Name: analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions
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This work was supported by the Natural Sciences and Engineering Council of Canada and the Ontario Ministry of Research and Innovation.

1. Preparativos para Cultura de Células <ol><li> Comprar ações disponíveis no mercado de células humanas. NOTA: Este protocolo utiliza carcinoma colorectal HCT116 e células primárias humanas renais proximais tubulares epiteliais (HRPTEC). </li><li> Adicione 500 ml de meio de cultura de HCT116: de Dulbecco Modified Eagle Médium (DMEM), meio de glucose / alta suplementado com 7,5% de Soro Fetal Bovino (FBS) e 1% de penicilina / estreptomicina (P / S). </li><li> Adicione 500 ml de meio de cultura de HRPTEC...

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	<li>Holcik, M., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+in+stress+and+apoptosis.">Translational control in stress and apoptosis.</a> Nat Rev Mol Cell Biol. 6 (4), 318-327 (2005).</li><li>Sonenberg, N., Hinnebusch, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+translation+initiation+in+eukaryotes:+mechanisms+and+biological+targets.">Regulation of translation initiation in eukaryotes: mechanisms and biological targets.</a> Cell. 136 (4), 731-745 (2009).</li><li>Gingras, A. C., Raught, B., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=eIF4+initiation+factors:+effectors+of+mRNA+recruitment+to+ribosomes+and+regulators+of+translation.">eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation.</a> Annu Rev Biochem. 68, 913-963 (1999).</li><li>Weingarten-Gabbay, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+genetics.+Systematic+discovery+of+cap-independent+translation+sequences+in+human+and+viral+genomes.">Comparative genetics. Systematic discovery of cap-independent translation sequences in human and viral genomes.</a> Science. 351 (6270), (2016).</li><li>Andreev, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+and+glucose+deprivation+induces+widespread+alterations+in+mRNA+translation+within+20+minutes.">Oxygen and glucose deprivation induces widespread alterations in mRNA translation within 20 minutes.</a> Genome Biol. 16, 90 (2015).</li><li>Ho, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+Reprogramming+of+Translation+Efficiencies+on+Oxygen+Stimulus.">Systemic Reprogramming of Translation Efficiencies on Oxygen Stimulus.</a> Cell Rep. 14 (6), 1293-1300 (2016).</li><li>Shatsky, I. N., Dmitriev, S. E., Andreev, D. E., Terenin, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome-wide+studies+uncover+the+diversity+of+modes+of+mRNA+recruitment+to+eukaryotic+ribosomes.">Transcriptome-wide studies uncover the diversity of modes of mRNA recruitment to eukaryotic ribosomes.</a> Crit Rev Biochem Mol Biol. 49 (2), 164-177 (2014).</li><li>Ho, J. J., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cap+for+Every+Occasion:+Alternative+eIF4F+Complexes.">A Cap for Every Occasion: Alternative eIF4F Complexes.</a> Trends Biochem Sci. , (2016).</li><li>Lin, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PHAS-I+as+a+link+between+mitogen-activated+protein+kinase+and+translation+initiation.">PHAS-I as a link between mitogen-activated protein kinase and translation initiation.</a> Science. 266 (5185), 653-656 (1994).</li><li>Richter, J. D., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cap-dependent+translation+by+eIF4E+inhibitory+proteins.">Regulation of cap-dependent translation by eIF4E inhibitory proteins.</a> Nature. 433 (7025), 477-480 (2005).</li><li>Tee, A. R., Tee, J. A., Blenis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+interaction+of+the+mammalian+eIF4E-related+protein+4EHP+with+4E-BP1.">Characterizing the interaction of the mammalian eIF4E-related protein 4EHP with 4E-BP1.</a> FEBS Lett. 564 (1-2), 58-62 (2004).