Les récepteurs couplés aux protéines G forment des complexes multimériques comprenant protéines G et effecteurs. Nous cherchons à caractériser de tels complexes comprenant les récepteurs opioïdes delta (DOR) et les canaux Kir3, qui nous sont d’intérêt vu leur implication dans l’analgésie des opioïdes. Des expériences d’immunopurification, de BRET et de liaison GTPgS ont été réalisées à l’intérieur de cellules HEK293 transfectées. Les canaux Kir3 ont été co-immunopurifiés avec les DOR, suggérant une interaction spontanée entre récepteur et effecteur. Des essais BRET ont corroboré que l’interaction était présente dans des cellules vivantes et nous ont permis d’identifier une interaction spontanée et spécifique entre DOR/Gg et Gg/Kir3, indiquant leur coexistence en un même complexe. Puisque l’activation du récepteur implique la présence de changements conformationnels à l’intérieur de celui-ci, nous étions intéressés à vérifier si l’information conformationnelle circule à partir du récepteur lié au ligand jusqu’à l’effecteur en aval. Ainsi, nous avons déterminé l’effet de différents ligands sur le signal BRET généré par les paires suivantes : DOR/Gbg, DOR/Kir3 et Kir3/Gbg. Nous avons constaté une modulation de l’interaction DOR/Gbg et Gbg/Kir3 suivant l’ordre d’efficacité des ligands à stimuler la protéine G, ce que nous n’avons pas observé entre DOR et Kir3. Donc, nous concluons que l’information conformationnelle circule du récepteur au canal Kir3 via la protéine Gbg. Ces résultats nous ont permis de développer un biosenseur BRET (EYFP-Gg2/Kir3.1-Rluc) qui pourrait être utilisé dans le criblage à haut débit afin de détecter de nouvelles molécules ayant une grande efficacité à activer les canaux Kir3. / G protein-coupled receptors form multimeric complexes comprising G protein and effectors. We want to characterize such complexes comprising delta opioid receptors (DOR) and Kir3 channels, which interest us due to their involvement in opioid analgesia. Immunopurification, BRET and GTPgS binding experiments were done in transfected HEK293 cells. Kir3 channels were co-immunopurified with DOR, implying a spontaneous interaction between the receptor and effector. BRET assays corroborated the presence of this interaction in living cells and allowed us to identify a spontaneous and specific interaction between DOR/Gg and Gg/Kir3, indicating their co-existence within the same complex. Since the activation of the receptor implies it undergoes conformational changes, we were interested in evaluating if the conformational information flows from the ligand-bound receptor until the downstream effector. Hence, we determined the effect of different ligands on the BRET signal that was generated by the following pairs: DOR/Gbg, DOR/Kir3 and Kir3/Gbg. We noticed a modulation of the DOR/Gbg and Gbg/Kir3 interactions that followed the order of efficacy of the ligands to activate the G protein, which we did not observe between DOR and Kir3. Therefore, we concluded that the conformational information flows from the receptor to the Kir3 channel via the Gbg protein. These results allowed us to develop a BRET biosensor (EYFP-Gg2/Kir3.1-Rluc), which could be used in high throughput screening to detect new molecules that activate Kir3 channels with high efficacy.

