The TCTP gene was inserted into the pMN155 plasmid, while the others were inserted into a pMC156 plasmid of the mNeptune-based BiFC system46 to construct the pMN155-

The 293T cells were cultured in the same way as the HeLa cells. The day before transfection, the 293T cells were plated evenly in 35 mm diameter glass-bottom dishes and grown 16− 18 h to 70−80% confluence. After that, the expression vectors were cotransfected into the cells using Lipofectamine 2000 C

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nonspecific binding proteins. Consequently, we identified 98 potential TCTP interacting partners (Table S2). Our results not only identified some of the known TCTP interacting proteins, but also discovered many novel ones. The raw data have been deposited in the publicly accessible database Peptide atlas (http://www.peptideatlas.org)55,56 and can be accessed with the identifier PASS00923 (http://www.peptideatlas.org/ PASS/PASS00923).

(Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The transacted 293T cells were incubated at 37 °C (5% CO2) for 5−6 h and then at 30 °C (5% CO2) for another 18 h. Hoechst 33342 (Beyotime Biotechnology) was used to stain the nuclei before imaging. The cells were imaged using an UltraVIEW VOX Confocal system (PerkinElmer, Fremont, CA, USA) and a 60×, 1.4 NA, oil immersion objective lens. The red fluorescence from the mNeptune channel was excited at 561 nm, while the nuclei were excited at 405 nm. The BiFC fluorescence signals in present study were detected in living cells.

Functional Categories and PPI Networks of TCTP Interacting Proteins

The PANTHER classification system was used to classify the identified TCTP interacting proteins according to biological processes, molecular functions, or cellular component. The GO classification analysis showed that binding (42.9%) was the largest functional group (Table S3), which implies that TCTP may act as a multifunctional protein through binding directly or indirectly to a series of molecules. The second largest group was structural molecule activity (36.7%). The GO biological processes classification results revealed that a majority of the identified TCTP binding proteins are involved in metabolic process (59.2%), cellular processes (30.6%), localization (16.3%), and cellular component organization or biogenesis (15.3%) (Table S3). GO cellular component analysis showed that most of the identified proteins were classified as cell part (34.7%), macromolecular complex (22.4%), or organelle (17%), which indicated that TCTP has diverse cellular functions (Table S3). GO enrichment analysis of the identified TCTP binding proteins also revealed that TCTP is involved in various cellular processes including translation, translational elongation, cellular macromolecule biosynthetic processes, and gene expression (Figure 1B and Table S4). The most over-represented function in the GO molecular function categories was structural molecule activity and binding (Figure 1B). These results further confirmed that TCTP is a vital regulator of cellular functions in a wide range of biological processes (Figure 1B). A PPI network of TCTP interacting proteins was generated using the STRING database, and the results showed that 93/98 (94.9%) proteins were involved in a single PPI network (Figure 2 and Table S5). The protein class analysis in PANTHER revealed that these proteins were implicated in a range of functional groups, such as nucleic acid binding, cytoskeletal proteins, and chaperones, which strongly suggested that the majority of the identified proteins are an integral part of the dynamic complex of TCTP (Figure 2 and Table S6). We generated a list of predicted TCTP interacting proteins (Table S7) by searching against GeneCards (http://www. genecards.org/). In Table S7, we highlighted the proteins commonly reported by our study and the GeneCards online database in purple. When we compared our identified TCTP interacting proteins with this online data set, few proteins were found in both sets. The differences between these two data sets were not unexpected, as many studies have shown that protein interaction data retrieved from different high-throughput technologies do not overlap to a significant degree and provide complementary types of information.57−59 These differences may primarily be a function of the experimental technologies used to identify these interactions. In addition, for all the TCTP interacting proteins identified in this study and searched against GeneCards, we retrieved the corresponding proteins’ cellular abundance data in HeLa cells from the study of Itzhak et al.60 Table S7 also includes the copy number/cell and abundance

Molecular Dynamics (MD) Simulations

The initial model of the TCTP module was prepared from the available crystal structure (PDB code 1YZ1, 99.42% sequence identity) via structural modeling using the SWISS-MODEL Server (http://swissmodel.expasy.org/). The structural model of the YBX11−129 region was created using the I-TASSER Server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). ZDOCK 3.0.2 docking program48 was used to perform a full rotation rigid-body docking to construct a putative model for the TCTP-YBX11−129 complex structure according to the instructions. The TCTP-YBX11−129 system was prepared for simulation using GROMACS 4.6.549 in conjunction with the GROMOS96(54a7) force field parameters.50 Each system was solvated in simple point charge water molecules51 in a triclinic box, with the box edges ∼1.0 nm from any atom of the protein, and additional Na+ ions were added to neutralize the charge of each system. Energy minimization for the solvated structures was carried out by the steepest descent method until the maximum force was less than 100 kJ/mol/nm. Next, all the simulations were performed under a constant temperature of 310 K, and the V-rescale algorithm48 was used with a temperature coupling time constant of 0.1 ps. The pressure was maintained at 1 bar by coupling the system to an isotropic pressure bath using a coupling constant of 2 ps. The van der Waals and electrostatic interactions used a cutoff at 1.2 nm, and long-range electrostatic interactions were handled using the particle mesh Ewald method52 with a fourth-order spline interpolation and a 0.12 nm Fourier grid spacing. All bond lengths were constrained using the LINCS algorithm.53 The SETTLE algorithm was used to constrain the geometry of the water molecules.54 Finally, each system was subjected to 70 ns of MD simulation, and the time step used in the simulation was 2 fs. All of the analyses were performed using the GROMACS suite of tools. The PyMOL Molecular Graphics System (version 1.7.2, http://www.pymol.org) was employed to present the structural results of this study.



