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

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 as...

<ol>
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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. 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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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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><a href="https://www.thermofisher.com/order/catalog/product/L3000015" target="_blank">L3000015</a></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>&#947;-Aminophenyl-m7GTP (C10-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) th...

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

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

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 ...

The regulation of the eIF4F complex signaling is associated with increased translation of mRNA subset that are involved in cancer proliferation and survival. Here we describe an eIF4E-eIF4G cell-based protein-protein interaction assays that enables us to assess drug-induced perturbation in eIF4F complex integrity in live cells. There is a growing interest in the development of modalities that efficiently target protein-protein interface.We envisioned that our eIF4E-eIF4G PPI assays will help in fostering these strategies and validate them. This assay will provide an optimal primary scheme for it to lead optimization of eIF4E-eIF4G inhibitors. Seeding the cell correctly and performing the transfer transfection are the most challenging aspect of this product since they may reflect directly on the suppression of the PPI reporting system.After thawing and counting cells, seed 6 well plates with HEK 293 cells, using 2 milliliters of standard growth medium per well. On the morning of day 2, for each planed transfection, dilute 9 microliters of liposome-based solution in a tube containing 125 microliters of reduced serum medium without phenol red. While the diluted liposomes incubate at room temperature for 5 minutes, prepare the master mix of DNA by diluting 3 micrograms of each plasmid in 125 microliters of reduced serum medium for each transfection tube.Add 12 microliters of enhancer reagents to the DNA master mix 2, then mix well. Immediately add this mixture to each tube of diluted liposomes in a ratio of 1 to 1. After incubating this DNA lipid complex for 15 minutes at room temperature, add the complex to each well of the 6 well plates.Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for 24 hours. On the morning of day 3, rinse each well with 1 milliliter of PBS, and add 0.3 milliliters of trypsin. Incubate the plates at 37 degrees Celsius for 5 minutes.After incubation, neutralize the trypsin by adding 2 milliliters to each well of the reduced serum medium without phenol red. Transfer the transfected cells into a 15 milliliter tube. Next, centrifuge the cells at 290x G for 5 minutes.Aspirate the medium, and re-suspend the cell pellet in 2 milliliters of the reduced serum medium. Seed the transfected HEK 293 cells in 96-well opaque plates, at a density of 30, 000 cells per well in 90 microliters of medium. To obtain 3 technical replicates for 3 different compounds within the same experiment, seed 60 wells.Exclude the wells on the edges. Immediately after seeding the transfected cells, add 10 microliters of 10%DMSO compound solution to each well. Prepare 1 millimolar compound stock solutions by dissolving each compound of interest in 100%DMSO.In order to have 3 replicates for each compound titration, use 8 microliters of the 1 millimolar compound stock solution. Perform a two-fold serial dilution of each stock compound solution in 8 wells of a 96-well PCR plate by pipetting 4 microliters of the 1 millimolar stock into 4 microliters of 100%DMSO for each titration point. Discard the excess 4 microliters after the last point of the two-fold serial dilution.Add 36 microliters of HPLC grade sterile water to each tube to prepare 40 microliters of 10x compound serial dilution solutions in 10%DMSO. Also, prepare a control. 10%DMS only stock solution in HPLC grade sterile water.Add 10 microliters 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%Incubate the plate at 37 degrees Celsius with 5%carbon dioxide for 3 hours. After 3 hours, start preparing the luciferase substrate reagent by combining 1 volume of substrate with 19 volumes of the dilution reagent. Use a multi-channel pipette to immediately add 25 microliters of the substrate reagent to each well of the 96 well plate with the cells.Shake the plate on an orbital shaker at 350 RPM, for 50 minutes at room temperature. To assess luminescence, place the plate on a plate reader. Set the mirror reader to luminescence and the emission filter to 455.Use a measurement height of 6.5 millimeters with a measurement time of 1 second. To assess cell viability, add 33 microliters of viability assay reagent to each well. After 15 minutes at room temperature, assess luminescence with the plate reader by setting the mirror reader to luminescence and the emission filter to 600.Use a measurement height of 6.5 millimeters and a measurement time of 1 second. Use the data from the plate reader to determine the IC 50 value of each compound, by curve-fitting the data to the 4-parameter fitting curve equation described in the manuscript. HEK 293 cells were transfected with the eIF4E-eIF4G complementation system, and then receded and treated with mTOR inhibitors.When luminescence was assessed 4 hours after treatment, PP242 and rapamycin both produced a dose-dependent inhibition of the signal. Neither PP242 nor rapamycin produced a significant decrease in cell viability, indicating that the decrease in luminescence in the eIF4E-eIF4G complementation system is not due to non-specific cell death, but rather to disruption of the EIF4E-4G interaction. Western blot analysis, following m7GTP pull down experiment, showed that 4EBP1 mediated disruption of endogenous eIF4E-eIF4G interaction correlates with the measured eIF4E-eIF4G assay signal.PP242 was a more potent inhibitor of total 4EBP1 phosphorylation than rapamycin. Both inhibitors showed an impact on mTOR signaling normally, with rapamycin being more active against mTORC1 substrates, and PP242 targeting both mTORC1 and mTORC2. The most critical aspect of this product are seeding the cells on the day of transaction, re-seeding the cells in a medium without phenol red, assessing the luminescence of the eIF4E-eIF4G complementation assay, and run in the viability assay on the same plate.A secondary viability assay can be performed to assess any drug of target and known specific effects.

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