Interactome Analysis Reveals that C1QBP (complement component 1, q subcomponent binding protein) Is Associated with Cancer Cell Chemotaxis and Metastasis

• DOI: 10.1074/mcp.m113.029413
• Publication Date: 2013-08-08T04:55:14Z
• Authors: Xiaofang Zhang; Fei Zhang; Lin Guo; Yanping Wang; Peng Zhang; Ruirui Wang; Ning Zhang; Ruibing Chen
• Keywords: Interactome; Complement; Component (thermodynamics)

The complement component 1, q subcomponent binding protein (C1QBP/p32/HABP1) is a ubiquitously expressed and multicompartmental cellular involved in various biological processes. In order to further understand its functions, we conducted proteomics analysis of interactome this study. An improved sample preparation mass spectrometric identification strategy was developed combining high-speed centrifugation, formaldehyde labeling, two-dimensional reverse-phase liquid chromatography. Using approach, identified 187 interacting proteins constructed highly connected network for C1QBP. Moreover, explored the interaction between C1QBP kinase C ζ, key regulator cell polarity migration. results indicated that regulated activity ζ modulated EGF-induced cancer chemotaxis. addition, required breast metastasis severe combined immunodeficiency mouse model. Furthermore, observed be overexpressed tissues, expression level closely linked with distant TNM stages. summary, as novel may serve therapeutic target treatment. 1The abbreviations used are: C1QBPcomplement proteincoIPco-immunoprecipitationEGFepidermal growth factorGSK3βglycogen synthase 3 βLC-MS/MSliquid chromatography coupled tandem spectrometryMSmass spectrometryPRKCZprotein ζRP-RP-LCdouble chromatographySCIDsevere immunodeficiencyUPLCultrahigh-performance an acidic binds C1q designated gC1q receptor (1Ghebrehiwet B. Lim B.L. Peerschke E.I. Willis A.C. Reid K.B. Isolation, cDNA cloning, overexpression 33-kD surface glycoprotein globular "heads" C1q.J. Exp. Med. 1994; 179: 1809-1821Crossref PubMed Scopus (316) Google Scholar). Its mature form contains 74 282 amino acid residues generated by removal N-terminal mitochondria targeting sequence (1 73 residues) during post-translational processing. It mainly distributed (2Muta T. Kang D. Kitajima S. 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Methods. 3: 135-139Crossref study, discovered contamination removed efficiently step reduce presence proteins. established (RP-LC). studied complexes associated data show (PRKCZ), pivotal modulator Mechanistic revealed mediates PRKCZ membrane. high tissues immunohistochemical staining, pathological close link Dithiothreitol (DTT), iodoacetamide, urea, formaldehyde, deuterated sodium cyanoborohydride, monoclonal antibody against γ-tubulin, Flag, anti-Flag antibody-conjugated agarose beads were purchased Sigma; antibodies C1QBP, PRKCZ, actin, phosphor-cofilin (Ser3) obtained Santa Cruz Biotechnology (Santa Cruz, CA); phosphor-PRKCZ (Thr410), GSK3β, phosphor-GSK3β (Ser9), cofilin bought Signaling Technology (Danvers, MA). Lipofectamine 2000, BCA reagents, A, G Invitrogen (Grand Island, NY). Enhanced chemiluminescence reagents Pierce (Rockford, IL). Protease inhibitor mixture tablets Roche Diagnostics (Indianapolis, IN). Sequencing-grade modified trypsin Promega (Madison, WI). LC-MS-grade water acetonitrile Merck (Whitehouse Station, NJ). Micro-Boyden chambers Neuro Probe (Gaithersburg, MD). Fibronectin recombinant EGF R&D Systems (Minneapolis, MN). complex I assay kits MitoSciences Co. Ltd. (Eugene, OR). SCID mice Wei Tong Hua Experimental Animal (Beijing, China). All lines American Type Culture Collection. Cells cultured DMEM supplemented 10% fetal bovine serum 1% glutamine Pen-Strep solution 37 °C 5% CO2. C1QBP-Flag amplified PCR vector pcDNA 3.1. plasmids then transfected HEK293T 2000. fusion confirmed Western blotting both anti-C1QBP antibodies. For knockdown, two StealthTM siRNAs (#1: 5′-GAAGGCCCTTGTGTTGGACTGTCAT-3′; #2: 5′-ACTGGCGAGTCTGAATGGAAGGATA-3′) scrambled siRNA synthesized Invitrogen; clones, shRNA plasmid containing #2 Genechem (Montreal, Quebec). silencing, (5′-GAGGAAGTGAGAGACATGTGT-3′) Genescript (Piscataway, Two 10-mm dishes 80% 90% confluence lysed Triton X-100 buffer (40 mm Tris, 120 NaCl, X-100, NaF, Na3VO4). same amount empty control. centrifuged 15,000 × g 4 15 min, pellets removed. cleanup, either 50,000 h incubated BenzonaseTM h. total concentrations measured assay, following procedure: extracts first precleared beads. Next, pulled down beads, samples. eluted 6 m urea. samples reduced incubation 10 DTT alkylated darkness 40 iodoacetamide. alkylation quenched added final concentration 50 mm. urea exchanged 25 bicarbonate means centrifugation 3-kDa ultrafiltration devices (Millipore, Billerica, overnight allow digestion complete. experiment performed described previously Briefly, cyanoborohydride digest Samples labeled 0.2 formaldehyde. mixed peptides vortexed quench reaction, 2 NH4OH immediately dried SpeedVac. reconstituted before HPLC separation. injected onto system (Waters, Milford, MA) high-pH C18 column (Phenomenex Gemini C18, 150 2.1 mm, μm) flow rate μl/min. 40-min gradient 5%–45% B (buffer A: ammonium formate, pH 10; B: acetonitrile). Fractions collected every min 60 min. Collected fractions SpeedVac 20 μl 0.1% formic acid. 5 each 11 subjected nano-ultrahigh-performance (UPLC)-MS/MS. A nanoUPLC (Waters) separate tryptic peptides. loaded trap flushed mobile phase (0.1% H2O) μl/min being delivered (C18, 0.075 1.7 μm). 