This work was supported by core budget from the p53lab (BMSI, A*STAR) and the JCO VIP grant (A*STAR).

HEK293 cell line was used for the protocol and was cultured in Dulbecco&#39;s Modified Eagle Medium supplemented with 10% Fetal Bovine Serum, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin. Cells were cultured at 37 &#176;C with 5% CO2 in a humidified environment.
1. Quantitative assessment of eIF4F complex disruption via eIF4E:eIF4G604-646 complementation assay
<ol>
	<li>Cell culture and transient transfection of eIF4E:eIF4G604-646 complementation assay
	<ol>
		<li>Use freshly thawed cells with less than 20 passages for all experiments. On day 1, determine the cell number using a standard cell counter and count the total viable cells using the Trypan blue exclusion method13.</li>
		<li>Seed 6-well plates with 0.9 - 1.2 x 106 of HEK 293 cells per well in 2 mL of standard growth medium. 
		NOTE: In order to achieve the best transfection efficiency within the cell lines indicated above, ensure that the plated cells are 70-90% confluent the day after seeding.</li>
		<li>On the morning of day 2, co-transfect cells with SubA-eIF4E and eIF4G604-646-SubB plasmid using a lipid-based transfection reagent (see Table of Materials) as described below.
		<ol>
			<li>Dilute 9 &#181;L of liposome-based solution in a tube containing 125 &#181;L of reduced serum medium without phenol red (see Table of Materials) for each transfection and incubate at room temperature for 5 min.</li>
			<li>Prepare master mix of DNA by diluting 3 &#181;g of each plasmid in 125 &#181;L of reduced serum medium for each transfection tube.</li>
			<li>Add 12 &#181;L of enhancer reagents to the DNA master mix tube, mix well and immediately add the DNA: enhancer master mix to each tube of diluted liposomes in a ratio 1:1. Incubate for 15 min at room temperature.</li>
			<li>Add the DNA-lipid complex to each well and incubate the cells in a 37 &#176;C incubator with 5% CO2 for 24 h.</li>
		</ol>
		</li>
		<li>On the morning of day 3, rinse each well with 1 mL of PBS.</li>
		<li>Remove the PBS and incubate cells in each well with 0.3 mL of trypsin for 5 min at 37 &#176;C.</li>
		<li>Neutralize trypsin by adding 2 mL of the reduced serum medium without phenol red containing 0% FCS in each well and transfer transfected cells into a 15 mL tube. 
		NOTE: Phenol red can interfere with the luciferase activity; therefore, cells must be handled from this step onward in medium without phenol red.</li>
		<li>Spin down cells for 5 min at 290 x g and aspirate the medium. Resuspend the cells in 2 mL of the reduced serum medium without phenol red containing 0% FCS. Count as described in step 1.1.</li>
		<li>Seed transfected HEK 293 cells in 96 well opaque plates at a density of 30,000 cells per well in 90 &#181;L of medium without phenol red containing 0% FCS. In order to obtain 3 technical replicates within the same experiment for 3 different compounds, seed 60 wells of the plates with cells excluding the wells on the edges.</li>
		<li>Immediately after seeding the transfected cells, add 10 &#181;L of 10% DMSO compound solution (see step 1.2).</li>
	</ol>
	</li>
	<li>Compound preparation
	<ol>
		<li>Prepare 1 mM compound stock solutions by dissolving the compound of interest in 100% DMSO (v/v). In order to have 3 replicates for each compound titration, use 8 &#181;L of the 1 mM compound stock solution. 
		NOTE: The volume of 1 mM stock compound used is more than what it is needed. This is done to take into account pipetting errors if any.</li>
		<li>Perform a 2-fold serial dilution of the stock compound solution with 4 &#181;L of the 1 mM stock into 4 &#181;L of 100% DMSO for each titration point. 
		NOTE: For a complete compound titration, perform 9 serial dilutions from the starting stock in 100% DMSO. If multiple compounds need to be tested, use a 96 well plate and a multichannel pipette to facilitate this step and subsequent dilutions.</li>