</li><li>Rom, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+4EHP,+a+novel+mammalian+eIF4E-related+cap-binding+protein.">Cloning and characterization of 4EHP, a novel mammalian eIF4E-related cap-binding protein.</a> J Biol Chem. 273 (21), 13104-13109 (1998).</li><li>Uniacke, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+oxygen-regulated+switch+in+the+protein+synthesis+machinery.">An oxygen-regulated switch in the protein synthesis machinery.</a> Nature. 486 (7401), 126-129 (2012).</li><li>Morita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+4EHP-GIGYF2+translational+repressor+complex+is+essential+for+mammalian+development.">A novel 4EHP-GIGYF2 translational repressor complex is essential for mammalian development.</a> Mol Cell Biol. 32 (17), 3585-3593 (2012).</li><li>Webb, N. R., Chari, R. V., DePillis, G., Kozarich, J. W., Rhoads, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+the+messenger+RNA+cap-binding+protein+using+a+new+affinity+medium.">Purification of the messenger RNA cap-binding protein using a new affinity medium.</a> Biochemistry. 23 (2), 177-181 (1984).</li><li>Kiriakidou, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+mRNA+m7G+cap+binding-like+motif+within+human+Ago2+represses+translation.">An mRNA m7G cap binding-like motif within human Ago2 represses translation.</a> Cell. 129 (6), 1141-1151 (2007).</li><li>Mazza, C., Segref, A., Mattaj, I. W., Cusack, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+induced+fit+recognition+of+an+m(7)GpppG+cap+analogue+by+the+human+nuclear+cap-binding+complex.">Large-scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap-binding complex.</a> EMBO J. 21 (20), 5548-5557 (2002).</li><li>Nojima, T., Hirose, T., Kimura, H., Hagiwara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+between+cap-binding+complex+and+RNA+export+factor+is+required+for+intronless+mRNA+export.">The interaction between cap-binding complex and RNA export factor is required for intronless mRNA export.</a> J Biol Chem. 282 (21), 15645-15651 (2007).</li><li>Pabis, M., Neufeld, N., Shav-Tal, Y., Neugebauer, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+properties+and+dynamic+localization+of+an+alternative+isoform+of+the+cap-binding+complex+subunit+CBP20.">Binding properties and dynamic localization of an alternative isoform of the cap-binding complex subunit CBP20.</a> Nucleus. 1 (5), 412-421 (2010).</li><li>Ruud, K. A., Kuhlow, C., Goss, D. J., Browning, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+novel+cap-binding+protein+from+Arabidopsis+thaliana.">Identification and characterization of a novel cap-binding protein from Arabidopsis thaliana.</a> J Biol Chem. 273 (17), 10325-10330 (1998).</li><li>Ptushkina, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+second+eIF4E+protein+in+Schizosaccharomyces+pombe+has+distinct+eIF4G-binding+properties.">A second eIF4E protein in Schizosaccharomyces pombe has distinct eIF4G-binding properties.</a> Nucleic Acids Res. 29 (22), 4561-4569 (2001).</li><li>Cho, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+paradigm+for+translational+control:+inhibition+via+5'-3'+mRNA+tethering+by+Bicoid+and+the+eIF4E+cognate+4EHP.">A new paradigm for translational control: inhibition via 5'-3' mRNA tethering by Bicoid and the eIF4E cognate 4EHP.</a> Cell. 121 (3), 411-423 (2005).</li><li>Dinkova, T. D., Keiper, B. D., Korneeva, N. L., Aamodt, E. J., Rhoads, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+of+a+small+subset+of+Caenorhabditis+elegans+mRNAs+is+dependent+on+a+specific+eukaryotic+translation+initiation+factor+4E+isoform.">Translation of a small subset of Caenorhabditis elegans mRNAs is dependent on a specific eukaryotic translation initiation factor 4E isoform.</a> Mol Cell Biol. 25 (1), 100-113 (2005).</li><li>Timpano, S., Uniacke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Cells+Cultured+Under+Physiological+Oxygen+Utilize+Two+Cap-binding+Proteins+to+Recruit+Distinct+mRNAs+for+Translation.">Human Cells Cultured Under Physiological Oxygen Utilize Two Cap-binding Proteins to Recruit Distinct mRNAs for Translation.