[Patient information booklet SOR SAVOIR PATIENT: understanding ovarian cancer, a guide for cancer patients and family members].
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Female↗
Biomedical subjects
M Véron
Publications and source records attributed to M Véron.
Inhibition of NEMO, the regulatory subunit of the IKK complex, induces apoptosis in high-risk myelodysplastic syndrome and acute myeloid leukemia.
In high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), blasts constitutively activate the antiapoptotic transcription factor nuclear factor-kappaB (NF-kappaB). Here, we show that this NF-kappaB activation relies on the constitutive activation of the IkappaB kinase (IKK) complex, which is formed by the IKKalpha, IKKbeta and IKKgamma/NF-kappaB essential modulator (NEMO) subunits. A cell-permeable peptide that mimics the leucine zipper subdomain of IKKgamma, thus preventing its oligomerization, inhibited the constitutive NF-kappaB activation and induced apoptotic cell death in a panel of human MDS and AML cell lines (P39, MOLM13, THP1 and MV4-11). Small interfering RNA-mediated knockdown of the p65 NF-kappaB subunit or the three IKK subunits including IKKgamma/NEMO also induced apoptotic cell death in P39 cells. Cell death induced by the IKKgamma/NEMO-antagonistic peptide involved the caspase-independent loss of the mitochondrial transmembrane potential as well as signs of outer mitochondrial membrane permeabilization with the consequent release of cytochrome c, apoptosis-inducing factor and endonuclease G. Primary bone marrow CD34(+) cells from high-risk MDS and AML patients also succumbed to the IKKgamma/NEMO-antagonistic peptide, but not to a mutated control peptide. Altogether, these data indicate that malignant cells in high-risk MDS and AML cells critically depend on IKKgamma/NEMO to survive. Moreover, our data delineate a novel procedure for their therapeutic removal, through inhibition of IKKgamma/NEMO oligomerization.
Acute Disease↗
Natural variation can significantly alter the sensitivity of influenza A (H5N1) viruses to oseltamivir.
Geographic spread of highly pathogenic avian H5N1 influenza viruses may give rise to an influenza pandemic. During the first months of a pandemic, control measures would rely mainly on antiviral drugs, such as the neuraminidase (NA) inhibitors oseltamivir and zanamivir. In this study, we compare the sensitivities to oseltamivir of the NAs of several highly pathogenic H5N1 viruses isolated in Asia from 1997 to 2005. The corresponding 50% inhibitory concentrations were determined using a standard in vitro NA inhibition assay. The K(m) for the substrate and the affinity for the inhibitor (K(i)) of NA were determined for a 1997 and a 2005 virus, using an NA inhibition assay on cells transiently expressing the viral enzyme. Our data show that the sensitivities of the NAs of H5N1 viruses isolated in 2004 and 2005 to oseltamivir are about 10-fold higher than those of earlier H5N1 viruses or currently circulating H1N1 viruses. Three-dimensional modeling of the N1 protein predicted that Glu248Gly and Tyr252His changes could account for increased sensitivity. Our data indicate that genetic variation in the absence of any drug-selective pressure may result in significant variations in sensitivity to anti-NA drugs. Although the clinical relevance of a 10-fold increase in the sensitivity of NA to oseltamivir needs to be investigated further, the possibility that sensitivity to anti-NA drugs could increase (or possibly decrease) significantly, even in the absence of treatment, underscores the need for continuous evaluation of the impact of genetic drift on this parameter, especially for influenza viruses with pandemic potential.
Antiviral Agents↗
Adenosine phosphonoacetic acid is slowly metabolized by NDP kinase.
NDP kinase catalyzes the last step in the phosphorylation of nucleotides. It is also involved in the activation by cellular kinases of nucleoside analogs used in antiviral therapies. Adenosine phosphonoacetic acid, a close analog of ADP already proposed as an inhibitor of ribonucleotide reductase, was found to be a poor substrate for human NDP kinase, as well as a weak inhibitor with an equilibrium dissociation constant of 0.6 mM to be compared to 0.025 mM for ADP. The X-ray structure of a complex of adenosine phosphonoacetic acid and the NDP kinase from Dictyostelium was determined to 2.0 A resolution showing that the analog adopts a binding mode similar to ADP, but that no magnesium ion is present at the active site. As ACP may also interfere with other cellular kinases, its potential as a drug targeting NDP kinase or ribonucleotide reductase is likely to be limited due to strong side effects. The design of new molecules with a narrower specificity and a stronger affinity will benefit from the detailed knowledge of the complex ACP-NDP kinase.
Adenosine↗
Peptidic determinants and structural model of human NDP kinase B (Nm23-H2) bound to single-stranded DNA.
