Este trabajo fue apoyado por el presupuesto básico del p53lab (BMSI, A*STAR) y la subvención VIP de JCO (A*STAR).

La línea celular HEK293 se utilizó para el protocolo y fue cultivada en el Medio águila modificada de Dulbecco complementado con 10% suero bovino fetal, 2 mM L-glutamina, y 100 penicilina U/mL/ estreptomicina. Las células se cultivaban a 37 °C con un 5% de CO2 en un ambiente humidificado.
1. Evaluación cuantitativa de la interrupción compleja del fei4F a través de eIF4E:eIF4G604-646 ensayo de complementación
<ol><li>Cultivo celular y transfección transitoria de eIF4E:eIF4G604-646 ensayo de complementación<ol>
<li>Utilice células recién descongeladas con menos de 20 pasajes para todos los experimentos. El día 1, determine el número de celda mediante un contador de celdas estándar y cuente las celdas viables totales mediante el método de exclusión azul Trypan13.</li>
		<li>Semillas 6-well placas con 0.9 - 1.2 x 106 de HEK 293 células por pozo en 2 mL de medio de crecimiento estándar. NOTA: Con el fin de lograr la mejor eficiencia de transfección dentro de las líneas celulares indicadas anteriormente, asegúrese de que las células chapadas sean 70-90% confluentes el día después de la siembra.</li>
		<li>En la mañana del día 2, co-transfectar células con SubA-eIF4E y eIF4G604-646-Plásmido SubB utilizando un reactivo de transfección basado en lípidos (ver Tabla de Materiales)como se describe a continuación.<ol>
<li>Diluir 9 μL de solución a base de liposomas en un tubo que contenga 125 μL de medio sérico reducido sin rojo fenol (ver Tabla de Materiales)para cada transfección e incubar a temperatura ambiente durante 5 minutos.</li>
			<li>Preparar la mezcla maestra de ADN diluyendo 3 μg de cada plásmido en 125 μL de medio sérico reducido para cada tubo de transfección.</li>
			<li>Añadir 12 μL de reactivos potenciadores al tubo de mezcla maestro del ADN, mezclar bien e inmediatamente añadir el ADN: mezcla maestra potenciador a cada tubo de liposomas diluidos en una relación 1:1. Incubar durante 15 min a temperatura ambiente.</li>
			<li>Añadir el complejo ADN-lípidos a cada pozo e incubar las células en una incubadora de 37 °C con 5% de CO2 para 24 h.</li>
		</ol>
</li>
		<li>En la mañana del día 3, enjuague cada pozo con 1 ml de PBS.</li>
		<li>Retire el PBS e incuba las células en cada pozo con 0,3 ml de trypsin durante 5 minutos a 37 °C.</li>
		<li>Neutralize la trippsina añadiendo 2 ml del medio sérico reducido sin rojo fenol que contenga un 0% de FCS en cada pozo y transfiera las células transfectadas a un tubo de 15 ml. NOTA: Phenol rojo puede interferir con la actividad luciferasa; por lo tanto, las células deben ser manejadas a partir de este paso hacia adelante en medio sin fenol rojo.</li>
		<li>Gire hacia abajo las células durante 5 minutos a 290 x g y aspire el medio. Resuspend las células en 2 ml del medio sérico reducido sin fenol rojo que contiene 0% FCS. Cuente como se describe en el paso 1.1.</li>
		<li>Células HEK 293 transfectadas por semillas en 96 placas opacas de pozo a una densidad de 30.000 células por pozo en 90 μL de medio sin fenol rojo que contenga un 0% de FCS. Con el fin de obtener 3 réplicas técnicas dentro del mismo experimento para 3 compuestos diferentes, sembrar 60 pozos de las placas con células excluyendo los pozos en los bordes.</li>
		<li>Inmediatamente después de sembrar las células transfectadas, agregue 10 μL de solución compuesta DMSO al 10% (ver paso 1.2).</li>
	</ol></li>
	<li>Preparación de compuestos<ol>
<li>Prepare soluciones de stock compuesto de 1 mM disolviendo el compuesto de interés en 100% DMSO (v/v). Para tener 3 réplicas para cada valoración compuesta, utilice 8 μL de la solución de stock compuesto de 1 mM. NOTA: El volumen de compuesto de stock de 1 mM utilizado es más de lo que se necesita. Esto se hace para tener en cuenta los errores de pipeteo si los hay.</li>
		<li>Realice una dilución serie de 2 veces la solución compuesta de stock con 4 μL de la caldo de 1 mM en 4 μL de 100% DMSO para cada punto de valoración. NOTA: Para una valoración compuesta completa, realice 9 diluciones en serie del stock inicial en 100% DMSO. Si es necesario probar varios compuestos, utilice una placa de pozo 96 y una pipeta multicanal para facilitar este paso y las diluciones posteriores.</li>
