Méthodologies de criblages d'interactions protéines-ligands par RMN (inhibitions de la Glms et de Bcl-xL)

La versatilité de la Résonance Magnétique Nucléaire (RMN) lui permet des applications dans des domaines variés. Cette versatilité en fait un instrument de première importance dans la recherche de traitements thérapeutiques. Elle permet de déterminer la structure et la dynamique des molécules en interactions. Nous avons utilisé la RMN sur deux protéines impliquées dans diverses pathologies : Bcl-xL, partiellement responsable de la dé cience en apoptose dans certains cancers, et la Glms, connnue pour provoquer des complications chez des patients atteints de diabète de type II et cible dans la lutte anti-bactérienne. Le but était d améliorer notre compréhension des interactions entre ces protéines et de nouveaux ligands capables d inhiber leurs activités. Ceux-ci sont soit extraits de plantes, dans le cas de Bcl-xL, soit de synthèse, dans le cas de Glms. Nos résultats ont permis de donner des orientations quant à l amélioration du pouvoir thérapeutique des ligands étudiés.The versatility of Nuclear Magnetic Resonance (NMR) allows several applications in various domains. This versatility makes it a tool of prime importance in the eld of therapeutic treatments research. It allows the determination of the structure and the dynamic of the interacting molecules. We used NMR on two proteins involved in diverse pathologies : Bcl-xL, partially responsible for the apoptosis de ciency for certain cancers, and the Glms, known to give complications to people affected with type II diabetes and target in the anti-microbial ght. The goal was to enhance our understanding of the interactions between those proteins and new molecules able to inhibit their activities. Those molecules are either extract from plants (Bcl-xL study), or synthesized (Glms study). Our results allowed to give orientations about the enhancements of the therapeutic effects of the studied molecules.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Hépatocarcinome: Pourquoi fermenter quand on peut respirer ?

WOS:000455202200018International audiencePour la troisième année, dans le cadre du module d’enseignement « Physiopathologie de la signalisation » proposé par l’université Paris-sud, les étudiants du Master « Biologie Santé » de l’université Paris-Saclay se sont confrontés à l’écriture scientifique. Ils ont sélectionné 8 articles scientifiques récents dans le domaine de la signalisation cellulaire présentant des résultats originaux, via des approches expérimentales variées, sur des thèmes allant des relations hôte-pathogène aux innovations thérapeutiques, en passant par la signalisation hépatique et le métabolisme. Après un travail préparatoire réalisé avec l’équipe pédagogique, les étudiants, organisés en binômes, ont ensuite rédigé, guidés par des chercheurs, une Nouvelle soulignant les résultats majeurs et l’originalité de l’article étudié. Ils ont beaucoup apprécié cette initiation à l’écriture d’articles scientifiques et, comme vous pourrez le lire, se sont investis dans ce travail avec enthousiasme ! Une de ces Nouvelles est publiée dans ce numéro, les autres le seront dans les prochains numéros de m/s

Crosstalk between TAp73 and TGF-β in fibroblast regulates iNOS expression and Nrf2-dependent gene transcription

International audienceInducible nitric oxide synthase (iNOS) activity produces anti-tumor and anti-microbial effects but also promotes carcinogenesis through mutagenic, immunosuppressive and pro-angiogenic mechanisms. The tumor suppressor p53 contributes to iNOS downregulation by repressing induction of the NOS2 gene encoding iNOS, thereby limiting NO-mediated DNA damages. This study focuses on the role of the p53 homologue TAp73 in the regulation of iNOS expression. Induction of iNOS by immunological stimuli was upregulated in immortalized MEFs from TAp73-/- mice, compared to TAp73+/+ fibroblasts. This overexpression resulted both from increased levels of NOS2 transcripts, and from an increased stability of the protein. Limitation of iNOS expression by TAp73 in wild-type cells is alleviated by TGF-β receptor I inhibitors, suggesting a cooperation between TAp73 and TGF-β in suppression of iNOS expression. Accordingly, downregulation of iNOS expression by exogenous TGF-β1 was impaired in TAp73-/- fibroblasts. Increased NO production in these cells resulted in a stronger, NO-dependent induction of Nrf2 target genes, indicating that the Nrf2-dependent adaptive response to nitrosative stress in fibroblasts is proportional to iNOS activity. NO-dependent induction of two HIF-1 target genes was also stronger in TAp73-deficient cells. Finally, the antimicrobial action of NO against Trypanosoma musculi parasites was enhanced in TAp73-/- fibroblasts. Our data indicate that tumor suppressive TAp73 isoforms cooperate with TGF-β to control iNOS expression, NO-dependent adaptive responses to stress, and pathogen proliferation

Caspase-dependent Proteolysis of Human Ribonucleotide Reductase Small Subunits R2 and p53R2 during Apoptosis

Ribonucleotide reductase (RnR) is a key enzyme synthesizing deoxyribonucleotides for DNA replication and repair. In mammals, the R1 catalytic subunit forms an active complex with either one of the two small subunits R2 and p53R2. Expression of R2 is S phase-specific and required for DNA replication. The p53R2 protein is expressed throughout the cell cycle and in quiescent cells where it provides dNTPs for mitochondrial DNA synthesis. Participation of R2 and p53R2 in DNA repair has also been suggested. In this study, we investigated the fate of the RnR subunits during apoptosis. The p53R2 protein was cleaved in a caspase-dependent manner in K-562 cells treated with inhibitors of the Bcr-Abl oncogenic kinase and in HeLa 229 cells incubated with TNF-α and cycloheximide. The cleavage site was mapped between Asp(342) and Asn(343). Caspase attack released a C-terminal p53R2 peptide of nine residues containing the conserved heptapeptide essential for R1 binding. As a consequence, the cleaved p53R2 protein was inactive. In vitro, purified caspase-3 and -8 could release the C-terminal tail of p53R2. Knocking down these caspases, but not caspase-2, -7, and -10, also inhibited p53R2 cleavage in cells committed to die via the extrinsic death receptor pathway. The R2 subunit was subjected to caspase- and proteasome-dependent proteolysis, which was prevented by siRNA targeting caspase-8. Knocking down caspase-3 was ineffective. Protein R1 was not subjected to degradation. Adding deoxyribonucleosides to restore dNTP pools transiently protected cells from apoptosis. These data identify RnR activity as a prosurvival function inactivated by proteolysis during apoptosis

Homo- and heteronuclear 2D NMR approaches to analyse a mixture of deuterated unlike/like stereoisomers using weakly ordering chiral liquid crystals

International audienceWe describe several homo- and heteronuclear 2D NMR strategies dedicated to the analysis of anisotropic 2H spectra of a mixture of dideuterated unlike/like stereoisomers with two remote stereogenic centers, using weakly orienting chiral liquid crystals. To this end, we propose various 2D correlation experiments, denoted ‘‘D(H)nD’’ or ‘‘D(H)nC’’ (with n = 1, 2), that involve two heteronuclear polarization transfers of INEPT-type with one or two proton relays. The analytical expressions of correlation signals for four pulse sequences reported here were calculated using the product-operators formalism for spin I = 1 and S = 1/2. The features and advantages of each scheme are presented and discussed. The efficiency of these 2D sequences is illustrated using various deuterated model molecules, dis-solved in organic solutions of polypeptides made of poly-gamma-benzyl-L-glutamate (PBLG) or poly-epslion-carbobenzyloxy-L-lysine (PCBLL) and NMR numerical simulations

Influence des inégalités socio-territoriales sur la mortalité en excès de patients atteints de SEP en France : une étude rétrospective observationnelle

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