RESULTS

Identification of TCTP Interacting Proteins by Co-IP and LC−MS/MS

In present study, we conducted a proteomics analysis to investigate proteins interacting with TCTP by IP from HeLa cells using anti-TCTP antibody, and then we separated them on an SDS-PAGE gel followed by in-gel digestion and LC− MS/MS (Figure 1A). The endogenous expression of TCTP in three different cell lines was confirmed by Western blot, and HeLa cells were used in our IP studies due to the high expression of TCTP (Figure S1). Control experiments were carried out using IgG (nonimmune) in parallel. Three biological replicates were analyzed. To reduce false positives, the proteins observed in the control group were treated as D

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detected in the TCTP immune complex and the HeLa cell lysate (input) but not in the control (Figure 3A). Moreover, the reciprocal Co-IP assay using specific antibodies for these proteins followed by Western blotting confirmed their binding to TCTP (Figure 3). Therefore, the in vitro evidence shows that TCTP binds with XRCC6, ACTB, TUBA1C, HSPA9, PRDX1, and YBX1 in HeLa cells. Validation of TCTP Interacting Proteins by BiFC Analysis

As an independent approach, the mNeptune-based BiFC system was applied to visualize the interactions of the six proteins with TCTP in living cells. The BiFC system is based on the reconstitution of two nonfluorescent fragments of a fluorescent protein.46 In this study, the encoding sequence of TCTP was fused to the N-terminal of mNeptune (mNeptune aa 1−155, MN155), and the encoding sequences of the ACTB, TUBA1C, HSPA9, PRDX1, XRCC6, and YBX1 proteins were each fused to the C-terminal of mNeptune (mNeptune aa 156C-terminal, MC156) separately. The coexpression of pXRCC6MC156, pACTB-MC156, pTUBA1C-MC156, pHSPA9MC156, pPRDX1-MC156, or pYBX1-MC156 and pMN155TCTP resulted in a bright red mNeptune fluorescence signal in the cytoplasm around the cellular chromosomes, which suggested that TCTP and XRCC6, ACTB, TUBA1C, HSPA9, PRDX1, and YBX1 all bind in the cytoplasm in vivo (Figure 4). Together, these results demonstrated that TCTP interacts with these proteins in vivo.

Figure 2. PPI analysis for the identified TCTP interacting proteins. The PPI network of TCTP interacting proteins was constructed using the STRING database v. 10.0. Ninety-three of the 98 TCTP interacting proteins are connected in this network and were classified into functional groups according to their GO categories. Yellow nodes in the network indicate proteins that were chosen for further validation.

percentile for the proteins identified and the other known interactors that were not identified in this study. The proteins identified in our study seem to be biased toward highly abundant proteins, and the ratio of low-abundance proteins in the unidentified interactors is much higher than that of the ones identified in this study.

Specific Interaction Region of TCTP and YBX1

X-ray crystallography has revealed that three domains of the human TCTP structure are highly conserved, including a mobile loop, a helical hairpin formed by two H2−H3 helices as well as a core of nine β-strands including a short H1 helix.62,63 To reveal the region of the TCTP protein that mediates the TCTP-YBX1 interaction, we first generated three split region fragments based on the crystallography of TCTP. As demonstrated in Figure 5, a bright red BiFC fluorescence signal could be observed with the coexpression of pYBX1MC156 and pMN155-TCTP1−83, but hardly any signal was seen with pMN155-TCTP84−132 or pMN155-TCTP133−172. Then we further generated two split region fragments of aa

Validation of TCTP Interacting Proteins by Reciprocal Co-IP

Immunoblot analysis was performed to validate six of the proteins identified by LC−MS/MS. On the basis of the bioinformatics analysis, six putative TCTP binding proteins (XRCC6, ACTB, TUBA1C, HSPA9, PRDX1, and YBX1) distributed in four groups in the PPI networks were selected for validation. XRCC6, which has previously been reported to be a TCTP interactor,61 was used as a positive control for the Co-IP and reciprocal Co-IP experiments. All six proteins were

Figure 3. Validation of the six selected TCTP interacting proteins by immunoblot analysis after co-IP assay. (A) Immunoprecipitation assays of TCTP, XRCC6, ACTB, TUBA1C, HSPA9, PRDX1, and YBX1 were performed for HeLa cell lysates. Immunoblot analysis demonstrated that the TCTP protein binds with these selected proteins. No band of TCTP was observed in the negative control (IgG). Input stands for the total cell lysate. Similarly, reverse immunoprecipitation assays confirmed the binding of (B) XRCC6, (C) ACTB, (D) TUBA1C, (E) HSPA9, (F) PRDX1, and (G) YBX1. IB, immunoblot; IP, immunoprecipitation; RIP, reverse immunoprecipitation. E

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Figure 4. Imaging of PPIs using far-red mNeptune-based BiFC in vivo. BiFC signals of 293T cells coexpressing TCTP and (A) XRCC6, (B) ACTB, (C) TUBA1C, (D) HSPA9, (E) PRDX1, or (F) YBX1, as well as the negative control. In the BiFC system, the fluorescence from the mNeptune channel was red, and the nucleus was stained with Hoechst 33342. The images were acquired by fluorescence microscopy under a 60× objective lens (scale bar, 15 μm).

Figure 5. We identified that the N-terminal aa 42−83 region of TCTP is the binding region of YBX1. (A) The secondary structure of TCTP. (B) Imaging the TCTP region-YBX1 interaction using far-red mNeptune-based BiFC in vivo. The nucleus was stained with Hoechst 33342. The images were acquired by fluorescence microscopy under a 60× objective lens (scale bar, 15 μm). (C) The crystal structure of human TCTP; the N-terminal 42−83 amino acid residues are shown in red. (D) Imaging the TCTP-YBX1 region interaction using far-red mNeptune-based BiFC in vivo.

TCTP1−83 and saw that the bright red BiFC fluorescence signal could also be observed with coexpression of pYBX1-MC156 and pMN155-TCTP42−83, but not with pMN155-TCTP1−41, indicating that YBX1 binds to the TCTP42−83 region.

Similarly, to investigate the specificity of the interaction and map the region of the YBX1 protein that mediates the TCTPYBX1 interaction, we split the coding gene of YBX1 into three regions according to its structure. We observed a bright red F

DOI: 10.1021/acs.jproteome.6b00556 J. Proteome Res. XXXX, XXX, XXX−XXX

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Journal of Proteome Research BiFC fluorescence signal with coexpression of pMN155-TCTP and pMC156-YBX11−50 and with coexpression of pMN155TCTP and pMC156-YBX151−129, which suggested that the aa 1−129 region of the YBX1 protein mediates the TCTP-YBX1 interaction.

