Molecular models of human P-glycoprotein in two different catalytic states

P-glycoprotein belongs to the family of ATP-binding cassette proteins which hydrolyze ATP to catalyse the translocation of their substrates through membranes. This protein extrudes a large range of components out of cells, especially therapeutic agents causing a phenomenon known as multidrug resistance. Because of its clinical interest, its activity and transport function have been largely characterized by various biochemical studies. In the absence of a high-resolution structure of P-glycoprotein, homology modeling is a useful tool to help interpretation of experimental data and potentially guide experimental studies.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Faire gras à Molène: dairy products and ruminant fats detected by lipid and isotopic analysis of pottery dating to the Final Neolithic-Early Bronze Age from the island site of Beg ar Loued (Molène, western Brittany, France): Faire gras à Molène : produits laitiers et graisses de ruminants détectés par l’analyse lipidique et isotopique des céramiques du Néolithique final et de l’âge du Bronze ancien du site insulaire de Beg ar Loued (Molène, Bretagne occidentale, France)

The subsistence strategies of early farming communities have been highlighted since the beginning of the Neolithic, thanks to numerous studies on lipid residues from ceramic vessels conducted in various parts of continental Europe. However, after the Early Neolithic, evidence of subsistence strategies along the northern Atlantic coast are still lacking, especially for island contexts. This paper presents the results of lipid residue analysis of 129 potsherds from Beg ar Loued (Molène, France), an island site dating primarily to the Early Bronze Age (c. 2700-2600 to 1800 BCE). Aiming to understand the use of vessels, vessel treatment and culinary practices on the settlement, analyses of visible charred residues, sherds and ceramic surfaces/coating layers were carried out using chromatographic (n = 174) and isotopic techniques (n = 24) after lipid extraction by solvent (n = 174) or acid methanolysis (n = 31). The results demonstrate the extensive use of terrestrial products (ruminant carcass and dairy) in pottery, including occasional plant products (with possible mixtures of different waxes), while the detection of aquatic products is limited. Thus, combined with evidence from faunal remains at the site, the results indicate that terrestrial resources like ruminant meat and dairy products were preferentially processed in vessels, and aquatic products mostly without the use of ceramics. These findings demonstrate the significance of lipid residue analysis for studying the role of pottery in food production and consumption at sites along the Atlantic coast. Les stratégies de subsistance des premières communautés agricoles ont été mises en évidence depuis le début du Néolithique grâce à de nombreuses études sur les résidus lipidiques des récipients en céramique menées dans diverses parties de l’Europe continentale. En revanche, très peu de données sont disponibles pour la fin du Néolithique et le début de l’âge du Bronze sur la côte atlantique, en particulier en contexte insulaire. Cet article présente les résultats de l’analyse de résidus lipidiques provenant de 129 fragments de poteries de Beg ar Loued (Molène, France), un site insulaire dont les principaux vestiges datent de l’âge du Bronze ancien (c. 2700-2600 à 1800 BCE). Dans le but de comprendre l’utilisation des récipients, les pratiques culinaires sur ce site et d’appréhender les techniques de finition des céramiques, des analyses de résidus visibles carbonisés, de tessons, et de surfaces/couches d’engobe ont été effectuées via des analyses chromatographiques (n = 174) et isotopiques (n = 24), après extraction des lipides par solvant (n = 174) ou méthanolyse acide (n = 31). Les résultats démontrent l’utilisation extensive de produits terrestres (carcasses de ruminants et produits laitiers), comprenant occasionnellement des produits végétaux (avec un mélange probable de différentes cires), alors que la détection des produits aquatiques est faible. Comparés aux données fauniques, ces résultats indiquent donc que les produits terrestres, tels que la viande de ruminant et les produits laitiers, sont transformés en utilisant des récipients en céramiques, tandis que les produits aquatiques semblent de préférence exploités sans avoir recours à une poterie. Ces résultats démontrent l’importance de l’analyse des résidus lipidiques pour connaître le rôle des récipients en céramique dans la production et la consommation d’aliments sur les sites de la côte atlantique

Mutation of Tyr137 of the universal Escherichia coli fimbrial adhesin FimH relaxes the tyrosine gate prior to mannose binding

The most prevalent diseases manifested by Escherichia coli are acute and recurrent bladder infections and chronic inflammatory bowel diseases such as Crohn's disease. E. coli clinical isolates express the FimH adhesin, which consists of a mannose-specific lectin domain connected via a pilin domain to the tip of type 1 pili. Although the isolated FimH lectin domain has affinities in the nanomolar range for all high-mannosidic glycans, differentiation between these glycans is based on their capacity to form predominantly hydrophobic interactions within the tyrosine gate at the entrance to the binding pocket. In this study, novel crystal structures of tyrosine-gate mutants of FimH, ligand-free or in complex with heptyl α - D - O -mannopyranoside or 4-biphenyl α - D - O- mannopyranoside, are combined with quantum-mechanical calculations and molecular-dynamics simulations. In the Y48A FimH crystal structure, a large increase in the dynamics of the alkyl chain of heptyl α - D - O -mannopyranoside attempts to compensate for the absence of the aromatic ring; however, the highly energetic and stringent mannose-binding pocket of wild-type FimH is largely maintained. The Y137A mutation, on the other hand, is the most detrimental to FimH affinity and specificity: (i) in the absence of ligand the FimH C-terminal residue Thr158 intrudes into the mannose-binding pocket and (ii) ethylenediaminetetraacetic acid interacts strongly with Glu50, Thr53 and Asn136, in spite of multiple dialysis and purification steps. Upon mutation, pre-ligand-binding relaxation of the backbone dihedral angles at position 137 in the tyrosine gate and their coupling to Tyr48 via the interiorly located Ile52 form the basis of the loss of affinity of the FimH adhesin in the Y137A mutant

