Comparative structure analyses of cystine knot-containing molecules with eight aminoacyl ring including glycoprotein hormones (GPH) alpha and beta subunits and GPH-related A2 (GPA2) and B5 (GPB5) molecules

<p>Abstract</p> <p>Background</p> <p>Cystine-knot (cys-knot) structure is found in a rather large number of secreted proteins and glycoproteins belonging to the TGFbeta and glycoprotein hormone (GPH) superfamilies, many of which are involved in endocrine control of reproduction. In these molecules, the cys-knot is formed by a disulfide (SS) bridge penetrating a ring formed by 8, 9 or 10 amino-acid residues among which four are cysteine residues forming two SS bridges. The glycoprotein hormones Follicle-Stimulating Hormone (FSH), Luteinizing Hormone (LH), Thyroid-Stimulating Hormone (TSH) and Chorionic Gonadotropin (CG) are heterodimers consisting of non-covalently associated alpha and beta subunits that possess cys-knots with 8-amino-acyl (8aa) rings. In order to get better insight in the structural evolution of glycoprotein hormones, we examined the number and organization of SS bridges in the sequences of human 8-aa-ring cys-knot proteins having 7 (gremlins), 9 (cerberus, DAN), 10 (GPA2, GPB5, GPHα) and 12 (GPHβ) cysteine residues in their sequence.</p> <p>Discussion</p> <p>The comparison indicated that the common GPH-alpha subunit exhibits a SS bridge organization ressembling that of DAN and GPA2 but possesses a unique bridge linking an additional cysteine inside the ring to the most N-terminal cysteine residue. The specific GPHbeta subunits also exhibit a SS bridge organization close to that of DAN but it has two additional C-terminal cysteine residues which are involved in the formation of the "seat belt" fastened by a SS "buckle" that ensures the stability of the heterodimeric structure of GPHs. GPA2 and GPB5 exhibit no cys residue potentially involved in interchain SS bridge and GPB5 does not possess a sequence homologous to that of the seatbelt in GPH β-subunits. GPA2 and GPB5 are thus not expected to form a stable heterodimer at low concentration in circulation.</p> <p>Summary</p> <p>The 8-aa cys-knot proteins GPA2 and GPB5 are expected to form a heterodimer only at concentrations above 0.1 microM: this would be consistent with a short-term paracrine role but not with an endocrine role after dilution in circulation. Consequently, GPA2 and GPB5 could exert separate endocrine roles either during development and/or during adult life of both vertebrates and invertebrates.</p

Effect of Pharmaceutical Potential Endocrine Disruptor Compounds on Protein Disulfide Isomerase Reductase Activity Using Di-Eosin-Oxidized-Glutathion

Background: Protein Disulfide Isomerase (PDI) in the endoplasmic reticulum of all cells catalyzes the rearrangement of disulfide bridges during folding of membrane and secreted proteins. As PDI is also known to bind various molecules including hormones such as estradiol and thyroxin, we considered the hypothesis that adverse effects of endocrinedisrupter compounds (EDC) could be mediated through their interaction with PDI leading to defects in membrane or secreted proteins. Methodology/Principal Findings: Taking advantage of the recent description of the fluorescence self quenched substrate di-eosin-oxidized-glutathion (DiE-GSSG), we determined kinetically the effects of various potential pharmaceutical EDCs on the in-vitro reductase activity of bovine liver PDI by measuring the fluorescence of the reaction product (E-GSH). Our data show that estrogens (ethynylestradiol and bisphenol-A) as well as indomethacin exert an inhibition whereas medroxyprogesteroneacetate and nortestosterone exert a potentiation of bovine PDI reductase activity. Conclusions: The present data indicate that the tested EDCs could not only affect endocrine target cells through nuclear receptors as previously shown, but could also affect these and all other cells by positively or negatively affecting PDI activity. The substrate DiE-GSSG has been demonstrated to be a convenient substrate to measure PDI reductase activity in the presence of various potential EDCs. It will certainely be usefull for the screening of potential effect of all kinds of chemical

GC/MS-based 13C metabolic flux analysis resolves the parallel and cyclic photomixotrophic metabolism of Synechocystis sp. PCC 6803 and selected deletion mutants including the Entner-Doudoroff and phosphoketolase pathways