lation.25,28 Among them, XRCC6 (Ku70) has been described to fulfill its functions in DNA double-strand break sensing and repairing by binding to TCTP.61,66,67 When DNA damage occurs, TCTP accumulates at the damage sites by colocalizing with XRCC6 and XRCC5 (Ku80) and forming complexes.61 The TCTP-XRCC5-XRCC6 complexes subsequently accumulate in the cytoplasm.61 The BiFC system imaging results in our study further confirmed the interaction of TCTP and XRCC6 as well as the localization of the TCTP-XRCC5-XRCC6 complexes (Figure 4A). These results show that the mNeptunebased BiFC system is highly reliable. The cytoskeletal protein class represents the second largest group (Figure 2). The cytoskeleton is a highly dynamic system comprising different groups of structural proteins including tubulin, actin, and intermediate filaments to form polymers and associated proteins with diverse regulatory functions.68 Additionally, cytoskeletal proteins also have activities in cell migration, mobility, apoptosis, and immunological synapse formation.68 TCTP has been reported to be associated with cytoskeleton proteins and related cellular processes.17,35,69 For example, TCTP is involved in regulating cell shape, probably via complex interactions with both F-actin and the microtubule cytoskeleton.17 TCTP also associates with microtubules during specific phases of the cell cycle by binding to tubulin.69 TCTP can release cofilin binding to G-actin and transfer active cofilin to F-actin, which increase the cofilin-activity cycle in invasive tumor cells.35 Fifteen cytoskeleton proteins were identified to be potential TCTP interacting proteins, including two actin proteins (ACTA1 and ACTB) and six tubulin proteins (TUBA3D, TUBA1C, TUBB, TUBA4A, TUBA8, and TUBB2A). ACTB and TUBA1C were further validated to be TCTP-binding proteins by our experiments (Figures 3 and 4C). The binding of TCTP and ACTB or TUBA1C may have similar functions as the TCTP−cytoskeleton protein interactions reported previously. The identification of new TCTP interacting cytoskeleton proteins sheds new light on the role of TCTP in cytoskeleton-related functions like cell morphology, tumorigenesis, cell proliferation, cell growth, and the cell cycle. Six chaperone proteins from the highly conserved HSP70 family were identified to be TCTP-interacting partners by MS (Figure 2). HSP70 family members have key regulatory roles in a variety of cellular stress responses.70,71 It has been reported that overexpression of HSPA9 can protect cells from oxidative damage.72,73 Interestingly, TCTP is upregulated during oxidative stress and has been implicated as an antioxidant protein.74−76 In the present study, both the immunoblot and BiFC assay results clearly demonstrated that HSPA9 is a TCTP-binding partner. Thus, it is likely that the HSPA9− TCTP complex can function together in resistance to intracellular oxidative stress. The HSPA9−TCTP complex may be involved in antiapoptotic processes, which is similar to the HSP27−TCTP complex.33 Also, the other four Hsp70s may have a similar mode of action as HSPA9. PRDX1 was also validated to be a TCTP-interacting protein in our study (Figures 3 and 4E). PRDX1 was the first antioxidant protein reported to protect proteins from inactivation through PPIs.77 When exposed to mild oxidative stress, PRDX1 is upregulated and binds to phosphatase and tensin homologue (PTEN) to protect it from oxidationinduced inactivation, and PRDX1 becomes reversibly oxidized.77 PTEN is a negative regulator of the serine/threonine kinase Akt, so the binding of PRDX1 and PTEN prevents Aktdriven oncogenesis.78 However, under high doses of oxidative

MD Simulation and Stability Analysis of TCTP and YBX11−129

To obtain further insight into the potential TCTP-YBX1 docking region, we used MD simulations to determine how TCTP interacts with YBX1. On the basis of the results of the BiFC analysis, the putative model of the TCTP-YBX11−129 complex structure was constructed using the ZDOCK docking program (http://zdock.umassmed.edu/). After 70 ns MD simulations, we observed that the YBX11−129 region had a tendency to embed into the TCTP42−83 region gradually during the entire course of simulations, and the TCTP-YBX11−129 complex that formed reached a relatively stable state in the period of 40−70 ns (Figure S2A). Consistently, the simulations also showed that the root−mean−square deviation and radius of gyration of the TCTP-YBX11−129 complex had lower conformational fluctuations and reached the equilibrium state gradually until 40 ns (Figure S2B,C). Together, these observations also suggest that TCTP forms a stable interaction with the YBX11−129 region.



DISCUSSION

Numerous studies show that TCTP is a protein with diverse cellular functions including proliferation, apoptosis, cell death, and tumorigenicity.64 The diversity of TCTP cellular functions relies on specific interactions with other proteins. However, many functions of TCTP are still unclear, and the exact mechanisms by which TCTP accomplishes its functions remain to be explored. To address this point, we employed a systematic proteomics study by LC−MS/MS analysis in combination with IP to investigate novel binding proteins of TCTP. As a result, a total of 98 potential TCTP binding proteins have been identified. Most of the 98 proteins are involved in a PPI network generated by STRING (Figure 2), which suggests that they are related to TCTP function. The protein functional classification system in PANTHER revealed that these proteins participate in a range of functional groups including nucleic acid binding, cytoskeletal proteins, chaperones, enzyme modulator, and transferase, which is consistent with the multifunctional nature of TCTP (Figure 2). Six proteins, ACTB, TUBA1C, XRCC6, HSPA9, PRDX1, and YBX1, which belong to four different functional groups, were selected for further validation. Of these six proteins, XRCC6 has already been shown to be a TCTP binding protein in a previous study.61 The interactions of TCTP and these six proteins were further validated in vitro and in vivo using immunoblot and BiFC assays, respectively. BiFC is a practical way to investigate PPIs in vivo due to its simplicity, high sensitivity, and lack of invasiveness.65 The recently developed mNeptune-based BiFC system was used in our study to image the interaction between TCTP and the six selected proteins in vivo.46 Both the immunoblot and mNeptune-based BiFC system imaging results demonstrated that TCTP interacts with these six proteins. In this study, 45 proteins were assigned to the nucleic acid binding proteins group, which represents the largest group, of which about one-third are transcription and translation regulatory proteins (Figure 2). These results indicate the essential role of TCTP in cellular gene expression reguG

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CONCLUSIONS In conclusion, we identified 98 potential TCTP interacting candidates by using a proteomics strategy. Bioinformatic analysis indicated that these proteins are involved in an extensive array of cellular processes, which provides new clues for deciphering TCTP function. Six representative proteins from different functional categories were further confirmed to be TCTP binding proteins using both in vitro and in vivo assays. The far-red mNeptune-based BiFC assay was used in confirming the predicted PPIs and was shown to be highly reliable. On the basis of our results, we propose several molecular mechanisms of TCTP and its interacting partners, useful for comprehensively investigating the diverse functions of TCTP. Moreover, the specific interaction regions of TCTP and YBX1 were also revealed by the BiFC assay, which is important for future investigation of the biological functions of the TCTP−YBX1 interaction. The systematic discovery of potential TCTP binding proteins also provides a valuable source for further in-depth studies of TCTP function.

