Robert Bellé

EIF4E/4E-BP dissociation and 4E-BP degradation in the first mitotic division of the sea urchin embryo

The mRNAs cap-binding protein eukaryotic translation initiation factor (eIF)4E is a major target for the regulation of translation initiation. eIF4E activity is controlled by a family of translation inhibitors, the eIF4E-binding proteins (4E-BPs). We have previously shown that a rapid dissociation of 4E-BP from eIF4E is related with the dramatic rise in protein synthesis that occurs following sea urchin fertilization. Here, we demonstrate that 4E-BP is destroyed shortly following fertilization and that 4E-BP degradation is sensitive to rapamycin, suggesting that proteolysis could be a novel means of regulating 4E-BP function. We also show that eIF4E/4E-BP dissociation following fertilization is sensitive to rapamycin. Furthermore, while rapamycin modestly affects global translation rates, the drug strongly inhibits cyclin B de novo synthesis and, consequently, precludes the completion of the first mitotic cleavage. These results demonstrate that, following sea urchin fertilization, cyclin B translation, and thus the onset of mitosis, are regulated by a rapamycin-sensitive pathway. These processes are effected at least in part through eIF4E/4E-BP complex dissociation and 4E-BP degradation.

Differences in the carcinogenic evaluation of glyphosate between the International Agency for Research on Cancer (IARC) and the European Food Safety Authority (EFSA)

The International Agency for Research on Cancer (IARC) Monographs Programme identifies chemicals, drugs, mixtures, occupational exposures, lifestyles and personal habits, and physical and biological agents that cause cancer in humans and has evaluated about 1000 agents since 1971. Monographs are written by ad hoc Working Groups (WGs) of international scientific experts over a period of about 12 months ending in an eight-day meeting. The WG evaluates all of the publicly available scientific information on each substance and, through a transparent and rigorous process,1 decides on the degree to which the scientific evidence supports that substances potential to cause or not cause cancer in humans. For Monograph 112,2 17 expert scientists evaluated the carcinogenic hazard for four insecticides and the herbicide glyphosate.3 The WG concluded that the data for glyphosate meet the criteria for classification as a probable human carcinogen . The European Food Safety Authority (EFSA) is the primary agency of the European Union for risk assessments regarding food safety. In October 2015, EFSA reported4 on their evaluation of the Renewal Assessment Report5 (RAR) for glyphosate that was prepared by the Rapporteur Member State, the German Federal Institute for Risk Assessment (BfR). EFSA concluded that ‘glyphosate is unlikely to pose a carcinogenic hazard to humans and the evidence does not support classification with regard to its carcinogenic potential’. Addendum 1 (the BfR Addendum) of the RAR5 discusses the scientific rationale for differing from the IARC WG conclusion. Serious flaws in the scientific evaluation in the RAR incorrectly characterise the potential for a carcinogenic hazard from exposure to glyphosate. Since the RAR is the basis for the European Food Safety Agency (EFSA) conclusion,4 it is critical that these shortcomings are corrected. EFSA concluded ‘that there is very limited evidence for an association between glyphosate-based formulations …

Free calcium in full grown Xenopus laevis oocyte following treatment with ionophore A 23187 or progesterone.

Free intracellular Ca2+ was monitored in isolated Xenopus laevis oocyte during induced maturation using the Ca2+ -sensitive luminescent protein, aequorin. Internal free Ca2+ was not precisely measured but data suggest it was quite low (in the micromolar range). No change in internal free Ca2+ was detected during maturation induced either by progesterone or by p-chloromercuribenzoate. By contrast, the ionophore A 23187 gave an increase in the free Ca2+ level when there was a raised external Ca2+ (10 mM), conditions which also induce oocyte maturation. About 3 h after progesterone or p-chloromercuribenzoate stimulation, the oocyte membrane potential decreased by about 50 mV while the membrane resistance increased transitorily.

Purification of a p47 phosphoprotein from Xenopus laevis oocytes and identification as an in vivo and in vitro p34cdc2 substrate

This paper describes the purification of a 47 kDa protein from Xenopus laevis oocytes that becomes phosphorylated when the oocytes undergo meiotic maturation. This protein (p47) is part of a high molecular mass complex containing at least two other proteins of molecular mass 30 and 36 kDa. This complex can be isolated from stage VI oocytes before maturation. We obtained a pattern for phosphopeptides in p47 phosphorylated in vivo very similar to that of the purified protein phosphorylated in vitro by p34cdc2 (a H1 kinase which is a component of the M‐phase promoting factor) and [γ‐32P]ATP. Therefore, the purified p47, already described as a marker of MPF activity, is the first reported in vivo substrate for the cell division control kinase.

A possible role for Mg2+ ions in the induction of meiotic maturation of Xenopus oocyte.

Progesterone induces in vitro the meiotic cell division of Xenopus full-grown oocytes. Microinjection into oocyte of a solution containing Mg2+ (20 mM) facilitates by one order of magnitude the dose of progesterone which induces 50% of germinal vesicle breakdown. Microinjected in the absence of hormone, Mg2+ and also Mn2+ can induce maturation with efficiencies of, respectively, 24% (SEM = 8; n = 13) and 70% (SEM = 6; n = 23). The dose-response curves of cation-induction of maturation show an optimum of 20 mM for Mg2+ and 15 mM for Mn2+ (pipet concentration); higher doses were less active. Cation-induction of maturation is inhibited when oocytes are preincubated with cholera toxin (500 ng/ml); nevertheless, it cannot be interpreted at the level of cAMP, since both Mg2+ and Mn2+ microinjections provoke an increase in the oocyte cAMP content. Mg2+ induction of maturation is more efficient when oocytes are incubated in trimethylamine at pH 8.2, which is known to increase intracellular pH suggesting an action at the level of alkali pH-sensitive enzymes. Altogether, our results indicate a positive role for Mg2+ ions in the induction of oocyte maturation and raise an attractive hypothesis about the respective roles of cAMP and Mg2+ changes involved in the mechanism of progesterone action. Our results also show that co-injection of 2-glycerophosphate and Mg2+ ions, which are both commonly used in the preparation of the MPF mitotic factors from dividing cells, induces oocyte maturation more efficiently than Mg2+ alone and drastically shortens the kinetics of germinal vesicle breakdown to 1 h 30 min to 2 h 30 min.

