FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1997895136> ?p ?o ?g. }

• W1997895136 endingPage "1122" @default.
• W1997895136 startingPage "1105" @default.
• W1997895136 abstract "The process of nucleocytoplasmic shuttling is mediated by karyopherins. Dysregulated expression of karyopherins may trigger oncogenesis through aberrant distribution of cargo proteins. Karyopherin subunit alpha-2 (KPNA2) was previously identified as a potential biomarker for nonsmall cell lung cancer by integration of the cancer cell secretome and tissue transcriptome data sets. Knockdown of KPNA2 suppressed the proliferation and migration abilities of lung cancer cells. However, the precise molecular mechanisms underlying KPNA2 activity in cancer remain to be established. In the current study, we applied gene knockdown, subcellular fractionation, and stable isotope labeling by amino acids in cell culture-based quantitative proteomic strategies to systematically analyze the KPNA2-regulating protein profiles in an adenocarcinoma cell line. Interaction network analysis revealed that several KPNA2-regulating proteins are involved in the cell cycle, DNA metabolic process, cellular component movements and cell migration. Importantly, E2F1 was identified as a potential novel cargo of KPNA2 in the nuclear proteome. The mRNA levels of potential effectors of E2F1 measured using quantitative PCR indicated that E2F1 is one of the “master molecule” responses to KPNA2 knockdown. Immunofluorescence staining and immunoprecipitation assays disclosed co-localization and association between E2F1 and KPNA2. An in vitro protein binding assay further demonstrated that E2F1 interacts directly with KPNA2. Moreover, knockdown of KPNA2 led to subcellular redistribution of E2F1 in lung cancer cells. Our results collectively demonstrate the utility of quantitative proteomic approaches and provide a fundamental platform to further explore the biological roles of KPNA2 in nonsmall cell lung cancer. The process of nucleocytoplasmic shuttling is mediated by karyopherins. Dysregulated expression of karyopherins may trigger oncogenesis through aberrant distribution of cargo proteins. Karyopherin subunit alpha-2 (KPNA2) was previously identified as a potential biomarker for nonsmall cell lung cancer by integration of the cancer cell secretome and tissue transcriptome data sets. Knockdown of KPNA2 suppressed the proliferation and migration abilities of lung cancer cells. However, the precise molecular mechanisms underlying KPNA2 activity in cancer remain to be established. In the current study, we applied gene knockdown, subcellular fractionation, and stable isotope labeling by amino acids in cell culture-based quantitative proteomic strategies to systematically analyze the KPNA2-regulating protein profiles in an adenocarcinoma cell line. Interaction network analysis revealed that several KPNA2-regulating proteins are involved in the cell cycle, DNA metabolic process, cellular component movements and cell migration. Importantly, E2F1 was identified as a potential novel cargo of KPNA2 in the nuclear proteome. The mRNA levels of potential effectors of E2F1 measured using quantitative PCR indicated that E2F1 is one of the “master molecule” responses to KPNA2 knockdown. Immunofluorescence staining and immunoprecipitation assays disclosed co-localization and association between E2F1 and KPNA2. An in vitro protein binding assay further demonstrated that E2F1 interacts directly with KPNA2. Moreover, knockdown of KPNA2 led to subcellular redistribution of E2F1 in lung cancer cells. Our results collectively demonstrate the utility of quantitative proteomic approaches and provide a fundamental platform to further explore the biological roles of KPNA2 in nonsmall cell lung cancer. Transportation of proteins and RNAs into (import) and out of (export) the nucleus occurs through the nuclear pore complex and is a vital event in eukaryotic cells. Nucleocytoplasmic shuttling of the large complex (>40 kDa) is mediated by an evolutionarily conserved family of transport factors, designated karyopherins (1Radu A. Blobel G. Moore M.S. Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins.Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 1769-1773Crossref PubMed Scopus (383) Google Scholar). The family of karyopherins, including importins and exportins, share limited sequence identity (15–25%) but adopt similar conformations. In human cells, at least 22 importin β and 6 importin α proteins have been identified to date (2Macara I.G. Transport into and out of the nucleus.Microbiol Mol. Biol. Rev. 2001; 65 (table of contents): 570-594Crossref PubMed Scopus (739) Google Scholar, 3Chook Y.M. Blobel G. Karyopherins and nuclear import.Curr. Opin. Struct. Biol. 2001; 11: 703-715Crossref PubMed Scopus (421) Google Scholar). Karyopherins cannot be classified solely based on their cargo repertoires, because many of these proteins are targeted by several different members of the karyopherin family (4Jäkel S. Gölich D. Importin beta, transportin, RanBP5 and RanBP7 mediate nuclear import of ribosomal proteins in mammalian cells.EMBO J. 1998; 17: 4491-4502Crossref PubMed Scopus (421) Google Scholar, 5Mosammaparast N. Jackson K.R. Guo Y. Brame C.J. Shabanowitz J. Hunt D.F. Pemberton L.F. Nuclear import of histone H2A and H2B is mediated by a network of karyopherins.J. Cell Biol. 2001; 153: 251-262Crossref PubMed Scopus (131) Google Scholar, 6Mühlhüusser P. Muller E.C. Otto A. Kutay U. Multiple pathways contribute to nuclear import of core histones.EMBO Rep. 2001; 2: 690-696Crossref PubMed Scopus (117) Google Scholar). The most well-established mechanism of nucleocytoplasmic shuttling is the classical nuclear import pathway in which classical nuclear localization signal (cNLS) 1The abbreviations used are:cNLSclassical nuclear localization signalKPNA2Karyopherin subunit alpha-2NSCLCnonsmall cell lung cancerMRNMRE11/RAD50/NBNDSBdouble-strand breakSILACstable isotope labeling with amino acids in cell culture2D LC-MS/MStwo-dimensional LC-MS/MSLCliquid chromatographySCXstrong cation exchangeRP18reverse phase 18FDRfalse discovery rateRT-PCRreverse transcription polymerase chain reactionqPCRreal-time quantitative PCRPIpropidium iodide.1The abbreviations used are:cNLSclassical nuclear localization signalKPNA2Karyopherin subunit alpha-2NSCLCnonsmall cell lung cancerMRNMRE11/RAD50/NBNDSBdouble-strand breakSILACstable isotope labeling with amino acids in cell culture2D LC-MS/MStwo-dimensional LC-MS/MSLCliquid chromatographySCXstrong cation exchangeRP18reverse phase 18FDRfalse discovery rateRT-PCRreverse transcription polymerase chain reactionqPCRreal-time quantitative PCRPIpropidium iodide.-containing cargo proteins transported into the nucleus are recognized by importin α/importin β heterodimers (7Lange A. Mills R.E. Lange C.J. Stewart M. Devine S.E. Corbett A.H. Classical nuclear localization signals: definition, function, and interaction with importin alpha.J. Biol. Chem. 2007; 282: 5101-5105Abstract Full Text Full Text PDF PubMed Scopus (856) Google Scholar). All importin β family members contain an N-terminal Ran-GTP-binding motif and selectively bind nucleoporins of the nuclear pore complex, whereas unusually, importin β interacts indirectly with hundreds of different cNLS-containing proteins via an adaptor protein, importin α. Importin α also acts independently through direct binding to cargo proteins without the requirement for importin β (8Kotera I. Sekimoto T. Miyamoto Y. Saiwaki T. Nagoshi E. Sakagami H. Kondo H. Yoneda Y. Importin alpha transports CaMKIV to the nucleus without utilizing importin beta.EMBO J. 2005; 24: 942-951Crossref PubMed Scopus (78) Google Scholar). The import and export processes of nucleocytoplasmic shuttle proteins are complicated, and dysregulated expression of karyopherin may have oncogenic effects resulting from the unusual distribution of cargo proteins. For example, aberrant nucleocytoplasmic localization of tumor suppressor proteins, such as PTEN, WT1, Arf, and p53, is involved in the development of several human cancers (9Runnebaum I.B. Kieback D.G. Mobus V.J. Tong X.W. Kreienberg R. Subcellular localization of accumulated p53 in ovarian cancer cells.Gynecol. Oncol. 1996; 61: 266-271Abstract Full Text PDF PubMed Scopus (25) Google Scholar, 10Nakatsuka S. Oji Y. Horiuchi T. Kanda T. Kitagawa M. Takeuchi T. Kawano K. Kuwae Y. Yamauchi A. Okumura M. Kitamura Y. Oka Y. Kawase I. Sugiyama H. Aozasa K. Immunohistochemical detection of WT1 protein in a variety of cancer cells.Mod. Pathol. 