stress, PTEN irreversibly dissociates from oxidized PRDX1 and becomes inactivated, which results in hyperactivation of Akt signaling.77−80 Notably, as a hallmark of stress response in tumor cells, high TCTP levels may provide a survival advantage under stress conditions.76 Under mild oxidative stress, TCTP was up-regulated in cells that survived the treatment.76 Conversely, when exposed to a strong oxidative hit, cancer cells caused a down-regulation of TCTP, followed by cell death.76 Therefore, the binding of TCTP and PRDX1 may also be involved in antioxidant pathways, and the TCTP−PRDX1 complex may prevent Akt-driven transformation by a similar mechanism as PTEN under mild oxidative stress. YBX1 can bind to DNA/RNA and functions as nucleic acid chaperone.81 It has been implicated in numerous cellular processes including DNA damage repair,82 transcription,83,84 translation,85,86 cell proliferation,83 differentiation,87 and stress responses88 in the nucleus and cytoplasm.47,89,90 In the present study, YBX1 was validated to be an interacting protein of TCTP by both immunoblotting and BiFC analysis (Figures 3 and 4F). To further explore the exact interacting regions of the two proteins, BiFC analyses of split fragments of both YBX1 and TCTP were performed. The BiFC results revealed that TCTP42−83 binds to YBX11−129 (Figure 5). Interestingly, YBX1 was shown to bind TCTP through its N-terminal and CSD domains (Figure 5). The N-terminal and CSD domains that jointly form binding regions are rather rare in the PPI of YBX1 and other proteins,90 which suggests a new functional mechanism of YBX1. Two highly conserved TCTP regions (TCTP45−55 and TCTP129−147) were reported previously that most likely mediate the interactions between TCTP and its binding partners.62 Notably, the TCTP45−55 signature was also seen in our results (TCTP42−83), which also verifies the reliability of the BiFC analysis. In addition, the findings of our MD simulations agreed with the results of the BiFC analysis (Figure S2). Revealing the specific interacting region of TCTP and YBX1 not only provides additional information about the YBX1 interaction spectrum, but also provides a starting point for further investigation of the biological relevance of the TCTP−YBX1 interaction. The pleiotropic functions of YBX1 and TCTP indicate that TCTP−YBX1 complexes may be involved in vital signaling pathways. YBX1 is closely related to the PI3K/Akt/mTOR signaling pathway, which regulates a series of tumorigenesis functions including proliferation, survival, metabolism, and metastasis.91 The expression of PIK3CA in basal-like breast cancer cells can be transcriptionally activated by YBX1.92 Serine phosphorylation of YBX1102, which is critical for its translocation, relies on Akt kinase activity.93−95 The inhibition of the PI3K pathway can also reduce the expression of YBX1.95 Additionally, a reduction in YBX1 leads to a marked decrease in mTOR protein levels.96 Interestingly, TCTP is also involved in PI3K/Akt/mTOR signaling. The translation of TCTP mRNA is regulated by PI3−K/Akt/mTOR signaling, and a positive feedback loop between TCTP and mTOR contributes to tumor formation.97,98 We propose that TCTP and YBX1 may work cooperatively in the PI3K/Akt/mTOR pathway by interacting with each other and forming complexes. We speculate that these TCTP−YBX1 complexes are associated with the PI3KAkt signaling pathway, which regulates tumorigenesis, malignant transformation, and mTOR signaling.99 The complexes may also regulate mTOR signaling directly and further influence the glycolysis pathway to regulate cell proliferation, cell growth, and the cell cycle.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.6b00556. Comparison of the TCTP expression level in HepG2, Hela and 293T cells (expression levels of TCTP confirmed by Western blotting); MD simulation of TCTP and YBX11−129 (PDF) List of primer sequences (XLS) Complete list of identified TCTP interacting proteins (XLS) GO classification of the identified TCTP interacting proteins (XLS) GO terms enriched in the identified TCTP binding proteins (XLS) List of protein−protein interaction details of identified TCTP interacting proteins (XLS) List of protein class in identified TCTP interacting proteins (XLS) List of predicted/known interacting proteins for TCTP (XLS)



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86-27-68780500. Fax: +86-27-68780500. *E-mail: [email protected]. Phone: +86-27-8719 9492. Fax: +8627-8719 9492. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the National Key Research and Development Program (2016YFA0501304), the State Key Laboratory of Freshwater Ecology and Biotechnology (Grant No. 2015FB10), the National Natural Science Foundation of China (Grant No. 31570829), and the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB14030202). H