		<li>Añadir 36 μL de agua estéril de grado HPLC para cada punto de la dilución serie de 2 veces para preparar un 40 μL de soluciones de trabajo de 10x en un 10% de DMSO (v/v) (es decir. 100 μM a 0,39 μM 10% serie de stock DMSO dará lugar a una solución final de 10 μM a 0,039 μM, en 1% DMSO cuando se agrega a las células). En cuanto al control del tratamiento, también prepare una solución de stock dmso del 10% en agua estéril de grado HPLC.</li>
		<li>Añadir 10 μL de soluciones de trabajo de 10x a las células de la placa opaca de 96 pozos para producir la concentración final prevista con una concentración dmso residual del 1% (v/v) en un volumen total de 100 μL e incubar durante 3 h a 37 °C con un 5% de CO atmosférico v/v2.</li>
	</ol></li>
	<li>Ensayo de complementación y viabilidad luciferasa<ol>
<li>Después de 3 h de drogado, comience a preparar el reactivo de sustrato luciferasa combinando 1 volumen de sustrato con 19 volúmenes del reactivo de dilución (consulte las instrucciones del fabricante). NOTA: El ensayo luciferasa se realiza utilizando un kit disponible comercialmente (véase Tabla de materiales).</li>
		<li>Utilice una pipeta multicanal para añadir inmediatamente 25 μL de reactivo de sustrato.</li>
		<li>Agitar la placa a 350 rpm durante 50 minutos en un agitador orbital a temperatura ambiente.</li>
		<li>Evalúe la luminiscencia utilizando un lector de placas. Para ello, ajuste el lector de espejos sobre la luminiscencia y el filtro de emisión en 455. Utilice una altura de medición de 6,5 mm con un tiempo de medición de 1 s.</li>
		<li>Con el fin de asesar la viabilidad de la célula, añadir 33 μL de reactivo de ensayo de viabilidad y luego volver a medir la luminiscencia de nuevo después de 15 min a temperatura ambiente, utilizando el lector de placas.</li>
		<li>Evalúe la luminiscencia con el lector de placas estableciendo el lector de espejos en Luminiscencia y el filtro de emisión en 600 nm. Utilice una altura de medición de 6,5 mm con un tiempo de medición de 1 s. NOTA: La viabilidad se evalúa midiendo el nivel intracelular de ATP en la lisis celular según las instrucciones del fabricante. Multiplexación es posible en este caso ya que el ensayo de viabilidad utiliza una luciferasa diferente con una longitud de onda de emisión diferente.</li>
		<li>Utilice los datos para determinar los valores IC50 de cada compuesto ajustando los datos a una ecuación de curva de ajuste de 4 parámetros: Y = ((A-D)/(1+((x/C)^B)))) + D Los valores numéricos D y A deben restringirse en el proceso de ajuste de curva: D = valor luminiscente en el eje Y para un asimptoto de curva mínimo o un nivel teórico mínimo de respuesta esperado del ensayo de complementación eIF4E:4G. A = valor luminiscente en el eje Y para asímptoto de curva máxima o respuesta máxima de nivel teórico esperada del ensayo de complementación eIF4E:4G. NOTA: El valor de la respuesta mínima se deriva del tratamiento de control 1% DMSO (v/v) y el valor de la respuesta máxima se deriva de la respuesta máxima de un inhibidor de mTOR de alta afinidad que se ha estancado sin ningún efecto sobre la viabilidad celular.</li>
	</ol></li>
</ol>2. Correlación eIF4E:eIF4G604-646 inhibición del ensayo con interrupción del complejo eIF4F en las células
<ol><li>Para confirmar que la señal que está midiendo el ensayo corresponde a la interrupción física de la interacción eIF4E-eIF4G por el compuesto, células de semillas como se describe en el paso 1.1.</li>
	<li>Al día siguiente, sustituir el medio por 1 ml de medio sérico reducido que no contenga fenol rojo e incubar durante 4 h por el compuesto de interés. Asegurar una concentración residual de DMSO del 1% (v/v) en un volumen total de 1 mL. NOTA: Con el fin de permitir fácilmente una correlación con el resultado, utilice concentraciones compuestas al principio, punto medio y punto final de la curva de valoración del ensayo de complemento medido.</li>
	<li>Lilas y realizóm7GTP tirar hacia abajo para aislar los complejos eIF4F y eIF4E:4EBP1. Los procedimientos detallados sobre cómo realizar el m7GTP pull down experiment se pueden encontrar en otro lugar14.</li>
	<li>Detecte los nivelesdeproteína eIF4G, eIF4E y 4EBP1 vinculados a m7 GTP mediante el análisis de manchas occidentales y, a continuación, correlacione con la interrupción del complejo eIF4F con la inhibición de la señal de ensayo de complemento.</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>

Los autores no tienen nada que revelar.

