ASIC1a oligomerization structure-function of its extracellular vestibule and functional characterization of αS243Pβɣ ENaC

Quatre cristaux du canal ASIC1a ont été publiés et soutiennent une stoechiométrie trimérique. Cependant, ces données contredisant de précédentes analyses fonctionnelles effectuées sur des canaux de la même famille, notre intérêt fut porté sur l'oligomérisation d'ASIC1a. Dans ce sens, un nouvel essai couplant la méthode d'analyse par substitution de cystéines (SCAM) avec l'utilisation de réactifs sulfhydryls bifonctionnels (crosslinkers) a été mis en place. Le but étant de stabiliser, puis sélectionner les canaux fonctionnels, pour ensuite les séparer selon leur taille par SDS-PAGE. Grâce à cette technique, nous avons démontré que le complexe stabilisé a une taille coïncidant avec une organisation tétramérique. En plus de son oligomérisation, le chemin emprunté par les ions pour traverser le canal n'est pas clairement défini dans ces structures. De ce fait, utilisant une approche électrophysiologique, nous avons étudié le lien entre la structure et la fonction du vestibule extracellulaire d'ASIC1a. Dans ce but, nous nous sommes intéressés l'accessibilité de cystéines spécifiques localisées dans ce vestibule pour des réactifs méthanethiosulfonates (MTS). Ainsi, nous avons pu corréler les cinétiques de modification de ces cystéines par les MTS avec les effets sur le courant sodique, et donc avoir des informations supplémentaires sur la voie empruntée par les ions. De plus, la simulation informatique de liaison de ces réactifs illustre le remplissage total de ce vestibule. Fonctionnellement, cette interaction ne perturbe pas le passage de ions, c'est pourquoi il nous apparaît probable que le vestibule présente une taille plus large que celle illustrée par les cristaux. Dans un deuxième temps, notre intérêt fut porté sur ENaC. Ce canal est composé des trois sous-unités (a, ß et y) et est exprimé dans divers épithéliums, dont les tubules des reins. Il participe à l'homéostasie sodique et est essentiellement régulé par voie hormonale via l'aldostérone et la Vasopressine, mais également par des sérines protéases ou le Na+. Nous avons étudié la répercussion fonctionnelle de la mutation aS243P, découverte chez un nouveau-né prématuré atteint de pseudohypoaldostéronisme de type 1. Cette maladie autosomale récessive se caractérise, généralement, par une hyponatrémie liée à d'importantes pertes de sel dans les urines, une hyperkaliémie, ainsi qu'un niveau élevé d'aldostérone. Tout d'abord aucune des expériences biochimiques et électrophysiologiques n'a pu démontrer un défaut d'expression ou une forte diminution de l'activité soutenant les données cliniques. Cependant, en challengeant aS243PßyENaC avec une forte concentration de Na+ externe, une hypersensibilité de canal fut observée. En effet, ni les phénomènes régulateurs de « feedback inhibition » ou de « Na+ self-inhibition » n'étaient semblables au canal sauvage. De ce fait, ils apparaissaient exacerbés en présence de la mutation, amenant ainsi à une diminution de la réabsorption de Na+. Ceci corrobore entièrement l'hyponatrémie diagnostiquée. Le rein d'un prématuré étant immature, la quantité de Na+ atteignant la partie distale du néphron est plus élevée, du fait que les autres mécanismes de réabsorption en amont ne sont probablement pas encore en place. Cette hypothèse est renforcée par l'existence d'un frère présentant la même mutation, mais qui, né à terme, ne présentait aucun signe d'hyponatrémie. - The main topic of my thesis is the structure-function relationship of the ENaC/Deg family of ion channels, namely the Acid-Sensing Ion Channel ASIC1a and the Epithelial Na Channel ENaC. The primary part of this research is dedicated to the structure of ASIC1a. Four channel crystals have been published, which support a trimeric stoichiometry, although these data contradict previous functional experiments on other ENaC/Deg members. We are therefore interested in ASIC1a oligomerization and have set up a new assay combining the Substituted- Cysteine Accessibility Method (SCAM) with Afunctional sulfhydryl reagents (crosslinkers) allowing its study. The aim was to first stabilize the channels, then select those that are functional and then resolve them according to their size on SDS-PAGE. We demonstrated that the stabilized complex has a molecular weight corresponding to a tetrameric stoichiometry. In addition to our interest in the oligomerization of the ENaC/Deg family of ion channels, we also wanted to investigate the thus far undefined way of permeation for these channels. Therefore, taking the advantage of a more electrophysiological approach, we studied the accessibility of specific cysteines for methanethiosulfonate reagents (MTS) and were able to correlate the MTS association kinetics on cysteine residues with Na+ currents. These results have given us an insight into ion permeation and our functional evidence indicates that the extracellular is larger than that depicted by the crystal structures. As a side project, we focused on ENaC, which is made up of three subunits (a, ß and y) and is expressed in various epithelia, especially in the distal nephron of the kidneys. It plays a role in Na+ homeostasis and is essentially regulated by hormones via aldosterone and vasopressin, but also by serine proteases or Na+. We have studied the functional impact of the aS243P mutation, discovered in a premature baby suffering from pseudohypoaldosteronism of type 1. This autosomal recessive disease is characterized by hyponatremia, hyperkalemia and high aldosterone levels. Firstly, neither biochemical nor electrophysiological experiments indicated an expression defect or a strong decrease in activity. However, challenging aS243PßyENaC with increased external Na+ concentration showed channel hypersensitivity. Indeed, both the "feedback inhibition" and the "Na+ self-inhibition" regulatory mechanisms are impaired, leading to a decrease in Na+ reabsorption, entirely supports the diagnosis. The kidneys in preterm infants are immature and Na+ levels reaching the distal nephron are higher than normally observed. We hypothesize that the upstream reabsorption machinery is unlikely to be sufficiently matured and this assumption is supported by an asymptomatic sibling carrying the same mutation, but born at term. - La cellule, unité fonctionnelle du corps humain, est délimitée par une membrane plasmique servant de barrière biologique entre les milieux intra et extracellulaires. Une communication entre cellules est indispensable pour un fonctionnement adéquat. Sa survie dépend, entre autres, du maintien de la teneur en ions dans chacun des milieux qui doivent pouvoir être réabsorbés, ou sécrétés, selon les besoins. Les protéines insérées dans la membrane forment un canal et sont un moyen de communication permettant spécifiquement à des ions tel que le sodium (Na+) de traverser. Le Na+ se trouve dans la plupart des aliments et le sel, et est spécifiquement réabsorbé au niveau des reins grâce au canal sodique épithélial ENaC. Cette réabsorption se fait de l'urine primaire vers l'intérieur de la cellule, puis est transporté vers le sang. Pour maintenir un équilibre, une régulation de ce canal est nécessaire. En effet, des dysfonctionnements impliquant la régulation ou l'activité d'ENaC lui-même sont à l'origine de maladies telles que la mucoviscidose, l'hypertension ou encore, le pseudohypoaldostéronisme (PHA). Cette maladie est caractérisée, notamment, par d'importantes pertes de sel dans les urines. Des pédiatres ont diagnostiqué un PHA chez un nouveau-né, ce dernier présentant une modification du canal ENaC, nous avons recréé cette protéine afin d'étudier l'impact de ce changement sur son activité. Nous avons démontré que la régulation d'ENaC était effectivement perturbée, conduisant ainsi à une forte réduction de la réabsorption sodique. Afin de développer des molécules capables de moduler l'activité de protéines. Il est nécessaire d'en connaître la structure. Celle du canal sodique sensible à l'acidification ASIC1, un canal cousin d'ENaC, est connue. Ces données structurales contredisant cependant les analyses fonctionnelles, nous nous sommes penchés une nouvelle fois sur ASIC1. Une protéine est une macromolécule biologique composée d'une chaîne d'acides aminés (aa). De l'enchaînement d'aa à la protéine fonctionnelle, quatre niveaux de structuration existent. Chaque aa donne une indication quant au repliement et plus particulièrement la cystéine. Arborant un groupe sulfhydryle (SH) capable de former une liaison spécifique et stable avec un autre SH, celle-ci est souvent impliquée dans la structure tridimensionnelle de la protéine. Ce type de liaison intervient également dans la stabilisation de la structure quaternaire, qui est l'association de plusieurs protéines identiques (homomère), ou pas (hétéromère). Dans cette partie, nous avons remplacé des aa par des cystéines à des endroits spécifiques. Le but était de stabiliser plusieurs homomères d'ASICl ensemble avec des réactifs créant des ponts entre deux SH. Ainsi, nous avons pu déterminer le nombre de protéines ASIC1 participant à la formation d'un canal fonctionnel. Nos résultats corroborent les données fonctionnelles soutenant un canal tétramérique. Nous avons également étudié l'accessibilité de ces nouvelles cystéines afin d'obtenir des informations supplémentaires sur la structure du chemin emprunté par le Na+ à travers ASIC1 et plus particulièrement du vestibule extracellulaire.