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colocalizes with TCTP to the mitotic spindle. Oncogene 2008, 27 (42), 5554−5566. (21) Johnson, T. M.; Antrobus, R.; Johnson, L. N. Plk1 activation by Ste20-like kinase (Slk) phosphorylation and polo-box phosphopeptide binding assayed with the substrate translationally controlled tumor protein (TCTP). Biochemistry 2008, 47 (12), 3688−96. (22) Huang, H.; Li, X.; Hu, G.; Zhuang, Z.; Liu, J.; Wu, D.; Yang, L.; Xu, X.; Huang, X.; Zhang, J.; Hong, W. X.; Yuan, J.; Gao, W.; Liu, Y. Poly(ADP-ribose) glycohydrolase silencing down-regulates TCTP and Cofilin-1 associated with metastasis in benzo(a)pyrene carcinogenesis. Am. J. Cancer Res. 2015, 5 (1), 155−67. (23) Roque, C. G.; Wong, H. H.; Lin, J. Q.; Holt, C. E. Tumor protein Tctp regulates axon development in the embryonic visual system. Development 2016, 143 (7), 1134−1148. (24) Wu, D.; Guo, Z.; Min, W.; Zhou, B.; Li, M.; Li, W.; Luo, D. Upregulation of TCTP expression in human skin squamous cell carcinoma increases tumor cell viability through anti-apoptotic action of the protein. Exp Ther Med. 2012, 3 (3), 437−442. (25) Cheng, X.; Li, J.; Deng, J.; Li, Z.; Meng, S.; Wang, H. Translationally controlled tumor protein (TCTP) downregulates Oct4 expression in mouse pluripotent cells. BMB Rep 2012, 45 (1), 20−5. (26) Sirois, I.; Raymond, M. A.; Brassard, N.; Cailhier, J. F.; Fedjaev, M.; Hamelin, K.; Londono, I.; Bendayan, M.; Pshezhetsky, A. V.; Hebert, M. J. Caspase-3-dependent export of TCTP: a novel pathway for antiapoptotic intercellular communication. Cell Death Differ. 2011, 18 (3), 549−62. (27) Miao, X.; Chen, Y. B.; Xu, S. L.; Zhao, T.; Liu, J. Y.; Li, Y. R.; Wang, J.; Zhang, J.; Guo, G. Z. TCTP overexpression is associated with the development and progression of glioma. Tumor Biol. 2013, 34 (6), 3357−61. (28) Chen, S. H.; Wu, P. S.; Chou, C. H.; Yan, Y. T.; Liu, H.; Weng, S. Y.; Yang-Yen, H. F. A knockout mouse approach reveals that TCTP functions as an essential factor for cell proliferation and survival in a tissue- or cell type-specific manner. Mol. Biol. Cell 2007, 18 (7), 2525− 32. (29) Tuynder, M.; Fiucci, G.; Prieur, S.; Lespagnol, A.; Geant, A.; Beaucourt, S.; Duflaut, D.; Besse, S.; Susini, L.; Cavarelli, J.; Moras, D.; Amson, R.; Telerman, A. Translationally controlled tumor protein is a target of tumor reversion. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (43), 15364−9. (30) Tuynder, M.; Susini, L.; Prieur, S.; Besse, S.; Fiucci, G.; Amson, R.; Telerman, A. Biological models and genes of tumor reversion: cellular reprogramming through tpt1/TCTP and SIAH-1. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (23), 14976−81. (31) Ge, F.; Zhang, L.; Tao, S. C.; Kitazato, K.; Zhang, Z. P.; Zhang, X. E.; Bi, L. J. Quantitative proteomic analysis of tumor reversion in multiple myeloma cells. J. Proteome Res. 2011, 10 (2), 845−55. (32) Lucibello, M.; Adanti, S.; Antelmi, E.; Dezi, D.; Ciafre, S.; Carcangiu, M. L.; Zonfrillo, M.; Nicotera, G.; Sica, L.; De Braud, F.; Pierimarchi, P. Phospho-TCTP as a therapeutic target of dihydroartemisinin for aggressive breast cancer cells. Oncotarget 2015, 6 (7), 5275−5291. (33) Baylot, V.; Katsogiannou, M.; Andrieu, C.; Taieb, D.; Acunzo, J.; Giusiano, S.; Fazli, L.; Gleave, M.; Garrido, C.; Rocchi, P. Targeting TCTP as a new therapeutic strategy in castration-resistant prostate cancer. Mol. Ther. 2012, 20 (12), 2244−56. (34) Tuynder, M.; Susini, L.; Prieur, S.; Besse, S.; Fiucci, G.; Amson, R.; Telerman, A. Biological models and genes of tumor reversion: Cellular reprogramming through tpt1/TCTP and SIAH-1. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (23), 14976−14981. (35) Tsarova, K.; Yarmola, E. G.; Bubb, M. R. Identification of a cofilin-like actin-binding site on translationally controlled tumor protein (TCTP). FEBS Lett. 2010, 584 (23), 4756−4760. (36) Langdon, J. M.; Vonakis, B. M.; MacDonald, S. M. Identification of the interaction between the human recombinant histamine releasing factor/translationally controlled tumor protein and elongation factor-1 delta (also known as elongation factor-1B beta). Biochim. Biophys. Acta, Mol. Basis Dis. 2004, 1688 (3), 232−236.

REFERENCES

(1) Yenofsky, R.; Cereghini, S.; Krowczynska, A.; Brawerman, G. Regulation of Messenger-Rna Utilization in Mouse ErythroleukemiaCells Induced to Differentiate by Exposure to Dimethylsulfoxide. Mol. Cell. Biol. 1983, 3 (7), 1197−1203. (2) Macdonald, S. M.; Rafnar, T.; Langdon, J.; Lichtenstein, L. M. Molecular-Identification of an Ige-Dependent Histamine-Releasing Factor. Science 1995, 269 (5224), 688−690. (3) Acunzo, J.; Baylot, V.; So, A.; Rocchi, P. TCTP as therapeutic target in cancers. Cancer Treat. Rev. 2014, 40 (6), 760−9. (4) Amson, R.; Pece, S.; Marine, J. C.; Di Fiore, P. P.; Telerman, A. TPT1/ TCTP-regulated pathways in phenotypic reprogramming. Trends Cell Biol. 2013, 23 (1), 37−46. (5) Bommer, U. A.; Thiele, B. J. The translationally controlled tumour protein (TCTP). Int. J. Biochem. Cell Biol. 2004, 36 (3), 379− 85. (6) Hong, S. T.; Choi, K. W. TCTP directly regulates ATM activity to control genome stability and organ development in Drosophila melanogaster. Nat. Commun. 2013, 4, 2986. (7) Koziol, M. J.; Gurdon, J. B. TCTP in development and cancer. Biochem. Res. Int. 2012, 2012, 105203. (8) Gu, X.; Yao, L.; Ma, G.; Cui, L.; Li, Y.; Liang, W.; Zhao, B.; Li, K. TCTP promotes glioma cell proliferation in vitro and in vivo via enhanced beta-catenin/TCF-4 transcription. Neuro Oncol 2014, 16 (2), 217−27. (9) Hsu, Y. C.; Chern, J. J.; Cai, Y.; Liu, M.; Choi, K. W. Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase. Nature 2007, 445 (7129), 785−8. (10) Chen, K.; Huang, C.; Yuan, J.; Cheng, H.; Zhou, R. Long-term artificial selection reveals a role of TCTP in autophagy in mammalian cells. Mol. Biol. Evol. 2014, 31 (8), 2194−211. (11) Lucibello, M.; Gambacurta, A.; Zonfrillo, M.; Pierimarchi, P.; Serafino, A.; Rasi, G.; Rubartelli, A.; Garaci, E. TCTP is a critical survival factor that protects cancer cells from oxidative stress-induced cell-death. Exp. Cell Res. 2011, 317 (17), 2479−89. (12) Susini, L.; Besse, S.; Duflaut, D.; Lespagnol, A.; Beekman, C.; Fiucci, G.; Atkinson, A. R.; Busso, D.; Poussin, P.; Marine, J. C.; Martinou, J. C.; Cavarelli, J.; Moras, D.; Amson, R.; Telerman, A. TCTP protects from apoptotic cell death by antagonizing bax function. Cell Death Differ. 2008, 15 (8), 1211−20. (13) Tsai, M. J.; Yang-Yen, H. F.; Chiang, M. K.; Wang, M. J.; Wu, S. S.; Chen, S. H. TCTP Is Essential for beta-Cell Proliferation and Mass Expansion During Development and beta-Cell Adaptation in Response to Insulin Resistance. Endocrinology 2014, 155 (2), 392−404. (14) Kaarbo, M.; Storm, M. L.; Qu, S.; Waehre, H.; Risberg, B.; Danielsen, H. E.; Saatcioglu, F. TCTP is an androgen-regulated gene implicated in prostate cancer. PLoS One 2013, 8 (7), e69398. (15) Jeon, H. J.; You, S. Y.; Park, Y. S.; Chang, J. W.; Kim, J. S.; Oh, J. S. TCTP regulates spindle microtubule dynamics by stabilizing polar microtubules during mouse oocyte meiosis. Biochim. Biophys. Acta, Mol. Cell Res. 2016, 1863 (4), 630−7. (16) Jaglarz, M. K.; Bazile, F.; Laskowska, K.; Polanski, Z.; Chesnel, F.; Borsuk, E.; Kloc, M.; Kubiak, J. Z. Association of TCTP with centrosome and microtubules. Biochem. Res. Int. 2012, 2012, 541906. (17) Bazile, F.; Pascal, A.; Arnal, I.; Le Clainche, C.; Chesnel, F.; Kubiak, J. Z. Complex relationship between TCTP, microtubules and actin microfilaments regulates cell shape in normal and cancer cells. Carcinogenesis 2009, 30 (4), 555−565. (18) Chen, K.; Chen, S.; Huang, C.; Cheng, H.; Zhou, R. TCTP increases stability of hypoxia-inducible factor 1alpha by interaction with and degradation of the tumour suppressor VHL. Biol. Cell 2013, 105 (5), 208−18. (19) Rho, S. B.; Lee, J. H.; Park, M. S.; Byun, H. J.; Kang, S.; Seo, S. S.; Kim, J. Y.; Park, S. Y. Anti-apoptotic protein TCTP controls the stability of the tumor suppressor p53. FEBS Lett. 2011, 585 (1), 29− 35. (20) Burgess, A.; Labbe, J. C.; Vigneron, S.; Bonneaud, N.; Strub, J. M.; Van Dorsselaer, A.; Lorca, T.; Castro, A. Chfr interacts and I