		<li>Add 36 &#181;L of HPLC grade sterile water for each point of the 2-fold serial dilution to prepare a 40 &#181;L of 10x working solutions in 10% DMSO (v/v) (i.e. 100 &#956;M to 0.39 &#956;M 10% DMSO stock series will lead to a final solution of 10 &#956;M to 0.039 &#956;M,in 1% DMSO when added to cells). As for treatment control, also prepare a 10% DMSO only stock solution in HPLC grade sterile water.</li>
		<li>Add 10 &#181;L of 10x working solutions to the cells in the 96 well opaque plate in order to yield the intended final concentration with a residual DMSO concentration of 1% (v/v) in a total volume of 100 &#181;L and incubate for 3 h at 37 &#176;C with 5% v/v atmospheric CO2.</li>
	</ol>
	</li>
	<li>Luciferase complementation and viability assay
	<ol>
		<li>After 3 h of drugging, start preparing the luciferase substrate reagent by combining 1 volume of substrate with 19 volumes of the dilution reagent (see manufacturer&#39;s instructions). 
		NOTE: The luciferase assay is performed using a commercially available kit (see Table of Materials).</li>
		<li>Use a multichannel pipette to immediately add 25 &#181;L of substrate reagent.</li>
		<li>Shake the plate at 350 rpm for 50 min on an orbital shaker at room temperature.</li>
		<li>Assess luminescence using a plate reader. To do so, set the mirror reader on luminescence and the emission filter on 455. Use a measurement height of 6.5 mm with a measurement time of 1 s.</li>
		<li>In order to asses cell viability, add 33 &#181;L of viability assay reagent and then re-measure luminescence again after 15 min at room temperature, using the plate reader.</li>
		<li>Assess luminescence with the plate reader by setting the mirror reader on Luminescence and the emission filter on 600 nm. Use a measurement height of 6.5 mm with a measurement time of 1 s. 
		NOTE: Viability is assessed by measuring the intracellular level of ATP upon cell lysis as per manufacturer instructions. Multiplexing is possible in this case since the viability assay uses a different luciferase with a different emission wavelength.</li>
		<li>Use the data to determine the IC50 values of each compound by curve fitting the data to a 4 parameter fitting curve equation: 
		Y = ((A-D)/(1+((x/C)^B))) + D 
		The numerical values D and A must be constrained in the curve fitting process: 
		D = luminescent value on the Y-axis for minimal curve asymptote or minimal theoretical level of response expected from the eIF4E:4G complementation assay. 
		A = luminescent value on the Y-axis for maximal curve asymptote or maximal theoretical level response expected from the eIF4E:4G complementation assay. 
		NOTE: The value for the minimal response is derived from the 1% DMSO (v/v) control treatment and the value for the maximal response is derived from the maximal response of a high affinity mTOR inhibitor that has plateaued with no effect on cell viability.</li>
	</ol>
	</li>
</ol>
2. Correlating eIF4E:eIF4G604-646 assay inhibition with eIF4F complex disruption in cells
<ol>
	<li>To confirm that the signal being measured by the assay corresponds to the physical disruption of eIF4E-eIF4G interaction by the compound, seed cells as described in step 1.1.</li>
	<li>The day after, replace the medium with 1 mL of reduced serum medium not containing phenol red and incubate for 4 h with the compound of interest. Ensure residual DMSO concentration of 1% (v/v) in a total volume of 1 mL. 
	NOTE: In order to easily allow a correlation with the result, use compound concentrations at the beginning, midpoint and endpoint of the measured complementation assay titration curve.</li>
	<li>Lyse cells and performed m7GTP pull down to isolate eIF4F and eIF4E:4EBP1 complexes. Detailed procedures on how to perform the m7GTP pull down experiment can be found elsewhere14.</li>
	<li>Detect m7GTP bound eIF4G, eIF4E and 4EBP1 protein levels by western blot analysis, and then correlate to eIF4F complex disruption with complementation assay signal inhibition.</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. 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The authors have nothing to disclose.

