Functional characterization of iron transporters Nramp1 and Nramp2 from Dictyostelium discoideum: a model of cellular iron homeostasis.

Background: Iron is an element necessary for all the organisms; its role in essential metabolism and cellular processes is fundamental for life. Studies on iron homeostasis, regulation and transport could be also done with a simplified model of professional phagocyte and pathogen host, Dyctiostelium discoideum (Dd). Aim: Characterize the different metal transport specificity and regulation of the two iron transporters of Dd, ddNramp1 and ddNramp2. Methods: Electrophysiological experiments with Two Electrode Voltage Clamp, X. laevis oocytes smoothies (XLOs) and immunolocalization experiments were performed to characterize these two divalent metal transporters and started with the synthesis of the two chimeric protein c-Nramp1 and c-Nramp2. Results: We demonstrated that c-Nramp1 is a sodium independent transporter with specificity for divalent transition metals, as mammal orthologs of SLC11 family, with maximal inward currents at acidic pH and a high affinity for substrate. We also detected a peculiar sodium leak current, mainly at neutral pH, that suggest the presence of a strong interaction in the binding site. We found that c-Nramp2 is electroneutral and transports divalent metal ions at all pH. Site directed mutagenesis on TMD6 restore the electrogenicity with small inward currents; XLOs analysis confirmed the electrophysiological results, these mutants are more functional than wild type c-Nramp2. Conclusions: Our findings on ddNramp1 contributed with novel insights on the mechanism of action of the mammalian ortholog, whereas similar studies on ddNramp2 suggested a role of the homologous proteins in protists, and helped to clarify the possible function of the contractile vacuole in divalent metal homeostasis

Design and Evaluation of a Peptidyl Fluorescent Chemosensor for Divalent Zinc

The rapid analysis of trace metal cations for environmental and biomedical applications is particularly demanding since it requires the specific recognition of a particular element in the presence of numerous closely related species. While remarkable progress has been achieved for inorganic analytes, such as Ca^(2+), there is still a significant need for the genesis of new fluorosensors. In light of the selectivity and avidity with which proteins bind divalent metal cations, we have chosen to use a polypeptide architecture as the framework for metal ion recognition.In addition, our ability to manipulate synthetic polypeptides allows the coordinated integration of fluorescent reporters for signal transduction within a metal-binding peptidyl construct. Herein we report the design, synthesis, and evaluation of a selective fluorescent chemosensor, sensitive to nanomolar concentrations of Zn^(2+)

Functional characterization of iron transporters Nramp1 and Nramp2 from Dictyostelium discoideum: a model of cellular iron homeostasis.

Background: Iron is an element necessary for all the organisms; its role in essential metabolism and cellular processes is fundamental for life. Studies on iron homeostasis, regulation and transport could be also done with a simplified model of professional phagocyte and pathogen host, Dyctiostelium discoideum (Dd). Aim: Characterize the different metal transport specificity and regulation of the two iron transporters of Dd, ddNramp1 and ddNramp2. Methods: Electrophysiological experiments with Two Electrode Voltage Clamp, X. laevis oocytes smoothies (XLOs) and immunolocalization experiments were performed to characterize these two divalent metal transporters and started with the synthesis of the two chimeric protein c-Nramp1 and c-Nramp2. Results: We demonstrated that c-Nramp1 is a sodium independent transporter with specificity for divalent transition metals, as mammal orthologs of SLC11 family, with maximal inward currents at acidic pH and a high affinity for substrate. We also detected a peculiar sodium leak current, mainly at neutral pH, that suggest the presence of a strong interaction in the binding site. We found that c-Nramp2 is electroneutral and transports divalent metal ions at all pH. Site directed mutagenesis on TMD6 restore the electrogenicity with small inward currents; XLOs analysis confirmed the electrophysiological results, these mutants are more functional than wild type c-Nramp2. Conclusions: Our findings on ddNramp1 contributed with novel insights on the mechanism of action of the mammalian ortholog, whereas similar studies on ddNramp2 suggested a role of the homologous proteins in protists, and helped to clarify the possible function of the contractile vacuole in divalent metal homeostasis

The Renaissance of Bacillosamine and Its Derivatives: Pathway Characterization and Implications in Pathogenicity

Prokaryote-specific sugars, including N,N′-diacetylbacillosamine (diNAcBac) and pseudaminic acid, have experienced a renaissance in the past decade because of their discovery in glycans related to microbial pathogenicity. DiNAcBac is found at the reducing end of oligosaccharides of N- and O-linked bacterial protein glycosylation pathways of Gram-negative pathogens, including Campylobacter jejuni and Neisseria gonorrhoeae. Further derivatization of diNAcBac results in the nonulosonic acid known as legionaminic acid, which was first characterized in the O-antigen of the lipopolysaccharide (LPS) in Legionella pneumophila. Pseudaminic acid, an isomer of legionaminic acid, is also important in pathogenic bacteria such as Helicobacter pylori because of its occurrence in O-linked glycosylation of flagellin proteins, which plays an important role in flagellar assembly and motility. Here, we present recent advances in the characterization of the biosynthetic pathways leading to these highly modified sugars and investigation of the roles that each plays in bacterial fitness and pathogenicity.National Institutes of Health (U.S.) (NIH BiotechnologyTraining Program T32-GM08334)National Institutes of Health (U.S.) (NIH Grant GM097241

Monitoring protein interactions and dynamics with solvatochromic fluorophores

Solvatochromic fluorophores possess emission properties that are sensitive to the nature of the local microenvironment. These dyes have been exploited in applications ranging from the study of protein structural dynamics to the detection of protein-binding interactions. Although the solvatochromic indole fluorophore of tryptophan has been utilized extensively for in vitro studies to advance our understanding of basic protein biochemistry, the emergence of new extrinsic synthetic dyes with improved properties, in conjunction with recent developments in site-selective methods to incorporate these chemical tools into proteins, now open the way for studies in more complex systems. Herein, we discuss recent technological advancements and their application in the design of powerful reporters, which serve critical roles in modern cell biology and assay development.National Institutes of Health (U.S.) (NIH Cell Migration Consortium (GM064346))National Science Foundation (U.S.) (CHE-0414243)National Institutes of Health (U.S.) (Interdepartmental Biotechnology Training Grant T32 GM08334)European Commission (Marie Curie Postdoctoral Fellowship Program

« De la musique, la littérature est le plus grand témoin ». Entretien avec Etienne Barilier

Dans le cadre de la série de présentations dont le présent volume conserve la trace, Etienne Barilier avait proposé une conférence sur la Flûte enchantée de Mozart. Mais les éditeurs de ce volume ont eu envie de retrouver derrière le conférencier l’écrivain mélomane, dont l’œuvre est véritablement irriguée par un rapport intime à la musique. N’était-il pas pertinent, sans sortir de la liste des conférenciers, d’envisager un propos d’une autre nature et de proposer à Etienne Barilier un entretien sur la place que la musique occupe dans son œuvre ? L’adhésion de l’intéressé à ce projet a donné lieu au présent article