Biomedical subjects

G Divita

Publications and source records attributed to G Divita.

At least 19 recordsLinked to original sources

PubMed1998

Kinetics of dimerization and interactions of p13suc1 with cyclin-dependent kinases.

The impact of p13suc1 on the conformation and regulation of cyclin-dependent kinases (cdks) and cyclins was investigated by spectroscopic and rapid kinetic approaches. In the absence of phosphorylation on cdks, p13suc1 formed stable complexes, mainly stabilized by hydrophobic interactions, specifically with cdk2 and cdc2. The presence of cyclin A, associated with cdk2 or cdc2, increased the stability of the interaction between cdk2 and p13suc1 by a factor of 2. However, cyclin A did not modify the association rate of p13suc1 to cdk2, but the dissociation rate, which was decreased 3-fold. Moreover, binding of p13suc1 to cdk2 resulted in a 2-fold decrease in the release of nucleotide from cdk2, indicating that p13suc1 induces a marked change in the structure of the nucleotide binding site of cdks. On the basis of the structure of cdk2/CksHs1 complex and on our kinetic results, we propose that the binding of Cks proteins to C-lobe of cdk2 is stabilized by the presence of cyclin A and that it may modify the orientation of the loop carrying residues 14 and 15 and their consequent access for dephosphorylation by cdc25 phosphatases. Finally, we have shown that dimerization of p13suc1 in the presence of zinc abolishes its interaction with cdks, which suggests that the binding of p13suc1 to cdk2 or cdk2/cyclin A may be regulated by dimerization of p13suc1 in vivo.

Amino Acid Sequence↗

PubMed1998

Interaction between the N-terminal domain of human DNA topoisomerase I and the arginine-serine domain of its substrate determines phosphorylation of SF2/ASF splicing factor.

Human DNA topoisomerase I, known for its DNA-relaxing activity, is possibly one of the kinases phosphorylating members of the SR protein family of splicing factors, in vivo. Little is known about the mechanism of action of this novel kinase. Using the prototypical SR protein SF2/ASF (SRp30a) as model substrate, we demonstrate that serine residues phosphorylated by topo I/kinase exclusively located within the most extended arginine-serine repeats of the SF2/ASF RS domain. Unlike other kinases such as cdc2 and SRPK1, which also phosphorylated serines at the RS domain, topo I/kinase required several SR dipeptide repeats. These repeats possibly contribute to a versatile structure in the RS domain thereby facilitating phosphorylation. Furthermore, far-western, fluorescence spectroscopy and kinase assays using the SF2/ASF mutants, demonstrated that kinase activity and binding were tightly coupled. Since the deletion of N-terminal 174 amino acids of Topo I destroys SF2/ASF binding and kinase activity but not ATP binding, we conclude that at least two distinct domains of Topo I are necessary for kinase activity: one in the C-terminal region contributing to the ATP binding site and the other one in the N-terminal region that allows binding of SF2/ASF.

Adenosine Triphosphate↗

PubMed1998

The C-terminal domain but not the tyrosine 723 of human DNA topoisomerase I active site contributes to kinase activity.

Human DNA topoisomerase I not only has DNA relaxing activity, but also splicing factors phosphorylating activity. Topo I shows strong preference for ATP as the phosphate donor. We used photoaffinity labeling with the ATP analogue [alpha-32P] 8-azidoadenosine-5'-triphosphate combined with limited proteolysis to characterize Topo I domains involved in ATP binding. The majority of incorporated analogue was associated with two fragments derived from N-terminal and C-terminal regions of Topo I, respectively. However, mutational analysis showed that deletion of the first 138 N-terminal residues, known to be dispensable for topoisomerase activity, did not change the binding of ATP or the kinase activity. In contrast, deletion of 162 residues from the C-terminal domain was deleterious for ATP binding, kinase and topoisomerase activities. Furthermore, a C-terminal tyrosine 723 mutant lacking topoisomerase activity is still able to bind ATP and to phosphorylate SF2/ASF, suggesting that the two functions of Topo I can be separated. These findings argue in favor of the fact that Topo I is a complex enzyme with a number of potential intra-cellular functions.

Adenosine Triphosphate↗

PubMed1998

Interactions of primary amphipathic vector peptides with membranes. Conformational consequences and influence on cellular localization.

The conformations of two peptides produced by the combinations of a nuclear localization sequence and a sequence issued from the fusion protein gp41 of HIV 1 have been analyzed both in solution and in membranes or in membrane mimicking environments. Both are shown to be nonordered in water, alpha-helical when incorporated into SDS micelles where the helical domain concerns the hydrophobic part of the peptides. Interactions with lipids induce the formation of beta-sheet and the lipid-peptide interactions are governed by the nature of the lipid polar headgroups. A monolayer study shows that replacement of the sequence separating the two sequences with an arginine favors the lipid-peptide interactions which may contribute to the understanding of the different, nuclear and membrane associated, cellular localizations of the peptides.