2006; 19: 804-814Crossref PubMed Scopus (249) Google Scholar, 11Perren A. Komminoth P. Saremaslani P. Matter C. Feurer S. Lees J.A. Heitz P.U. Eng C. Mutation and expression analyses reveal differential subcellular compartmentalization of PTEN in endocrine pancreatic tumors compared to normal islet cells.Am. J. Pathol. 2000; 157: 1097-1103Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 12Colombo E. Martinelli P. Zamponi R. Shing D.C. Bonetti P. Luzi L. Volorio S. Bernard L. Pruneri G. Alcalay M. Pelicci P.G. Delocalization and destabilization of the Arf tumor suppressor by the leukemia-associated NPM mutant.Cancer Res. 2006; 66: 3044-3050Crossref PubMed Scopus (125) Google Scholar, 13Lu W. Pochampally R. Chen L. Traidej M. Wang Y. Chen J. Nuclear exclusion of p53 in a subset of tumors requires MDM2 function.Oncogene. 2000; 19: 232-240Crossref PubMed Scopus (69) Google Scholar, 14Flamini G. Curigliano G. Ratto C. Astone A. Ferretti G. Nucera P. Sofo L. Sgambato A. Boninsegna A. Crucitti F. Cittadini A. Prognostic significance of cytoplasmic p53 overexpression in colorectal cancer. An immunohistochemical analysis.Eur. J. Cancer. 1996; 32A: 802-806Abstract Full Text PDF PubMed Scopus (55) Google Scholar). classical nuclear localization signal Karyopherin subunit alpha-2 nonsmall cell lung cancer MRE11/RAD50/NBN double-strand break stable isotope labeling with amino acids in cell culture two-dimensional LC-MS/MS liquid chromatography strong cation exchange reverse phase 18 false discovery rate reverse transcription polymerase chain reaction real-time quantitative PCR propidium iodide. classical nuclear localization signal Karyopherin subunit alpha-2 nonsmall cell lung cancer MRE11/RAD50/NBN double-strand break stable isotope labeling with amino acids in cell culture two-dimensional LC-MS/MS liquid chromatography strong cation exchange reverse phase 18 false discovery rate reverse transcription polymerase chain reaction real-time quantitative PCR propidium iodide. Karyopherin subunit alpha-2 (KPNA2) belongs to the karyopherin family and delivers numerous cargo proteins to the nucleus, followed by translocation back to cytoplasmic compartments in a Ran-GTP-dependent manner (15Goldfarb D.S. Corbett A.H. Mason D.A. Harreman M.T. Adam S.A. Importin alpha: a multipurpose nuclear-transport receptor.Trends Cell Biol. 2004; 14: 505-514Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). One proposed hypothesis is that KPNA2 plays opposing roles in oncogenesis through modulation of proper subcellular localization of particular cargo proteins (16Teng S.C. Wu K.J. Tseng S.F. Wong C.W. Kao L. Importin KPNA2, NBS1, DNA repair and tumorigenesis.J. Mol. Histol. 2006; 37: 293-299Crossref PubMed Scopus (50) Google Scholar). For example, KPNA2 mediates the nuclear transport of NBN (also known as NBS1 or Nibrin), a component of the MRE11/RAD50/NBN (MRN) complex involved in double-strand break (DSB) repair, DNA recombination, cell cycle checkpoint control, and maintenance of DNA integrity and genomic stability. Nuclear NBN generally acts as a tumor suppressor protein (17Carney J.P. Maser R.S. Olivares H. Davis E.M. Le Beau M. Yates 3rd, J.R. Hays L. Morgan W.F. Petrini J.H. The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response.Cell. 1998; 93: 477-486Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar, 18Lee J.H. Paull T.T. ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.Science. 2005; 308: 551-554Crossref PubMed Scopus (1073) Google Scholar, 19Tauchi H. Kobayashi J. Morishima K. van Gent D.C. Shiraishi T. Verkaik N.S. vanHeems D. Ito E. Nakamura A. Sonoda E. Takata M. Takeda S. Matsuura S. Komatsu K. Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells.Nature. 2002; 420: 93-98Crossref PubMed Scopus (238) Google Scholar, 20Wu X. Ranganathan V. Weisman D.S. Heine W.F. Ciccone D.N. O'Neill T.B. Crick K.E. Pierce K.A. Lane W.S. Rathbun G. Livingston D.M. Weaver D.T. ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response.Nature. 