Isoform B of human NDP kinase (NDPK-B) was previously identified as a transcription factor stimulating in vitro and ex vivo the transcription of the c-myc oncogene, which involves this enzyme in carcinogenesis. We have studied the enzymatic properties of NDPK-B in the presence of several single-stranded oligonucleotides. We show that the oligonucleotides are competitive inhibitors of the catalytic activity, indicating that the active site acts as a binding template for the anchorage of the oligonucleotide. Furthermore, the presence of a guanine at the 3'-end of several different aptamers increases its affinity 10-fold. To define the surface of the protein contacting the DNA within the nucleoprotein complex, we used single nanosecond laser pulses as the cross-linking reagent and MALDI-TOF mass spectrometry to identify cross-linked peptides purified from proteolytic digests of the cross-linked complex. Using 11-mer and 30-mer single-stranded oligonucleotides, the same three different nucleopeptides were identified after irradiation of the complexes, indicating a common binding mode for these two aptamers. Taken together, these results allowed us to propose a structural model of NDPK-B bound to single-stranded DNA.
Amino Acid Sequence↗
Mechanism of phosphoryl transfer by nucleoside diphosphate kinase pH dependence and role of the active site Lys16 and Tyr56 residues.
Nucleoside diphosphate (NDP) kinase phosphorylates nucleoside diphosphates with little specificity for the base and the sugar. Although nucleotide analogues used in antiviral therapies are also metabolized to their triphosphate form by NDP kinase, their lack of the 3'-hydroxyl of the ribose, which allows them to be DNA chain terminators, severely impairs the catalytic efficiency of NDP kinase. We have analyzed the kinetics parameters of several mutant NDP kinases modified on residues (Lys16, Tyr56, Asn119) interacting with the gamma-phosphate and/or the 3'-OH of the Mg2+-ATP substrate. We compared the relative contributions of the active-site residues and the substrate 3'-OH for point mutations on Lys16, Tyr56 and Asn119. Analysis of additional data from pH profiles identify the ionization state of these residues in the enzyme active form. X-ray structure of K16A mutant NDP kinase shows no detectable rearrangement of the residues of the active site.
Adenosine Triphosphate↗
Structural basis for activation of alpha-boranophosphate nucleotide analogues targeting drug-resistant reverse transcriptase.
AIDS chemotherapy is limited by inadequate intracellular concentrations of the active triphosphate form of nucleoside analogues, leading to incomplete inhibition of viral replication and the appearance of drug-resistant virus. Drug activation by nucleoside diphosphate kinase and inhibition of HIV-1 reverse transcriptase were studied comparatively. We synthesized analogues with a borano (BH(3)(-)) group on the alpha-phosphate, and found that they are substrates for both enzymes. X-ray structures of complexes with nucleotide diphosphate kinase provided a structural basis for their activation. The complex with d4T triphosphate displayed an intramolecular CH.O bond contributing to catalysis, and the R(p) diastereoisomer of thymidine alpha-boranotriphosphate bound like a normal substrate. Using alpha-(R(p))-boranophosphate derivatives of the clinically relevant compounds AZT and d4T, the presence of the alpha-borano group improved both phosphorylation by nucleotide diphosphate kinase and inhibition of reverse transcription. Moreover, repair of blocked DNA chains by pyrophosphorolysis was reduced significantly in variant reverse transcriptases bearing substitutions found in drug-resistant viruses. Thus, the alpha-borano modification of analogues targeting reverse transcriptase may be of generic value in fighting viral drug resistance.
Anti-HIV Agents↗
Role of nucleoside diphosphate kinase in the activation of anti-HIV nucleoside analogs.
Nucleoside analogs are currently used in antiretrovirus therapies. The best known example is AZT one of the first drug to be used for the treatment of AIDS. However, only the triphosphate derivatives of these compounds act as substrates of the viral reverse transcriptase. Since they do not enter cells, nucleoside analogs are administered and phosphorylated by cellular kinases. The last step in this phosphorylation pathway is catalyzed by nucleoside diphosphate (NDP) kinase. The incorporation of the nucleoside triphosphates into nascent viral DNA chain results in termination of the elongation process. We have performed kinetics studies of the phosphorylation reaction by NDP kinase of dideoxynucleoside diphosphates such as 2',3'-dideoxy-3'-azidothymidine diphosphate (AZT-DP) and 2',3'-dideoxy-2',3'-didehydrothymidine diphosphate (d4T-DP). We show that the catalytic efficiency is strongly decreased and, therefore, that the reaction step catalyzed by NDP kinase constitutes a bottleneck in the processing pathway of anti-HIV compounds. In addition, the affinity of the analogs in the absence of catalysis was determined using a catalytically inactive NDP kinase mutant, showing a reduction of affinity by a factor of 2 to 30, depending on the analog. The structure of NDP kinase provides a structural explanation for these results. Indeed, all nucleoside analogs acting as chain terminators must lack a 3'-OH in the nucleotide deoxyribose. Unfortunately, this same substitution is detrimental for their capacity to be phosphorylated by NDP kinase. This defines the framework for the design of new nucleoside analogs with increased efficiency in antiretroviral therapies.
