Name: monitoring eif4f assembly by measuring eif4e-eif4g interaction in live cells
Uploaded: 2020-05-01T13:30:00.000+00:00
Duration: 8 min 47 s
Description: Watch this Scientific Journal Video about Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells at JoVE.com

Este trabalho foi apoiado pelo orçamento central do p53lab (BMSI, A*STAR) e da concessão VIP JCO (A*STAR).

A linha celular HEK293 foi utilizada para o protocolo e foi cultivada no Medium De Águia Modificada de Dulbecco, suplementada com soro bovino fetal de 10%, 2 mM L-glutamina e 100 penicilina/estreptomicina U/mL. As células foram cultivadas a 37 °C com 5% de CO2 em um ambiente umidificado.
1. Avaliação quantitativa da interrupção do complexo eIF4F via ensaio de complementação eIF4E:eIF4G604-646
<ol><li>Ensaio de complementação de cultura celular e transfecção transitória de eIF4E:eIF4G604-646 ensaio de complementação<ol>
<li>Use células recém-descongeladas com menos de 20 passagens para todos os experimentos. No primeiro dia, determine o número de célula usando um contador de células padrão e conte as células viáveis totais usando o método de exclusão azul Trypan13.</li>
		<li>Sementes 6-bem placas com 0.9 - 1.2 x 106 de células HEK 293 por poço em 2 mL de meio de crescimento padrão. NOTA: Para alcançar a melhor eficiência de transfecção dentro das linhas celulares indicadas acima, certifique-se de que as células banhadas estejam 70-90% confluentes no dia seguinte à semeadura.</li>
		<li>Na manhã do dia 2, células co-transfeito com SubA-eIF4E e eIF4G604-646-Plasmid SubB usando um reagente de transfecção à base de lipídio (ver Tabela de Materiais) conforme descrito abaixo.<ol>
<li>Diluir 9 μL de solução à base de lipossom em um tubo contendo 125 μL de meio soro reduzido sem vermelho fenol (ver Tabela de Materiais) para cada transfecção e incubar à temperatura ambiente por 5 minutos.</li>
			<li>Prepare a mistura mestre do DNA diluindo 3 μg de cada plasmídeo em 125 μL de meio sérico reduzido para cada tubo de transfecção.</li>
			<li>Adicione 12 μL de reagentes de melhorador ao tubo de mistura mestre de DNA, misture bem e adicione imediatamente o DNA: melhore a mistura mestre de um tubo de lipossomos diluídos em uma proporção 1:1. Incubar por 15 minutos em temperatura ambiente.</li>
			<li>Adicione o complexo DNA-lipídide a cada poço e incuba as células em uma incubadora de 37 °C com 5% de CO2 por 24 h.</li>
		</ol>
</li>
		<li>Na manhã do dia 3, enxágue cada poço com 1 mL de PBS.</li>
		<li>Remova o PBS e incuba as células em cada poço com 0,3 mL de trippsina por 5 min a 37 °C.</li>
		<li>Neutralizar a trippsina adicionando 2 mL do meio de soro reduzido sem vermelho fenol contendo 0% de FCS em cada poço e transfira células transfeminadas em um tubo de 15 mL. NOTA: O vermelho fenol pode interferir na atividade da luciferase; portanto, as células devem ser manuseadas a partir desta etapa em meio sem vermelho fenol.</li>
		<li>Gire as células por 5 min a 290 x g e aspire o meio. Resuspend as células em 2 mL do meio sérico reduzido sem vermelho fenol contendo 0% de FCS. Conte como descrito na etapa 1.1.</li>
		<li>Sementes transfeinou células HEK 293 em 96 placas bem opacas a uma densidade de 30.000 células por poço em 90 μL de médio sem vermelho fenol contendo 0% de FCS. Para obter 3 réplicas técnicas dentro do mesmo experimento para 3 compostos diferentes, semente 60 poços das placas com células excluindo os poços nas bordas.</li>
		<li>Imediatamente após a semeadura das células transfeinadas, adicione 10 μL de solução composta DMSO de 10% (ver passo 1.2).</li>
	</ol></li>
	<li>Preparação composta<ol>
<li>Prepare soluções de estoque composto de 1 mM dissolvendo o composto de juros em DMSO 100% (v/v). Para ter 3 réplicas para cada titulação composta, use 8 μL da solução de estoque composto de 1 mM. NOTA: O volume do composto de estoque de 1 mM utilizado é mais do que o necessário. Isso é feito para levar em conta os erros de pipetação, se houver.</li>
		<li>Realize uma diluição serial de 2 vezes da solução composta de estoque com 4 μL do estoque de 1 mM em 4 μL de 100% DMSO para cada ponto de titulação. NOTA: Para uma titulação composta completa, realize 9 diluições seriais do estoque inicial em 100% DMSO. Se vários compostos precisarem ser testados, use uma placa de 96 poços e uma pipeta multicanal para facilitar esta etapa e diluições subsequentes.</li>