7%–45% acetonitrile) over 90 nano-electrospray ionization LTQ Orbitrap spectrometer (Thermo Electron Corp.). operated data-dependent mode initial Fourier transform scan recorded range m/z 350–2000 eight most abundant ions automatically selected collision-activated dissociation. spray voltage set 2.0 kV. normalized collision energy 35% MS/MS. Raw searched International Index database (release version 3.83) 93,289 entries SEQUEST algorithm embedded Discoverer 1.3 Software parameters search: ppm precursor error tolerance; Da fragment static modifications carbamidomethylation all cysteine residues, dimethylation (Δ28 Da), deuterated-formaldehyde (Δ32 Da) lysines N terminus; flexible modification oxidation methionine residues; one missed cleavage site allowed. reversed evaluate discovery rate, <0.05 filtering criterion only more considered, grouped treated one. Quantitation software Ratios control calculated based extract ion areas (n = 3). ratio ≥10 cutoff threshold. Gene ontology annotation information acquired through 1.3. Pathway DAVID. Known searching databases VisAnt. integrated Excel imported Cytoscape v2.8.3 visualization. plated 12-well plates sterile glass coverslips, allowed grow 24 h, starved serum-free medium least After stimulation ng/ml °C, fixed 4% paraformaldehyde PBS (pH 7.4) room temperature, permeabilized 0.2% triton/PBS blocked 3% BSA temperature. primary stained Alexa Fluor 488 546-conjugated DAPI nuclear staining. Coverslips counterstained DAPI, mounted ProLong® Gold antifade visualized confocal laser scanning microscopy (Leica TCS SP5). migrate 8 wound-healing paraformaldehyde, anti-PRKCZ, anti-Giantin, anti-γ-tubulin Co-localization efficiency Image J (NIH, Bethesda, total, images analyzed. micro-Boyden suspended (DMEM BSA, HEPES). 2.5 104 upper chambers, lower chambers. 10-μm polyvinyl pyrrolidone-free polycarbonate filter membrane placed humidified incubator, membranes stained. number migrating counted under light 400× (high-powered field). seeded six-well plate formed monolayer. straight scratch had made 10-μl pipette tip, minimum (containing 0.5% FBS) 37% incubator 0, 3, 6, 9, 12, Then wound distances microscopy. F-actin measurement Tsuboi (23Tsuboi Wiskott-Aldrich syndrome mammalian verprolins plays monocyte Immunol. 176: 6576-6585Crossref (34) 106 coverslips. simulated period time °C. Next Alexa-fluro 568 conjugated phalloidin measurement, bounded extracted methanol content microplate fluorescence reader excitation wavelength 578 nm emission 600 nm. Results content, where (F-actint − F-actint0)/F-actint0 (fluorescencet fluorescencet0)/fluorescencet0. experimental studies, shC1QBP (equal clone cells) 100 ml tail veins 6). nodes macroscopically after 7 weeks. lungs formalin paraffin. Serial sections analyzed H&E staining verify micrometastasis. animal accordance guidelines Institutional Care Use Committee. 145 formalin-fixed, paraffin-embedded invasive 17 hyperplasia lobular Department Breast Cancer, Tianjin Medical University Cancer Institute Hospital. use tissue approved Ethics Committee University. Immunohistochemical (24Zhang Guo Cao Hao mTOR Rictor interacts metastasis.Cancer 70: 9360-9370Crossref (108) 25Nesrine Sellami Doghri Rifi Raies Mezlini Testicular synovial sarcoma: case report.Cancer 2012; 9: 274-276PubMed deparaffinized xylene rehydrated graded series ethanol solution. antigen retrieval citrate buffer, anti-human HRP-conjugated antibody. addition 3,3-diaminobenzidine buffer. place negative controls. low scored semi-quantitatively experienced pathologists. statistical SPSS 17.0 software. relationships status clinical chi-squared test, p value less than 0.05 considered statistically significant. Typically, sample-preparation assays, debris precipitated (∼ min). wide fragments supernatant (Fig. 1A). decrease sensitivity detection. As shown Fig. 1B, extending duration greatly supernatant; however, certain remained. remaining decreased increasing speed, no g. We compared centrifugation—in case, added. 1C, decreased, not centrifugation. Incubation could increase (data shown). might degradation affect identification. High-speed 1D, affected BenzonaseTM, slightly nanoUPLC-MS/MS, 230 group, 267 143 3; 1E). cytoskeleton (i.e. tubulin, myosin). large ribosomal isoforms groups, contribute interfere detection actual Most these 1F). discussed above, when used, subsequent analysis. coverage identification, RP-RP-LC-MS/MS applied. offline separation 10. online comparison one-dimensional supplemental S2. result, ∼4-fold groups achieved LC-MS/MS LC-MS/MS. Howev