Use of an in-house approach to study the three-dimensional structures of various outer membrane proteins: structure of the alcaligin outer membrane transporter FauA from Bordetella pertussis.

Bordetella pertussis is the bacterial agent of whooping cough in humans. Under iron-limiting conditions, it produces the siderophore alcaligin. Released to the extracellular environment, alcaligin chelates iron, which is then taken up as a ferric alcaligin complex via the FauA outer membrane transporter. FauA belongs to a family of TonB-dependent outer membrane transporters that function using energy derived from the proton motive force. Using an in-house protocol for membrane-protein expression, purification and crystallization, FauA was crystallized in its apo form together with three other TonB-dependent transporters from different organisms. Here, the protocol used to study FauA is described and its three-dimensional structure determined at 2.3 A resolution is discussed.

Selected configuration interaction with truncation energy error and application to the Ne atom

Selected configuration interaction (SCI) for atomic and molecular electronic structure calculations is reformulated in a general framework encompassing all CI methods. The linked cluster expansion is used as an intermediate device to approximate CI coefficients BK of disconnected configurations (those that can be expressed as products of combinations of singly and doubly excited ones) in terms of CI coefficients of lower-excited configurations where each K is a linear combination of configuration-state-functions (CSFs) over all degenerate elements of K. Disconnected configurations up to sextuply excited ones are selected by Brown's energy formula, ΔEK=(E-HKK)BK2/(1-BK2), with BK determined from coefficients of singly and doubly excited configurations. The truncation energy error from disconnected configurations, Δdis, is approximated by the sum of ΔEKS of all discarded Ks. The remaining (connected) configurations are selected by thresholds based on natural orbital concepts. Given a model CI space M, a usual upper bound ES is computed by CI in a selected space S, and EM=E S+ΔEdis+δE, where δE is a residual error which can be calculated by well-defined sensitivity analyses. An SCI calculation on Ne ground state featuring 1077 orbitals is presented. Convergence to within near spectroscopic accuracy (0.5 cm-1) is achieved in a model space M of 1.4× 109 CSFs (1.1 × 1012 determinants) containing up to quadruply excited CSFs. Accurate energy contributions of quintuples and sextuples in a model space of 6.5 × 1012 CSFs are obtained. The impact of SCI on various orbital methods is discussed. Since ΔEdis can readily be calculated for very large basis sets without the need of a CI calculation, it can be used to estimate the orbital basis incompleteness error. A method for precise and efficient evaluation of ES is taken up in a companion paper

Crystal structure of NLRC4 reveals its autoinhibition mechanism.

Nucleotide-binding and oligomerization domain-like receptor (NLR) proteins oligomerize into multiprotein complexes termed inflammasomes when activated. Their autoinhibition mechanism remains poorly defined. Here, we report the crystal structure of mouse NLRC4 in a closed form. The adenosine diphosphate-mediated interaction between the central nucleotide-binding domain (NBD) and the winged-helix domain (WHD) was critical for stabilizing the closed conformation of NLRC4. The helical domain HD2 repressively contacted a conserved and functionally important α-helix of the NBD. The C-terminal leucine-rich repeat (LRR) domain is positioned to sterically occlude one side of the NBD domain and consequently sequester NLRC4 in a monomeric state. Disruption of ADP-mediated NBD-WHD or NBD-HD2/NBD-LRR interactions resulted in constitutive activation of NLRC4. Together, our data reveal the NBD-organized cooperative autoinhibition mechanism of NLRC4 and provide insight into its activation.

Development and validation of AMANDA, a new algorithm for selecting highly relevant regions in Molecular Interaction Fields

Descriptors based on Molecular Interaction Fields (MIF) are highly suitable for drug discovery, but their size (thousands of variables) often limits their application in practice. Here we describe a simple and fast computational method that extracts from a MIF a handful of highly informative points (hot spots) which summarize the most relevant information. The method was specifically developed for drug discovery, is fast, and does not require human supervision, being suitable for its application on very large series of compounds. The quality of the results has been tested by running the method on the ligand structure of a large number of ligand-receptor complexes and then comparing the position of the selected hot spots with actual atoms of the receptor. As an additional test, the hot spots obtained with the novel method were used to obtain GRIND-like molecular descriptors which were compared with the original GRIND. In both cases the results show that the novel method is highly suitable for describing ligand-receptor interactions and compares favorably with other state-of-the-art methods.

Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity.

