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Front Genet
2014 May 06;5:117. doi: 10.3389/fgene.2014.00117.
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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.
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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 kinaseFRAP/mTOR.
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 koff1 from unfertilized value (1 to 100-fold) and the parameter time change (1 to 15 min). (B) Ranging klys4E-BP from unfertilized value (1–100-fold) and the parameter time change (1–15 min). (C) Changing koff1 and klys4E-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 koff1 and klys4E-BP when the parameter time change is fixed to 5 min (kcat4E-BP and koff1 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 (koff1, kon2, kon1, koff2) at fertilization. klys4E-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 koff1, kon2, kon1, koff2. 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 klys4E-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.
Abiko,
Binding preference of eIF4E for 4E-binding protein isoform and function of eIF4E N-terminal flexible region for interaction, studied by SPR analysis.
2007, Pubmed
Abiko,
Binding preference of eIF4E for 4E-binding protein isoform and function of eIF4E N-terminal flexible region for interaction, studied by SPR analysis.
2007,
Pubmed Bellé,
Model of cap-dependent translation initiation in sea urchin: a step towards the eukaryotic translation regulation network.
2010,
Pubmed
,
Echinobase Bellé,
Identification of a new isoform of eEF2 whose phosphorylation is required for completion of cell division in sea urchin embryos.
2011,
Pubmed
,
Echinobase Brunn,
The mammalian target of rapamycin phosphorylates sites having a (Ser/Thr)-Pro motif and is activated by antibodies to a region near its COOH terminus.
1997,
Pubmed Burnett,
RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1.
1998,
Pubmed Calzone,
BIOCHAM: an environment for modeling biological systems and formalizing experimental knowledge.
2006,
Pubmed Cormier,
Cap-dependent translation and control of the cell cycle.
2003,
Pubmed Cormier,
eIF4E association with 4E-BP decreases rapidly following fertilization in sea urchin.
2001,
Pubmed
,
Echinobase Costache,
Dephosphorylation of eIF2α is essential for protein synthesis increase and cell cycle progression after sea urchin fertilization.
2012,
Pubmed
,
Echinobase Epel,
The initiation of development at fertilization.
1990,
Pubmed
,
Echinobase Gingras,
Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.
1999,
Pubmed Gingras,
Regulation of translation initiation by FRAP/mTOR.
2001,
Pubmed Gingras,
eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation.
1999,
Pubmed Gosselin,
The translational repressor 4E-BP called to order by eIF4E: new structural insights by SAXS.
2011,
Pubmed Haghighat,
Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E.
1995,
Pubmed Hinnebusch,
Molecular mechanism of scanning and start codon selection in eukaryotes.
2011,
Pubmed Jackson,
Termination and post-termination events in eukaryotic translation.
2012,
Pubmed Jackson,
The mechanism of eukaryotic translation initiation and principles of its regulation.
2010,
Pubmed Jackson,
Cap-dependent and cap-independent translation: operational distinctions and mechanistic interpretations.
1995,
Pubmed Joshi,
Phylogenetic analysis of eIF4E-family members.
2005,
Pubmed Mader,
The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins.
1995,
Pubmed Mizuno,
Importance of C-terminal flexible region of 4E-binding protein in binding with eukaryotic initiation factor 4E.
2008,
Pubmed Morales,
Translational control genes in the sea urchin genome.
2006,
Pubmed
,
Echinobase Oulhen,
A variant mimicking hyperphosphorylated 4E-BP inhibits protein synthesis in a sea urchin cell-free, cap-dependent translation system.
2009,
Pubmed
,
Echinobase Oulhen,
After fertilization of sea urchin eggs, eIF4G is post-translationally modified and associated with the cap-binding protein eIF4E.
2007,
Pubmed
,
Echinobase Oulhen,
eIF4E-binding proteins are differentially modified after ammonia versus intracellular calcium activation of sea urchin unfertilized eggs.
2010,
Pubmed
,
Echinobase Parrington,
Flipping the switch: how a sperm activates the egg at fertilization.
2007,
Pubmed
,
Echinobase Pause,
Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function.
1994,
Pubmed Pyronnet,
Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnk1 to phosphorylate eIF4E.
1999,
Pubmed Rousseau,
The eIF4E-binding proteins 1 and 2 are negative regulators of cell growth.
1996,
Pubmed Salaün,
eIF4E/4E-BP dissociation and 4E-BP degradation in the first mitotic division of the sea urchin embryo.
2003,
Pubmed
,
Echinobase Salaün,
Embryonic-stage-dependent changes in the level of eIF4E-binding proteins during early development of sea urchin embryos.
2005,
Pubmed
,
Echinobase Salaün,
Signal transduction pathways that contribute to CDK1/cyclin B activation during the first mitotic division in sea urchin embryos.
2004,
Pubmed
,
Echinobase Sonenberg,
The mRNA 5' cap-binding protein eIF4E and control of cell growth.
1998,
Pubmed Umenaga,
Identification and function of the second eIF4E-binding region in N-terminal domain of eIF4G: comparison with eIF4E-binding protein.
2011,
Pubmed von der Haar,
Folding transitions during assembly of the eukaryotic mRNA cap-binding complex.
2006,
Pubmed Yanagiya,
Translational homeostasis via the mRNA cap-binding protein, eIF4E.
2012,
Pubmed