		<li>Adicione 36 μL de água estéril de grau HPLC para cada ponto da diluição serial de 2 vezes para preparar um 40 μL de soluções de trabalho de 10x em 10% DMSO (v/v) (ou seja. 100 μM a 0,39 μM série de ações DMSO 10% levará a uma solução final de 10 μM a 0,039 μM, em 1% DMSO quando adicionado às células). Quanto ao controle de tratamento, também prepare uma solução de estoque 10% DMSO somente em água estéril de grau HPLC.</li>
		<li>Adicione 10 μL de soluções de trabalho de 10x às células na placa 96 bem opaca, a fim de produzir a concentração final pretendida com uma concentração de DMSO residual de 1% (v/v) em um volume total de 100 μL e incubar por 3h a 37 °C com 5% v/v de CO atmosférico2.</li>
	</ol></li>
	<li>Ensaio de complementação e viabilidade da luciferase<ol>
<li>Após 3h de drogagem, comece a preparar o reagente do substrato de luciferase combinando 1 volume de substrato com 19 volumes do reagente de diluição (veja as instruções do fabricante). NOTA: O ensaio de luciferase é realizado utilizando um kit comercialmente disponível (ver Tabela de Materiais).</li>
		<li>Use uma pipeta multicanal para adicionar imediatamente 25 μL de reagente de substrato.</li>
		<li>Agite a placa a 350 rpm por 50 min em um agitador orbital à temperatura ambiente.</li>
		<li>Avalie a luminescência usando um leitor de placas. Para isso, coloque o leitor de espelhos em luminescência e o filtro de emissão em 455. Use uma altura de medição de 6,5 mm com um tempo de medição de 1 s.</li>
		<li>Para avaliar a viabilidade celular, adicione 33 μL de reagente de ensaio de viabilidade e, em seguida, remendo a luminescência novamente após 15 minutos à temperatura ambiente, usando o leitor de placas.</li>
		<li>Avalie a luminescência com o leitor de placas definindo o leitor de espelhos na Luminescência e o filtro de emissão em 600 nm. Use uma altura de medição de 6,5 mm com um tempo de medição de 1 s. NOTA: A viabilidade é avaliada medindo o nível intracelular de ATP sobre a lise celular de acordo com as instruções do fabricante. Multiplexing é possível neste caso, uma vez que o ensaio de viabilidade usa uma luciferase diferente com um comprimento de onda de emissão diferente.</li>
		<li>Use os dados para determinar os valores IC50 de cada composto por curva encaixando os dados em uma equação de curva de ajuste de 4 parâmetros: Y = ((A-D)/(1+((x/C)^B)) + D Os valores numéricos D e A devem ser restringidos no processo de montagem da curva: D = valor luminescente no eixo Y para assíntototo de curva mínima ou nível teórico mínimo de resposta esperado do ensaio de complementação eIF4E:4G. A = valor luminescente no eixo Y para assíntotototo de curva máxima ou resposta de nível teórico máximo esperada do ensaio de complementação eIF4E:4G. NOTA: O valor para a resposta mínima é derivado do tratamento de controle DMSO (v/v) de 1% e o valor para a resposta máxima é derivado da resposta máxima de um inibidor mTOR de alta afinidade que tem nivelado sem efeito sobre a viabilidade celular.</li>
	</ol></li>
</ol>2. Correlacionando eIF4E:eIF4G604-646 ensaio inibição com interrupção complexa eIF4F em células
<ol><li>Para confirmar que o sinal que está sendo medido pelo ensaio corresponde à interrupção física da interação eIF4E-eIF4G pelo composto, células de sementes como descrito na etapa 1.1.</li>
	<li>No dia seguinte, substitua o meio por 1 mL de meio de soro reduzido não contendo fenol vermelho e incubar por 4h com o composto de juros. Assegurar concentração de DMSO residual de 1% (v/v) em um volume total de 1 mL. NOTA: Para permitir facilmente uma correlação com o resultado, utilize concentrações compostas no início, ponto médio e ponto final da curva de titulação do ensaio de complementação medido.</li>
	<li>As células lyse e realizaram m7GTP puxar para baixo para isolar os complexos eIF4F e eIF4E:4EBP1. Procedimentos detalhados sobre como realizar o experimento de retirada m7GTP podem ser encontrados em outros lugares14.</li>
	<li>Detecte os níveis de proteína eIF4G, eIF4E e 4EBP1 vinculados a m7GTP por análise de manchas ocidentais e, em seguida, correlacionar-se com a interrupção do complexo eIF4F com a inibição do sinal de ensaio de complementação.</li>
</ol>