2000; 405: 477-482Crossref PubMed Scopus (373) Google Scholar). Inhibition or blockage of the interactions between KPNA2 and NBN results in reduction of DSB repair, cell cycle checkpoint signaling and radiation-induced nuclear focus accumulation (21Tseng S.F. Chang C.Y. Wu K.J. Teng S.C. Importin KPNA2 is required for proper nuclear localization and multiple functions of NBS1.J. Biol. Chem. 2005; 280: 39594-39600Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), signifying inhibition of the tumor suppressor function of nuclear NBN, as it loses interactions with KPNA2. In addition, a recent study showed that cytoplasmic NBN plays an oncogenic role via binding and activation of the PI3-kinase/AKT pathway and promotes tumorigenesis (22Chen Y.C. Su Y.N. Chou P.C. Chiang W.C. Chang M.C. Wang L.S. Teng S.C. Wu K.J. Overexpression of NBS1 contributes to transformation through the activation of phosphatidylinositol 3-kinase/Akt.J. Biol. Chem. 2005; 280: 32505-32511Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Therefore, KPNA2 appears to be a major determinant of the subcellular localization and the biological functions of its cargo proteins, such as NBN. We previously identified and validated KPNA2 as a potential biomarker for nonsmall cell lung cancer (NSCLC) by integration of cancer cell secretome and tissue transcriptome data sets (23Wang C.I. Wang C.L. Wang C.W. Chen C.D. Wu C.C. Liang Y. Tsai Y.H. Chang Y.S. Yu J.S. Yu C.J. Importin subunit alpha-2 is identified as a potential biomarker for non-small cell lung cancer by integration of the cancer cell secretome and tissue transcriptome.Int. J. Cancer. 2011; 128: 2364-2372Crossref PubMed Scopus (101) Google Scholar). We detected KPNA2 overexpression in lung cancer tissues and showed that cancer cells with poor differentiation and high mitosis are independent determinants for nuclear KPNA2 expression in NSCLC. Data obtained from exogenous expression and KPNA2 knockdown experiments supported its involvement in cellular growth and motility of lung cancer cells. KPNA2 is also overexpressed in several cancer tissues, including breast cancer, esophageal squamous cell carcinoma, ovarian cancer, bladder cancer, and prostate cancer, and may be associated with tumor invasiveness (24Dahl E. Kristiansen G. Gottlob K. Klaman I. Ebner E. Hinzmann B. Hermann K. Pilarsky C. Dürst M. Klinkhammer-Schalke M. Blaszyk H. Knuechel R. Hartmann A. Rosenthal A. Wild P.J. Molecular profiling of laser-microdissected matched tumor and normal breast tissue identifies karyopherin alpha2 as a potential novel prognostic marker in breast cancer.Clin. Cancer Res. 2006; 12: 3950-3960Crossref PubMed Scopus (135) Google Scholar, 25Sakai M. Sohda M. Miyazaki T. Suzuki S. Sano A. Tanaka N. Inose T. Nakajima M. Kato H. Kuwano H. Significance of karyopherin-{alpha} 2 (KPNA2) expression in esophageal squamous cell carcinoma.Anticancer Res. 2010; 30: 851-856PubMed Google Scholar, 26Jensen J.B. Munksgaard P.P. Sørensen C.M. Fristrup N. Birkenkamp-Demtroder K. Ulhoi B.P. Jensen K.M. Orntoft T.F. Dyrskjot L. High expression of karyopherin-alpha2 defines poor prognosis in non-muscle-invasive bladder cancer and in patients with invasive bladder cancer undergoing radical cystectomy.Eur. Urol. 2011; 59: 841-848Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 27Zheng M. Tang L. Huang L. Ding H. Liao W.T. Zeng M.S. Wang H.Y. Overexpression of karyopherin-2 in epithelial ovarian cancer and correlation with poor prognosis.Obstet. Gynecol. 2010; 116: 884-891Crossref PubMed Scopus (68) Google Scholar, 28Mortezavi A. Hermanns T. Seifert H.H. Baumgartner M.K. Provenzano M. Sulser T. Burger M. Montani M. Ikenberg K. Hofstädter F. Hartmann A. Jaggi R. Moch H. Kristiansen G. Wild P.J. KPNA2 expression is an independent adverse predictor of biochemical recurrence after radical prostatectomy.Clin. Cancer Res. 2011; 17: 1111-1121Crossref PubMed Scopus (86) Google Scholar). Moreover, KPNA2 is possibly involved in the regulation of cell proliferation, differentiation, DNA repair, and migration (16Teng S.C. Wu K.J. Tseng S.F. Wong C.W. Kao L. Importin KPNA2, NBS1, DNA repair and tumorigenesis.J. Mol. Histol. 2006; 37: 293-299Crossref PubMed Scopus (50) Google Scholar, 23Wang C.I. Wang C.L. Wang C.W. Chen C.D. Wu C.C. Liang Y. Tsai Y.H. Chang Y.S. Yu J.S. Yu C.J. Importin subunit alpha-2 is identified as a potential biomarker for non-small cell lung cancer by integration of the cancer cell secretome and tissue transcriptome.Int. J. Cancer. 