Background Cyanobacteria receive huge interest as green catalysts. While exploiting energy from sunlight, they co-utilize sugar and CO2. This photomixotrophic mode enables fast growth and high cell densities, opening perspectives for sustainable biomanufacturing. The model cyanobacterium Synechocystis sp. PCC 6803 possesses a complex architecture of glycolytic routes for glucose breakdown that are intertwined with the CO2-fixing Calvin-Benson-Bassham (CBB) cycle. To date, the contribution of these pathways to photomixotrophic metabolism has remained unclear. Results Here, we developed a comprehensive approach for 13C metabolic flux analysis of Synechocystis sp. PCC 6803 during steady state photomixotrophic growth. Under these conditions, the Entner-Doudoroff (ED) and phosphoketolase (PK) pathways were found inactive but the microbe used the phosphoglucoisomerase (PGI) (63.1%) and the oxidative pentose phosphate pathway (OPP) shunts (9.3%) to fuel the CBB cycle. Mutants that lacked the ED pathway, the PK pathway, or phosphofructokinases were not affected in growth under metabolic steady-state. An ED pathway-deficient mutant (Δeda) exhibited an enhanced CBB cycle flux and increased glycogen formation, while the OPP shunt was almost inactive (1.3%). Under fluctuating light, ∆eda showed a growth defect, different to wild type and the other deletion strains. Conclusions The developed approach, based on parallel 13C tracer studies with GC–MS analysis of amino acids, sugars, and sugar derivatives, optionally adding NMR data from amino acids, is valuable to study fluxes in photomixotrophic microbes to detail. In photomixotrophic cells, PGI and OPP form glycolytic shunts that merge at switch points and result in synergistic fueling of the CBB cycle for maximized CO2 fixation. However, redirected fluxes in an ED shunt-deficient mutant and the impossibility to delete this shunt in a GAPDH2 knockout mutant, indicate that either minor fluxes (below the resolution limit of 13C flux analysis) might exist that could provide catalytic amounts of regulatory intermediates or alternatively, that EDA possesses additional so far unknown functions. These ideas require further experiments

Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1–2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly

PLoS Biol

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as "catabolite repression," allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named "metabolic contest" for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This "metabolic contest" depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met

Electron holes in oxide type cuprate supraconductors. Cristal chemical study of a new family of compounds A3Te2O<6X2 (A=Sr, Ba and X=Cl, Br)

Certaines propriétés des cuprates supraconducteurs restent aujourd'hui encore mystérieuses pour le monde scientifique. Ce travail vise a éclaircir l'une d'entre-elles: la nature de leurs porteurs de charge. Une approche théorique a été effectuée sur le composé La2CuO4 puis étendue a des composés de type cuprate supraconducteur exempts de cuivre. Elle consiste en des calculs de potentiel de Madelung et de diagrammes de structure de bandes. Les composes "TlBa2ZnO5" et "TlBa2MgO5" ayant servi de modèles pour ces calculs ont ensuite été préparés puis caractérisés par dosage iodométrique, diffraction des rayons X, spectrométrie Raman, MET et RPE. Les résultats alors obtenus mettent en évidence l'existence de trous d'électrons sur les atomes d'oxygène de ces composés, notes O dans le texte. Nous avons alors tenté d'expliquer le comportement encore inconnu de ces porteurs de charge. Une deuxième étude a été menée en parallèle de la précédente. Elle nous a permis de mettre a jour une nouvelle famille d'oxyhalogenures de tellure et d'alcalino-terreux de formulation A3Te2O6X2. Le composé pour lequel A=Sr et X=Cl a fait l'objet d'une détermination structurale complète. Les composés pour lesquels A=Sr, Ba et X=Br ont été isolés.non disponibl

Electron holes in oxide type cuprate supraconductors. Cristal chemical study of a new family of compounds A3Te2O<6X2 (A=Sr, Ba and X=Cl, Br)