<ol>
	<li>Silvera, D., Formenti, S. C., Schneider, R. J. <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+cancer.">Translational control in cancer.</a> Nature Reviews Cancer. 10 (4), 254-266 (2010).</li><li>Gebauer, F., Hentze, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanism+of+translational+control.">Molecular Mechanism of translational control.</a> Nature Reviews Molecular Cell Biology. 10, 827-835 (2004).</li><li>Bhat, 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=Targeting+the+translation+machinery+in+cancer.">Targeting the translation machinery in cancer.</a> Nature Reviews Drug Discovery. 14 (4), 261-278 (2015).</li><li>Pelletier, J., Graff, J., Ruggero, 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=Targeting+the+eIF4F+translation+initiation+complex:+a+critical+nexus+for+cancer+development.">Targeting the eIF4F translation initiation complex: a critical nexus for cancer development.</a> Cancer Research. 75 (2), 250-263 (2015).</li><li>Montanaro, L., Pandolfi, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+mRNA+translation+in+oncogenesis:+the+role+of+eIF4E.">Initiation of mRNA translation in oncogenesis: the role of eIF4E.</a> Cell Cycle. 11, 1387-1389 (2004).</li><li>Merrick, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=eIF4E:+A+retrospective.">eIF4E: A retrospective.</a> Journal of Biological Chemistry. 290 (40), 24091-24099 (2015).</li><li>Marcotrigiano, J., Gingras, A. C., Sonenberg, N., Burley, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap-dependent+translation+initiation+in+eukaryotes+is+regulated+by+a+molecular+mimic+of+eIF4G.">Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G.</a> Molecular Cell. 3 (6), 707-716 (1999).</li><li>Umenaga, Y., Paku, K. S., In, Y., Ishida, T., Tomoo, K. <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+function+of+the+second+eIF4E-binding+region+in+N-terminal+domain+of+eIF4G:+comparison+with+eIF4E-binding+protein.">Identification and function of the second eIF4E-binding region in N-terminal domain of eIF4G: comparison with eIF4E-binding protein.</a> Biochemical and Biophysical Research Communication. 414 (3), 462-467 (2011).</li><li>Gr&#252;ner, 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=The+Structures+of+eIF4E-eIF4G+Complexes+Reveal+an+Extended+Interface+to+Regulate+Translation+Initiation.">The Structures of eIF4E-eIF4G Complexes Reveal an Extended Interface to Regulate Translation Initiation.</a> Molecular Cell. 64 (3), 467-479 (2016).</li><li>Gingras, A. C., 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=Hierarchical+phosphorylation+of+the+translation+inhibitor+4E-BP1.">Hierarchical phosphorylation of the translation inhibitor 4E-BP1.</a> Genes and Development. 15 (21), 2852-2864 (2001).</li><li>Dixon, A. 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=NanoLuc+Complementation+Reporter+Optimized+for+Accurate+Measurement+of+Protein+Interactions+in+Cells.">NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells.</a> ACS Chemical Biology. 11 (2), 400-408 (2016).</li><li>Sun, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTOR+kinase+inhibitors+as+potential+cancer+therapeutic+drugs.">mTOR kinase inhibitors as potential cancer therapeutic drugs.</a> Cancer Letters. 340 (1), 1-8 (2013).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+exclusion+test+of+cell+viability.">Trypan blue exclusion test of cell viability.</a> Current Protocols in Immunology. , (2001).</li><li>Sekiyama, 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=Molecular+mechanism+of+the+dual+activity+of+4EGI-1:+Dissociating+eIF4G+from+eIF4E+but+stabilizing+the+binding+of+unphosphorylated+4E-BP1.">Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1.</a> Proceedings of the National Academy of Science U. S. A. 112 (30), e4036-e4045 (2015).</li><li>Muller, D., 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=4E-BP+restrains+eIF4E+phosphorylation.">4E-BP restrains eIF4E phosphorylation.</a> Translation. 1 (2), e25819 (2013).</li><li>Frosi, Y., Usher, R., Lian, D. T. G., Lane, D. P., Brown, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+flux+in+signalling+pathways+through+measurements+of+4EBP1-mediated+eIF4F+complex+assembly.">Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly.</a> BMC Biology. (1), 40 (2019).</li><li>Ran, X., Gestwicki, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+protein-protein+interactions+(PPIs):+An+analysis+of+scaffold+choices+and+buried+surface+area.">Inhibitors of protein-protein interactions (PPIs): An analysis of scaffold choices and buried surface area.</a> Current Opinion in Chemical Biology. 44, 75-86 (2018).</li></ol>