As an approved vaccine adjuvant for use in humans, alum has vast health implications, but, as it is a crystal, questions remain regarding its mechanism. Furthermore, little is known about the target cells, receptors, and signaling pathways engaged by alum. Here we report that, independent of inflammasome and membrane proteins, alum binds dendritic cell (DC) plasma membrane lipids with substantial force. Subsequent lipid sorting activates an abortive phagocytic response that leads to antigen uptake. Such activated DCs, without further association with alum, show high affinity and stable binding with CD4(+) T cells via the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1). We propose that alum triggers DC responses by altering membrane lipid structures. This study therefore suggests an unexpected mechanism for how this crystalline structure interacts with the immune system and how the DC plasma membrane may behave as a general sensor for solid structures.

Different internalization properties of the alpha1a- and alpha1b-adrenergic receptor subtypes: the potential role of receptor interaction with beta-arrestins and AP50.

The internalization properties of the alpha1a- and alpha1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the alpha1b-AR displayed robust agonist-induced endocytosis, the alpha1a-AR did not. Constitutive internalization of the alpha1a-AR was negligible, whereas the alpha1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the alpha1-AR subtypes with beta-arrestins 1 and 2 as well as with the AP50 subunit of the clathrin adaptor complex AP2. The results from both coimmunoprecipitation experiments and beta-arrestin translocation assays indicated that the agonistinduced interaction of the alpha1a-AR with beta-arrestins was much weaker than that of the alpha1b-AR. In addition, the alpha1a-AR did not bind AP50. The alpha1b-AR mutant M8, lacking the main phosphorylation sites in the receptor C tail, was unable to undergo endocytosis and was profoundly impaired in binding beta-arrestins despite its binding to AP50. In contrast, the alpha1b-AR mutant DeltaR8, lacking AP50 binding, bound beta-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that beta-arrestin 2 plays a prominent role in alpha1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the alpha1a- and alpha1b-AR and provide evidence that the lack of significant endocytosis of the alpha1a-AR is linked to its poor interaction with beta-arrestins as well as with AP50. We also provide evidence that the integrity of the phosphorylation sites in the C tail of the alpha1b-AR is important for receptor/beta-arrestin interaction and that this interaction is the main event triggering receptor internalization.

Model structure of human APOBEC3G.

BACKGROUND: APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) has antiretroviral activity associated with the hypermutation of viral DNA through cytosine deamination. APOBEC3G has two cytosine deaminase (CDA) domains; the catalytically inactive amino-terminal domain of APOBEC3G (N-CDA) carries the Vif interaction domain. There is no 3-D structure of APOBEC3G solved by X-ray or nuclear magnetic resonance. METHODOLOGY/PRINCIPAL FINDINGS: We predicted the structure of human APOBEC3G based on the crystal structure of APOBEC2. To assess the model structure, we evaluated 48 mutants of APOBEC3G N-CDA that identify novel variants altering DeltaVif HIV-1 infectivity and packaging of APOBEC3G. Results indicated that the key residue D128 is exposed at the surface of the model, with a negative local electrostatic potential. Mutation D128K changes the sign of that local potential. In addition, two novel functionally relevant residues that result in defective APOBEC3G encapsidation, R122 and W127, cluster at the surface. CONCLUSIONS/SIGNIFICANCE: The structure model identifies a cluster of residues important for packaging of APOBEC3G into virions, and may serve to guide functional analysis of APOBEC3G.

Targeting of DNA polymerase to the adenovirus origin of DNA replication by interaction with nuclear factor I.

Efficient initiation by the DNA polymerase of adenovirus type 2 requires nuclear factor I (NFI), a cellular sequence-specific transcription factor. Three functions of NFI--dimerization, DNA binding, and activation of DNA replication--are colocalized within the N-terminal portion of the protein. To define more precisely the role of NFI in viral DNA replication, a series of site-directed mutations within the N-terminal domain have been generated, thus allowing the separation of all three functions contained within this region. Impairment of the dimerization function prevents sequence-specific DNA binding and in turn abolishes the NFI-mediated activation of DNA replication. NFI DNA-binding activity, although necessary, is not sufficient to activate the initiation of adenovirus replication. A distinct class of NFI mutations that abolish the recruitment of the viral DNA polymerase to the origin also prevent the activation of replication. Thus, a direct interaction of NFI with the viral DNA polymerase complex is required to form a stable and active preinitiation complex on the origin and is responsible for the activation of replication by NFI.