Les récepteurs couplés aux protéines G forment des complexes multimériques comprenant protéines G et effecteurs. Nous cherchons à caractériser de tels complexes comprenant les récepteurs opioïdes delta (DOR) et les canaux Kir3, qui nous sont d’intérêt vu leur implication dans l’analgésie des opioïdes. Des expériences d’immunopurification, de BRET et de liaison GTPgS ont été réalisées à l’intérieur de cellules HEK293 transfectées. Les canaux Kir3 ont été co-immunopurifiés avec les DOR, suggérant une interaction spontanée entre récepteur et effecteur. Des essais BRET ont corroboré que l’interaction était présente dans des cellules vivantes et nous ont permis d’identifier une interaction spontanée et spécifique entre DOR/Gg et Gg/Kir3, indiquant leur coexistence en un même complexe. Puisque l’activation du récepteur implique la présence de changements conformationnels à l’intérieur de celui-ci, nous étions intéressés à vérifier si l’information conformationnelle circule à partir du récepteur lié au ligand jusqu’à l’effecteur en aval. Ainsi, nous avons déterminé l’effet de différents ligands sur le signal BRET généré par les paires suivantes : DOR/Gbg, DOR/Kir3 et Kir3/Gbg. Nous avons constaté une modulation de l’interaction DOR/Gbg et Gbg/Kir3 suivant l’ordre d’efficacité des ligands à stimuler la protéine G, ce que nous n’avons pas observé entre DOR et Kir3. Donc, nous concluons que l’information conformationnelle circule du récepteur au canal Kir3 via la protéine Gbg. Ces résultats nous ont permis de développer un biosenseur BRET (EYFP-Gg2/Kir3.1-Rluc) qui pourrait être utilisé dans le criblage à haut débit afin de détecter de nouvelles molécules ayant une grande efficacité à activer les canaux Kir3. / G protein-coupled receptors form multimeric complexes comprising G protein and effectors. We want to characterize such complexes comprising delta opioid receptors (DOR) and Kir3 channels, which interest us due to their involvement in opioid analgesia. Immunopurification, BRET and GTPgS binding experiments were done in transfected HEK293 cells. Kir3 channels were co-immunopurified with DOR, implying a spontaneous interaction between the receptor and effector. BRET assays corroborated the presence of this interaction in living cells and allowed us to identify a spontaneous and specific interaction between DOR/Gg and Gg/Kir3, indicating their co-existence within the same complex. Since the activation of the receptor implies it undergoes conformational changes, we were interested in evaluating if the conformational information flows from the ligand-bound receptor until the downstream effector. Hence, we determined the effect of different ligands on the BRET signal that was generated by the following pairs: DOR/Gbg, DOR/Kir3 and Kir3/Gbg. We noticed a modulation of the DOR/Gbg and Gbg/Kir3 interactions that followed the order of efficacy of the ligands to activate the G protein, which we did not observe between DOR and Kir3. Therefore, we concluded that the conformational information flows from the receptor to the Kir3 channel via the Gbg protein. These results allowed us to develop a BRET biosensor (EYFP-Gg2/Kir3.1-Rluc), which could be used in high throughput screening to detect new molecules that activate Kir3 channels with high efficacy.

Deregulation of protein kinases is associated with many diseases making them important targets for therapeutic intervention. Kinases can switch between active and inactive conformations that can be targeted by type 1 or type 2 inhibitors respectively. One of the most relevant conformational switches is the ‘in’ and ‘out’ movement of the ATP/Mg2+ binding motif DFG. Factors modulating the conformational equilibria such as the residue environment of regulatory motifs remain poorly understood despite their importance for drug discovery. In this thesis, the first model system tested the hypothesis that accessibility of the DFG-out conformation is restricted by the energetic cost of transition between the in and out states. CDK2 was chosen as a target that was thought to have an inaccessible DFG-out conformation, and several point mutations were introduced to promote this conformational transition. Detailed biochemical and biophysical characterisation illustrated that the mutants bound type 2 inhibitors more potently than the wild type. In addition, the wild-type CDK2 was shown to bind type 2 inhibitors in the absence, but not in the presence, of cyclin. The first known CDK2 co-crystal structure in the DFG-out conformation was solved, opening the door to a new class of CDK2 inhibitors. In the second project, site-directed mutagenesis was used to explore the residues determining inhibitor selectivity between PIM1 and PIM2. Evaluation of ligand binding to the variants and comparison of PIM1 and PIM2 crystal structures showed that flexibility of the phosphate-binding loop was the dominant factor determining the differences in their affinities for ATP and small molecule inhibitors. These studies illustrate that residues contributing to kinase conformational equilibria can be just as important for inhibitor binding as contact residues formed in the ligand complex.