2011; 128: 2364-2372Crossref PubMed Scopus (101) Google Scholar, 29Umegaki N. Tamai K. Nakano H. Moritsugu R. Yamazaki T. Hanada K. Katayama I. Kaneda Y. Differential regulation of karyopherin alpha 2 expression by TGF-beta1 and IFN-gamma in normal human epidermal keratinocytes: evident contribution of KPNA2 for nuclear translocation of IRF-1.J. Invest. Dermatol. 2007; 127: 1456-1464Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 30Noetzel E. Rose M. Bornemann J. Gajewski M. Knüchel R. Dahl E. Nuclear transport receptor karyopherin-alpha2 promotes malignant breast cancer phenotypes in vitro.Oncogene. 2012; 31: 2101-2114Crossref PubMed Scopus (66) Google Scholar, 31Hall M.N. Griffin C.A. Simionescu A. Corbett A.H. Pavlath G.K. Distinct roles for classical nuclear import receptors in the growth of multinucleated muscle cells.Dev. Biol. 2011; 357: 248-258Crossref PubMed Scopus (26) Google Scholar). However, the molecular pathways regulated by KPNA2 in lung cancer are yet to be elucidated. Here, we applied stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic technology (32Zhu H. Pan S. Gu S. Bradbury E.M. Chen X. Amino acid residue specific stable isotope labeling for quantitative proteomics.Rapid Commun. Mass Spectrom. 2002; 16: 2115-2123Crossref PubMed Scopus (188) Google Scholar), in combination with gene knockdown and subcellular fractionation, to analyze KPNA2 siRNA-induced differentially expressed protein profiles in an adenocarcinoma cell line and explored the molecular mechanisms of KPNA2-mediated regulation in lung cancer. The CL1-5 human lung cancer cell line was derived from one man with poorly differentiated lung adenocarcinoma and kindly provided by Professor P.C. Yang (Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan). Cells were maintained in RPMI 1640 with 10% fetal bovine serum plus antibiotics at 37 °C at a humidified atmosphere of 95% air/5% CO2 (33Chu Y.W. Yang P.C. Yang S.C. Shyu Y.C. Hendrix M.J. Wu R. Wu C.W. Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line.Am. J. Respir. Cell Mol. Biol. 1997; 17: 353-360Crossref PubMed Scopus (375) Google Scholar). For SILAC experiments, CL1-5 cells were maintained in lysine-depleted RPMI 1640 (Invitrogen, Grand Island, NY) supplemented with 10% dialyzed fetal bovine serum (Invitrogen, Carlsbad, CA), and 0.1 mg/ml heavy [U-13C6]l-lysine (Invitrogen) or 0.1 mg/ml light l-lysine (Invitrogen). Every 3–4 days, cells were split and media replaced with the corresponding light or heavy labeling medium. In approximately six doubling times, cells achieved almost 100% incorporation of isotopic labeling amino acids and were subjected to small interfering RNA treatments. Gene knockdown of KPNA2 was performed as described previously (23Wang C.I. Wang C.L. Wang C.W. Chen C.D. Wu C.C. Liang Y. Tsai Y.H. Chang Y.S. Yu J.S. Yu C.J. Importin subunit alpha-2 is identified as a potential biomarker for non-small cell lung cancer by integration of the cancer cell secretome and tissue transcriptome.Int. J. Cancer. 2011; 128: 2364-2372Crossref PubMed Scopus (101) Google Scholar). Briefly, 19-nucleotide RNA duplexes for targeting human KPNA2 were synthesized and annealed by Dharmacon (Thermo Fisher Scientific, Lafayette, CO). In brief, CL1-5 cells cultured in stable isotope labeling medium were transfected with control siRNA (Heavy) and KPNA2 pooled siRNA (Light) (GAAAUGAGGCGUCGCAGAA, GAAGCUACGUGGACAAUGU, AAUCUUACCUGGACACUUU, GUAAAUUGGUCUGUUGAUG)), respectively using Lipofectamine RNAiMAX reagents (Invitrogen) according to the protocol provided by the manufacturer. At 48 h after transfection, cell lysates were prepared for Western blotting to determine gene knockdown efficacy. CL1-5 cells transfected with control siRNA or KPNA2 siRNA were mixed at an equal ratio (1:1) and subjected to a subcellular fractionation using the ProteoJETTM Cytoplasmic and Nuclear Protein Extraction Kit K0311 (Fermentas, Canada), according to the manufacturer's instructions. The efficacy of fractionation was determined via Western blotting using GAPDH as the cytosolic control and Lamin B as the nuclear control protein. In-solution digestion