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E Jacquet

Publications and source records attributed to E Jacquet.

13 recordsLinked to original sources
PubMed1992

A mouse CDC25-like product enhances the formation of the active GTP complex of human ras p21 and Saccharomyces cerevisiae RAS2 proteins.

GDP-dissociation stimulators (GDSs) are the key element for the regeneration of the active state of ras proteins, but despite intensive investigations, little is so far known about their functional and structural properties, particularly in mammals. A growing number of genes from various organisms have been postulated to encode GDSs on the basis of sequence similarity with the Saccharomyces cerevisiae CDC25 gene, whose product acts as a GDS of RAS proteins. However, except for CDC25 and the related SDC25 C-domain, no biochemical evidence of ras GDS activity for these CDC25-like proteins has yet been available. We show that the product of a recently isolated mouse CDC25-like gene (CDC25Mm) can strongly enhance (more than 1000 times) the GDP release from both human c-Ha-ras p21 and yeast RAS2 in vitro. As a consequence, the CDC25Mm induces a rapid formation of the biologically active Ras.GTP complex. This GDS is much more active on the GDP than on the GTP complex and has a narrow substrate specificity, since it was found to be inactive on several ras-like proteins. The mouse GDS can efficiently substitute for yeast CDC25 in an in vitro adenylylcyclase assay on RAS2 cdc25 yeast membranes. Our results show that a cloned GDP to GTP exchange factor of mammalian ras belongs to the novel family of CDC25-like proteins.

Adenylyl Cyclases↗
PubMed1992

Mutations of Ha-ras p21 that define important regions for the molecular mechanism of the SDC25 C-domain, a guanine nucleotide dissociation stimulator.

The SDC25 C-domain is a very active guanine nucleotide dissociation stimulator (GDS) isolated from Saccharomyces cerevisiae which acts equally well on Ha-ras p21 and yeast RAS2. These properties make the SDC25 C-domain a suitable tool to study the basic mechanism of a GDS. The action of the SDC25 C-domain was analysed by mutation of structurally important regions of p21. Substitutions that influence the coordination of Mg2+.GDP or the interaction of the guanine ring were found to stimulate the intrinsic dissociation of GDP and suppress the action of the SDC25 C-domain. No relevant effects were observed with mutations in the phosphate binding loop L1 or by deleting the last 23 C-terminal residues of p21. Substitutions in the switch region 1 (loop L2) and 2 (loop L4) of p21 strongly impaired the action of this GDS; however, we show that this effect is not related to a decreased affinity of the SDC25 C-domain for the mutated p21. No functional competition could be found between this GDS and the catalytic domain of the human GTPase activating protein (GAP). This indicates that GDS and GAP bind to different sites of the p21.nucleotide complex, even though the same mutations in loops L2 and L4 regions affect the activity of both effectors. Since these two regions appear not to be involved directly in the interaction with GDS, we conclude that the negative effect induced by their mutation is related to their function as switches of selective conformations during the GDP to GTP exchange reaction catalysed by GDS.

Escherichia coli↗
PubMed1990

Fluoroaluminates do not affect the guanine-nucleotide binding centre of the peptide chain elongation factor EF-Tu.

EF-Tu is often referred to as a model for guanine-nucleotide-binding regulatory proteins (G-proteins), since X-ray diffraction analysis of its GTP-binding domain shows a detailed location of the 'consensus' amino acid sequences involved in nucleotide binding. Fluoroaluminates are thought to mimick the gamma-phosphate in the GTPase centre on account of their activating effect on a variety of GTP binding proteins. In the case of EF-Tu, we could find no such effects on the basis of at least three independent functional assays. We did notice, however, complicating interactions between free nucleotides, fluoroaluminates and other ligands. By consequence, if indeed AlF4- behaves as a gamma-phosphate analogue in G-proteins, then EF-Tu must have a different GDP/GTP binding site, despite of the conserved consensus sequences.

Aluminum↗
PubMed1989

Substitution of Val20 by Gly in elongation factor Tu. Effects on the interaction with elongation factors Ts, aminoacyl-tRNA and ribosomes.

Substitution of V20 by G in the consensus element G18HVDHGK24 of EF-Tu (referred to as EF-TuG20) strongly influences the interaction with GDP as well as the GTPase activity [Jacquet, E. & Parmeggiani, A. (1988) EMBO J. 7, 2861-2867]. In an extension of this work we describe additional properties of the mutated factor, paying particular attention to the interaction with the macromolecular ligands. Our results show that the conformational transitions induced by the mutation strongly favor the regeneration of the active complex EF-TuG20.GTP, almost as effectively as with wild-type EF-Tu in the presence of elongation factor Ts. Addition of elongation factor Ts further enhances the rate of the GDP to GTP exchange of the mutated factor. Remarkably, EF-TuG20.GDP can support the enzymatic binding of aminoacyl-tRNA to ribosome.mRNA at low MgCl2 concentration, an effect that with wild-type EF-Tu can only occur in the presence of kirromycin. Our results show that EF-TuG20.GDP shares common features with the GTP-like conformation induced by kirromycin on wild-type EF-Tu. The ability of the ribosome to activate the EF-TuG20 center for GTP hydrolysis is strongly decreased, while the stimulation by aminoacyl-tRNA is conserved. The ribosomal activity is partially restored by addition of aminoacyl-tRNA plus poly(U), showing that codon/anticodon interaction contribute to correct the anomalous interaction between ternary complex and ribosomes. The impaired activity of EF-TuG20 in poly(Phe) synthesis is related to the degree of defective GTP hydrolysis and, most interestingly, it is characterized by a striking increase of the fidelity of translation at high MgCl2 concentration. This effect probably depends on a more selective recognition of the ternary complex by ribosome.mRNA, as a consequence of a longer pausing of EF-TuG20 on the ribosome. In conclusion, position 20 in EF-Tu is important for coordinating the allosteric mechanisms controlling the action of EF-Tu and its ligands.