Os autores não têm nada a revelar.

O método descrito neste artigo utiliza um ensaio de complementação baseado em luciferase para monitorar quantitativamente o conjunto complexo eIF4F através da medição direta da interação eIF4G-eIF4E em células vivas. Fornecemos detalhes para o uso do sistema de complementação eIF4E-eIF4G e também mostramos que o sistema é extremamente preciso na medição da dissociação mediada por medicamentos 4EBP1 da interação eIF4E-eIF4G16. Para facilitar o throughput deste ensaio, a configura...

O controle da expressão genética desempenha um papel fundamental na correta execução de programas celulares, como a proliferação do crescimento e a diferenciação. Um mecanismo de controle regulatório pode ser exercido tanto no nível da transcrição genética quanto no nível da tradução mRNA. Na última década, tornou-se cada vez mais evidente que o controle translacional pela modulação do processo de iniciação em vez das etapas posteriores de alongamento e término pode regular finamente a síntese de subconjuntos específicos de proteínas que desempenham uma ampla gama de funções biológicas.
O aumento da tradução das mRNAs envolvidas na sobrevivência, respostas anti-autofágicas e anti-apoptóticas foram implicadas em vários cânceres e também foram causadores mente ligadas à ativação aberrante ou à expressão dos fatores de iniciação da tradução1.
O complexo eIF4F é um regulador mestre da iniciação da tradução. Ao vincular a estrutura da tampa no final de 5' dos mRNAs, o eIF4F está impulsionando o recrutamento inicial mRNA-ribossomo e, por sua vez, aumentando a eficiência de tradução mRNA de mRNAs eucarióticas mal traduzidas2. A tradução mediada eIF4F de mRNAs relacionadas ao câncer tem sido relatada para muitos modelos de câncer que abrigam ativação aberrante de vias RAS/MAPK ou AKT/TOR, sugerindo que as células cancerígenas reregulam o eIF4F para impulsionar sua própria atividade pró-neoplásica. A interrupção deste loop de avanço alimentar inibindo a formação complexa eIF4F é, portanto, uma estratégia terapêutica muito promissora3,4.
O complexo eIF4F consiste em (i) eIF4E, a subunidade de ligação de tampa do eIF4F que interage com a estrutura da tampa encontrada no UTR de 5' de mRNA, (ii) eIF4A, o RNA helicase e (iii) eIF4G, a proteína do andaime que interage com o eIF4A e o eIF4E e que eventualmente recruta a subunidade ribossômica 40S5. a associação eIF4G com o eIF4E é o passo limitante de taxas para a montagem de complexos funcionais eIF4F e é regulada negativamente pelas proteínas de ligação eIF4E (4EBPs, membros 1, 2 e 3))6. Competindo com a vinculação eIF4G ao eIF4E através de uma interface que consiste em sequências de ligação eIF4E canônicas e canônicas7,8,9 (região que abrange aa 604-646 em eIF4E humano), o 4EBP reduz o pool de eIF4E ativamente envolvido na tradução e prevenção da formação do complexo eIF4F. A interação entre proteínas e proteínas é regulada principalmente pelo alvo mamífero da fosforilação mediada pela rapamicina (mTOR) de 4EBP. Após estímulos mitogênicos, o mTOR fosforila diretamente os membros da família de proteínas 4E-BP, diminuindo sua associação com o eIF4E e, assim, promovendo a interação eIF4E-eIF4G e a formação de complexos funcionais eIF4F10.