Anti-HIV Agents↗
The binding mode of human nucleoside diphosphate kinase B to single-strand DNA.
In this paper, we studied the interaction of the human isoform B of nucleoside diphosphate kinase (NDP kinase B) with the nuclease hypersensitive element (NHE) present in the promoter element of the c-myc oncogene. The DNA-binding properties of NDP kinase B and other NDP kinases are compared and the nucleotide requirement for binding are discussed. Using quantitative methods, we identified the DNA-binding sites on the protein and we proposed a structural model for a complex of one hexameric NDP kinase B with an oligonucleotide.
DNA, Single-Stranded↗
Health effects of work schedules in healthcare professions.
Increasing variety in working patterns and the appearance of new forms of shift schedules in the different occupational sectors, including health services, have raised great concern about the quality of working life and job performance. The aim of this paper is to present a systematic review of the literature on the effects on health of irregular schedules in healthcare professionals. Computer and manual searches of databases, and discussion with experts, were used to identify relevant studies. No conclusive evidence was found to favour any particular work system, although there is evidence that extended workdays (9-12 h) should be avoided as much as possible. There is need for carefully designed studies in order to evaluate the long-term consequences of work schedules in healthcare workers.
Health Personnel↗
Single strand DNA specificity analysis of human nucleoside diphosphate kinase B.
Nucleoside diphosphate kinases (NDP kinases) form a family of oligomeric enzymes present in all organisms. Eukaryotic NDP kinases are hexamers composed of identical subunits (approximately 17 kDa). A distinctive property of human NDPK-B encoded by the gene nm23-H2 is its ability to stimulate the gene transcription. This property is independent of its catalytic activity and is possibly related to the role of this protein in cellular events including differentiation and tumor metastasis. In this paper, we report the first characterization of human NDPK-B.DNA complex formation using a filter-binding assay and fluorescence spectroscopy. We analyzed the binding of several oligonucleotides mimicking the promoter region of the c-myc oncogene including variants in sequence, structure, and length of both strands. We show that NDPK-B binds to single-stranded oligonucleotides in a nonsequence specific manner, but that it exhibits a poor binding activity to double-stranded oligonucleotides. This indicates that the specificity of recognition to DNA is a function of the structural conformation of DNA rather than of its specific sequence. Moreover, competition experiments performed with all nucleotides provide evidence for the contribution of the six active sites in the DNA.protein complex formation. We propose a mechanism through which human NDPK-B could stimulate transcription of c-myc or possibly other genes involved in cellular differentiation.
Binding Sites↗
Nucleophilic activation by positioning in phosphoryl transfer catalyzed by nucleoside diphosphate kinase.
The nonenzymatic reaction of ATP with a nucleophile to generate ADP and a phosphorylated product proceeds via a dissociative transition state with little bond formation to the nucleophile. Consideration of the dissociative nature of the nonenzymatic transition state leads to the following question: To what extent can the nucleophile be activated in enzymatic phosphoryl transfer? We have addressed this question for the NDP kinase reaction. A mutant form of the enzyme lacking the nucleophilic histidine (H122G) can be chemically rescued for ATP attack by imidazole or other exogenous small nucleophiles. The ATP reaction is 50-fold faster with the wild-type enzyme, which has an imidazole nucleophile positioned for reaction by a covalent bond, than with H122G, which employs a noncovalently bound imidazole nucleophile [(kcat/KM)ATP]. Further, a 4-fold advantage for imidazole positioned in the nucleophile binding pocket created by the mutation is suggested from comparison of the reaction of H122G and ATP with an imidazole versus a water nucleophile, after correction for the intrinsic reactivities of imidazole and water toward ATP in solution. X-ray structural analysis shows no detectable rearrangement of the residues surrounding His 122 upon mutation to Gly 122. The overall rate effect of approximately 10(2)-fold for the covalent imidazole nucleophile relative to water is therefore attributed to positioning of the nucleophile with respect to the reactive phosphoryl group. This is underscored by the more deleterious effect of replacing ATP with AlphaTauPgammaS in the wild-type reaction than in the imidazole-rescued mutant reaction, as follows. For the wild-type, AlphaTauPgammaS presumably disrupts positioning between nucleophile and substrate, resulting in a large thio effect of 300-fold, whereas precise alignment is already disrupted in the mutant because there is no covalent bond to the nucleophile, resulting in a smaller thio effect of 10-fold. In summary, the results suggest a catalytic role for activation of the nucleophile by positioning in phosphoryl transfer catalyzed by NDP kinase.
