Biomedical subjects

Patrick Cormier

Publications and source records attributed to Patrick Cormier.

16 recordsLinked to original sources

PubMed2007

After fertilization of sea urchin eggs, eIF4G is post-translationally modified and associated with the cap-binding protein eIF4E.

Release of eukaryotic initiation factor 4E (eIF4E) from its translational repressor eIF4E-binding protein (4E-BP) is a crucial event for the first mitotic division following fertilization of sea urchin eggs. Finding partners of eIF4E following fertilization is crucial to understand how eIF4E functions during this physiological process. The isolation and characterization of cDNA encoding Sphaerechinus granularis eIF4G (SgIF4G) are reported. mRNA of SgIF4G is present as a single 8.5-kb transcript in unfertilized eggs, suggesting that only one ortholog exists in echinoderms. The longest open reading frame predicts a sequence of 5235 nucleotides encoding a deduced polypeptide of 1745 amino acids with a predicted molecular mass of 192 kDa. Among highly conserved domains, SgIF4G protein possesses motifs that correspond to the poly(A) binding protein and eIF4E protein-binding sites. A specific polyclonal antibody was produced and used to characterize the SgIF4G protein in unfertilized and fertilized eggs by SDS-PAGE and western blotting. Multiple differentially migrating bands representing isoforms of sea urchin eIF4G are present in unfertilized eggs. Fertilization triggers modifications of the SgIF4G isoforms and rapid formation of the SgIF4G-eIF4E complex. Whereas rapamycin inhibits the formation of the SgIF4G-eIF4E complex, modification of these SgIF4G isoforms occurs independently from the rapamycin-sensitive pathway. Microinjection of a peptide corresponding to the eIF4E-binding site derived from the sequence of SgIF4G into unfertilized eggs affects the first mitotic division of sea urchin embryos. Association of SgIF4G with eIF4E is a crucial event for the onset of the first mitotic division following fertilization, suggesting that cap-dependent translation is highly regulated during this process. This hypothesis is strengthened by the evidence that microinjection of the cap analog m(7)GDP into unfertilized eggs inhibits the first mitotic division.

Amino Acid Sequence↗

PubMed2006

The genome of the sea urchin Strongylocentrotus purpuratus.

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.

Animals↗

PubMed2006

The genomic repertoire for cell cycle control and DNA metabolism in S. purpuratus.

A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model.

Amino Acid Sequence↗

PubMed2006

Hypoxia and DNA-damaging agent bleomycin both increase the cellular level of the protein 4E-BP.

The 4E-binding proteins (4E-BPs) regulate the cap-dependent eukaryotic initiation factor 4E (eIF4E). The level of 4E-BP protein is regulated during early development of sea urchin embryos. Fertilization leads to the rapid disappearance of the protein that reappears later in development. We show that two important cellular stresses, hypoxia and bleomycin prolonged checkpoint mobilization provoked the overexpression of the protein 4E-BP in developing sea urchin embryos. Hypoxia resulted after 1 h in a reversible gradual increase in the protein 4E-BP level. At 20 h, the protein 4E-BP had reached the level existing in the unfertilized eggs. Bleomycin used as a DNA-damaging agent for checkpoint activation, provoked cell cycle inhibition and after prolonged exposure (20 h), induced the expression of the protein 4E-BP. The effect of bleomycin on 4E-BP protein overexpression was dose-dependent between 0.4 and 1.2 mM. The role of the overexpression of the protein 4E-BP is discussed in relation with cellular stress responses.

Animals↗

PubMed2006

Translational control genes in the sea urchin genome.

Sea urchin eggs and early cleavage stage embryos provide an example of regulated gene expression at the level of translation. The availability of the sea urchin genome offers the opportunity to investigate the "translational control" toolkit of this model system. The annotation of the genome reveals that most of the factors implicated in translational control are encoded by nonredundant genes in echinoderm, an advantage for future functional studies. In this paper, we focus on translation factors that have been shown or suggested to play crucial role in cell cycle and development of sea urchin embryos. Addressing the cap-binding translational control, three closely related eIF4E genes (class I, II, III) are present, whereas its repressor 4E-BP and its activator eIF4G are both encoded by one gene. Analysis of the class III eIF4E proteins in various phyla shows an echinoderm-specific amino acid substitution. Furthermore, an interaction site between eIF4G and poly(A)-binding protein is uncovered in the sea urchin eIF4G proteins and is conserved in metazoan evolution. In silico screening of the sea urchin genome has uncovered potential new regulators of eIF4E sharing the common eIF4E recognition motif. Taking together, these data provide new insights regarding the strong requirement of cap-dependent translation following fertilization. The genome analysis gives insights on the complexity of eEF1B structure and motifs of functional relevance, involved in the translational control of gene expression at the level of elongation. Finally, because deregulation of translation process can lead to diseases and tumor formation in humans, the sea urchin orthologs of human genes implicated in human diseases and signaling pathways regulating translation were also discussed.