Amino Acid Sequence↗

PubMed1998

Design of carrier peptide-oligonucleotide conjugates with rapid membrane translocation and nuclear localization properties.

Peptides containing a hydrophobic motif associated with a nuclear localization signal separated by various linkers were synthesized in solid phase. The hydrophobic sequence corresponds either to a signal peptide sequence or to a fragment of the fusion peptide of GP41 while the hydrophilic sequence is that of a nuclear localization signal. The C-termini of these peptides bear a cysteamide group that was linked to a fluorescent probe. This allowed the cellular localization of the probe to be determined as a function of the peptide sequences. The labeled peptides were then incubated with fibroblasts. Using N-biotinylated derivatives we confirmed by indirect immunofluorescence that the observed localizations corresponds to those of the peptides. The presence of a linker appears to play a role in the cellular localization. One of these peptides was successfully used to target fluorescent oligodeoxynucleotides into living cells demonstrating improved cell delivery of peptide-oligodeoxynucleotide conjugates.

Amino Acid Sequence↗

PubMed1998

Evaluating lexical variant generation to improve information retrieval.

Techniques for managing lexical variation constitute an integral part of information retrieval systems. We report on a series of experiments aimed at evaluating LVG, a lexical variant management tool which addresses the particular problems involved in matching health related vocabularies to concepts in the Unified Medical Language System (UMLS) Metathesaurus. Experiments conducted on data from the Large Scale Vocabulary Test indicate the effectiveness of this approach to managing biomedical information.

Evaluation Studies as Topic↗

PubMed1997

Conformations of primary amphipathic carrier peptides in membrane mimicking environments.

Two peptides designed for drug delivery were generated by the combination of a signal peptide with a nuclear localization sequence and are shown to facilitate the cellular internalization of small molecules which are covalently linked to these peptides. In order to understand the mechanism of internalization, the conformations of the peptides were investigated through different approaches both in solution and in membrane-mimicking environments. These peptides are highly versatile and adopt different conformational states depending on their environment. While in a disordered form in water, they adopt an alpha-helical structure in TFE and in the presence of micelles of SDS or DPC. The structured domain encompasses the hydrophobic part of the peptides, whereas the charged C-termini remain unstructured. In contrast, in the presence of lipids and whatever the nature of the phosphate headgroup, the two peptides mainly adopt an antiparallel beta-sheet form and embed in the lipidic cores. This result suggests that the beta-sheet is responsible for the translocation through the cellular membranes but also questions the conformational state of signal peptides when associated to hydrophilic sequences.

Amino Acid Sequence↗

PubMed1997

Study of the substrate-binding properties of bovine liver adenosine kinase and inhibition by fluorescent nucleoside analogues.

Adenosine kinase (AK) catalyzes the phosphorylation of adenosine to AMP with ATP as phosphate donor. Intrinsic fluorescence of bovine liver AK was shown previously to be a sensitive probe to quantify the binding of substrates to the enzyme [Elaloui, A., Divita, G., Maury, G., Imbach, J.-L. & Goody, R. S. (1994) Eur. J Biochem. 221, 839-846]. AK contains two catalytic, sites: a high-affinity site, which binds adenosine and AMP selectively; and a site for ATP and ADP. In the present work, these two sites were characterized by combining the quenching of protein fluorescence induced by the binding of the ligands and the fluorescence enhancement observed upon binding of the N-methylanthraniloyl-derivated nucleotides or adenosine. A new fluorescent analog of adenosine, 5'-N-methylanthraniloyl-adenosine, was synthesized and shown to bind selectively to the high-affinity adenosine-binding site with an affinity similar to that of adenosine (Kd 1 microM). In contrast, 2'(3')-N-methylanthraniloyl derivatives of ATP, adenosine (5')tetraphospho(5')adenosine (Ap4A), and adenosine (5')pentaphospho(5')adenosine (Ap5A), bind to the enzyme at the ATP site. Methylantraniloyl derivatives of ATP and adenosine were used as tools for selective characterization of a series of adenosine analogues. The bisubstrate inhibitors Ap4A and Ap5A bind to the ATP site with high affinity and apparently not to the adenosine site, thus acting more as ATP analogues than true bisubstrate ligands. The binding properties of a series of adenosine analogues were strongly dependent on the structural modifications on adenosine. The analogues modified at positions 2' or 3' show similar affinities for AK as that of adenosine, whereas adenosine analogues modified at the base present a relatively low affinity for the enzyme.