was performed with the protocols described below. Briefly, the protein mixture (100 μg) was dissolved in 50 mm NH4HCO3, reduced with 10 mm dithiothreitol (DTT) for 30 min at 56 °C and alkylated using 30 mm iodoacetamide in the dark for 45 min at room temperature. Next, 30 mm DTT was added to quench iodoacetamide for 30 min at room temperature. The solution was diluted fivefold with 25 mm NH4HCO3, and trypsin added at a ratio of 1:50 and digested at 37 °C overnight. Micro-columns packed with SOURCE 15RPC (30 μl bed volume) were regenerated with series of steps: 100% acetonitrile (ACN)/0.1% formic acid (FA), 80% ACN/0.1%FA, 50% ACN/0.1% FA, and final equilibration with 0.1%FA. Samples were acidified with 0.1% FA and desalted using the micro-column via brief centrifugation at 3000 rpm at room temperature for 2 min. Peptides bound to resin were eluted using 25% ACN/0.1%FA, 50% ACN/0.1%FA and 80% ACN/0.1%FA (three bed volumes each). Peptides prepared from in-solution digestion were separated and analyzed via the online two-dimensional liquid chromatography-mass spectrometry (2D LC-MS/MS) technique using a strong cation exchange (SCX) and reverse-phase 18 (RP18) nanoscale liquid chromatography system coupled with a LTQ-Orbitrap mass spectrometer (Thermo Fisher, San Jose, CA) (34Li Y. Yu J. Wang Y. Griffin N.M. Long F. Shore S. Oh P. Schnitzer J.E. Enhancing identifications of lipid-embedded proteins by mass spectrometry for improved mapping of endothelial plasma membranes in vivo.Mol. Cell. Proteomics. 2009; 8: 1219-1235Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Briefly, equal mixtures of SILAC peptides were injected into an in-house packed SCX column (Luna SCX, 5 μm, 0.5 × 150 mm, Phenomenex) and fractionated into 30 fractions using a 45 h continuous ammonium chloride gradient in the presence of 30% ACN and 0.1% FA. Each effluent of SCX fraction (1 μl/min) was continuously mixed with a stream of 0.1% FA/H2O (50 μl/min), and peptides were trapped on a RP column (Source 15 RPC, 0.5 × 5 mm, GE Healthcare) and separated using coupled BEH C18 chromatography (1.7 μm, 0.1 × 120 mm, Waters) with an acetonitrile gradient in 0.1% FA performed on a Dionex UltiMate 3000 nano LC system. MS/MS analysis was performed on a LTQ-Orbitrap mass spectrometer with a nanoelectrospray ion source (Proxeon Biosystems). Full-scan MS spectra (m/z 430–m/z 2000) were acquired in the Orbitrap mass analyzer at a resolution of 60,000 at m/z 400. The lock mass calibration feature was enabled to improve mass accuracy. The most intense ions (up to 12) with a minimal signal intensity of 20,000 were sequentially isolated for MS/MS fragmentation in the order of intensity of precursor peaks in the linear ion trap using collision-induced dissociation energy of 35%, Q activation at 0.25, activation time of 30 ms, and isolation width of 2.0. Targeted ions with m/z ± 30 ppm were selected for MS/MS once and dynamically excluded for 50s. All MS and MS/MS data were analyzed and processed with Quant.exe in the MaxQuant environment (version 1. 0. 13. 8), developed by Professor M. Mann for peptide identification and quantification analyses (35Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9154) Google Scholar). The top six of fragment ions per 100 Da of each MS/MS spectrum were extracted for a protein database search using the Mascot search engine (version 2.2.03, Matrix Science) against the concatenated Swiss-Prot v.56 human forward and reverse protein sequence data set with a set of common contaminant proteins (total 45500 entries). The search parameters were set as follows: carbamidomethylation (C) as the fixed modification, oxidation (M), N-acetyl (protein), and pyro-Glu/Gln (N-term) as variable modifications, 5 ppm for MS tolerance, 0.5 Da for MS/MS tolerance, and 2 for missing cleavage. The SILAC label (K) was set as either none, fixed or variable modifications, depending on whether the precursor ion was determined as light, heavy or uncertain label by the MS feature detection algorithm of MaxQuant, respectively, to generate three Mascot search results. The peptides and proteins identified in all search results were further analyzed with Identity.exe using following criteria: six for minimum peptide length, one for minimum unique peptides for the assigned protein. The posterior error probability of peptides identified in forward and reverse databases was used to rank and determine the false discovery rate (FDR) for statistical evaluation (35Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9154) Google Scholar, 36Cox J. Matic I. Hilger M. Nagaraj N. Selbach M. Olsen J.V. Mann M. A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics.Nat. Protoc. 