Amino Acid Sequence↗

PubMed2006

Cellular coexistence of two high molecular subsets of eEF1B complex.

The elongation factor eEF1B involved in protein translation was found to contain two isoforms of the eEF1Bdelta subunit in sea urchin eggs. The eEF1Bdelta2 isoform differs from eEF1Bdelta1 by a specific insert of 26 amino acids. Both isoforms are co-expressed in the cell and likely originate from a unique gene. The feature appears universal in metazoans as judged from in silico analysis in EST-databanks. The eEF1B components were co-immunoprecipitated by specific eEF1Bdelta2 antibodies. Quantification of the proteins in immunoprecipitates and on immunoblots demonstrates that eEF1Bdelta1 and eEF1Bdelta2 proteins are present in two subsets of eEF1B complex. We discuss and propose a model for the different subsets of eEF1B complex concomitantly present in the cell.

Animals↗

PubMed2006

eEF1B: At the dawn of the 21st century.

Translational regulation of gene expression in eukaryotes can rapidly and accurately control cell activity in response to stimuli or when rapidly dividing. There is increasing evidence for a key role of the elongation step in this process. Elongation factor-1 (eEF1), which is responsible for aminoacyl-tRNA transfer on the ribosome, is comprised of two entities: a G-protein named eEF1A and a nucleotide exchange factor, eEF1B. The multifunctional nature of eEF1A, as well as its oncogenic potential, is currently the subject of a number of studies. Until recently, less work has been done on eEF1B. This review describes the macromolecular complexity of eEF1B, its multiple phosphorylation sites and numerous cellular partners, which lead us to suggest an essential role for the factor in the control of gene expression, particularly during the cell cycle.

Animals↗

PubMed2006

[eIF4E and developmental decisions: when translation drives the development].

Regulation of mRNA translation is an important regulatory step in gene expression. During embryonic development, mRNA translation is tightly regulated to produce the protein at the right place, at the right time. The eukaryotic initiation factor 4E (eIF4E) is a major target for the regulation of cap-dependent translation, that plays a key role during embryogenesis including gametogenesis, fertilization and establishment of embryonic axes. In this review, we describe recent advances illustrating the importance of the translational regulator eIF4E and its partners in developmental decisions. double dagger.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

A glyphosate-based pesticide impinges on transcription.

Widely spread chemicals used for human benefits may exert adverse effects on health or the environment, the identification of which are a major challenge. The early development of the sea urchin constitutes an appropriate model for the identification of undesirable cellular and molecular targets of pollutants. The widespread glyphosate-based pesticide affected sea urchin development by impeding the hatching process at millimolar range concentration of glyphosate. Glyphosate, the active herbicide ingredient of Roundup, by itself delayed hatching as judged from the comparable effect of different commercial glyphosate-based pesticides and from the effect of pure glyphosate addition to a threshold concentration of Roundup. The surfactant polyoxyethylene amine (POEA), the major component of commercial Roundup, was found to be highly toxic to the embryos when tested alone and therefore could contribute to the inhibition of hatching. Hatching, a landmark of early development, is a transcription-dependent process. Correlatively, the herbicide inhibited the global transcription, which follows fertilization at the 16-cell stage. Transcription inhibition was dose-dependent in the millimolar glyphosate range concentration. A 1257-bp fragment of the hatching enzyme transcript from Sphaerechinus granularis was cloned and sequenced; its transcription was delayed by 2 h in the pesticide-treated embryos. Because transcription is a fundamental basic biological process, the pesticide may be of health concern by inhalation near herbicide spraying at a concentration 25 times the adverse transcription concentration in the sprayed microdroplets.

Animals↗

PubMed2004

Formulated glyphosate activates the DNA-response checkpoint of the cell cycle leading to the prevention of G2/M transition.