</a> J Biol Chem. , (2016).</li><li>Dings, J., Meixensberger, J., Jager, A., Roosen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+experience+with+118+brain+tissue+oxygen+partial+pressure+catheter+probes.">Clinical experience with 118 brain tissue oxygen partial pressure catheter probes.</a> Neurosurgery. 43 (5), 1082-1095 (1998).</li><li>Le, Q. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+tumor+oxygenation+and+gene+expression+in+patients+with+early+stage+non-small+cell+lung+cancers.">An evaluation of tumor oxygenation and gene expression in patients with early stage non-small cell lung cancers.</a> Clin Cancer Res. 12 (5), 1507-1514 (2006).</li><li>Muller, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+desflurane+and+isoflurane+on+intestinal+tissue+oxygen+pressure+during+colorectal+surgery.">Effects of desflurane and isoflurane on intestinal tissue oxygen pressure during colorectal surgery.</a> Anaesthesia. 57 (2), 110-115 (2002).</li><li>Brooks, A. J., Eastwood, J., Beckingham, I. J., Girling, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+tissue+partial+pressure+of+oxygen+and+carbon+dioxide+during+partial+hepatectomy.">Liver tissue partial pressure of oxygen and carbon dioxide during partial hepatectomy.</a> Br J Anaesth. 92 (5), 735-737 (2004).</li><li>Muller, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renocortical+tissue+oxygen+pressure+measurements+in+patients+undergoing+living+donor+kidney+transplantation.">Renocortical tissue oxygen pressure measurements in patients undergoing living donor kidney transplantation.</a> Anesth Analg. 87 (2), 474-476 (1998).</li><li>Richardson, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+skeletal+muscle+intracellular+oxygenation:+the+impact+of+ambient+oxygen+availability.">Human skeletal muscle intracellular oxygenation: the impact of ambient oxygen availability.</a> J Physiol. 571 (Pt 2), 415-424 (2006).</li><li>Harrison, J. S., Rameshwar, P., Chang, V., Bandari, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+saturation+in+the+bone+marrow+of+healthy+volunteers.">Oxygen saturation in the bone marrow of healthy volunteers.</a> Blood. 99 (1), 394 (2002).</li><li>Gleadle, J., Ratcliffe, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hypoxia. , (2001).</li><li>Gluckman, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+reconstitution+in+a+patient+with+Fanconi's+anemia+by+means+of+umbilical-cord+blood+from+an+HLA-identical+sibling.">Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical-cord blood from an HLA-identical sibling.</a> N Engl J Med. 321 (17), 1174-1178 (1989).</li><li>Parrinello, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+sensitivity+severely+limits+the+replicative+lifespan+of+murine+fibroblasts.">Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts.</a> Nat Cell Biol. 5 (8), 741-747 (2003).</li><li>Jewell, U. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+HIF-1alpha+in+response+to+hypoxia+is+instantaneous.">Induction of HIF-1alpha in response to hypoxia is instantaneous.</a> FASEB J. 15 (7), 1312-1314 (2001).</li><li>Newby, D., Marks, L., Lyall, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissolved+oxygen+concentration+in+culture+medium:+assumptions+and+pitfalls.">Dissolved oxygen concentration in culture medium: assumptions and pitfalls.</a> Placenta. 26 (4), 353-357 (2005).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc Natl Acad Sci U S A. 76 (9), 4350-4354 (1979).</li><li>Haghighat, A., Mader, S., Pause, A., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repression+of+cap-dependent+translation+by+4E-binding+protein+1:+competition+with+p220+for+binding+to+eukaryotic+initiation+factor-4E.">Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E.</a> EMBO J. 14 (22), 5701-5709 (1995).