Biomedical subjects

L Thion

Publications and source records attributed to L Thion.

7 recordsLinked to original sources

PubMed2001

Role of the ATP-binding site of SopA protein in partition of the F plasmid.

SopA belongs to a large family of bacterial partition protein ATPases. It helps stabilize the F plasmid by acting as the primary repressor of transcription of the sopAB operon, preventing the destabilizing effects of Sop protein excess. It is also thought to act directly in the F partition mechanism. We have examined the role of SopA in partition and repression by observing the consequences of replacing an invariant ATP-binding site lysine, K120, by glutamine or arginine. Circular dichroism studies of the purified mutant proteins revealed no major differences from wild-type, but in the presence of ADP or ATP each protein showed a characteristic spectrum which suggested a distinct conformational change. The K120Q mutant retained most of the wild-type ATPase activity while the K120R mutant lost it. In neither case was the residual activity stimulated by SopB, as occurs for wild-type SopA. The strength of sop promoter repression by the mutant SopA proteins alone was comparable to that resulting from SopB-enhancement of wild-type SopA, but SopB enhanced repression by the mutant SopA proteins either slightly (K120R) or not at all (K120Q). Mini-Fs in which the sop operon was controlled by a constitutive promoter were destabilized by the mutations, demonstrating the need for SopA and its ATP-binding site in the partition process. The K120R mini-F was lost at the same rate as a mini-F lacking the sopC centromere, the K120Q mutant was lost faster. SopAK120R at high levels was more effective than SopA(+) in disrupting the partition complex, whereas SopAK120Q did not disrupt it at all. These results suggest that one function of SopA in the partition mechanism is to break the paired plasmid structure to allow F molecules to segregate to daughter cells.

Adenosine Diphosphate↗

PubMed2001

Identification of the enzymatic active site of tobacco caffeoyl-coenzyme A O-methyltransferase by site-directed mutagenesis.

Animal catechol O-methyltransferases and plant caffeoyl-coenzyme A O-methyltransferases share about 20% sequence identity and display common structural features. The crystallographic structure of rat liver catechol O-methyltransferase was used as a template to construct a homology model for tobacco caffeoyl-coenzyme A O-methyltransferase. Integrating substrate specificity data, the three-dimensional model identified several amino acid residues putatively involved in substrate binding. These residues were mutated by a polymerase chain reaction method and wild-type and mutant enzymes were each expressed in Escherichia coli and purified. Substitution of Arg-220 with Thr resulted in the total loss of enzyme activity, thus indicating that Arg-220 is involved in the electrostatic interaction with the coenzyme A moiety of the substrate. Changes of Asp-58 to Ala and Gln-61 to Ser were shown to increase K(m) values for caffeoyl coenzyme A and to decrease catalytic activity. Deletions of two amino acid sequences specific for plant enzymes abolished activity. The secondary structures of the mutants, as measured by circular dichroism, were essentially unperturbed as compared with the wild type. Similar changes in circular dichroism spectra were observed after addition of caffeoyl coenzyme A to the wild-type enzyme and the substitution mutants but not in the case of deletion mutants, thus revealing the importance of these sequences in substrate-enzyme interactions.

Adenine↗

PubMed2001

Reactivity at the interface of chiral amphiphilic dendrimers. High asymmetric reduction by NaBH(4) of various prochiral ketones.

New amphiphilic dendrimers derived from PAMAM and D-gluconolactone were found to induce chirality in the reduction of prochiral ketones by NaBH(4), in heterogeneous (THF) and homogeneous (water) conditions. The third generation of these amphiphilic dendrimers, G(3)G, was found to be a good chiral ligand for the reduction of various prochiral ketones in heterogeneous conditions. Even with substrates well-known to give poor results (especially linear ketones), good enantioselectivities were obtained. It is also important to notice that under heterogeneous conditions (THF) the dendrimer could be recovered by filtration, regenerated, and recycled (up to 10 times), leading to reproducible results in asymmetric reduction of ketones. We have also discussed the reduction of acetophenone in water. Evidence is presented that the selectivity is dominated by the architecture of the dendrimer and some supramolecular ordering in the position of the ketone at the chiral solvating interface. The results obtained showed a correlation between stereoselectivity of the reduction and the compact character of the dendritic particles.

Journal Article↗

PubMed2001

Atypical binding of the neuronal POU protein N-Oct3 to noncanonical DNA targets. Implications for heterodimerization with HNF-3 beta.

The capacity of POU proteins to recognize different DNA sequences and to bind target DNA in the form of monomers, cooperative dimers or heterodimers is important in relation to their transcriptional regulatory properties. The N-Oct3 neuron-specific protein binds to an octamer-like sequence (AAATAATGC) within the (-102/-72) neuronal promoter region of the human aromatic L-amino acid decarboxylase (AADC) gene. In this atypical case the POUh and POUs tetrameric subsites are spaced one nucleotide apart and in switched order as compared with the consensus octamer. Moreover this POU binding motif overlaps the hepatocyte nuclear factor HNF-3 beta binding site (TGCTCAGTAAA) which itself contains a heptamer-like sequence (CTCAGTA). Using the isolated DNA binding domains (DBD) of the two proteins, it is shown that, when binding to this unusual recognition sequence, N-Oct3 either exhibits noncooperative homodimerization or allows the simultaneous binding of the second transcription activator HNF-3 beta. CD studies indicate that the binding of N-Oct3 monomers/dimers and N-Oct3-HNF-3 beta heterodimers to the DNA induces conformational changes of both protein and DNA. Partial proteolysis/MALDI-MS was used in conjunction with molecular modelling to show that the protein conformational change resulting from binary N-Oct3/DNA complex formation occurs within the linker peptide joining the POUs and POUh subdomains. Furthermore, modelling the N-Oct3/HNF-3 beta/DNA ternary complex predicts a nucleotide rearrangement in the overlap region and an interaction between both transcription factors. In the light of our findings, which illustrate both site-dependent and site-independent protein and DNA conformational changes, general implications for the allosteric function of DNA response elements in transcriptional regulation are discussed.

