Crystal structures of an Extracytoplasmic Solute Receptor from a TRAP transporter in its open and closed forms reveal a helix-swapped dimer requiring a cation for alpha-keto acid binding.

International audienceBACKGROUND: The import of solutes into the bacterial cytoplasm involves several types of membrane transporters, which may be driven by ATP hydrolysis (ABC transporters) or by an ion or H+ electrochemical membrane potential, as in the tripartite ATP-independent periplasmic system (TRAP). In both the ABC and TRAP systems, a specific periplasmic protein from the ESR family (Extracytoplasmic Solute Receptors) is often involved for the recruitment of the solute and its presentation to the membrane complex. In Rhodobacter sphaeroides, TakP (previously named SmoM) is an ESR from a TRAP transporter and binds alpha-keto acids in vitro. RESULTS: We describe the high-resolution crystal structures of TakP in its unliganded form and as a complex with sodium-pyruvate. The results show a limited "Venus flytrap" conformational change induced by substrate binding. In the liganded structure, a cation (most probably a sodium ion) is present and plays a key role in the association of the pyruvate to the protein. The structure of the binding pocket gives a rationale for the relative affinities of various ligands that were tested from a fluorescence assay. The protein appears to be dimeric in solution and in the crystals, with a helix-swapping structure largely participating in the dimer formation. A 30 A-long water channel buried at the dimer interface connects the two ligand binding cavities of the dimer. CONCLUSION: The concerted recruitment by TakP of the substrate group with a cation could represent a first step in the coupled transport of both partners, providing the driving force for solute import. Furthermore, the unexpected dimeric structure of TakP suggests a molecular mechanism of solute uptake by the dimeric ESR via a channel that connects the binding sites of the two monomers

Territoires et IHM Distribuées : Raffinement de Règles et d'une Méthode de Conception de Jeux Multi-Dispositifs

National audienceThe multitude of different interaction devices and the importance of their spread alongside various people allow to propose distributed interfaces (interactions). Territoriality rules have to be applied in order to ensure the adequation of information position and their access constraints (personal information, privacy, etc.). The navigability between different interfaces has to be assured. Illustrated by four designs of distributed application, this article proposes a feedback which allow to refine a design methodology.La multitude des supports d'interaction et l'ampleur de leur propagation auprès d'un public varié permet de proposer des interfaces (ou interactions) distribuées. Les règles de territorialité doivent s'appliquer afin de garantir une adéquation de la position de l'information à son but et à ses contraintes d'accès (informations personnelles, informations privées, etc.). Il faut s'assurer également de la navigabilité entre les différentes interfaces. Cet article propose un retour d'expérience permettant de raffiner une méthode de conception, sur la base d'illustration de quatre conceptions d'application distribuées

Eschar and neck lymphadenopathy caused by Francisella tularensis after a tick bite: a case report

<p>Abstract</p> <p>Introduction</p> <p>In 25 to 35% of cases, the aetiological agent of scalp eschar and neck lymphadenopathy after a tick bite remains undetermined. To date, <it>Rickettsia slovaca</it>, <it>Rickettsia raoultii </it>and more recently <it>Bartonella henselae </it>have been associated with this syndrome.</p> <p>Case presentation</p> <p>A four-year-old Caucasian boy was admitted to hospital with fever, vomiting and abdominal pain. On physical examination, an inflammatory and suppurating eschar was seen on the scalp, with multiple enlarged cervical lymph nodes on both sides. Although no tick was found in this scalp lesion, a diagnosis of tick-borne lymphadenopathy was suggested, and explored by serology testing and polymerase chain reaction of a biopsy from the eschar. <it>Francisella tularensis </it>DNA was found in the skin biopsy and the serology showed titres consistent with tularaemia.</p> <p>Conclusion</p> <p>This is, to the best of our knowledge, the first reported case of scalp eschar and neck lymphadenopathy after tick bite infection caused by <it>F. tularensis.</it></p

Phosphate Starvation Triggers Production and Secretion of an Extracellular Lipoprotein in Caulobacter crescentus