The method described in this article utilizes a luciferase-based complementation assay to quantitatively monitor eIF4F complex assembly through direct measurement of eIF4G-eIF4E interaction in live cells. We have provided details for use of eIF4E-eIF4G complementation system and we have also showed that the system is extremely accurate in measuring drug-induced 4EBP1-mediated dissociation of eIF4E-eIF4G interaction16. In order to facilitate the throughput of this&#160;assay, the experimental setup described in this article has been designed for a 96 well microplate format usage.
For optimal results, two critical steps should be considered when performing the assay. First, the transfection efficiency between experiments should remain similar. This can be ensured through the use of low passage number cells, and by rigorously counting cells on the day of seeding. Cell confluency should also be assessed before DNA transfection is carried out, as it is not recommended to transfect cells with lipid-based transfection reagent if cell confluency is less than 70-90%. Second, it is important to multiplex the complementation assay with a cell viability assay. Some compounds may impact the luciferase signal primarily by decreasing the number of viable cells through deleterious effects. It is, therefore, important to measure the viability of the cell immediately after the eIF4E:eIF4G604-646 complementation assay to address off-target and non-specific effects.
Protein-protein interfaces, such the one between eIF4E and eIF4G, that are devoid of hydrophobic clefts and are relatively large and planar, are generally considered to be &#34;un-druggable&#34; by conventional small molecule therapeutics (&#60;500 MW)17. Thus, there is a growing interest in the development of novel modalities that efficiently interact with these type of surfaces (e.g., macrocyclic and peptidomimetic compounds). However, many of these novel modalities are not innately able to cross the cell membrane and engage their target. To circumvent these issues, many research groups are conducting research into new chemical optimization and cellular delivery strategies. We envision that the eIF4E:eIF4G604-646 live cell PPI assay and other similar PPI derived assays will play a pivotal role in fostering these strategies and validating them.

Control of gene expression plays a pivotal role in the correct execution of cellular programs such as growth proliferation and differentiation. A regulatory control mechanism can be exerted either at the level of gene transcription or at the level of mRNA translation. In the last decade, it has become increasingly evident that translational control by modulation of the initiation process rather than the later steps of elongation and termination can finely regulate synthesis of specific subsets of proteins that play a wide range of biological functions.
Increased translation of mRNAs involved in survival, anti-autophagic and anti-apoptotic responses have been implicated in several cancers and have also been causatively linked to either aberrant activation or over expression of translation initiation factors1.
The eIF4F complex is a master regulator of translation initiation. By binding the cap-structure on the 5&#39; end of mRNAs, eIF4F is driving initial mRNA-ribosome recruitment and in turn increasing mRNA translation efficiency of weakly translated eukaryotic mRNAs2. eIF4F mediated translation of cancer-related mRNAs has been reported for many cancer models harboring aberrant activation of RAS/MAPK or AKT/TOR pathways, suggesting that cancer cells upregulate eIF4F to boost their own pro-neoplastic activity. Disruption of this feed-forward loop by inhibiting eIF4F complex formation is thereby a very promising therapeutic strategy3,4.
The eIF4F complex consists of (i) eIF4E, the cap-binding subunit of eIF4F that interacts with the cap structure found at the 5&#39; UTR of mRNA, (ii) eIF4A, the RNA helicase and (iii) eIF4G, the scaffold protein that interacts with both eIF4A and eIF4E and which eventually recruits the 40S ribosomal subunit5. eIF4G association with eIF4E is the rate-limiting step for the assembly of functional eIF4F complexes and it is negatively regulated by the eIF4E binding proteins (4EBPs, members 1, 2 and 3))6. By competing with eIF4G binding to eIF4E through an interface that consists of canonical and non-canonical eIF4E binding sequences7,8,9 (region spanning aa 604-646 on human eIF4E), 4EBP reduces the pool of eIF4E actively involved in translation and preventing eIF4F complex formation. Interplay of these protein-protein interactions is mainly regulated by the mammalian target of rapamycin (mTOR)-mediated phosphorylation of 4EBP. Upon mitogenic stimuli, mTOR directly phosphorylates the members of the 4E-BP protein family, decreasing their association with eIF4E and, thereby, promoting eIF4E-eIF4G interaction and formation of functional eIF4F complexes10.
Despite the great effort in developing compounds targeting eIF4F complex integrity, the lack of assays measuring direct disruption of eIF4E-eIF4G interaction in live cells has limited the search for cellular active hit compounds. We have applied a luciferase assay based on a coelenterazine analog (e.g., Nanoluc-based complementation assay) to monitor in real time the status of eIF4F integrity through the eIF4E-eIF4G interaction. The luciferase complementation protein system consists of an 18 kDa protein fragment (SubA) and 11 amino acid peptide fragment (SubB) optimized for minimal self-association and stability11.&#160;Once expressed as a fusion product with the human full length eIF4E and the eIF4E interaction domain from human eiF4G1 (aa 604-646), the two interacting proteins will bring the SubA and SubB fragment into close proximity of each other and will induce the formation of the active luciferase that, in presence of a cell permeable substrate, will eventually generate a bright luminescent signal (Figure 1). We have reported elsewhere the construction and validation of the eIF4E:eIF4G604-646 complementation system16.
Here, we describe how the eIF4E:eIF4G604-646 complementation system (available upon request) can be applied to accurately measure 4EBP1-mediated eIF4E-eIF4G disruption in live cells. Additionally, we demonstrate its utility by measuring the effects of several mTOR inhibitors that are currently under clinical trials as potential cancer therapeutic drugs12. Because off-target&#160;effects often mask drug-specific activity, we also describe how the versatility of the eIF4E:eIF4G604-646 system measurement can be extended with orthogonal measurements of cellular viability to take these into account.