Dans notre laboratoire rémois, le travail sur les sucres est à la base de toutes les recherches, que ce soit pour la synthèse de molécules d'intérêt biologique telles que des analogues du KRN 7000 (un glycolipide présentant une activité antitumorale), ou encore la mise en place de stratégies de synthèse (avec par exemple la stratégie NOE : addition nucléophile stéréosélective suivie d'une ouverture intramoléculaire régiosélective d'un époxyde). Mon trravail a consisté à étudier la flexibilité, et donc les conformations stables ou moins stables, de petites molécules que sont les C-furanosides, le but étant d'établir un certain nombre de règles permettant d'anticiper les questions concernant à la fois l'entrée et la pose de la structure dans un site actif. J'ai donc débuté avec une étude théorique dans le vide, puis j'ai considéré le milieu solvaté, l'objectif à long terme étant la prise en compte d'un site actif. Durant ces études, jai aussi eu la possibilité de réaliser un certain nombre de travaux en synthèse organique, allant jusqu'à l'établissement d'une voie de synthèse vers différents C-furanosides possédant un bras alcyne pouvant être personnalisé par notre partenaire lyonnais via une réaction de chimie click, pour la synthèse de molécules actives contre le diabète. / In our laboratory (in Reims), working on sugars is the base of all researches, whether for biologically interesting molecules synthesis as analogues of KRN 7000 (a glycolipid showing antitumor effects), or for the development of strategies for synthesis (for example the NEO stragegy : stereoselective nucleophilic addition followed by a regioselective intramolecular epoxide opening). My work was about studying the flexibility, and consequently stable and less stable conformations of small molecules : C-furanosides, the aim being the establishment of several rules anticipating the questions concerning the entry and the pose of the structure in an active site. So I have started with a theoretical study in vacuum, then I have considered solvation. The long term goal being the consideration of an active site. During these stdies, I have also realized several works in the field of organic chemistry, going to the establishment of a synthetic pathway to different C-furanosides with an alkyne arm, which could be functionalized by our partner (in Lyon) with a click reaction, for the synthesis of bioactive molecules against diabetes.

The gas-phase hydrogen/deuterium (H/D) exchange of protonated di- and tripeptides containing a basic amino acid residue has been studied with a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Bimolecular reactions are monitored as a function of time providing exchange efficiencies and temporal distributions for the peptide ions. Results from these experiments indicated that position of the basic residue within the peptide (i.e. N-terminal, internal, or C-terminal) influences gas-phase H/D exchange, suggesting unique peptide ion conformations. The FT-ICR mass spectrometer employed for these gas-phase H/D exchange studies was modified from its original design. Instrument modifications include development of an internal matrix assisted laser desorption ionization (MALDI) source for peptide protonation. In addition, a two-section cell was utilized, allowing control of ion motion and factors affecting gas-phase ion molecule reactions. Systems investigated in these gas-phase H/D exchange studies are peptides containing the same amino acid residues but different sequences. These sequence isomers display dissimilar reaction efficiencies and temporal distributions for deuterium incorporation depending on the primary structure of the peptide ion. Specifically, [M+H]+ peptide ions containing a N-terminal basic residue demonstrate unique H/D exchange behavior when compared to their internal and C-terminal counterparts. These differences are attributed to dissimilar intramolecular bridging interactions involved with inductive stabilization of the charge site. Gas-phase H/D exchange of peptide sequence isomers was also probed with various deuterium reagents. Findings suggest that different reagents also influence H/D exchange reaction rate efficiencies and temporal distributions. These dissimilarities are ascribed to relative gas-phase basicity and proposed mechanistic exchange differences for the deuterium reagents.