</li><li>Pyronnet, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+eukaryotic+translation+initiation+factor+4G+(eIF4G)+recruits+mnk1+to+phosphorylate+eIF4E.">Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnk1 to phosphorylate eIF4E.</a> EMBO J. 18 (1), 270-279 (1999).</li><li>Okumura, F., Zou, W., Zhang, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ISG15+modification+of+the+eIF4E+cognate+4EHP+enhances+cap+structure-binding+activity+of+4EHP.">ISG15 modification of the eIF4E cognate 4EHP enhances cap structure-binding activity of 4EHP.</a> Genes Dev. 21 (3), 255-260 (2007).</li><li>Kedersha, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+ternary+complex+(eIF2-GTP-tRNA(i)(Met))-deficient+preinitiation+complexes+are+core+constituents+of+mammalian+stress+granules.">Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules.</a> Mol Biol Cell. 13 (1), 195-210 (2002).</li><li>Gu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+structure,+mechanism,+and+regulation+of+scavenger+mRNA+decapping+activity.">Insights into the structure, mechanism, and regulation of scavenger mRNA decapping activity.</a> Mol Cell. 14 (1), 67-80 (2004).</li><li>Szczepaniak, S. A., Zuberek, J., Darzynkiewicz, E., Kufel, J., Jemielity, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+resins+containing+enzymatically+resistant+mRNA+cap+analogs--a+new+tool+for+the+analysis+of+cap-binding+proteins.">Affinity resins containing enzymatically resistant mRNA cap analogs--a new tool for the analysis of cap-binding proteins.</a> RNA. 18 (7), 1421-1432 (2012).</li><li>Joshi, B., Cameron, A., Jagus, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mammalian+eIF4E-family+members.">Characterization of mammalian eIF4E-family members.</a> Eur J Biochem. 271 (11), 2189-2203 (2004).</li></ol>

A análise de proteínas de ligação de PAC em células humanas expostas a condições fisiológicas de oxigénio pode permitir a identificação de novos factores de iniciação de tradução regulada de oxigénio. A afinidade destes factores para a tampa 5 &#39;do mRNA ou de outras proteínas associadas a tampa pode ser medida pela força da sua associação a 7 m-GTP ligados esferas de agarose. Uma limitação desta técnica é que ele mede o potencial de ligação a tampa de proteínas pós-lise, mas é ...

Controle de translação está a emergir como um passo igualmente importante para a regulação da transcrição na expressão do gene, especialmente durante períodos de estresse celular 1. Um ponto focal de controlo de tradução é para o passo limitante da velocidade de início em que os primeiros passos da síntese de proteínas envolve a ligação do factor eucariótico de iniciação 4E (eIF4E) para o 7-metilguanosina (m 7 GTP) 5 &#39;tampão de mRNAs 2 . eIF4E é parte de um complexo trimérico chamado eIF4F que inclui elF4A, uma helicase de ARN, e eIF4G, uma proteína andaime necessário para o recrutamento de outros factores de tradução e os ribossomas 40S 3. Sob condições fisiológicas normais, a grande maioria dos mRNAs são traduzidas por um mecanismo dependente da tampa, mas sob períodos de stress celular, aproximadamente 10% de mRNAs humanos contêm 5 &#39;UTRs que podem permitir a tradução independente de cap iniciação 1,4. tradução dependente de Cap tem sido historicamente sinônimoous com eIF4F, no entanto, variações específicas de estresse dos eIF4F tornaram-se um trending topic 5-8. Várias tensões celulares causar actividade eIF4E a ser reprimido através do alvo de mamífero do complexo rapamicina 1 (mTORC1). Esta quinase torna-se prejudicada sob estresse, o que resulta no aumento da atividade de um dos seus alvos, proteína do 4E de ligação (4E-BP). Não fosforilada 4E-BP liga-se a eIF4E e bloqueia a sua capacidade de interagir com eIF4G fazendo com que a repressão da tradução dependente de tampão 9,10. Curiosamente, um homólogo de eIF4E chamado eIF4E2 (ou 4EHP) tem uma afinidade muito menor para 4E-BP 11, permitindo que ele talvez para evadir a repressão mediada por