2009; 4: 698-705Crossref PubMed Scopus (621) Google Scholar). First, peptide and protein identifications with FDR less than 1% were accepted. The peptides shared (not unique for leading proteins) between multiple leading proteins were assigned to one of them (the first one) as razor peptides. And then, proteins with at least two ratio counts generated from unique and razor peptides were considered as quantified proteins for further analysis (detailed information is summarized in supplemental Tables S3 and S4). The median value of SILAC ratios was calculated as protein abundance (KPNA2 siRNA/control siRNA ratio) to minimize the effects of outlier values. Finally, global median normalization was applied for recalculating protein abundance (normalized protein ratio) to reduce the system error from sample preparation in each experiment. After quantification analysis, differentially expressed proteins of interest were converted into gene symbols and uploaded into MetaCore Version 6.5 build 27009 (GeneGo, St. Joseph, MI) for biological network building. MetaCore consists of curated protein interaction networks based on manually annotated and regularly updated databases. The databases describe millions of relationships between proteins based on publications on proteins and small molecules. These relationships include direct protein interactions, transcriptional regulation, binding, enzyme-substrate interactions, and other structural or functional relationships. In the present study, we used a variant of the shortest paths algorithm in Analyze Networks to map the hypothetical networks of uploaded proteins by default settings. The relevant network maps are analyzed based on relative enrichment of the uploaded proteins and also relative saturation of networks with canonical pathways. In this workflow the networks are prioritized based on the number of fragments of canonical pathways on the networks. Total RNA was extracted from control siRNA and KPNA2 siRNA-transfected CL1-5 cells using TRIzol reagent (Invitrogen). Subsequently, cDNA was synthesized via reverse transcription polymerase chain reaction (RT-PCR) with the SuperscriptIIIkit (Invitrogen). Real-time quantitative PCR (qPCR) was performed on a 20-μl reaction mixture containing 750 nm forward and reverse primers, varying amounts of template and 1× Power SYBR Green reaction mix (Applied Biosystems, Foster City, CA). The primers used in this study were designed using Primer Express Software (Applied Biosystems) and the sequences listed in supplemental Table S1. SYBR Green fluorescence intensity was determined using the ABI PRISM 7500 detection system (Applied Biosystems), and the gene level normalized against that of the beta-actin control gene. Cells transfected with control siRNA or KPNA2 siRNA for 48 h were harvested by trypsinization. For cell cycle analysis, cells were fixed in 70% ice-cold ethanol comprising 2 mg/ml RNase for 30 min, and ultimately stained with propidium iodide (PI, 50 mg/ml) for 10 min. The fluorescence of PI in control siRNA or KPNA2 siRNA-transfected cells was determined using flow cytometric analysis (BectonDickinson FACScan SYSTEM, Mountain View, CA). We counted the percentage of cells in the sub-G0/G1, G0/G1, S and G2/M phases using CellQuestTM programs. For expression of E2F1 in mammalian and E. coli cells, the open reading frame of E2F1 was obtained from" @default.
• W1997895136 created "2016-06-24" @default.
• W1997895136 creator A5014847684 @default.
• W1997895136 creator A5022647839 @default.
• W1997895136 creator A5023583881 @default.
• W1997895136 creator A5031354828 @default.
• W1997895136 creator A5043228667 @default.
• W1997895136 creator A5055247770 @default.
• W1997895136 creator A5087598894 @default.
• W1997895136 date "2012-11-01" @default.