The gas-phase hydrogen/deuterium (H/D) exchange of protonated di- and tripeptides containing a basic amino acid residue has been studied with a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Bimolecular reactions are monitored as a function of time providing exchange efficiencies and temporal distributions for the peptide ions. Results from these experiments indicated that position of the basic residue within the peptide (i.e. N-terminal, internal, or C-terminal) influences gas-phase H/D exchange, suggesting unique peptide ion conformations. The FT-ICR mass spectrometer employed for these gas-phase H/D exchange studies was modified from its original design. Instrument modifications include development of an internal matrix assisted laser desorption ionization (MALDI) source for peptide protonation. In addition, a two-section cell was utilized, allowing control of ion motion and factors affecting gas-phase ion molecule reactions. Systems investigated in these gas-phase H/D exchange studies are peptides containing the same amino acid residues but different sequences. These sequence isomers display dissimilar reaction efficiencies and temporal distributions for deuterium incorporation depending on the primary structure of the peptide ion. Specifically, [M+H]+ peptide ions containing a N-terminal basic residue demonstrate unique H/D exchange behavior when compared to their internal and C-terminal counterparts. These differences are attributed to dissimilar intramolecular bridging interactions involved with inductive stabilization of the charge site. Gas-phase H/D exchange of peptide sequence isomers was also probed with various deuterium reagents. Findings suggest that different reagents also influence H/D exchange reaction rate efficiencies and temporal distributions. These dissimilarities are ascribed to relative gas-phase basicity and proposed mechanistic exchange differences for the deuterium reagents.

No description available.

Sequence analyses show that deletions of 10 and 12 bp occur at homologous sites in a domain that is rich in alternating purines and pyrimidines (Pu/Py) in B42 and EXT, two cloned variants of a complex satellite DNA. A 3-bp deletion occurs 27 bp upstream from the site of the specific deletions in B42 and RU, a third cloned satellite variant that has not suffered the 10-bp deletion. Under torsional stress, the Pu/Py-rich domain adopts a Z-conformation as shown by (i) inhibition of cutting at a BssHII site that accounts for built2 5 of a 15-bp tract of pure Pu/Py in the domain; (ii) binding of polyclonal and monoclonal anti-Z-DNA antibodies to the domain; and (iii) antibody stabilization and subsequent relaxation of the Z-region.

No description available.

It is becoming increasingly clear that long-range control of gene expression is mediated through direct physical interactions between genes and regulatory elements, either intra- or interchromosomally. In addition to transcriptional initiation, formation of active chromatin hubs seem to be crucial for increased transcriptional efficiency as well as insulation from neighbouring heterochromatic environment. Regulatory factors apparently have an important role in organization of such functional modules in a development and differentiated- dependent fashion. The relevance of trans-acting factors in the ‘choice’ process of X-Chromosome Inactivation (XCI) was highlighted by our observations where CTCF was shown to occupy a homologous position on the active mouse and human Xist/XIST promoters and its binding affinity was altered in familial cases of opposite skewed X-inactivation patterns. The paradigm of genomic imprinting, i.e. the Igf2-H19 locus, manifests its imprinted states through the H19 Imprinting Control Region (ICR). The repression of the maternal Igf2 allele depends on the insulator properties of the H19 ICR when this interacts with CTCF. The studies here detected a novel kind of CTCF-dependent tightly closed pocket- like higher order structure exclusively on maternal allele which was found to be essential for imprinted Igf2 expression as well as maintenance of precise epigenetic marks at various Differentially Methylated Regions (DMRs) across this locus. Despite the highly condensed state of the mitotic chromosome, the insulator protein CTCF was found to constitutively occupy its known target sites. Furthermore, pivotal CTCF-dependent long-range regulatory loops within Igf2-H19 locus were found to survive mitotic compaction and such mechanisms might serve as a novel kind of epigenetic memory to minimize transcriptional chaos and to reset proper expression domains in the daughter cells as soon as cells exit mitosis. Our observations also suggest that the epigenetic reprogramming of H19 ICR during spermatogenesis is initiated by a CTCF-dependent recruitment of chromatin remodeling factor Lsh to the H19 ICR followed by completion of the imprint acquisition process by a replacement of CTCF with its closely related paralogue termed BORIS. Overall, this thesis unravels the novel roles for CTCF as an architectural factor in the organization of higher order chromatin conformations and transcriptional regulation.