Allosteric Site↗

PubMed1998

Plasma membrane depolarization-activated calcium channels, stimulated by microtubule-depolymerizing drugs in wild-type Arabidopsis thaliana protoplasts, display constitutively large activities and a longer half-life in ton 2 mutant cells affected in the organization of cortical microtubules.

Depolarization-activated plasma membrane calcium channels have been suggested to play prominent roles in signal perception and transduction processes during growth and development of higher plants. The existence of such channels has recently been established in higher plant cells. However, patch-clamp experiments have shown that their activity is very low and decreases very rapidly after the establishment of the whole-cell configuration, due most probably to protein-protein interactions involving microtubules. The present study takes advantage of the existence of Arabidopsis thaliana mutants referred to as ton 2 mutants reported to be affected in their microtubule organization, to address the physiological relevance of such a hypothesis based on a pharmacological approach. Patch-clamp studies showed that depolarization-activated calcium channel activities in ton 2 protoplasts were 10-fold higher and their relative half-life three-times longer than in wild-type protoplasts. In addition, oryzalin and colchicine, which disrupt the microtubule organization, stimulated and stabilized calcium channel activities in wild-type but remained ineffective on ton 2 protoplasts. However, although the microtubules appeared important in the regulation of calcium channels in A. thaliana, immunocytological staining of tubulin demonstrated that there was no visible difference in the general organization of microtubule networks or in the amount of microtubules bound to the plasma membrane in ton 2 and wild-type protoplasts. It is suggested that the down-regulation of calcium channels implicating microtubules involves additional component(s) corresponding probably to gene product(s) defective in ton 2 mutant cells.

Arabidopsis↗

PubMed1997

Organization of cytoskeleton controls the changes in cytosolic calcium of cold-shocked Nicotiana plumbaginifolia protoplasts.

Using Nicotiana plumbaginifolia constitutively expressing the recombinant bioluminescent calcium indicator, aequorin, it has been previously demonstrated that plant cells react to cold-shock by an immediate rise in cytosolic calcium. Such an opportune system has been exploited to address the regulatory pathway involved in the calcium response. For this purpose, we have used protoplasts derived from N. plumbaginifolia leaves that behave as the whole plant but with a better reproducibility. By both immunodetecting cytoskeletal components on membrane ghosts and measuring the relative change in cytosolic calcium, we demonstrate that the organization of the cytoskeleton has profound influences on the calcium response. The disruption of the microtubule meshwork by various active drugs, such as colchicin, oryzalin and vinblastin, leads to an important increase in the cytosolic calcium (up to 400 nM) in cold-shocked protoplasts over control. beta-Lumicolchicin, an inactive analogue of colchicin, is ineffective either on cytoplasmic calcium increase or on microtubule organization. A microfilament disrupting drug, cytochalasin D, exerts a slight stimulatory effect, whereas the simultaneous disruption of microtubule and microfilament meshworks results in a dramatic increase in the calcium response to cold-shock. The results described in the present paper illustrate the role of the intracellular organization and, more specifically, the role of cytoskeleton in controlling the intensity of calcium response to an extracellular stimulus.

Actin Cytoskeleton↗

PubMed1996

Activation of plasma membrane voltage-dependent calcium-permeable channels by disruption of microtubules in carrot cells.

Plasma membrane-bound voltage-dependent calcium channels may couple the perception of an initial stimulus to a regulated pathway for calcium influx. The activities of these channels have been shown to be very low and highly unstable but may be recruited by large-predepolarizing pulses, according to a process referred to as recruitment. By combining pharmacological and electrophysiological approaches, we demonstrate in the present paper that the cytoskeleton plays an important role in the regulation of the activity and stability of voltage-dependent calcium channels during whole-cell patch-clamp experiments on carrot protoplasts. Whereas drugs affecting the organization of the microfilament network have no measurable effect, the manipulation of the microtubule network elicits important changes. Thus, the addition of colchicine or oryzalin, which are known to disrupt microtubule organization, leads to a 6-10-fold increase in calcium channel activities and half-life. In contrast, stabilization of the microtubules by taxol has no effect on any of these parameters. The data obtained suggest that interactions of microtubules and voltage-dependent calcium channels by either direct or indirect mechanisms inhibit channel activities and decrease their half-life. In contrast, the disruption of the network overcomes such an inhibitory effect and allows the activation of calcium channels. It is speculated that under normal physiological conditions these protein-protein interactions may work in a reversible manner and contribute to signal transduction in higher plants.

Calcium Channels↗