stress. Com efeito, caracterizada inicialmente como um repressor da tradução devido à sua falta de interacção com eIF4G 12, eIF4E2 inicia a tradução de centenas de ARNm que contêm elementos de resposta a hipoxia de ARN na sua UTR 3 &#39;durante o stress hipóxico 6,13. Este i activaçãos alcançado através de interações com ligação eIF4G3, RNA motivo de proteína 4 e 2α o fator inducible hipoxia (HIF) para constituir um complexo eIF4F hipóxica ou eIF4F H 6,13. Como um repressor em condições normais, se liga com eIF4E2 GIGYF2 e ZNF598 14. Estes complexos foram, em parte, identificados através de m 7 resinas GTP de afinidade de agarose ligadas. Este método clássico 15 é padrão na área de tradução e é os ensaios de melhor e mais comumente utilizado técnica para isolar complexos de ligação de PAC em puxar para baixo e in vitro de ligação 16-19. À medida que a maquinaria de tradução dependente de tampão está a emergir como flexível e adaptável com partes inter- mudando 6-8,13, este método é uma ferramenta poderosa para identificar rapidamente novas proteínas de ligação ao tampão envolvidas na resposta ao stress. Além disso, as variações na eIF4F poderia ter implicações amplas como vários sistemas modelo eucarióticos parecem usar um homólogo eIF4E2 para as respostas ao estresse taiscomo A. thaliana 20, S. Pombe 21, 22 de D. melanogaster e C. elegans 23. As evidências sugerem que variações no eIF4F pode não ser estritamente limitado a condições de estresse, mas estar envolvidos na fisiologia normal 24. O fornecimento de oxigénio para os tecidos (em extremidades capilares) ou dentro dos tecidos (medida através de microeléctrodos) varia de 2-6% no cérebro 25, 3-12% nos pulmões 26, 3,5-6% no intestino 27, 4% em o fígado 28, 7-12% no rim 29, 4% no músculo 30, e 6-7% na medula óssea 31. Células e mitocôndrias contêm menos de 1,3% de oxigênio 32. Estes valores são muito mais próximos à hipoxia do que a do ar ambiente, quando as células são cultivadas por rotina. Isto sugere que o que foram previamente pensado como processos celulares específicas de hipóxia pode ser relevante em um ambiente fisiológico. Curiosamente, eIF4F e H eIF4F </sup&gt; participar activamente na iniciação da tradução de piscinas distintas ou classes de mRNAs em várias linhas de células humanas diferentes expostas ao oxigénio fisiológica ou &quot;physioxia&quot; 24. Baixo oxigênio também impulsiona o desenvolvimento fetal adequado 33 e células geralmente têm taxas mais elevadas de proliferação, a expectativa de vida mais longos, menos danos ao DNA e menos respostas de estresse geral em physioxia 34. Portanto, é provável H eIF4F um factor-chave na expressão de genes selecionados em condições fisiológicas. Aqui, nós fornecemos um protocolo para células de cultura em condições de oxigénio fisiológicas fixos ou em um intervalo de flutuação dinâmica que é provável mais representativo do microambiente do tecido. Uma vantagem deste método é que as células são lisadas dentro da estação de trabalho hipóxia. Não é claro como muitas vezes a transição a partir da cultura de células hipóxicas à lise das células é realizada em outros protocolos. As células são muitas vezes removidos em primeiro lugar a partir de uma pequena incubadora ser hipoxiatona lise, mas esta exposição ao oxigénio poderia afectar vias bioquímicas como a resposta celular ao oxigénio é rápido (uma ou duas min) 35. Certas proteínas de ligação PAC necessitem de interações com uma segunda base ou pode hidrolisar o GTP m 7, portanto, alguns interagentes cap podem ser desperdiçada no processo de purificação. Agarose ligada aos análogos cap resistentes enzimaticamente pode ser substituído neste protocolo. Explorando a atividade e composição do eIF4F H e outras variações de eIF4F através do método descrito aqui vai lançar luz sobre os mecanismos de expressão genética intrincados que as células utilizam durante as condições fisiológicas ou respostas ao estresse.