El método descrito en este artículo utiliza un ensayo de complementación basado en luciferasa para monitorear cuantitativamente el montaje complejo eIF4F a través de la medición directa de la interacción eIF4G-eIF4E en células vivas. Hemos proporcionado detalles para el uso del sistema de complementación eIF4E-eIF4G y también hemos demostrado que el sistema es extremadamente preciso en la medición de la disociación mediada por 4EBP1 inducida por fármacos de la interacción eIF4E-eIF4G16</sup...

El control de la expresión génica desempeña un papel fundamental en la correcta ejecución de programas celulares como la proliferación del crecimiento y la diferenciación. Se puede ejercer un mecanismo de control reglamentario ya sea a nivel de transcripción genética o a nivel de traducción de ARNm. En la última década, se ha hecho cada vez más evidente que el control traslacional mediante la modulación del proceso de iniciación en lugar de los pasos posteriores de alargamiento y terminación puede regular finamente la síntesis de subconjuntos específicos de proteínas que desempeñan una amplia gama de funciones biológicas.
El aumento de la traducción de los MRNAs implicados en la supervivencia, las respuestas anti-autofágicas y anti-apoptóticas se ha implicado en varios tipos de cáncer y también se ha relacionado causalmente con la activación aberrante o con la expresión excesiva de los factores de iniciación a la traducción1.
El complejo eIF4F es un regulador maestro de la iniciación de la traducción. Al enlazar la estructura de la tapa en el extremo de 5' de losRNAs, eIF4F está impulsando la contratación inicial de ARNm-ribosoma y, a su vez, aumentando la eficiencia de traducción de ARNm de ARNM eucariota débilmente traducida2. Se ha notificado una traducción mediada por eIF4F de los mRNAs relacionados con el cáncer para muchos modelos de cáncer que albergan la activación aberrante de las vías RAS/MAPK o AKT/TOR, lo que sugiere que las células cancerosas upregulan eIF4F para impulsar su propia actividad pro-neoplástica. La interrupción de este bucle avance mediante la inhibición de la formación compleja eIF4F es por lo tanto una estrategia terapéutica muy prometedora3,4.
El complejo eIF4F consiste en (i) eIF4E, la subunidad de unión a la tapa de eIF4F que interactúa con la estructura de la tapa que se encuentra en el 5' UTR de ARNm, (ii) eIF4A, la helicasa de ARN y (iii) eIF4G, la proteína andamio que interactúa tanto con eIF4A como con eIF4E y que finalmente recluta el 40S ribosomal subu5nit. La asociación eIF4G con eIF4E es el paso limitante de velocidad para el montaje de complejos funcionales eIF4F y está regulada negativamente por las proteínas de unión eIF4E (4EBPs, miembros 1, 2 y 3))6. Al competir con el enlace eIF4G a eIF4E a través de una interfaz que consiste en secuencias de enlace eIF4E canónicas y no canónicas7,8,9 (región que abarca aa 604-646 en eIF4E humano), 4EBP reduce el grupo de eIF4E involucrado activamente en la traducción y prevención de la formación compleja eIF4F. La interacción entre estas interacciones proteína-proteína está regulada principalmente por el objetivo de los mamíferos de la fosforilación mediada por la rapamicina (mTOR) de 4EBP. Tras los estímulos mitogénicos, mTOR fosforila directamente a los miembros de la familia proteica 4E-BP, disminuyendo su asociación con eIF4E y, por lo tanto, promoviendo la interacción eIF4E-eIF4G y la formación de complejos funcionales eIF4F10.
A pesar del gran esfuerzo en el desarrollo de compuestos dirigidos a la integridad compleja eIF4F, la falta de ensayos que miden la interrupción directa de la interacción eIF4E-eIF4G en las células vivas ha limitado la búsqueda de compuestos de golpe activo celular. Hemos aplicado un ensayo luciferasa basado en un análogo de coelenterazina (por ejemplo, ensayo de complementación basado en Nanoluc) para monitorear en tiempo real el estado de integridad de eIF4F a través de la interacción eIF4E-eIF4G. El sistema de proteínas de complementación luciferasa consiste en un fragmento de proteína de 18 kDa (SubA) y 11 fragmentos de péptido de aminoácidos (SubB) optimizados para una mínima auto-asociación y estabilidad11. Una vez expresado como un producto de fusión con el eIF4E de longitud completa humana y el dominio de interacción eIF4E del eiF4G1 humano (aa 604-646), las dos proteínas que interactúan acercarán el fragmento SubA y SubB entre sí e inducirán la formación de la luciferasa activa que, en presencia de un sustrato permeable celular, eventualmente generará una señal luminiscente brillante(Figura 1). Hemos informado en otros lugares de la construcción y validación del sistema de complementación eIF4E:eIF4G604-646 16.
Aquí, describimos cómo el sistema de complementación eIF4E:eIF4G604-646 (disponible bajo petición) se puede aplicar para medir con precisión la interrupción eIF4E-eIF4G mediada por 4EBP1 en las células vivas. Además, demostramos su utilidad midiendo los efectos de varios inhibidores de mTOR que actualmente están bajo ensayos clínicos como posibles fármacos terapéuticos contra el cáncer12. Debido a que los efectos fuera del objetivo a menudo enmascaran la actividad específica de drogas, también describimos cómo la versatilidad de la medición del sistema eIF4E:eIF4G604-646 se puede ampliar con mediciones ortogonales de viabilidad celular para tenerlas en cuenta.