Adenosine↗

PubMed1997

A new peptide vector for efficient delivery of oligonucleotides into mammalian cells.

The development of antisense and gene therapy has focused mainly on improving methods for oligonucleotide and gene delivery into cells. In the present work, we describe a potent new strategy for oligonucleotide delivery based on the use of a short peptide vector, termed MPG (27 residues), which contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T-antigen. The formation of peptide vector/oligonucleotide complexes was investigated by measuring changes in intrinsic tryptophan fluorescence of peptide and of mansyl-labelled oligonucleotides. MPG exhibits relatively high affinity for both single- and double-stranded DNA in a nanomolar range. Based on both intrinsic and extrinsic fluorescence titrations, it appears that the main binding between MPG and oligonucleotides occurs through electrostatic interactions, which involve the basic-residues of the peptide vector. Further peptide/peptide interactions also occur, leading to a higher MPG/oligonucleotide ratio (in the region of 20/1), which suggests that oligonucleotides are most likely coated with several molecules of MPG. Premixed complexes of peptide vector with single or double stranded oligonucleotides are delivered into cultured mammalian cells in less than 1 h with relatively high efficiency (90%). This new strategy of oligonucleotide delivery into cultured cells based on a peptide vector offers several advantages compared to other commonly used approaches of delivery including efficiency, stability and absence of cytotoxicity. The interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and crossing of the plasma membrane. The mechanism of cell delivery of oligonucleotides by MPG does not follow the endosomal pathway, which explains the rapid and efficient delivery of oligonucleotides in the nucleus. As such, we propose this peptide vector as a powerful tool for potential development in gene and antisense therapy.

3T3 Cells↗

PubMed1997

Interactions of cyclins with cyclin-dependent kinases: a common interactive mechanism.

The formation of cdk-cyclin complexes has been investigated at the molecular level and quantified using spectroscopic approaches. In the absence of phosphorylation, cdk2, cdc2, and cdk7 form highly stable complexes with their "natural" cyclin partners with dissociation constants in the nanomolar range. In contrast, nonphosphorylated cdc2-cyclin H, cdk2-cyclin H, and cdk7-cyclin A complexes present a 25-fold lower stability. On the basis of both the structure of the cdk2-cyclin A complex and on our kinetic results, we suggest that interaction of any cyclin with any cdk involves the same hydrophobic contacts and induces a marked conformational change in the catalytic cleft of the cdks. Although cdks bind ATP strongly, they remain in a catalytically inactive conformation. In contrast, binding of the cyclin induces structural rearrangements which result in the selective reorientation of ATP, a concomitant 3-fold increase in its affinity, and a 5-fold decrease of its release from the active site of cdks.

Adenosine Triphosphate↗

PubMed1997

[New strategy for RNA vectorization in mammalian cells. Use of a peptide vector].

A major barrier for gene delivery is the low permeability of nucleic acids to cellular membranes. The development of antisenses and gene therapy has focused mainly on improving methods of oligonucleotide or gene delivery to the cell. In this report we described a new strategy for RNA cell delivery, based on a short single peptide. This peptide vector is derived from both the fusion domain of the gp41 protein of HIV and the nuclear localization sequence of the SV40 large T antigen. This peptide vector localizes rapidly to the cytoplasm then to the nucleus of human fibroblasts (HS-68) within a few minutes and exhibits a high affinity for a single-stranded mRNA encoding the p66 subunit of the HIV-1 reverse transcriptase (in a 100 nM range). The peptide/RNA complex formation involves mainly electrostatic interactions between the basic residues of the peptide and the charges on the phosphate group of the RNA. In the presence of the peptide-vector fluorescently-labelled mRNA is delivered into the cytoplasm of mammalian cells (HS68 human fibroblasts) in less than 1 h with a relatively high efficiency (80%). This new concept based on a peptide-derived vector offers several advantages compared to other compounds commonly used in gene delivery. This vector is highly soluble and exhibits no cytotoxicity at the concentrations used for optimal gene delivery. This result clearly supports the fact that this peptide vector is a powerful tool and that it can be used widely, as much for laboratory research as for new applications and development in gene and/or antisense therapy.

Cells, Cultured↗

PubMed1997

Conducting the NLM/AHCPR Large Scale Vocabulary Test: a distributed Internet-based experiment.