DOI: 10.1021/acs.jproteome.6b00556 J. Proteome Res. XXXX, XXX, XXX−XXX

Article

Journal of Proteome Research (37) Nagano-Ito, M.; Ichikawa, S. Biological effects of Mammalian translationally controlled tumor protein (TCTP) on cell death, proliferation, and tumorigenesis. Biochem. Res. Int. 2012, 2012, 204960. (38) Liu, H.; Peng, H. W.; Cheng, Y. S.; Yuan, H. S.; Yang-Yen, H. F. Stabilization and enhancement of the antiapoptotic activity of mcl-1 by TCTP. Mol. Cell. Biol. 2005, 25 (8), 3117−26. (39) Zhang, D.; Li, F.; Weidner, D.; Mnjoyan, Z. H.; Fujise, K. Physical and functional interaction between myeloid cell leukemia 1 protein (MCL1) and fortilin - The potential role of MCL1 as a fortilin chaperone. J. Biol. Chem. 2002, 277 (40), 37430−37438. (40) Gnanasekar, M.; Dakshinamoorthy, G.; Ramaswamy, K. Translationally controlled tumor protein is a novel heat shock protein with chaperone-like activity. Biochem. Biophys. Res. Commun. 2009, 386 (2), 333−337. (41) Rho, S. B.; Lee, J. H.; Park, M. S.; Byun, H. J.; Kang, S.; Seo, S. S.; Kim, J. Y.; Park, S. Y. Anti-apoptotic protein TCTP controls the stability of the tumor suppressor p53. FEBS Lett. 2011, 585 (1), 29− 35. (42) Jung, J.; Kim, H. Y.; Kim, M.; Sohn, K.; Kim, M.; Lee, K. Translationally controlled tumor protein induces human breast epithelial cell transformation through the activation of Src. Oncogene 2011, 30 (19), 2264−2274. (43) Mi, H. Y.; Dong, Q.; Muruganujan, A.; Gaudet, P.; Lewis, S.; Thomas, P. D. PANTHER version 7: improved phylogenetic trees, orthologs and collaboration with the Gene Ontology Consortium. Nucleic Acids Res. 2010, 38, D204−D210. (44) Maere, S.; Heymans, K.; Kuiper, M. BiNGO: a Cytoscape plugin to assess overrepresentation of Gene Ontology categories in Biological Networks. Bioinformatics 2005, 21 (16), 3448−3449. (45) Franceschini, A.; Szklarczyk, D.; Frankild, S.; Kuhn, M.; Simonovic, M.; Roth, A.; Lin, J. Y.; Minguez, P.; Bork, P.; von Mering, C.; Jensen, L. J. STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 2013, 41 (D1), D808−D815. (46) Han, Y.; Wang, S. F.; Zhang, Z. P.; Ma, X. H.; Li, W.; Zhang, X. W.; Deng, J. Y.; Wei, H. P.; Li, Z. Y.; Zhang, X. E.; Cui, Z. Q. In vivo imaging of protein-protein and RNA-protein interactions using novel far-red fluorescence complementation systems. Nucleic Acids Res. 2014, 42, e103. (47) Kohno, K.; Izumi, H.; Uchiumi, T.; Ashizuka, M.; Kuwano, M. The pleiotropic functions of the Y-box-binding protein, YB-1. BioEssays 2003, 25 (7), 691−698. (48) Bussi, G.; Donadio, D.; Parrinello, M. Canonical sampling through velocity rescaling. J. Chem. Phys. 2007, 126 (1), 014101. (49) Van Der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. GROMACS: fast, flexible, and free. J. Comput. Chem. 2005, 26 (16), 1701−18. (50) Stocker, U.; van Gunsteren, W. F. Molecular dynamics simulation of hen egg white lysozyme: a test of the GROMOS96 force field against nuclear magnetic resonance data. Proteins: Struct., Funct., Genet. 2000, 40 (1), 145−53. (51) Price, D. J.; Brooks, C. L., 3rd A modified TIP3P water potential for simulation with Ewald summation. J. Chem. Phys. 2004, 121 (20), 10096−103. (52) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. A Smooth Particle Mesh Ewald Method. J. Chem. Phys. 1995, 103 (19), 8577−8593. (53) Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. LINCS: A linear constraint solver for molecular simulations. J. Comput. Chem. 1997, 18 (12), 1463−1472. (54) Miyamoto, S.; Kollman, P. A. Settle - an Analytical Version of the Shake and Rattle Algorithm for Rigid Water Models. J. Comput. Chem. 1992, 13 (8), 952−962. (55) Farrah, T.; Deutsch, E. W.; Omenn, G. S.; Campbell, D. S.; Sun, Z.; Bletz, J. A.; Mallick, P.; Katz, J. E.; Malmstrom, J.; Ossola, R.; Watts, J. D.; Lin, B. A. Y.; Zhang, H.; Moritz, R. L.; Aebersold, R. A High-Confidence Human Plasma Proteome Reference Set with Estimated Concentrations in PeptideAtlas. Mol. Cell. Proteomics 2011, 10 (9), M110.006353.