<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

Se ha demostrado que la formación del complejo eIF4F es el nodo descendente clave para la convergencia de vías de señalización que a menudo se someten a activación oncogénica en humanos. eIF4F es un complejo de unión a la tapa que participa en la fase de reclutamiento del ARNm-ribosoma de iniciación de traducción. En muchos modelos celulares y preclínicos de cáncer, la desregulación del eIF4F conduce a una mayor traducción de subconjuntos específicos de ARNm que están involucrados en la proliferación y supervivencia del cáncer. eIF4F es un complejo hetero-trimérico construido a partir de la subunidad de unión a la tapa eIF4E, el eIF4A helicaso y el subunit de andamios eIF4G. Fundamental para el montaje de complejos activos eIF4F es la interacción proteína-proteína entre las proteínas eIF4E y eIF4G. En este artículo, describimos un protocolo para medir el ensamblado eIF4F que supervisa el estado de la interacción eIF4E-eIF4G en celdas vivas. El ensayo de interacción proteína-proteína basado en células eIF4e:4G también permite evaluar con precisión y fiabilidad los cambios inducidos por fármacos en la integridad compleja del fei4F. Vislumbramos que este método puede aplicarse para verificar la actividad de compuestos disponibles comercialmente o para un mayor cribado de nuevos compuestos o modalidades que interrumpan eficientemente la formación del complejo eIF4F.

Con el fin de validar la sensibilidad del sistema de complementación eIF4E:eIF4G604-646, se evaluó la inhibición mediada de 4EBP1 del conjunto complejo eIF4F mediante inhibidores de mTOR. Al inhibir la fosforilación dependiente de la quinasa mTORC1 de la familia de proteínas 4EBP, la inhibición del mTOR mejora la asociación 4EBP1 a eIF4E y, por lo tanto, el desmontaje eIF4F15. Se probaron dos clases de inhibidor mecanicísticamente diferente de la...

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

Aquí, presentamos un protocolo para medir la interacción eIF4E-eIF4G en células vivas que permitiría al usuario evaluar la perturbación inducida por fármacos de dinámicas complejas eIF4F en formatos de cribado.

Monitorización del conjunto eIF4F midiendo la interacción eIF4E-eIF4G en 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 vistas.  A*STAR (Agency for Science, Technology and Research). Aquí, presentamos un protocolo para medir la interacción eIF4E-eIF4G en células vivas que permitiría al usuario evaluar la perturbación inducida por fármacos de dinámicas complejas eIF4F en formatos de cribado.

Video: 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.

Análisis de proteínas Cap-unión en células humanas expuestas a condiciones de oxígeno fisiológicas

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

Investigación

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.

Análisis en tiempo real del Factor de transcripción vinculante, transcripción, traducción y rotación para mostrar los eventos globales durante la activación 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.

Un ensayo de imágenes de molécula única in vitro para el análisis de cinética de traducción dependiente de la tapa

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.

Seguimiento de las actividades de quinasa y fosfatasa través del ciclo celular por Proporcional FRET

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

Biología

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.

Cuantificación de Efferocytosis por microscopía de fluorescencia unicelular

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

Inmunología e Infección

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.

Identificación de los socios interacción de proteínas en tándem de purificación de afinidad

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 Microscopía de Monitoreo en tiempo real de eventos de señalización en las células vivas Uso de biosensores unimoleculares

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.

Identificación de proteínas compañeros de interacción en células de mamífero utilizando SILAC-inmunoprecipitación cuantitativos Proteómica

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 modelo para identificar Traducción Deterioro

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.

Una alta resolución Método de Seguimiento activación fosforilación dependiente de IRF3

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