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		<tr height="26">
			<td height="26" style="height:26px;width:380px;">&#947;-aminophenyl-m7GTP agarose C10-linked beads</td>
			<td style="width:151px;">Jena Bioscience</td>
			<td style="width:165px;">AC-1555</td>
			<td style="width:205px;">Agarose-linked m7GTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm culture dish</td>
			<td>Corning</td>
			<td>877222</td>
			<td>10-cm culture dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 mm culture dish</td>
			<td>Thermofisher</td>
			<td>130183</td>
			<td>15-cm culture dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AEBSF Hydrochloride</td>
			<td>ACROS Organics</td>
			<td>A0356829</td>
			<td>AEBSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose Beads</td>
			<td>Jena Bioscience&#160;</td>
			<td>AC-0015</td>
			<td>Agarose bead control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenol Blue</td>
			<td>Fisher</td>
			<td>BP112-25</td>
			<td>Component of SDS-PAGE loading buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL Centrifuge Tubes</td>
			<td>FroggaBio</td>
			<td>1210-00S</td>
			<td>Used to centrifuge small volumes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Conical Centrifuge Tubes</td>
			<td>Fisher</td>
			<td>1495970C</td>
			<td>Used in culturing primary cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Defined trypsin inhibitor</td>
			<td>Fisher</td>
			<td>R007100</td>
			<td>DTI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreital</td>
			<td>Fisher</td>
			<td>BP172-25</td>
			<td>DTT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epithelial cell medium (complete kit)</td>
			<td>ScienCell</td>
			<td>4101</td>
			<td>Includes serum and growth factor supplements)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td>Component of SDS-PAGE loading buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mM Guanosine 5&#39;-triphosphate, 1 mL</td>
			<td>Jena Bioscience</td>
			<td>272076-0251M</td>
			<td>GTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCT116 colorectal carcinoma</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>Human cancer cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human renal proximal tubular epithelial cells</td>
			<td>ATCC</td>
			<td>PCS-400-010</td>
			<td>HRPTEC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone DMEM/High Glucose</td>
			<td>GE Life Sciences</td>
			<td>SH30022.01</td>
			<td>Standard media for human cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone Penicillin-Streptomycin solution</td>
			<td>GE Life Sciences</td>
			<td>SV30010</td>
			<td>Antibiotic component of DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">H35 HypOxystation</td>
			<td>Hypoxygen</td>
			<td>N/A</td>
			<td>Hypoxia workstation</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Igepal CA-630</td>
			<td>MP Biomedicals</td>
			<td>2198596</td>
			<td>Detergent component of lysis buffer</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Monopotassium phosphate</td>
			<td>Fisher</td>
			<td>P288-500</td>
			<td>KH2PO4</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Potassium chloride</td>
			<td>Fisher</td>
			<td>P217-500</td>
			<td>KCl</td>
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		<tr height="25">
			<td height="25" style="height:25px;">Magnesium chloride</td>
			<td>Fisher</td>
			<td>M33-500</td>
			<td>MgCl2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Fisher</td>
			<td>BP358-10</td>
			<td>NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium fluoride</td>
			<td>Fisher</td>
			<td>5299-100</td>
			<td>NaF (phosphatase inhibitor component of lysis buffer)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Disodium phosphate</td>
			<td>Fisher</td>
			<td>5369-500</td>
			<td>Na2HPO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Grade Fetal Bovine Serum</td>
			<td>Seradigm</td>
			<td>1500-500</td>
			<td>FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail (100 x)</td>
			<td>Cell Signalling</td>
			<td>58715</td>
			<td>Component of lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Dodecyl Sulfate</td>
			<td>Fisher</td>
			<td>BP166-100</td>
			<td>SDS</td>
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		<tr height="26">
			<td height="26" style="height:26px;">Sodium Orthovanadate</td>
			<td>Sigma</td>
			<td>56508</td>
			<td>Na3VO4</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>Fisher</td>
			<td>BP152-5</td>
			<td>Component of buffers</td>
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		<tr height="21">
			<td height="21" style="height:21px;">0.05% Trypsin-EDTA (1x)</td>
			<td>Life Technologies</td>
			<td>2500-067</td>
			<td>Trypsin used to detach adherent cells</td>
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	</tbody>
</table>

analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Análise da Capacidade Cap-obrigatório em resposta a Oxygen de eIF4E e eIF4E2 em uma m 7 GTP de afinidade de coluna As Figuras 1 e 2 representam Western blot de m 7 GTP purificação por afinidade típica de duas importantes proteínas de ligação do tampão em resposta a flutuações de oxigênio em duas linhas de células humanas: células primárias humanas renais proxi...

