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 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.
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 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' 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. 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 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.
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:eIF4G604-646 complementation assay
2. Correlating eIF4E:eIF4G604-646 assay inhibition with eIF4F complex disruption in cells
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 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 ± 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: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.
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 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: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 ± 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.
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: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 "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:eIF4G604-646 live cell PPI assay and other similar PPI derived assays will play a pivotal role in fostering these strategies and validating them.
The authors have nothing to disclose.
This work was supported by core budget from the p53lab (BMSI, A*STAR) and the JCO VIP grant (A*STAR).
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 |