Certaines propriétés des cuprates supraconducteurs restent aujourd'hui encore mystérieuses pour le monde scientifique. Ce travail vise a éclaircir l'une d'entre-elles: la nature de leurs porteurs de charge. Une approche théorique a été effectuée sur le composé La2CuO4 puis étendue a des composés de type cuprate supraconducteur exempts de cuivre. Elle consiste en des calculs de potentiel de Madelung et de diagrammes de structure de bandes. Les composes "TlBa2ZnO5" et "TlBa2MgO5" ayant servi de modèles pour ces calculs ont ensuite été préparés puis caractérisés par dosage iodométrique, diffraction des rayons X, spectrométrie Raman, MET et RPE. Les résultats alors obtenus mettent en évidence l'existence de trous d'électrons sur les atomes d'oxygène de ces composés, notes O dans le texte. Nous avons alors tenté d'expliquer le comportement encore inconnu de ces porteurs de charge. Une deuxième étude a été menée en parallèle de la précédente. Elle nous a permis de mettre a jour une nouvelle famille d'oxyhalogenures de tellure et d'alcalino-terreux de formulation A3Te2O6X2. Le composé pour lequel A=Sr et X=Cl a fait l'objet d'une détermination structurale complète. Les composés pour lesquels A=Sr, Ba et X=Br ont été isolés.non disponibl

Trous d' électrons dans des oxydes de type cuprate supraconducteur. Etude cristallochimique d'une nouvelle famille d' oxyhalogènure de tellure et d' alcalinoterreux A3Te2O6X2 (A=Sr, Ba et X=Cl, Br

Certaines propriétés des cuprates supraconducteurs restent aujourd'hui encore mystérieuses pour le monde scientifique. Ce travail vise a éclaircir l'une d'entre-elles: la nature de leurs porteurs de charge. Une approche théorique a été effectuée sur le composé La2CuO4 puis étendue a des composés de type cuprate supraconducteur exempts de cuivre. Elle consiste en des calculs de potentiel de Madelung et de diagrammes de structure de bandes. Les composes "TlBa2ZnO5" et "TlBa2MgO5" ayant servi de modèles pour ces calculs ont ensuite été préparés puis caractérisés par dosage iodométrique, diffraction des rayons X, spectrométrie Raman, MET et RPE. Les résultats alors obtenus mettent en évidence l'existence de trous d'électrons sur les atomes d'oxygène de ces composés, notes O dans le texte. Nous avons alors tenté d'expliquer le comportement encore inconnu de ces porteurs de charge. Une deuxième étude a été menée en parallèle de la précédente. Elle nous a permis de mettre a jour une nouvelle famille d'oxyhalogenures de tellure et d'alcalino-terreux de formulation A3Te2O6X2. Le composé pour lequel A=Sr et X=Cl a fait l'objet d'une détermination structurale complète. Les composés pour lesquels A=Sr, Ba et X=Br ont été isolés

Trous d' électrons dans des oxydes de type cuprate supraconducteur. Etude cristallochimique d'une nouvelle famille d' oxyhalogènure de tellure et d' alcalinoterreux A3Te2O6X2 (A=Sr, Ba et X=Cl, Br

Certaines propriétés des cuprates supraconducteurs restent aujourd'hui encore mystérieuses pour le monde scientifique. Ce travail vise a éclaircir l'une d'entre-elles: la nature de leurs porteurs de charge. Une approche théorique a été effectuée sur le composé La2CuO4 puis étendue a des composés de type cuprate supraconducteur exempts de cuivre. Elle consiste en des calculs de potentiel de Madelung et de diagrammes de structure de bandes. Les composes "TlBa2ZnO5" et "TlBa2MgO5" ayant servi de modèles pour ces calculs ont ensuite été préparés puis caractérisés par dosage iodométrique, diffraction des rayons X, spectrométrie Raman, MET et RPE. Les résultats alors obtenus mettent en évidence l'existence de trous d'électrons sur les atomes d'oxygène de ces composés, notes O dans le texte. Nous avons alors tenté d'expliquer le comportement encore inconnu de ces porteurs de charge. Une deuxième étude a été menée en parallèle de la précédente. Elle nous a permis de mettre a jour une nouvelle famille d'oxyhalogenures de tellure et d'alcalino-terreux de formulation A3Te2O6X2. Le composé pour lequel A=Sr et X=Cl a fait l'objet d'une détermination structurale complète. Les composés pour lesquels A=Sr, Ba et X=Br ont été isolés.non disponibl