Apesar do grande esforço no desenvolvimento de compostos voltados para a integridade complexa eIF4F, a falta de ensaios que medem a interrupção direta da interação eIF4E-eIF4G em células vivas limitou a busca por compostos ativos celulares. Aplicamos um ensaio de luciferase baseado em um analógico coelenterazina (por exemplo, ensaio de complementação baseado em nanoluc) para monitorar em tempo real o status da integridade eIF4F através da interação eIF4E-eIF4G. O sistema de proteína de complementação da luciferase consiste em um fragmento de proteína de 18 kDa (SubA) e 11 fragmentos de peptídeos de aminoácidos (SubB) otimizados para auto-associação mínima e estabilidade11. Uma vez expressos como um produto de fusão com o eIF4E de comprimento total humano e o domínio de interação eIF4E do eiF4G1 humano (aa 604-646), as duas proteínas interativas trarão o fragmento SubA e SubB para uma proximidade entre si e induzirão a formação da luciferase ativa que, na presença de um substrato permeável celular, eventualmente gerará um sinal luminoso brilhante (Figura 1). Reportamos em outros lugares a construção e validação do sistema de complementação eIF4E:eIF4G604-646 16.
Aqui, descrevemos como o sistema de complementação eIF4E:eIF4G604-646 (disponível mediante solicitação) pode ser aplicado para medir com precisão a interrupção eIF4E-eIF4G mediada em células vivas. Além disso, demonstramos sua utilidade medindo os efeitos de vários inibidores de mTOR que estão atualmente sob testes clínicos como potenciais medicamentos terapêuticos de câncer12. Como os efeitos fora do alvo muitas vezes mascaram atividade específica de drogas, também descrevemos como a versatilidade da medição do sistema eIF4E:eIF4G604-646 pode ser estendida com medições ortogonais de viabilidade celular para levar isso em conta.

<table><tbody><tr><td>293FT cells</td><td>Thermo Fisher Scientific</td><td>R70007</td><td></td></tr><tr><td>Cell culture microplate 96 well, F-Bottom</td><td>greiner bio-one</td><td>655083</td><td></td></tr><tr><td>Cell titer Glo 2.0</td><td>PROMEGA</td><td>G9241</td><td></td></tr><tr><td>Envision Multilabel Reader</td><td>PerkinElmer</td><td>not applcable</td><td></td></tr><tr><td>Finnpipette F2 Multichannel Pipettes 12-channels 30-300 ml</td><td>Thermo Fisher Scientific</td><td>4662070</td><td></td></tr><tr><td>Finnpipette F2 Multichannel Pipettes 12-channels 5-50 ml</td><td>Thermo Fisher Scientific</td><td>4662050</td><td></td></tr><tr><td>FUGENE6</td><td>PROMEGA</td><td>E2692</td><td></td></tr><tr><td>Lipofectamine 3000</td><td>Thermo Fisher Scientific</td><td>&lt;a href=&quot;https://www.thermofisher.com/order/catalog/product/L3000015&quot; target=&quot;_blank&quot;&gt;L3000015&lt;/a&gt;</td><td></td></tr><tr><td>NanoBiT PPI Starter Systems</td><td>PROMEGA</td><td>N2014</td><td></td></tr><tr><td>Optimem I Reduced Serum Mediun, no phenol red</td><td>Thermo Fisher Scientific</td><td>11058021</td><td></td></tr><tr><td>Orbital shaker</td><td>Eppendorf</td><td>not appicable</td><td></td></tr><tr><td>&amp;gamma;-Aminophenyl-m&lt;sup&gt;7&lt;/sup&gt;GTP (C&lt;sub&gt;10&lt;/sub&gt;-spacer)-Agarose</td><td>Jena Bioscience</td><td>AC-155S</td><td></td></tr></tbody></table>