Adenosine Diphosphate↗
Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.
The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit.
Animals↗
3'-Phosphorylated nucleotides are tight binding inhibitors of nucleoside diphosphate kinase activity.
Nucleoside diphosphate (NDP) kinase catalyzes the phosphorylation of ribo- and deoxyribonucleosides diphosphates into triphosphates. NDP kinase is also involved in malignant tumors and was shown to activate in vitro transcription of the c-myc oncogene by binding to its NHE sequence. The structure of the complex of NDP kinase with bound ADP shows that the nucleotide adopts a different conformation from that observed in other phosphokinases with an internal H bond between the 3'-OH and the beta-O made free by the phosphate transfer. We use intrinsic protein fluorescence to investigate the inhibitory and binding potential of nucleotide analogues phosphorylated in 3'-OH position of the ribose to both wild type and F64W mutant NDP kinase from Dictyostelium discoideum. Due to their 3'-phosphate, 5'-phosphoadenosine 3'-phosphate (PAP) and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) can be regarded as structural analogues of enzyme-bound ADP. The KD of PAPS (10 microM) is three times lower than the KD of ADP. PAPS also acts as a competitive inhibitor toward natural substrates during catalysis, with a KI in agreement with binding data. The crystal structure of the binary complex between Dictyostelium NDP kinase and PAPS was solved at 2.8-A resolution. It shows a new mode of nucleotide binding at the active site with the 3'-phosphate of PAPS located near the catalytic histidine, at the same position as the gamma-phosphate in the transition state. The sulfate group is directed toward the protein surface. PAPS will be useful for the design of high affinity drugs targeted to NDP kinases.
Animals↗
Coupling between catalysis and oligomeric structure in nucleoside diphosphate kinase.
A dimeric Dictyostelium nucleoside diphosphate kinase has been stabilized by the double mutation P100S-N150stop which targets residues involved in the trimer interface (Karlsson, A., Mesnildrey, S., Xu, Y., Moréra, S., Janin, J., and Veron, M. (1996) J. Biol. Chem. 271, 19928-19934). The reassociation of this dimeric form into a hexamer similar to the wild-type enzyme is induced by the presence of a nucleotide substrate. Equilibrium sedimentation and gel filtration experiments, as well as enzymatic activity measurements, show that reactivation of the enzyme closely parallels its reassociation. A phosphorylatable intermediate with low activity participates in the association pathway while the dimeric form is shown totally devoid of enzymatic activity. Our results support the hypothesis that different oligomeric species of nucleoside diphosphate kinase are involved in different cellular processes where the enzymatic activity is not required.
Adenosine Triphosphate↗
Reaction of 2',3'-dideoxynucleotides triphosphates with recombinant human nucleoside diphosphate kinase.
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Adenosine Diphosphate↗
Functional analysis of the catalytic subunit of Dictyostelium PKA in vivo.
The catalytic subunit of the cAMP-dependent protein kinase (PKA) from Dictyostelium discoideum contains several domains, including an unusually long N-terminal extension preceding a highly conserved catalytic core. We transformed the aggregationless PkaC-null strain with several deletion constructs of both domains. Strains transformed with genes expressing catalytically-inactive polypeptides could not rescue development. Cotransformation with constructs encoding the N-terminal extension and the catalytic core, both unable to rescue development by themselves, yielded transformants able to proceed to late development. A 27-amino acid long hydrophobic region, immediately upstream of the catalytic core, was found indispensable for PKA function. A putative role of this sequence in the acquisition of the active conformation of the protein is discussed.
Amino Acid Sequence↗
The in vitro DNA binding properties of NDP kinase are related to its oligomeric state.
Genetic and biochemical evidences suggest that the enzymatic activity of NDP kinase is necessary but not sufficient for its biological function. While the human NDPK-B binds specifically single-strand polypyrimidines sequences, the hexameric enzyme from Dictyostelium does not. We demonstrated by electrophoretic mobility shift assay and filter binding assay that a dimeric mutant from Dictyostelium binds to an oligodesoxynucleotide while the wild-type does not. These data suggest that the differences in the DNA binding properties of several eucaryotic NDP kinases might be correlated to the differences in the stability of their hexameric structure.
Animals↗