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

Summary

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.

Abstract

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

Introduction

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' 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 strategy^3,4.

The eIF4F complex consists of (i) eIF4E, the cap-binding subunit of eIF4F that interacts with the cap structure found at the 5' 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 sequences^7,8,9 (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. 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:eIF4G^604-646 complementation system¹⁶.

Here, we describe how the eIF4E:eIF4G^604-646 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 effects often mask drug-specific activity, we also describe how the versatility of the eIF4E:eIF4G^604-646 system measurement can be extended with orthogonal measurements of cellular viability to take these into account.

Protocol

HEK293 cell line was used for the protocol and was cultured in Dulbecco'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 °C with 5% CO₂ in a humidified environment.

1. Quantitative assessment of eIF4F complex disruption via eIF4E:eIF4G^604-646 complementation assay

1. Cell culture and transient transfection of eIF4E:eIF4G^604-646 complementation assay
   1. 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.
   2. 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.
   3. On the morning of day 2, co-transfect cells with SubA-eIF4E and eIF4G^604-646-SubB plasmid using a lipid-based transfection reagent (see Table of Materials) as described below.
      1. Dilute 9 µL of liposome-based solution in a tube containing 125 µL of reduced serum medium without phenol red (see Table of Materials) for each transfection and incubate at room temperature for 5 min.
      2. Prepare master mix of DNA by diluting 3 µg of each plasmid in 125 µL of reduced serum medium for each transfection tube.
      3. Add 12 µL 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.
      4. Add the DNA-lipid complex to each well and incubate the cells in a 37 °C incubator with 5% CO₂ for 24 h.
   4. On the morning of day 3, rinse each well with 1 mL of PBS.
   5. Remove the PBS and incubate cells in each well with 0.3 mL of trypsin for 5 min at 37 °C.
   6. 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.
   7. 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.
   8. Seed transfected HEK 293 cells in 96 well opaque plates at a density of 30,000 cells per well in 90 µL 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.
   9. Immediately after seeding the transfected cells, add 10 µL of 10% DMSO compound solution (see step 1.2).
2. Compound preparation
   1. 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 µL 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.
   2. Perform a 2-fold serial dilution of the stock compound solution with 4 µL of the 1 mM stock into 4 µL 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.
   3. Add 36 µL of HPLC grade sterile water for each point of the 2-fold serial dilution to prepare a 40 µL of 10x working solutions in 10% DMSO (v/v) (i.e. 100 μM to 0.39 μM 10% DMSO stock series will lead to a final solution of 10 μM to 0.039 μM,in 1% DMSO when added to cells). As for treatment control, also prepare a 10% DMSO only stock solution in HPLC grade sterile water.
   4. Add 10 µL 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 µL and incubate for 3 h at 37 °C with 5% v/v atmospheric CO₂.
3. Luciferase complementation and viability assay
   1. 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's instructions).
      NOTE: The luciferase assay is performed using a commercially available kit (see Table of Materials).
   2. Use a multichannel pipette to immediately add 25 µL of substrate reagent.
   3. Shake the plate at 350 rpm for 50 min on an orbital shaker at room temperature.
   4. 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.
   5. In order to asses cell viability, add 33 µL of viability assay reagent and then re-measure luminescence again after 15 min at room temperature, using the plate reader.
   6. 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.
   7. 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.

2. Correlating eIF4E:eIF4G^604-646 assay inhibition with eIF4F complex disruption in cells

1. 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.
2. 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.
3. 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.
4. 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.

Representative Results

In order to validate the sensitivity of the eIF4E:eIF4G^604-646 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 catalytic activity.

HEK293 cells were transfected with the eIF4E:eIF4G^604-646 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 ± 0.04 µM, while an IC50 of 6.88 ± 0.88 µM 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:eIF4G^604-646 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:eIF4G^604-646 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:eIF4G^604-646 system can accurately measure eIF4F complex assembly in living cells.

Figure 1: eIF4E:eIF4G^604-646 complementation system. Schematic representation showing how the interaction of protein X (eIF4E) and protein Y (eIF4G^604-646) enables SubA and SubB fusions to come into proximity with each other and reconstitute the active luciferase. Please click here to view a larger version of this figure.

Figure 2: 4EBP1-mediated disruption of eIF4F complex. (A) PP242 and rapamycin eIF4E:eIF4G^604-646 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 ± SD (n=3). This figure has been modified from¹⁶. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Discussion

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 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:eIF4G^604-646 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 "un-druggable" by conventional small molecule therapeutics (<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:eIF4G^604-646 live cell PPI assay and other similar PPI derived assays will play a pivotal role in fostering these strategies and validating them.

Materials

Name	Company	Catalog Number	Comments
293FT cells	Thermo Fisher Scientific	R70007
Cell culture microplate 96 well, F-Bottom	greiner bio-one	655083
Cell titer Glo 2.0	PROMEGA	G9241
Envision Multilabel Reader	PerkinElmer	not applcable
Finnpipette F2 Multichannel Pipettes 12-channels 30-300 ml	Thermo Fisher Scientific	4662070
Finnpipette F2 Multichannel Pipettes 12-channels 5-50 ml	Thermo Fisher Scientific	4662050
FUGENE6	PROMEGA	E2692
Lipofectamine 3000	Thermo Fisher Scientific	L3000015
NanoBiT PPI Starter Systems	PROMEGA	N2014
Optimem I Reduced Serum Mediun, no phenol red	Thermo Fisher Scientific	11058021
Orbital shaker	Eppendorf	not appicable
γ-Aminophenyl-m7GTP (C10-spacer)-Agarose	Jena Bioscience	AC-155S