Life in oligotrophic environments necessitates quick adaptive responses to a sudden lack of nutrients. Secretion of specific degradative enzymes into the extracellular medium is a means to mobilize the required nutrient from nearby sources. The aquatic bacterium Caulobacter crescentus must often face changes in its environment such as phosphate limitation. Evidence reported in this paper indicates that under phosphate starvation, C. crescentus produces a membrane surface-anchored lipoprotein named ElpS subsequently released into the extracellular medium. A complete set of 12 genes encoding a type II secretion system (T2SS) is located adjacent to the elpS locus in the C. crescentus genome. Deletion of this T2SS impairs release of ElpS in the environment, which surprisingly remains present at the cell surface, indicating that the T2SS is not involved in the translocation of ElpS to the outer membrane but rather in its release. Accordingly, treatment with protease inhibitors prevents release of ElpS in the extracellular medium suggesting that ElpS secretion relies on a T2SS-secreted protease. Finally, secretion of ElpS is associated with an increase in alkaline phosphatase activity in culture supernatants, suggesting a role of the secreted protein in inorganic phosphate mobilization. In conlusion, we have shown that upon phosphate starvation, C. crescentus produces an outer membrane bound lipoprotein, ElpS, which is further cleaved and released in the extracellular medium in a T2SS-dependent manner. Our data suggest that ElpS is associated with an alkaline phosphatase activity, thereby allowing the bacterium to gather inorganic phosphates from a poor environment

Work meaningfulness as a key enhancer of ethical values in business

Despite abundant research on work meaningfulness, the link between work meaningfulness and general ethical attitude at work has not been discussed so far. In this article, we propose a theoretical framework to explain how work meaningfulness contributes to enhanced ethical behavior. We argue that by providing a way for individuals to relate work to one's personal core values and identity, work meaningfulness leads to affective commitment – the involvement of one's cognitive, emotional, and physical resources. This, in turn, leads to engagement and so facilitates the integration of one's personal values in the daily work routines, and so reduces the risk of unethical behavior. On the contrary, anomie, that is, the absence of meaning and consequently of personal involvement, will lead to lower rational commitment rather than affective commitment, and consequently to disengagement and a-morality. We conclude with implications for the management of ethical attitudes

Caractérisation structurale et fonctionnelle de la sous-unité périplasmique TaKP d'un transporteur TRAP chez Rhodobacter sphaeroides