Ownership structure, customer satisfaction and brand equity

This paper studies the interaction between ownership structure, taken as a proxy for shareholders commitment, and customer satisfaction - the main driver of consumer loyalty - and their impact on a firm s brand equity. The results show that customer satisfaction has a positive direct effect on brand equity but an indirect negative one because of reductions in ownership concentration. This latter effect emerges when managers are mainly customer-oriented. Such result gives out a warning signal that highlights the perverse effect of implementing policies, focused excessively on satisfying customers at the expense of shareholders, on a firm s brand equity. The empirical analysis uses an incomplete panel data comprising 69 firms from 11 nations, for the period 2002-2005.

Brain structure in movement disorders: a neuroimaging perspective.

Purpose of review: An overview of recent advances in structural neuroimaging and their impact on movement disorders research is presented. Recent findings: Novel developments in computational neuroanatomy and improvements in magnetic resonance image quality have brought further insight into the pathophysiology of movement disorders. Sophisticated automated techniques allow for sensitive and reliable in-vivo differentiation of phenotype/genotype related traits and their interaction even at presymptomatic stages of disease. Summary: Voxel-based morphometry consistently demonstrates well defined patterns of brain structure changes in movement disorders. Advanced stages of idiopathic Parkinson's disease are characterized by grey matter volume decreases in basal ganglia. Depending on the presence of cognitive impairment, volume changes are reported in widespread cortical and limbic areas. Atypical Parkinsonian syndromes still pose a challenge for accurate morphometry-based classification, especially in early stages of disease progression. Essential tremor has been mainly associated with thalamic and cerebellar changes. Studies on preclinical Huntington's disease show progressive loss of tissue in the caudate and cortical thinning related to distinct motor and cognitive phenotypes. Basal ganglia volume in primary dystonia reveals an interaction between genotype and phenotype such that brain structure changes are modulated by the presence of symptoms under the influence of genetic factors. Tics in Tourette's syndrome correlate with brain structure changes in limbic, motor and associative fronto-striato-parietal circuits. Computational neuroanatomy provides useful tools for in-vivo assessment of brain structure in movement disorders, allowing for accurate classification in early clinical stages as well as for monitoring therapy effects and/or disease progression.

Shaping the interaction landscape of bioactive molecules.

MOTIVATION: Most bioactive molecules perform their action by interacting with proteins or other macromolecules. However, for a significant fraction of them, the primary target remains unknown. In addition, the majority of bioactive molecules have more than one target, many of which are poorly characterized. Computational predictions of bioactive molecule targets based on similarity with known ligands are powerful to narrow down the number of potential targets and to rationalize side effects of known molecules. RESULTS: Using a reference set of 224 412 molecules active on 1700 human proteins, we show that accurate target prediction can be achieved by combining different measures of chemical similarity based on both chemical structure and molecular shape. Our results indicate that the combined approach is especially efficient when no ligand with the same scaffold or from the same chemical series has yet been discovered. We also observe that different combinations of similarity measures are optimal for different molecular properties, such as the number of heavy atoms. This further highlights the importance of considering different classes of similarity measures between new molecules and known ligands to accurately predict their targets. CONTACT: olivier.michielin@unil.ch or vincent.zoete@unil.ch SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BAFF, APRIL and their receptors: structure, function and signaling.

BAFF, APRIL and their receptors play important immunological roles, especially in the B cell arm of the immune system. A number of splice isoforms have been described for both ligands and receptors in this subfamily, some of which are conserved between mouse and human, while others are species-specific. Structural and mutational analyses have revealed key determinants of receptor-ligand specificity. BAFF-R has a strong selectivity for BAFF; BCMA has a higher affinity for APRIL than for BAFF, while TACI binds both ligands equally well. The molecular signaling events downstream of BAFF-R, BCMA and TACI are still incompletely characterized. Survival appears to be mediated by upregulation of Bcl-2 family members through NF-kappaB activation, degradation of the pro-apototic Bim protein, and control of subcellular localization of PCKdelta. Very little is known about other signaling events associated with receptor engagement by BAFF and APRIL that lead for example to B cell activation or to CD40L-independent Ig switch.

Ferromagnetic instabilities in neutron matter at finite temperature with the Skyrme interaction

The properties of spin-polarized neutron matter are studied at both zero and finite temperature using Skyrme-type interactions. It is shown that the critical density at which ferromagnetism takes place decreases with temperature. This unexpected behavior is associated to an anomalous behavior of the entropy that becomes larger for the polarized phase than for the unpolarized one above a certain critical density. This fact is a consequence of the dependence of the entropy on the effective mass of the neutrons with different third spin component. A new constraint on the parameters of the effective Skyrme force is derived if this behavior is to be avoided.