A glyphosate containing pesticide impedes at 10 mM glyphosate the G2/M transition as judged from analysis of the first cell cycle of sea urchin development. We show that formulated glyphosate prevented dephosphorylation of Tyr 15 of the cell cycle regulator CDK1/cyclin B in vivo, the end point target of the G2/M cell cycle checkpoint. Formulated glyphosate had no direct effect on the dual specific cdc25 phosphatase activity responsible for Tyr 15 dephosphorylation. At a concentration that efficiently impeded the cell cycle, formulated glyphosate inhibited the synthesis of DNA occurring in S phase of the cell cycle. The extent of the inhibition of DNA synthesis by formulated glyphosate was correlated with the effect on the cell cycle. We conclude that formulated glyphosate's effect on the cell cycle is exerted at the level of the DNA-response checkpoint of S phase. The resulting inhibition of CDK1/cyclin B Tyr 15 dephosphorylation leads to prevention of the G2/M transition and cell cycle progression.

Animals↗

PubMed2004

Signal transduction pathways that contribute to CDK1/cyclin B activation during the first mitotic division in sea urchin embryos.

In sea urchins, fertilization triggers a rapid rise in protein synthesis necessary for activation of CDK1/cyclin B, the universal cell cycle regulator. It has been shown that FRAP/mTOR is required for eIF4E release from the translational repressor 4E-BP, a process that occurs upstream of de novo cyclin B synthesis. Here, we investigate whether PI 3-kinase acts independently or upstream from FRAP/mTOR in the signal transduction pathway that links fertilization to the activation of the CDK1/cyclin B complex in sea urchin egg. We found that wortmannin, a potent inhibitor of PI 3-kinase, partially inhibited the global increase in protein synthesis triggered by fertilization. Furthermore, wortmannin treatment induced partial inhibition of cyclin B translation triggered by fertilization, in correlation with an intermediate effect of the drug on 4E-BP degradation and on the dissociation of the 4E-BP/eIF4E complex induced by fertilization. Our results presented here suggest that PI 3-kinase activity is required for completion of mitotic divisions of the sea urchin embryo. Incubation of eggs with wortmannin or microinjection of wortmannin or LY 294002 affects drastically mitotic divisions induced by fertilization. In addition, we found that wortmannin treatment inhibits dephosphorylation of the tyrosine inhibitory site of CDK1. Taken together, these data suggest that PI 3-kinase acts upstream of at least two independent targets that function in the CDK1/cyclin B activation triggered by fertilization of sea urchin oocytes. We discuss the significance of these results concerning the cascade of reactions that impinge upon the activation of the CDK1/cyclin B complex that follows sea urchin oocyte fertilization.

Animals↗

PubMed2003

M-phase regulation of the recruitment of mRNAs onto polysomes using the CDK1/cyclin B inhibitor aminopurvalanol.

Translation under the control of the universal cell cycle regulator CDK1/cyclin B was investigated during the first cell cycle in sea urchin embryos. The CDK1/cyclin B inhibitor aminopurvalanol arrested embryos at the G2/M transition. Polysomal mRNAs were purified from control and arrested embryos, and screened for specific mRNA recruitment or release at M-phase by subtractive hybridization. The polysomal repartition of clones issued from this screen was analyzed. Three specific mRNAs were selectively recruited onto polysomes at M-phase. Conversely, two other specific mRNAs were released from polysomes. The isolation of these translationally regulated mRNAs gives now important tools for insights into the regulation of protein synthesis by the cell cycle regulator CDK1-cyclin B.

Animals↗

PubMed2003

eIF4E/4E-BP dissociation and 4E-BP degradation in the first mitotic division of the sea urchin embryo.

The mRNA's 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.

Animals↗

PubMed2003

Cap-dependent translation and control of the cell cycle.

The control of gene expression at the translational level has emerged in the past decade as an important aspect of cell growth, proliferation and malignant transformation. Translation is primarily regulated at the initiation step, and mitogen-dependent signaling pathways converge to modulate the activity of translation initiation factors. In most tumors tested, at least one translation initiation factor is overexpressed and overexpression of translation initiation factors often provokes transformation. Malignant transformation could be caused by the increased translation of a subset of mRNAs encoding important proteins which are required for cell growth and proliferation. These mRNAs usually possess regulatory sequences that render their translation more sensitive to changes in the activity of translation initiation factors. In this chapter, we describe recent advances illustrating the importance of translation in cell cycle progression and cell transformation. Control of translation initiation may represent an excellent target for antitumor drugs.

Animals↗

PubMedDate not supplied

Translational control during mitosis.

Translation is now recognized as an important process in the regulation of gene expression. During the cell cycle, translation is tightly regulated. Protein synthesis is necessary for entry into and progression through mitosis and conversely, modifications of translational activity are observed during the cell cycle. This review focuses on translational control during mitosis (or M-phase) and the role of CDK1/cyclin B, the universal cell cycle regulator implicated in the G2/M transition, in protein synthesis regulation.

CDC2 Protein Kinase↗