Allosteric Regulation↗
PubMed1988

Structure-function relationships in the GTP binding domain of EF-Tu: mutation of Val20, the residue homologous to position 12 in p21.

Val20 of elongation factor Tu (EF-Tu), one of the best-characterized GTP binding proteins, is a variable residue within the consensus motif G-X-X-X-X-G-K involved in the interaction with the phosphates of GDP/GTP. To investigate the structure-function relationships of EF-Tu, which is widely used as a model protein, Val20 has been substituted by Gly using oligonucleotide-directed mutagenesis. The most important effects are: (i) a strong reduction of the intrinsic GTPase activity, (ii) a remarkable enhancement of the association and dissociation rates of EF-TuGly20-GDP, mimicking the effect of elongation factor Ts (EF-Ts) and (iii) the inability of ribosomes to influence the intrinsic GTPase of EF-Tu uncoupled from poly(Phe) synthesis. EF-TuGly20 can sustain poly(Phe) synthesis, albeit at a much lower rate than wild-type EF-TuVal20. As with the latter, poly(Phe) synthesis by EF-TuGly20 is inhibited by the antibiotic kirromycin, but differs remarkably in that it is largely independent of the presence of EF-Ts. According to primary sequence alignment, position 20 is homologous to position 12 of ras protein p21. As in p21, this position in EF-Tu is critical, influencing specifically the GDP/GTP interaction as well as other functions. The effect of the mutation displays diversities but also similarities with the situation reported for p21 having the corresponding residues in position 12. The differences observed with two homologous residues, Gly20 and Gly12 in EF-Tu and p21 respectively, show the importance of a variable residue in a consensus element in defining specific functions of GTP binding proteins.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

[Centric relation].

Explore the source record for details and available documents.

Dental Occlusion, Centric↗
PubMed1981

[beta-Lipoprotein serum levels in the months preceeding pregnancy toxemia and its relationship with the Cetrimonium test (author's transl)].

beta-Lipoprotein determination and selective serum protein precipitation with Cetrimonium bromide (Badin's Cetrimonium test) were carried out retroactively (after delivery) on sera selected out of 700 sera collected from women at 4-5 months and 6-7 months of pregnancy. The selected sera were those from 36 women in the first group and 37 in the second group, who had ended pregnancy with toxemia. The sera were matched to sera collected at the same period from women whose pregnancy had been normal until the end. In both the 4-5 month and the 6-7 month groups, women with toxemia at the end of pregnancy had significantly higher levels of beta-lipoprotein and higher Cetrimonium tests but the two data were not correlated. The information they provide on the abnormal condition preceeding toxemia symptoms are different : hyperlipoproteinemia would be a predisposing factor and the Cetrimonium test an evidence of some latent inflammatory process.

Cetrimonium↗
PubMedDate not supplied

Structure-function relationships of elongation factor Tu as studied by mutagenesis.

We have modified elongation factor Tu (EF-Tu) from Escherichia coli via mutagenesis of its encoding tufA gene to study its function-structure relationships. The isolation of the N-terminal half molecule of EF-Tu (G domain) has facilitated the analysis of the basic EF-Tu activities, since the G domain binds the substrate GTP/GDP, catalyzes the GTP hydrolysis and is not exposed to the allosteric constraints of the intact molecule. So far, the best studied region has been the guanine nucleotide-binding pocket defined by the consensus elements typical for the GTP-binding proteins. In this area most substitutions were carried out in the G domain and were found to influence GTP hydrolysis. In particular, the mutation VG20 (in both G domain and EF-Tu) decreases this activity and enhances the GDP to GTP exchange; PT82 induces autophosphorylation of Thr82 and HG84 strongly affects the GTPase without altering the interaction with the substrate. SD173, a residue interacting with (O)6 of the guanine, abolishes the GTP and GDP binding activity. Substitution of residues Gln114 and Glu117, located in the proximity of the GTP binding pocket, influences respectively the GTPase and the stability of the G domain, whereas the double replacement VD88/LK121, located on alpha-helices bordering the GTP-binding pocket, moderately reduces the stability of the G domain without greatly affecting GTPase and interaction with GTP(GDP). Concerning the effect of ligands, EF-TuVG20 supports a lower poly(Phe) synthesis but is more accurate than wild-type EF-Tu, probably due to a longer pausing on the ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