(56) Desiere, F.; Deutsch, E. W.; King, N. L.; Nesvizhskii, A. I.; Mallick, P.; Eng, J.; Chen, S.; Eddes, J.; Loevenich, S. N.; Aebersold, R. The PeptideAtlas project. Nucleic Acids Res. 2006, 34, D655−D658. (57) Yu, X.; Ivanic, J.; Memisevic, V.; Wallqvist, A.; Reifman, J. Categorizing biases in high-confidence high-throughput proteinprotein interaction data sets. Mol. Cell. Proteomics 2011, 10 (12), M111.012500. (58) von Mering, C.; Krause, R.; Snel, B.; Cornell, M.; Oliver, S. G.; Fields, S.; Bork, P. Comparative assessment of large-scale data sets of protein-protein interactions. Nature 2002, 417 (6887), 399−403. (59) Lievens, S.; Eyckerman, S.; Lemmens, I.; Tavernier, J. Largescale protein interactome mapping: strategies and opportunities. Expert Rev. Proteomics 2010, 7 (5), 679−90. (60) Itzhak, D. N.; Tyanova, S.; Cox, J.; Borner, G. H. Global, quantitative and dynamic mapping of protein subcellular localization. eLife 2016, 5, e16950. (61) Zhang, J.; de Toledo, S. M.; Pandey, B. N.; Guo, G. Z.; Pain, D.; Li, H.; Azzam, E. I. Role of the translationally controlled tumor protein in DNA damage sensing and repair. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (16), E926−E933. (62) Thaw, P.; Baxter, N. J.; Hounslow, A. M.; Price, C.; Waltho, J. P.; Craven, C. J. Structure of TCTP reveals unexpected relationship with guanine nucleotide-free chaperones. Nat. Struct. Biol. 2001, 8 (8), 701−704. (63) Susini, L.; Besse, S.; Duflaut, D.; Lespagnol, A.; Beekman, C.; Fiucci, G.; Atkinson, A. R.; Busso, D.; Poussin, P.; Marine, J. C.; Martinou, J. C.; Cavarelli, J.; Moras, D.; Amson, R.; Telerman, A. TCTP protects from apoptotic cell death by antagonizing bax function. Cell Death Differ. 2008, 15 (8), 1211−1220. (64) Amson, R.; Pece, S.; Marine, J. C.; Di Fiore, P. P.; Telerman, A. TPT1/ TCTP-regulated pathways in phenotypic reprogramming. Trends Cell Biol. 2013, 23 (1), 37−46. (65) Anderie, I.; Schmid, A. In vivo visualization of actin dynamics and actin interactions by BiFC. Cell Biol. Int. 2007, 31 (10), 1131− 1135. (66) Wang, W.; Lu, Y. J.; Stemmer, P. M.; Zhang, X. M.; Bi, Y. Y.; Yi, Z. P.; Chen, F. The proteomic investigation reveals interaction of mdig protein with the machinery of DNA double-strand break repair. Oncotarget 2015, 6 (29), 28269−28281. (67) Gullo, C.; Au, M.; Feng, G.; Teoh, G. The biology of Ku and its potential oncogenic role in cancer. Biochim. Biophys. Acta, Rev. Cancer 2006, 1765 (2), 223−234. (68) Petrasek, J.; Schwarzerova, K. Actin and microtubule cytoskeleton interactions. Curr. Opin. Plant Biol. 2009, 12 (6), 728− 734. (69) Gachet, Y.; Tournier, S.; Lee, M.; Lazaris-Karatzas, A.; Poulton, T.; Bommer, U. A. The growth-related, translationally controlled protein P23 has properties of a tubulin binding protein and associates transiently with microtubules during the cell cycle. J. Cell Sci. 1999, 112 (Pt 8), 1257−71. (70) Murphy, M. E. The HSP70 family and cancer. Carcinogenesis 2013, 34 (6), 1181−1188. (71) Daugaard, M.; Rohde, M.; Jaattela, M. The heat shock protein 70 family: Highly homologous proteins with overlapping and distinct functions. FEBS Lett. 2007, 581 (19), 3702−3710. (72) Liu, Y.; Liu, W.; Song, X. D.; Zuo, J. Effect of GRP75/mthsp70/ PBP74/mortalin overexpression on intracellular ATP level, mitochondrial membrane potential and ROS accumulation following glucose deprivation in PC12 cells. Mol. Cell. Biochem. 2005, 268 (1−2), 45−51. (73) Orsini, F.; Migliaccio, E.; Moroni, M.; Contursi, C.; Raker, V. A.; Piccini, D.; Martin-Padura, I.; Pelliccia, G.; Trinei, M.; Bono, M.; Puri, C.; Tacchetti, C.; Ferrini, M.; Mannucci, R.; Nicoletti, I.; Lanfrancone, L.; Giorgio, M.; Pelicci, P. G. The life span determinant p66Shc localizes to mitochondria where it associates with mitochondrial heat shock protein 70 and regulates trans-membrane potential. J. Biol. Chem. 2004, 279 (24), 25689−25695. (74) Oikawa, K.; Ohbayashi, T.; Mimura, J.; Fujii-Kuriyama, Y.; Teshima, S.; Rokutan, K.; Mukai, K.; Kuroda, M. Dioxin stimulates J