• W1997895136 modified "2023-10-08" @default.
• W1997895136 title "Quantitative Proteomics Reveals Regulation of Karyopherin Subunit Alpha-2 (KPNA2) and Its Potential Novel Cargo Proteins in Nonsmall Cell Lung Cancer" @default.
• W1997895136 cites W1255890544 @default.
• W1997895136 cites W1499561015 @default.
• W1997895136 cites W1633875338 @default.
• W1997895136 cites W1732244037 @default.
• W1997895136 cites W1966947732 @default.
• W1997895136 cites W1967301597 @default.
• W1997895136 cites W1968191899 @default.
• W1997895136 cites W1970471513 @default.
• W1997895136 cites W1972318805 @default.
• W1997895136 cites W1972455404 @default.
• W1997895136 cites W1974667981 @default.
• W1997895136 cites W1977039418 @default.
• W1997895136 cites W1977696113 @default.
• W1997895136 cites W1978353502 @default.
• W1997895136 cites W1978798894 @default.
• W1997895136 cites W1989700964 @default.
• W1997895136 cites W1991427702 @default.
• W1997895136 cites W1992366122 @default.
• W1997895136 cites W1996868718 @default.
• W1997895136 cites W2003592398 @default.
• W1997895136 cites W2005692005 @default.
• W1997895136 cites W2006195578 @default.
• W1997895136 cites W2009398566 @default.
• W1997895136 cites W2009440524 @default.
• W1997895136 cites W2009750102 @default.
• W1997895136 cites W2010631202 @default.
• W1997895136 cites W2011370737 @default.
• W1997895136 cites W2012966977 @default.
• W1997895136 cites W2013165066 @default.
• W1997895136 cites W2013561492 @default.
• W1997895136 cites W2017284082 @default.
• W1997895136 cites W2021384128 @default.
• W1997895136 cites W2046064533 @default.
• W1997895136 cites W2049485403 @default.
• W1997895136 cites W2050818286 @default.
• W1997895136 cites W2051494528 @default.
• W1997895136 cites W2055607178 @default.
• W1997895136 cites W2056724496 @default.
• W1997895136 cites W2059023802 @default.
• W1997895136 cites W2059238555 @default.
• W1997895136 cites W2060291259 @default.
• W1997895136 cites W2063632686 @default.
• W1997895136 cites W2067030969 @default.
• W1997895136 cites W2069562617 @default.
• W1997895136 cites W2071707853 @default.
• W1997895136 cites W2074817241 @default.
• W1997895136 cites W2074891103 @default.
• W1997895136 cites W2077260273 @default.
• W1997895136 cites W2080752012 @default.
• W1997895136 cites W2084089807 @default.
• W1997895136 cites W2085553911 @default.
• W1997895136 cites W2085648246 @default.
• W1997895136 cites W2087979658 @default.
• W1997895136 cites W2088992580 @default.
• W1997895136 cites W2090258042 @default.
• W1997895136 cites W2093159000 @default.
• W1997895136 cites W2095934571 @default.
• W1997895136 cites W2097565501 @default.
• W1997895136 cites W2099454327 @default.
• W1997895136 cites W2101087680 @default.
• W1997895136 cites W2101430080 @default.
• W1997895136 cites W2102934843 @default.
• W1997895136 cites W2103670184 @default.
• W1997895136 cites W2111312305 @default.
• W1997895136 cites W2114542812 @default.
• W1997895136 cites W2116960596 @default.
• W1997895136 cites W2119876924 @default.
• W1997895136 cites W2120365943 @default.
• W1997895136 cites W2123642928 @default.
• W1997895136 cites W2125824349 @default.
• W1997895136 cites W2140568660 @default.
• W1997895136 cites W2144718087 @default.
• W1997895136 cites W2150015686 @default.
• W1997895136 cites W2150627415 @default.
• W1997895136 cites W2151692695 @default.
• W1997895136 cites W2155148117 @default.
• W1997895136 cites W2156613325 @default.
• W1997895136 cites W2156961771 @default.
• W1997895136 cites W2158869723 @default.
• W1997895136 cites W2159099039 @default.
• W1997895136 cites W2159177482 @default.
• W1997895136 cites W2168300502 @default.
• W1997895136 cites W2170851763 @default.
• W1997895136 cites W2791873134 @default.
• W1997895136 doi "https://doi.org/10.1074/mcp.m111.016592" @default.
• W1997895136 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3494185" @default.

• next