Watch this Scientific Journal Video about Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions at JoVE.com

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

Análise de proteínas Cap-ligação em células humanas expostas a condições de oxigênio fisiológicos

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

The overall goal of this experiment is to capture cap-binding proteins in conditions of low oxygen. This method utilizes an agarose linked seven methyl-guanosine cap analog to investigate cap-binding factors and their interacting partners. This method will help answer key questions in the field of protein synthesis such as identifying novel oxygen regulated factors involved in cap dependent translation.The main advantage of this technique is that cells are kept in a hypoxia workstation up until the point of lysis. The implications of this technique extend towards cancer therapy because cancer cells within a tumor are exposed to very low levels of oxygen and translational control plays a key role in tumor progression. We first had the idea for this method when we read a recent publication from Corona's colleagues reporting that tissue oxygenation is actually closer to what we call hypoxia rather than the normaxia that cells are routinely cultured in.Begin this procedure will cell culture as described in the text protocol. After counting the cells using a hemocytometer feed 150 millimeter cultured dishes containing 15 milliliters of complete medium to obtain a cell density of 2500 to 5000 cells per square centimeter. Use two 150 millimeter dishes for each cap-binding assay.Place the seeded culture dishes in the incubator at 37 degrees celsius and a five percent carbon dioxide atmosphere. After the cells have incubated at least 24 hours place the cells into a hypoxia workstation for the desired time. Then, set the workstation to the appropriate physioxia.Pre-warm one X phosphate buffered saline, or PBS, and trypsin in a 37 degree celsius water bath for 30 minutes. Aliquot 980 microliters of lysis buffer into a 1.5 microliter centrifuge tube for each cap-binding assay being performed. 10 microliters of 100X protease inhibitor cocktail and 10 microliters of 4-benzenesulfonyl fluoride hydrocloride to the 980 microliters of lysis buffer on ice.Discard the media from each 150 millimeter dish in a waste container within the hypoxia workstation. Then, wash the cells with three to four milliliters of warm one X PBS and discard the liquid. Add one milliliter of warm 05 percent trypsin EDTA to each dish and let the dishes sit for two minutes or until the cells are no longer adhered to the plate.Using a pipette transfer the cells of one plate to a 1.5 milliliter micro-centrifuge tube. Repeat as needed so that each plate of cells is transferred to a separate 1.5 milliliter micro-centrifuge tube. Centrifuge the micro-centrifuge tubes at 6000 times g for 90 seconds to pellet the cells.Following centrifugation aspirate the trypsin using a pipette without disturbing the pellet. Wash the cells with warm one X PBS by gently pipetting 200 microliters of PBS into the tube just above the pellet. Then aspirate the PBS without disturbing the pellet.Next, pipette 500 microliters from the one milliliter prepared lysis buffer solution onto the first cellular pellet. Re-suspend the pellet entirely by pipetting up and down. Combine the re-suspended cells with the second cell pellet and re-suspend the second pellet by pipetting up and down.Repeat this process until all pellets have been combined and re-suspended for each sample. Next, combine the lysate with the remaining 500 microliters of lysis buffer in a 1.5 liter micro-centrifuge tube. Remove the samples from the hypoxia workstation.Lyse the cells using gentle agitation by rotating the samples at four degrees celsisus for 1.5 to two hours. Following incubation, centrifuge the lysed cells at 1200 times g for 15 minutes at four degrees celsius to remove cellular debris. Transfer the lysate to a new 1.5 milliliter micro-centrifuge tube and discard the pellet.Reserve a portion of the