Embryonic-stage-dependent changes in the level of eIF4E-binding proteins during early development of sea urchin embryos

The eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) inhibit translation initiation by binding eIF4E and preventing recruitment of the translation machinery to mRNA. We have previously shown that fertilization of sea urchin eggs triggers eIF4E–4E-BP complex dissociation and 4E-BP degradation. Here, we show that microinjection of eIF4E-binding motif peptide into unfertilized eggs delays the onset of the first mitosis triggered by fertilization, demonstrating that dissociation of the eIF4E–4E-BP complex is functionally important for the first mitotic division in sea urchin embryos. We also show by gel filtration analyses that eIF4E is present in unfertilized eggs as an 80 kDa molecular mass complex containing 4E-BP and a new 4E-BP of 40 kDa. Fertilization triggers the dissociation of eIF4E from these two 4E-BPs and triggers the rapid recruitment of eIF4E into a high-molecular-mass complex. Release of eIF4E from the two 4E-BPs is correlated with a decrease in the total level of both 4E-BPs following fertilization. Abundance of the two 4E-BPs has been monitored during embryonic development. The level of the two proteins remains very low during the rapid cleavage stage of early development and increases 8 hours after fertilization. These results demonstrate that these two 4E-BPs are down- and upregulated during the embryonic development of sea urchins. Consequently, these data suggest that eIF4E availability to other partners represents an important determinant of the early development of sea urchin embryos.

In vitro facilitation of Xenopus oocyte maturation by subthreshold doses of progesterone

Following progesterone treatment, a significant lag (4–8 hr) in the induction of germinal vesicle breakdown (GVBD) is observed in amphibian oocytes. Preincubation of Xenopus oocytes in the presence of subthreshold doses of progesterone decreases the lag (1–3 hr) and, therefore, facilitates oocyte maturation. Progesterone facilitation of GVBD is a dose-dependent reversible phenomenon. On the other hand, it is also reported that cyclic-AMP phosphodiesterase inhibitors increase the lag (8–15 hr) between progesterone stimulation and germinal vesicle breakdown.

Multiple Phosphorylation Sites and Quaternary Organization of Guanine-Nucleotide Exchange Complex of Elongation Factor-1 (EF-1βγδ/ValRS) Control the Various Functions of EF-1α

The eukaryotic guanine-nucleotide exchange factor commonly called elongation factor-1βγδ (EF-1βγδ), comprises four different subunits including valyl-tRNA synthetase (EF-1βγδ/ValRS). The factor is multiply-phosphorylated by three different protein kinases, protein kinase C, casein kinase II and cyclin dependent kinase 1 (CDK1). EF-1βγδ/ValRS is organized as a macromolecular complex for which we propose a new structural model. Evidence that EF-1βγδ/ValRS is a sophisticated supramolecular complex containing many phosphorylation sites, makes it a potential regulator of any of the functions of its partner EF-1α, not only involved in protein synthesis elongation, but also in many other cellular functions.

Polyamine levels during Xenopus laevis oogenesis: a role in oocyte competence to meiotic resumption

The results presented here show that a decrease in the concentration of total polyamines, due to a decrease in putrescine and spermine, occurs during oogenesis in Xenopus laevis. The microinjection of spermine or spermidine decreases the hormonal responsiveness (maturation) of the fully-grown oocytes. This effect is synergistic with that already described for the microinjection of casein kinase II (Mulner-Lorillon, O. et al. (1987) Eur. J. Biochem. 171, 107-117), a polyamine dependent enzyme. Therefore a decrease in polyamine concentration, via its effect on endogeneous casein kinase II, could constitute one of the molecular changes required for the acquisition of competence to mature.

Early increase of a 105,000-dalton phosphoprotein during meiotic maturation of Xenopus laevis oocyte

In ovo [32P] phosphoproteins were analyzed during meiotic maturation of Xenopus laevis oocytes. A phosphoprotein of 105,000-dalton was found to increase early (one hour) after progesterone induction of meiosis. The pure heat-stable inhibitor (PKI) of cAMP-dependent protein kinase, which induces maturation, was microinjected into oocytes. Again the early increase in the 105,000-dalton [32P] phosphoprotein occurred. The burst in protein phosphorylation, which takes place at the period of germinal vesicle breakdown, was quantitatively and qualitatively comparable in progesterone and PKI-stimulated oocytes. In order to confirm the inverse relationship between the 105,000 dalton [32P] phosphoprotein increase and cAMP-dependent protein kinase activity, purified C-subunit of the kinase has been microinjected into oocytes. C-subunit which inhibits maturation did not increase significantly the 105,000-dalton [32P] phosphoprotein whereas it increased the total level of in ovo phosphorylation. Enucleation experiments favour the localization of the 105,000-dalton protein in both the oocyte cytoplasm and nucleus. Furthermore the progesterone-induced increase in the phosphorylation of the 105,000-dalton protein was found in the cytoplasmic compartment after oocyte enucleation.