fbl-Typing of Staphylococcus lugdunensis: A Frontline Tool for Epidemiological Studies, but Not Predictive of Fibrinogen Binding Ability

Staphylococcus lugdunensis is increasingly recognized as a potent pathogen, responsible for severe infections with an outcome resembling that of Staphylococcus aureus. Here, we developed and evaluated a tool for S. lugdunensis typing, using DNA sequence analysis of the repeat-encoding region (R-domain) in the gene encoding the fibrinogen (Fg)-binding protein Fbl (fbl-typing). We typed 240 S. lugdunensis isolates from various clinical and geographical origins. The length of the R-domain ranged from 9 to 52 repeats. fbl-typing identified 54 unique 18-bp repeat sequences and 92 distinct fbl-types. The discriminatory power of fbl-typing was higher than that of multilocus sequence typing (MLST) and equivalent to that of tandem repeat sequence typing. fbl-types could assign isolates to MLST clonal complexes with excellent predictive power. The ability to promote adherence to immobilized human Fg was evaluated for 55 isolates chosen to reflect the genetic diversity of the fbl gene. We observed no direct correlation between Fg binding ability and fbl-types. However, the lowest percentage of Fg binding was observed for isolates carrying a 5′-end frameshift mutation of the fbl gene and for those harboring fewer than 43 repeats in the R-domain. qRT-PCR assays for some isolates revealed no correlation between fbl gene expression and Fg binding capacity. In conclusion, this study shows that fbl-typing is a useful tool in S. lugdunensis epidemiology, especially because it is an easy, cost-effective, rapid and portable method (http://fbl-typing.univ-rouen.fr/). The impact of fbl polymorphism on the structure of the protein, its expression on the cell surface and in virulence remains to be determined

Systematic Mutational Analysis of the Intracellular Regions of Yeast Gap1 Permease

The yeast general amino acid permease Gap1 is a convenient model for studying the intracellular trafficking of membrane proteins. Present at the plasma membrane when the nitrogen source is poor, it undergoes ubiquitin-dependent endocytosis and degradation upon addition of a good nitrogen source, e.g. ammonium. It comprises 12 transmembrane domains (TM) flanked by cytosol-facing N- and C-terminal tails (NT, CT). The NT of Gap1 contains the acceptor lysines for ubiquitylation and its CT includes a sequence essential to exit from the endoplasmic reticulum (ER).Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Concentration Dependent Ion Selectivity in VDAC: A Molecular Dynamics Simulation Study

The voltage-dependent anion channel (VDAC) forms the major pore in the outer mitochondrial membrane. Its high conducting open state features a moderate anion selectivity. There is some evidence indicating that the electrophysiological properties of VDAC vary with the salt concentration. Using a theoretical approach the molecular basis for this concentration dependence was investigated. Molecular dynamics simulations and continuum electrostatic calculations performed on the mouse VDAC1 isoform clearly demonstrate that the distribution of fixed charges in the channel creates an electric field, which determines the anion preference of VDAC at low salt concentration. Increasing the salt concentration in the bulk results in a higher concentration of ions in the VDAC wide pore. This event induces a large electrostatic screening of the charged residues promoting a less anion selective channel. Residues that are responsible for the electrostatic pattern of the channel were identified using the molecular dynamics trajectories. Some of these residues are found to be conserved suggesting that ion permeation between different VDAC species occurs through a common mechanism. This inference is buttressed by electrophysiological experiments performed on bean VDAC32 protein akin to mouse VDAC

Interplays between copper and Mycobacterium tuberculosis GroEL1

The recalcitrance of pathogenic Mycobacterium tuberculosis, the agent of tuberculosis, to eradication is due to various factors allowing bacteria to escape from stress situations. The mycobacterial chaperone GroEL1, overproduced after macrophage entry and under oxidative stress, could be one of these key players. We previously reported that GroEL1 is necessary for the biosynthesis of phthiocerol dimycocerosate, a virulence-associated lipid and for reducing antibiotic susceptibility. In the present study, we showed that GroEL1, bearing a unique C-terminal histidine-rich region, is required for copper tolerance during Mycobacterium bovis BCG biofilm growth. Mass spectrometry analysis demonstrated that GroEL1 displays high affinity for copper ions, especially at its C-terminal histidine-rich region. Furthermore, the binding of copper protects GroEL1 from destabilization and increases GroEL1 ATPase activity. Altogether, these findings suggest that GroEL1 could counteract copper toxicity, notably in the macrophage phagosome, and further emphasizes that M. tuberculosis GroEL1 could be an interesting antitubercular target