supernatant to be used as whole cell lysate input control for western blot analysis. To prepare for the cap-binding assay use scissors to remove the tip of the pipette to facilitate collection of the bead slurry. For each sample transfer 50 microliters of the blank agarose bead control slurry and 50 microliters of the m7GTP agarose C10-linked beads slurry to separate 1.5 milliliter micro-centrifuge tubes.Then, pellet the beads by centrifuging the slurry at 500 times g for 30 seconds. Remove the supernatant carefully and re-suspend the beads in 500 microliters of TBS. After repeating these steps pellet the beads at 500 times g for 30 seconds and remove the supernatant.Next, transfer the supernatant containing the lysate to the 1.5 milliliter micro-centrifuge tube containing the blank agarose beads. Incubate for 10 minutes at four degrees celsius with gentle agitation. After 10 minutes, pellet the blank agarose beads by centrifuging at 500 times g for 30 seconds.Following centrifugation transfer the lysate that doesn't bind to the beads to the 1.5 milliliter micro-centrifuge tube containing the m7GTP agarose C10-linked beads. Wash the blank agarose beads by re-suspending the beads in 500 microliters of lysis buffer, pelleting the beads by centrifuging at 500 time g for 30 seconds, and discarding the supernatant. Repeat this four times for a total of five washes.Re-suspend the blank agarose beads in one X SDS-PAGE sample buffer. And boil the beads for 90 seconds at 95 degrees celsius. Store the sample at 20 degrees celsius for future western blot analysis to observe weather the protein of interest binds non-specifically to the bead.Incubate the lysate with the m7GTP agarose C10-linked beads with gentle agitation for one hour at four degrees celsuis to capture cap-binding proteins. Following agitation, pellet the m7GTP agarose C10-linked beads by centrifuging at 500 times g for 30 seconds. After discarding the supernatant, wash the m7GTP agarose C10-linked beads as before.Re-suspend the beads in 600 microliters of lysis buffer and add GTP to a final concentration of one millimolar. Incubate the m7GTP agarose C10-linked beads plus one millimolar GTP with gentle agitation for one hour at four degrees celsius. This will dissociate proteins that non-specifically interact with m7GTP.Pellet the m7GTP agarose C10-linked beads by centrifuging at 500 time g for 30 seconds. Transfer the supernatant to a new 1.5 milliliter micro-centrifuge tube, and add 200 microliters of four X SDS-PAGE sample buffer. After washing the beads as before, re-suspend the beads in one X SDS-PAGE sample buffer and boil at 95 degrees celsius for 90 seconds.Shown here are representative western blots of a typical m7GTP affinity purification of two major cap-binding proteins in response to oxygen fluctuations in primary human renal proximal tubule epithelial cells. The cell lysates were exposed to eight percent, five percent, three percent, or one percent oxygen for 24 hours. The lanes include 10 percent of the whole cell lysate as the input, a GTP washed to measure the specificity for m7GTP, and the proteins bound to m7GTP beads after the GTP wash.Data from at least three independent experiments were quantified by Image J and expressed as relative density units relative to 10 percent input of the whole cell lysate. Results indicate that human cells cultured under physiological oxygen utilize two cap-binding proteins to recruit distinct mRNAs for translation. After watching this video you should have a good understanding of how to capture cap-binding proteins in low oxygen conditions.While attempting this procedure it's important to remember to keep cells in the hypoxia workstation up until the point of lysis, as oxygen can rapidly alter the biochemical properties of these translation initiation factors. Following this procedure other methods like polysome fractionation can be performed to answer additional questions such as which cap-binding proteins associate with active translation under conditions of low oxygen.

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