The Large Scale Vocabulary Test, sponsored by the National Library of Medicine (NLM) and the Agency for Health Care Policy and Research (AHCPR), was conducted to determine the extent to which a combination of existing health-related terminologies cover vocabulary needed in health care information systems. The test was conducted over the Internet using a sophisticated World Wide Web interface with over 60 participants and over 40,000 terms submitted. This paper discusses the issues encountered in the design and execution of the experiment, including the design of the interface and the issues of recruitment, training, and guidance of remote participants over the Internet. Test data are currently undergoing expert review. Upon completion of the expert review, the results of the test will be fully reported.

Computer Communication Networks↗

PubMed1996

Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I.

Several metazoan splicing factors are characterized by ribonucleoprotein (RNP) consensus sequences and arginine-serine repeats (RS domain) which are essential for their function in splicing. These include members of the SR-protein family (SC35, SF2/ASF), the U1 small nuclear (sn) RNP protein (U1-70K) and the U2 snRNP auxiliary factor (U2AF). SR proteins are phosphorylated in vivo and the phosphorylation state of U1-70K's RS domain influences its splicing activity. Here we report the purification of a protein kinase that is specific for SR proteins and show that it is DNA topoisomerase I. This enzyme lacks a canonical ATP-binding motif but binds ATP with a dissociation constant of 50 nM. Camptothecin and derivatives, known to be specific inhibitors of DNA topoisomerase I, strongly inhibit the kinase activity in the presence of DNA and affect the phosphorylation state of SR proteins. Thus, DNA topoisomerase I may well be one of the SR protein kinases operating in vivo.

Adenosine Triphosphate↗

PubMed1996

Escherichia coli isocitrate dehydrogenase kinase/phosphatase. Overproduction and kinetics of interaction with its substrates by using intrinsic fluorescence and fluorescent nucleotide analogues.

The aceK gene of Escherichia coli, which encodes the isocitrate dehydrogenase kinase/phosphatase (IDH K/P), was cloned in the pQE30 expression vector to overproduce a protein tagged with six histidine residues at its N-terminus. By using a one-step chromatographic procedure, the IDH K/P was purified to near homogeneity. The IDH K/P, which contains nine Trp residues, exhibited a characteristic intrinsic tryptophan fluorescence with a low maximal emission at 326 nm. The low value of the Stern-Volmer quenching constant in the presence of acrylamide (Ksv = 2.1 M-1) indicated that the tryptophan residues were deeply buried in the protein. Furthermore, the intrinsic tryptophan fluorescence was very sensitive to the binding of nucleotide. The quenching of protein fluorescence induced by the binding of nucleotide together with an increased intrinsic fluorescence of fluorescent nucleotide analogues, methylanthraniloyl-derivatives ADP, ATP, GDP and GTP and adenosine-5'-triphosphoro-1-(5-sulfonic-acid) naphthylamidate, were used to investigate the interaction with IDH K/P. The IDH K/P dimer was shown to contain two identical nucleotide binding sites, one on each subunit, with a Kd in the range of 1.7-2.5 microM for unmodified ADP or ATP and of 2.5-3.7 microM for fluorescently labelled nucleotides. In contrast, the affinity for GDP or GTP was 10-fold lower than for adenine nucleotides. The nucleotide binding site was located within residues 315-340 by using limited proteolysis of IDH K/P by endoproteinase Lys-C. Only one main site of cleavage was obtained: the peptide bond K346-E347 which was strongly protected in the presence of ATP.

Adenosine Triphosphatases↗

PubMed1995

Conformational stability of dimeric HIV-1 and HIV-2 reverse transcriptases.

The dissociation of dimeric reverse transcriptase (RT) of the human immunodeficiency virus (HIV) types 1 and 2 has been investigated using acetonitrile as a dissociating agent. The equilibrium transitions were monitored by combining different approaches (fluorescence spectroscopy, polymerase activity assay, and size-exclusion HPLC). The dissociation of RT induced a complete loss of polymerase activity and a 25% increase of the intrinsic fluorescence. It is fully reversible, and the midpoints of the equilibrium transition curves are dependent on the concentration of the enzyme used, suggesting a two-state transition model for the dissociation of RT in which dimers are in equilibrium with folded monomers. For both RTs, the heterodimeric form is more stable against dissociating agents and different pH than the corresponding homodimeric form. Moreover, heterodimeric HIV-2 RT exhibits a higher stability than HIV-1 RT, with a free energy of dissociation of 12.1 kcal/mol at pH 6.5 and 25 degrees C, instead of 10 kcal/mol for HIV-1 RT. The binding of a primer/template induces a marked conformational change in both RTs, shown by the lower accessibility of the tryptophans to quenchers and the increase in tryptophan heterogeneity, and stabilized the dimeric form of both RTs (10-100-fold). The central role of hydrophobic interactions in dimer formation has been revealed by the 30% increase of exposure of the tryptophan cluster to quenchers upon dissociation of RT and the binding of 4 equiv of 1-anilino-8-naphthalenesulfonate to the dissociated enzymes.