DOI: 10.1021/acs.jproteome.6b00556 J. Proteome Res. XXXX, XXX, XXX−XXX

Article

Journal of Proteome Research synthesis and secretion of IgE-dependent histamine-releasing factor. Biochem. Biophys. Res. Commun. 2002, 290 (3), 984−7. (75) Rupec, R. A.; Poujol, D.; Kaltschmidt, C.; Messer, G. Isolation of a hypoxia-induced cDNA with homology to the mammalian growthrelated protein p23. Oncol Res. 1998, 10 (2), 69−74. (76) Lucibello, M.; Gambacurta, A.; Zonfrillo, M.; Pierimarchi, P.; Serafino, A.; Rasi, G.; Rubartelli, A.; Garaci, E. TCTP is a critical survival factor that protects cancer cells from oxidative stress-induced cell-death. Exp. Cell Res. 2011, 317 (17), 2479−2489. (77) Neumann, C. A.; Cao, J. X.; Manevich, Y. Peroxiredoxin 1 and its role in cell signaling. Cell Cycle 2009, 8 (24), 4072−4078. (78) Stambolic, V.; Suzuki, A.; de la Pompa, J. L.; Brothers, G. M.; Mirtsos, C.; Sasaki, T.; Ruland, J.; Penninger, J. M.; Siderovski, D. P.; Mak, T. W. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 1998, 95 (1), 29−39. (79) Backman, S. A.; Ghazarian, D.; So, K.; Sanchez, O.; Wagner, K. U.; Hennighausen, L.; Suzuki, A.; Tsao, M. S.; Chapman, W. B.; Stambolic, V.; Mak, T. W. Early onset of neoplasia in the prostate and skin of mice with tissue-specific deletion of Pten. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (6), 1725−1730. (80) Stambolic, V.; Tsao, M. S.; Macpherson, D.; Suzuki, A.; Chapman, W. R.; Mak, T. W. High incidence of breast and endometrial neoplasia resembling human Cowden syndrome in pten(±) mice. Cancer Res. 2000, 60 (13), 3605−3611. (81) Skabkin, M. A.; Evdokimova, V.; Thomas, A. A. M.; Ovchinnikov, L. P. The major messenger ribonucleoprotein particle protein p50 (YB-1) promotes nucleic acid strand annealing. J. Biol. Chem. 2001, 276 (48), 44841−44847. (82) Izumi, H.; Imamura, T.; Nagatani, G.; Ise, T.; Murakami, T.; Uramoto, H.; Torigoe, T.; Ishiguchi, H.; Yoshida, Y.; Nomoto, M.; Okamoto, T.; Uchiumi, T.; Kuwano, M.; Funa, K.; Kohno, K. Y boxbinding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3 ′-> 5 ′ exonuclease activity. Nucleic Acids Res. 2001, 29 (5), 1200−1207. (83) Ladomery, M.; Sommerville, J. A Role for Y-Box Proteins in Cell-Proliferation. BioEssays 1995, 17 (1), 9−11. (84) Ohga, T.; Uchiumi, T.; Makino, Y.; Koike, K.; Wada, M.; Kuwano, M.; Kohno, K. Direct involvement of the Y-box binding protein YB-1 in genotoxic stress-induced activation of the human multidrug resistance 1 gene. J. Biol. Chem. 1998, 273 (11), 5997−6000. (85) Evdokimova, V. M.; Kovrigina, E. A.; Nashchekin, D. V.; Davydova, E. K.; Hershey, J. W. B.; Ovchinnikov, L. P. The major core protein of messenger ribonucleoprotein particles (p50) promotes initiation of protein biosynthesis in vitro. J. Biol. Chem. 1998, 273 (6), 3574−3581. (86) Sommerville, J. Activities of cold-shock domain proteins in translation control. BioEssays 1999, 21 (4), 319−325. (87) Song, Y. J.; Lee, H. YB1/p32, a nuclear Y-box binding protein 1, is a novel regulator of myoblast differentiation that interacts with Msx1 homeoprotein. Exp. Cell Res. 2010, 316 (4), 517−529. (88) Lu, Z. H.; Books, J. T.; Ley, T. J. YB-1 is important for late-stage embryonic development, optimal cellular stress responses, and the prevention of premature senescence. Mol. Cell. Biol. 2005, 25 (11), 4625−4637. (89) Matsumoto, K.; Wolffe, A. P. Gene regulation by Y-box proteins: coupling control of transcription and translation. Trends Cell Biol. 1998, 8 (8), 318−323. (90) Eliseeva, I. A.; Kim, E. R.; Guryanov, S. G.; Ovchinnikov, L. P.; Lyabin, D. N. Y-box-binding protein 1 (YB-1) and its functions. Biochemistry 2011, 76 (13), 1402−1433. (91) Dazert, E.; Hall, M. N. mTOR signaling in disease. Curr. Opin. Cell Biol. 2011, 23 (6), 744−755. (92) Astanehe, A.; Finkbeiner, M. R.; Hojabrpour, P.; To, K.; Fotovati, A.; Shadeo, A.; Stratford, A. L.; Lam, W. L.; Berquin, I. M.; Duronio, V.; Dunn, S. E. The transcriptional induction of PIK3CA in tumor cells is dependent on the oncoprotein Y-box binding protein-1. Oncogene 2009, 28 (25), 2406−2418. (93) Sutherland, B. W.; Kucab, J.; Wu, J.; Lee, C.; Cheang, M. C. U.; Yorida, E.; Turbin, D.; Dedhar, S.; Nelson, C.; Pollak, M.; Grimes, H.

L.; Miller, K.; Badve, S.; Huntsman, D.; Blake-Gilks, C.; Chen, M.; Pallen, C. J.; Dunn, S. E. Akt phosphorylates the Y-box binding protein 1 at Ser102 located in the cold shock domain and affects the anchorage-independent growth of breast cancer cells. Oncogene 2005, 24 (26), 4281−4292. (94) Basaki, Y.; Hosoi, F.; Oda, Y.; Fotovati, A.; Maruyama, Y.; Oie, S.; Ono, M.; Izumi, H.; Kohno, K.; Sakai, K.; Shimoyama, T.; Nishio, K.; Kuwano, M. Akt-dependent nuclear localization of Y-box-binding protein 1 in acquisition of malignant characteristics by human ovarian cancer cells. Oncogene 2007, 26 (19), 2736−2746. (95) Sinnberg, T.; Sauer, B.; Holm, P.; Spangler, B.; Kuphal, S.; Bosserhoff, A.; Schittek, B. MAPK and PI3K/AKT mediated YB-1 activation promotes melanoma cell proliferation which is counteracted by an autoregulatory loop. Experimental Dermatology 2012, 21 (4), 265−270. (96) Lee, C.; Dhillon, J.; Wang, M. Y. C.; Gao, Y. Y.; Hu, K. J.; Park, E.; Astanehe, A.; Hung, M. C.; Eirew, P.; Eaves, C. J.; Dunn, S. E. Targeting YB-1 in HER-2 Overexpressing Breast Cancer Cells Induces Apoptosis via the mTOR/STAT3 Pathway and Suppresses Tumor Growth in Mice. Cancer Res. 2008, 68 (21), 8661−8666. (97) Bommer, U. A.; Iadevaia, V.; Chen, J.; Knoch, B.; Engel, M.; Proud, C. G. Growth-factor dependent expression of the translationally controlled tumour protein TCTP is regulated through the PI3-K/ Akt/mTORC1 signalling pathway. Cell. Signalling 2015, 27 (8), 1557− 68. (98) Kobayashi, D.; Hirayama, M.; Komohara, Y.; Mizuguchi, S.; Wilson Morifuji, M.; Ihn, H.; Takeya, M.; Kuramochi, A.; Araki, N. Translationally controlled tumor protein is a novel biological target for neurofibromatosis type 1-associated tumors. J. Biol. Chem. 2014, 289 (38), 26314−26. (99) Covarrubias, A. J.; Aksoylar, H. I.; Horng, T. Control of macrophage metabolism and activation by mTOR and Akt signaling. Semin. Immunol. 2015, 27 (4), 286−296.

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