<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

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 activation in humans. eIF4F is a cap-binding complex involved in the mRNA-ribosome recruitment phase of translation initiation. In many cellular and pre-clinical model of cancers, the deregulation of eIF4F leads to increased translation of specific mRNA subsets that are involved in cancer proliferation and survival. eIF4F is a hetero-trimeric complex built from the cap-binding subunit eIF4E, the helicase eIF4A and the scaffolding subunit eIF4G. Critical for the assembly of active eIF4F complexes is the protein-protein interaction between eIF4E and eIF4G proteins. In this article, we describe a protocol to measure eIF4F assembly that monitors the status of eIF4E-eIF4G interaction in live cells. The eIF4e:4G cell-based protein-protein interaction assay also allows drug induced changes in eIF4F complex integrity to be accurately and reliably assessed. We envision that this method can be applied for verifying the activity of commercially available compounds or for further screening of novel compounds or modalities that efficiently disrupt formation of eIF4F complex.

In order to validate the sensitivity of the eIF4E:eIF4G604-646 complementation system, 4EBP1 mediated inhibition of eIF4F complex assembly was assessed by using mTOR inhibitors. By inhibiting mTORC1 kinase dependent phosphorylation of the 4EBP protein family, mTOR inhibition enhances 4EBP1 association to eIF4E and, therefore, eIF4F disassembly15. Two classes of mechanistically different inhibitor of mTOR kinases were tested: rapalogs (e.g., Rapamycin) that are allosteric inhibitors of mTORC1 but not mTORC2 and ATP competitive-based inhibitors (e.g., PP242) that are designed to specifically inhibits both mTORC1 and mTORC2 kinase&#160;catalytic activity.
HEK293 cells were transfected with the eIF4E:eIF4G604-646 complementation system as described in step 1. After 24 h of transfection, cells were re-seeded and treated with the mTOR inhibitors PP242 and rapamycin (as described in step 1.2). Four hours after the treatment, luminescence was assessed, as described previously, followed by cell viability. As shown in Figure 2, PP242 produces a dose-dependent inhibition of the signal with a calculated IC50 of 0.72 &#177; 0.04 &#181;M, while an IC50 of 6.88 &#177; 0.88 &#181;M is derived for rapamycin (Figure 2A). Plates were then multiplexed for cellular viability assay (Figure 2D). This analysis shows that neither PP242 nor rapamycin produces a significant decrease in cell viability, proving that the decrease in luminescence in the eIF4E:eIF4G604-646 complementation system is not due to nonspecific cell death but rather through disruption of the eIF4E:4G interaction.
A m7GTP pull down experiment performed by incubating untransfected cells with compound concentrations that correspond to the beginning, mid and end points of the measured titration curve in Figure 2A show that 4EBP1-mediated disruption of endogenous eIF4E-eIF4G interaction correlates with the measured eIF4E:eIF4G604-646 assay signal (Figure 2B, 2C). Consistent with these results, PP242 is shown to be a more potent inhibitor of total 4EBP1 phosphorylation than rapamycin under the experimental conditions tested in HEK 293 cells (Figure 3A), while both inhibitors showed an impact to mTOR signaling normally, with rapamycin being more active against mTORC1 substrates and PP242 targeting both mTORC1 and mTORC2 (Figure 3B).
Taken together, these results showed that PP242 is more effective in disrupting eIF4F complex formation than rapamycin in HEK293 cells and further demonstrate that the eIF4E:eIF4G604-646 system can accurately measure eIF4F complex assembly in living cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60850/60850fig1.jpg" /> 
