Modelization of the regulation of protein synthesis following fertilization in sea urchin shows requirement of two processes: a destabilization of eIF4E:4E-BP complex and a great stimulation of the 4E-BP-degradation mechanism, both rapamycin-sensitive

Abstract

Fertilization of sea urchin eggs involves an increase in protein synthesis associated with a decrease in the amount of the translation initiation inhibitor 4E-BP. A highly simple reaction model for the regulation of protein synthesis was built and was used to simulate the physiological changes in the total 4E-BP amount observed during time after fertilization. Our study evidenced that two changes occurring at fertilization are necessary to fit with experimental data. The first change was an 8-fold increase in the dissociation parameter (koff1) of the eIF4E:4E-BP complex. The second was an important 32.5-fold activation of the degradation mechanism of the protein 4E-BP. Additionally, the changes in both processes should occur in 5 min time interval post-fertilization. To validate the model, we checked that the kinetic of the predicted 4.2-fold increase of eIF4E:eIF4G complex concentration at fertilization matched the increase of protein synthesis experimentally observed after fertilization (6.6-fold, SD = 2.3, n = 8). The minimal model was also used to simulate changes observed after fertilization in the presence of rapamycin, a FRAP/mTOR inhibitor. The model showed that the eIF4E:4E-BP complex destabilization was impacted and surprisingly, that the mechanism of 4E-BP degradation was also strongly affected, therefore suggesting that both processes are controlled by the protein kinase FRAP/mTOR.

Keywords: deterministic model; mechanisms of fertilization; sea urchin embryos; translation simulation; translational control.

Figure 1

Surface plasmon resonance Interaction of eIF4E and 4E-BP. Each sensorgram, relative refractive index (μRIU) as a function of time, was obtained after injection of eIF4E from 4.37 μM to 53.9 nM according to a three-fold dilution range. Overlapped signals were fitted using a single equilibrium kinetic isotherm (solid red lines).

Figure 2

Determination of the degradation rate of 4E-BP in unfertilized eggs. Unfertilized eggs were incubated in the presence of 100 μM emetine. The amount of total 4E-BP remaining upon time was determined from immunoblotting experiments as indicated in the material and methods section. The values from 8 independent experiments are plotted as black cross. Simulation using the model (reactions R1, R2, and R5) leading to the best fit is shown as red line. The total amount of 4E-BP is expressed as % of initial value.

Figure 3

Changes in 4E-BP total amount and protein synthesis in vivo after fertilization. (A) 4E-BP was measured by immunoblotting and densitometric quantification as described in the material and methods section. Mean values from 10 independent experiments are plotted as a function of time. The total amount of 4E-BP is expressed as % of initial value. (B) Protein synthesis was determined from incorporation into proteins of [³⁵S]methionine at different times after fertilization as indicated in the Materials and Methods section. Mean values of protein accumulation from 11 independent determinations are plotted as a function of time in arbitrary unit (UA).

Figure 4

Simulation of fertilization changes on 4E-BP using the minimal model. Best fits (red curves) compared to experimental data (black cross) are shown: (A) Ranging k_off1 from unfertilized value (1 to 100-fold) and the parameter time change (1 to 15 min). (B) Ranging k_lys4E-BP from unfertilized value (1–100-fold) and the parameter time change (1–15 min). (C) Changing k_off1 and k_lys4E-BP from unfertilized value (1–100-fold) and the parameter time change (1–15 min). (D) Color representation of the fit according to the change of k_off1 and k_lys4E-BP when the parameter time change is fixed to 5 min (k_cat4E-BP and k_off1 ratio are the ratio between the value reached after fertilization and the unfertilized value). The color scale on the right was associated to distance to data (sum of square residual) obtained in the simulations. The white cross corresponds to the best fit. The total amount of 4E-BP is expressed as % of initial value.

Figure 5

Simulations performed varying the indicated parameters (k_off1, k_on2, k_on1, k_off2) at fertilization. k_lys4E-BP was fixed as 32.5-fold increase compared to unfertilized eggs. Parameter time change from unfertilized values was set at 5 min. The best fits (red line) for each parameter are shown (A–D) as compared to experimental data (black cross). The total amount of 4E-BP is expressed as % of initial value.

Figure 6

Distance to data calculations from simulations performed varying together all of the parameters k_off1, k_on2, k_on1, k_off2. Each indicated parameter varied by steps from 1/32 to 32-fold compared to unfertilized values in such a way that a 8-fold increase in KD₁/KD₂ was maintained constant (this gives 10434 possibilities). The k_lys4E-BP was fixed as 32.5-fold increase compared to unfertilized eggs and the parameter time change was fixed to 5 min. For each possible ratio (fertilized value vs. unfertilized value), the mean and SD of the distances to data were reported. Green bar corresponds to region where simulation fits well.

Figure 7

Simulation of the concentration changes occurring at fertilization. The concentration changes of the indicated constituents were calculated and plotted vs. time after fertilization using the parameters depicted in Table 1; “eIF4E,” “eIF4G,” and “4E-BP” are the concentrations of the free forms of these proteins; “eIF4E:eIF4G” and “eIF4E:4E-BP” correspond to the concentrations of the complexed forms; “Total 4E-BP” represents the total concentration of 4E-BP (free and complexed). Normalization was done with 100% corresponding to the initial concentration of total 4E-BP, i.e., 3.67 μM (see Table 1). The kinetic of accumulation of protein was calculated from the kinetic of eIF4E:4E-BP changes and is shown in relative units (black curve).

Figure 8

Simulation of fertilization changes on 4E-BP in the presence of rapamycin. Simulated curve (red line) obtained with optimized parameters (see text) compared to experimental data (black cross). The total amount of 4E-BP is expressed as % of initial value.