Les transporteurs TRAP sont de type actif secondaire, et sont composés de trois sous-unités, parmi lesquelles deux sont membranaires. La troisième sous-unité est soluble dans le périplasme chez les bactéries Gram négatif. Elle est également appelée ESR (Extracytoplasmic Solute Receptor) car elle fixe le composé importé. Ces transporteurs sont peu caractérisés à ce jour. Cette thèse est consacrée à l étude structurale et fonctionnelle de l ESR du transporteur TRAP TakPQM chez les deux souches Rhodobacter sphaeroides f. sp. denitrificans IL106 et Rhodobacter sphaeroides 2.4.1. Le phe notype d un mutant de transposition de la souche IL106 nous a d abord conduits à faire l hypothèse d un lien possible entre l ESR TakP et l import de sélénite. La caractérisation du phénotype d un mutant de délétion nous a ensuite permis d infirmer cette hypothèse. Par ailleurs, par le phénotypage de mutants de délétion et le suivi in vitro des interactions protéine-soluté par spectrométrie de fluorescence, nous avons montré que TakP de 2.4.1 fixe le pyruvate. Nous avons cristallisé la protéine et obtenu la structure tridimensionnelle de TakP de 2.4.1 par diffraction des rayons X, en absence et en présence de pyruvate. Cette structure d ESR est la première montrant une structure quaternaire dimérique. La fixation du substrat se fait par l intermédiaire d un ion sodium, pouvant avoir un rôle dans l énergisation du transport. Enfin, grâce au même type d expériences répétées sur des mutants et sur la protéine TakP de la souche IL106, les résultats obtenus suggèrent un rôle de TakP dans l import d a-cétoglutarate et non de pyruvate pour cette souche. TakP de IL106 et de 2.4.1 ne se différencient que par 4 acides aminés, dont l un (l arginine 242 chez IL106 et la glycine 242 chez 2.4.1) se situe à la charnière des deux domaines de la protéine et pourrait être impliqué indirectement dans la spécificité de substrat. Nous avons validé cette hypothèse en montrant que le mutant R242G de TakP IL106 est capable in vitro de fixer le pyruvate contrairement à la protéine sauvage.TRAP transporters are secondary active transporters. They are composed of three subunits, two of which are inserted in the membrane. The third one is soluble in the periplasm of Gram negativ bacteria, and is also called ESR (Extracytoplasmic Solute Receptor) because it binds the compound to be imported. These transporters are poorly characterized to date. This work aims at characterizing the structure and function of the ESR from the TRAP transporter TakPQM in the two strains Rhodobacter sphaeroides f. sp. denitrificans IL106 and Rhodobacter sphaeroides 2.4.1. According to the phenotype of a transposition mutant of the strain IL106, we first made the hypothesis that there was a link between the ESR TakP and selenite import. The phenotypic characterization of a deletion mutant enabled us to finally reject this hypothesis. Furthermore, by studying the phenotype of deletion mutants and solute-protein interactions in vitro by fluorescence spectroscopy, we demonstrated that TakP from 2.4.1 binds pyruvate. We cristallised the protein and obtained the tridimensional structure of TakP from 2.4.1 by X-ray diffraction, alone or in the presence of pyruvate. This ESR structure is the first one to show a dimeric quaternary fold. The binding of the substrate is mediated by a sodium ion which could play a part in the transport energisation. Eventually, we repeated the same type of experiments on deletion mutants and on the protein TakP from strain IL106, which results indicate that TakP from IL106 binds a-ketoglutarate and not pyruvate. Only four amino acids differ between TakP from IL106 and from 2.4.1, and one of them (arginine 242 in IL106 and glycine 242 in 2.4.1) is part of the hinge between the two domains of TakP and could indirectly interfere in its substrate specificity. We have shown that the mutant R242G of TakP IL106 is able to bind pyruvate in vitro, contrary to the wild-type protein.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Hsp27 as a negative regulator of cytochrome C release.

We previously showed that Hsp27 protects against apoptosis through its interaction with cytosolic cytochrome c. We have revisited this protective activity in murine cell lines expressing different levels of Hsp27. We report that Hsp27 also interferes, in a manner dependent on level of expression, with the release of cytochrome c from mitochondria. Moreover, a decreased level of endogenous Hsp27, which sensitized HeLa cells to apoptosis, reduced the delay required for cytochrome c release and procaspase 3 activation. The molecular mechanism regulating this function of Hsp27 is unknown. In our cell systems, Hsp27 is mainly cytosolic and only a small fraction of this protein colocalized with mitochondria. Moreover, we show that only a very small fraction of cytochrome c interacts with Hsp27, hence excluding a role of this interaction in the retention of cytochrome c in mitochondria. We also report that Bid intracellular relocalization was altered by changes in Hsp27 level of expression, suggesting that Hsp27 interferes with apoptotic signals upstream of mitochondria. We therefore investigated if the ability of Hsp27 to act as an expression-dependent modulator of F-actin microfilaments integrity was linked to the retention of cytochrome c in mitochondria. We show here that the F-actin depolymerizing agent cytochalasin D rapidly induced the release of cytochrome c from mitochondria and caspase activation. This phenomenon was delayed in cells pretreated with the F-actin stabilizer phalloidin and in cells expressing a high level of Hsp27. This suggests the existence of an apoptotic signaling pathway linking cytoskeleton damages to mitochondria. This pathway, which induces Bid intracellular redistribution, is negatively regulated by the ability of Hsp27 to protect F-actin network integrity. However, this upstream pathway is probably not the only one to be regulated by Hsp27 since, in staurosporine-treated cells, phalloidin only partially inhibited cytochrome c release and caspase activation. Moreover, in etoposide-treated cells, Hsp27 still delayed the release of cytochrome c from mitochondria and Bid intracellular redistribution in conditions where F-actin was not altered