Figure 1: eIF4E:eIF4G604-646 complementation system. Schematic representation showing how the interaction of protein X (eIF4E) and protein Y (eIF4G604-646) enables SubA and SubB fusions to come into proximity with each other and&#160;reconstitute the active luciferase. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60850/60850fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60850/60850fig2.jpg" /> 
Figure 2: 4EBP1-mediated disruption of eIF4F complex. (A) PP242 and rapamycin eIF4E:eIF4G604-646 assay titration modelling the interaction between eIF4E and eIF4G in transfected HEK 293 cells. (B,C) Western blot analysis showing endogenous level of eIF4E, eIF4G and 4EBP1 in HEK 293 extracts and in m7GTP pull down after incubation of cultured cells with different concentration of PP242 or Rapamycin respectively. (D) Treated cells in A where multiplexed for cell viability and luminescence assessed. All values represent mean &#177; SD (n=3). This figure has been modified from16. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60850/60850fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60850/60850fig3.jpg" /> 
Figure 3: Differential effect of mTOR inhibition on 4EBP1 phoshorylation. (A) Western blot analysis of phosphorylation status of 4EBP1 in non-transfected HEK293 cells treated with indicated concentration of PP242 and rapamycin. (B) Western blot analysis of AKT and S6 phosphorylation status in non-transfected HEK293 cells treated with either the dual MTORC1/2 active site inhibitors PP242, or the allosteric inhibitor of mTORC1 Rapamycin. Beta actin was visualized for loading control as well as total 4EBP1, AKT and S6. This figure has been modified from16. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60850/60850fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Here, we present a protocol to measure eIF4E-eIF4G interaction in live cells that would enable the user to evaluate drug induced perturbation of eIF4F complex dynamics in screening formats.

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 ...

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Universidad San Sebastian

Research

JoVE Journal

Cancer Research

3.0K Views.  A*STAR (Agency for Science, Technology and Research). Here, we present a protocol to measure eIF4E-eIF4G interaction in live cells that would enable the user to evaluate drug induced perturbation of eIF4F complex dynamics in screening formats.

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.

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

Genetics

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.

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.

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

Biochemistry

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.

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

Biology

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.

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

Immunology and Infection

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.

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.

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.

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.

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.

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

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

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Chapters in this video

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

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

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

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

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Identification of Protein Interacting Partners Using Tandem Affinity Purification

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

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

Xenopus laevis as a Model to Identify Translation Impairment

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