Acetonitriles↗

PubMed1995

Interface peptides as structure-based human immunodeficiency virus reverse transcriptase inhibitors.

Reverse transcriptases from both human immunodeficiency viruses type 1 and 2 are obligatory dimers. A tryptophan-rich repeat motif that is highly conserved between these proteins, as well as in the reverse transcriptase from simian immunodeficiency virus, has been postulated to be involved in hydrophobic subunit interactions. A synthetic 19-mer peptide covering part of this tryptophan repeat motif was recently shown to inhibit human immunodeficiency viruses type 1 reverse transcriptase subunit dimerization (Divita, G., Restle, T., Goody, R. S., Chermann, J.-C., and Baillon, J. G. (1994) J. Biol. Chem. 269, 13080-13083). In the present study, we show that the same peptide can also inhibit human immunodeficiency virus type 2 reverse transcriptase subunit dimerization, suggesting that the same inhibitors might be used as agents against both viruses as well as against variants of human immunodeficiency virus type 1 that differ from the variant against which they were developed. Under appropriate experimental conditions, e.g. at acidic pH, this peptide is also able to induce the dissociation of the enzyme from human immunodeficiency virus type 1.

Amino Acid Sequence↗

PubMed1995

Human immunodeficiency virus reverse transcriptase substrate-induced conformational changes and the mechanism of inhibition by nonnucleoside inhibitors.

A combination of transient kinetic and equilibrium titration methods has been used to show that both primer/template and nucleotide binding to human immunodeficiency virus type 1 (HIV-1) reverse transcriptase are two-step processes. In both cases, after initial formation of relatively weakly bound states, isomerization reactions lead to tightly bound states. In the case of deoxynucleotide binding to the reverse transcriptase-primer/template complex, the second step in the interaction is rate-limiting in the overall reaction during processive polymerization. Discrimination against incorrect nucleotides occurs both in the initial weak binding and in the second step but is purely kinetic in the second step (as opposed to thermodynamic in the first step). Nonnucleoside inhibitors have a relatively small effect on nucleotide-binding steps (overall affinity is reduced by a factor of ca. 10), while the affinity of the primer/template duplex is increased by at least a factor of 10. The major effect of nonnucleoside inhibitors is on the chemical step (nucleotide transfer).

Antiviral Agents↗

PubMed1995

Dimerization kinetics of HIV-1 and HIV-2 reverse transcriptase: a two step process.

The dimerization processes of the human immunodeficiency virus (HIV) types 1 and 2 reverse transcriptase (RTs) from their subunits have been investigated using a number of complementary approaches (fluorescence spectroscopy, size exclusion-HPLC and polymerase activity assay). The formation of the native heterodimeric form of HIV-1 and HIV-2 RT occurs in a two step process. The first step is a concentration-dependent association of the two subunits (p66 and p51) to give a heterodimeric intermediate, which slowly isomerizes to the "mature" heterodimeric form of the enzyme. For both RTs, the first step behaves as a second order reaction with similar association rate constants (in the range of 2 x 10(4) to 4 x 10(4) M-1 s-1). This initial dimerization results in a 25% quenching of the intrinsic fluorescence and a 30% decrease in the accessibility of the tryptophan hydrophobic cluster to solvent as revealed by iodide quenching experiments and by monitoring the binding of 1-anilino-8-naphthalenesulphonate. The formation of the intermediate-RT form appears to involve hydrophobic regions of the subunits containing tryptophan residues. This intermediate form is devoid of polymerase activity, but is able to bind primer/template with high affinity. The final stage of the mature RT-heterodimer formation occurs in a slow first order reaction, which is 12-fold faster for HIV-2 (1.2 h-1) than HIV-1 RT (0.1 h-1). At micromolar concentrations, this slow isomerization constitutes the rate limiting step of the RT maturation and the structural change involved appears to be partly associated with the catalytic site, as shown using fluorescent labelled primer/template. On the basis of both the presently available X-ray structure of the HIV-1 RT and the predicted structure of HIV-2 RT, the thumb subdomain of the p51 subunit seems to be involved in this maturation step, which is probably the interaction of this domain with the RNAse H domain of the large subunit. The placement of the fingers subdomain of p51 in the palm subdomain of the p66 subunit may also be associated with formation of mature heterodimeric RTs.

Anilino Naphthalenesulfonates↗