monitoring eif4f assembly by measuring eif4e-eif4g interaction in live cells

A formação do complexo eIF4F tem se mostrado o nó fundamental a jusante para a convergência de vias de sinalização que muitas vezes sofrem ativação oncogênica em humanos. O eIF4F é um complexo de vinculação de tampas envolvido na fase de recrutamento mRNA-ribossomo de iniciação de tradução. Em muitos modelos celulares e pré-clínicos de cânceres, a desregulamentação do eIF4F leva ao aumento da tradução de subconjuntos específicos de mRNA que estão envolvidos na proliferação e sobrevivência do câncer. eIF4F é um complexo hetero-trimérico construído a partir da subunidade de ligação de tampa eIF4E, do eIF4A helicase e da subunidade de andaimes eIF4G. Fundamental para a montagem de complexos eIF4F ativos é a interação proteína-proteína entre proteínas eIF4E e eIF4G. Neste artigo, descrevemos um protocolo para medir o conjunto eIF4F que monitora o status da interação eIF4E-eIF4G em células vivas. O ensaio de interação proteína-proteína baseado em células eIF4e:4G também permite que as alterações induzidas por medicamentos na integridade complexa eIF4F sejam avaliadas com precisão e confiabilidade. Prevemos que este método possa ser aplicado para verificar a atividade de compostos disponíveis comercialmente ou para uma triagem adicional de novos compostos ou modalidades que interrompam eficientemente a formação do complexo eIF4F.

Para validar a sensibilidade do sistema de complementação eIF4E:eIF4G604-646, a inibição mediada de 4EBP1 do conjunto complexo eIF4F foi avaliada por meio de inibidores mTOR. Ao inibir a fosforilação dependente mTORC1 quinase da família de proteínas 4EBP, a inibição do mTOR aumenta a associação 4EBP1 para eIF4E e, portanto, a desmontagem eIF4F15. Duas classes de inibidores mecanicamente diferentes de quinases mTOR foram testadas: rapalogs (po...

Watch this Scientific Journal Video about Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells at JoVE.com

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Aqui, apresentamos um protocolo para medir a interação eIF4E-eIF4G em células vivas que permitiria ao usuário avaliar a perturbação induzida por drogas da dinâmica complexa eIF4F em formatos de triagem.

Monitorando o conjunto eIF4F medindo a interação eIF4E-eIF4G em células vivas

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic ...

monitoring-eif4f-assembly-measuring-eif4e-eif4g-interaction-live

Research

JoVE Journal

Cancer Research

3.0K visualizações.  A*STAR (Agency for Science, Technology and Research). Aqui, apresentamos um protocolo para medir a interação eIF4E-eIF4G em células vivas que permitiria ao usuário avaliar a perturbação induzida por drogas da dinâmica complexa eIF4F em formatos de triagem.

Vídeo: Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

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.

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 ...

Genética

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Análise em tempo real do fator de transcrição, ligação, transcrição, tradução e volume de negócios para exibir eventos globais durante a ativação celular

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Um ensaio de imagem de molécula única in vitro para a análise de cinética de tradução dependente da tampa

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Bioquímica

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

Atividades de monitoramento quinases e fosfatases através do ciclo celular por raciométrica FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Biologia

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Quantificação de Efferocytosis por microscopia de fluorescência de célula única

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Imunologia e Infecção

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Identificação de parceiros de proteína Interagir utilizando Tandem purificação por afinidade

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET Microscopia para Monitoramento em Tempo Real de eventos de sinalização em células vivas utilizando biossensores unimolecular

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Identificação de parceiros de interação de proteínas em células de mamíferos utilizando SILAC-imunoprecipitação quantitativos Proteômica

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis como um modelo para identificar Tradução Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

Uma resolução alta Método de Monitoramento de Ativação fosforilação dependente de IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...