Therapeutic Blockade of LIGHT Interaction With Herpesvirus Entry Mediator and Lymphotoxin β Receptor Attenuates In Vivo Cytotoxic Allogeneic Responses.

BACKGROUND: Tumor necrosis factor/tumor necrosis factor receptor superfamily members conform a group of molecular interaction pathways of essential relevance during the process of T-cell activation and differentiation toward effector cells and particularly for the maintenance phase of the immune response. Specific blockade of these interacting pathways, such as CD40-CD40L, contributes to modulate the deleterious outcome of allogeneic immune responses. We postulated that antagonizing the interaction of LIGHT expression on activated T cells with its receptors, herpesvirus entry mediator and lymphotoxin β receptor, may decrease T cell-mediated allogeneic responses. METHODS: A flow cytometry competition assay was designed to identify anti-LIGHT monoclonal antibodies capable to prevent the interaction of mouse LIGHT with its receptors expressed on transfected cells. An antibody with the desired specificity was evaluated in a short-term in vivo allogeneic cytotoxic assay and tested for its ability to detect endogenous mouse LIGHT. RESULTS: We provide evidence for the first time that in mice, as previously described in humans, LIGHT protein is rapidly and transiently expressed after T-cell activation, and this expression was stronger on CD8 T cells than on CD4 T cells. Two anti-LIGHT antibodies prevented interactions of mouse LIGHT with its two known receptors, herpesvirus entry mediator and lymphotoxin β receptor. In vivo administration of anti-LIGHT antibody (clone 10F12) ameliorated host antidonor short-term cytotoxic response in wild type B6 mice, although to a lesser extent than that observed in LIGHT-deficient mice. CONCLUSION: The therapeutic targeting of LIGHT may contribute to achieve a better control of cytotoxic responses refractory to current immunosuppressive drugs in transplantation.

Broadening the horizon--level 2.5 of the HUPO-PSI format for molecular interactions.

BACKGROUND: Molecular interaction Information is a key resource in modern biomedical research. Publicly available data have previously been provided in a broad array of diverse formats, making access to this very difficult. The publication and wide implementation of the Human Proteome Organisation Proteomics Standards Initiative Molecular Interactions (HUPO PSI-MI) format in 2004 was a major step towards the establishment of a single, unified format by which molecular interactions should be presented, but focused purely on protein-protein interactions. RESULTS: The HUPO-PSI has further developed the PSI-MI XML schema to enable the description of interactions between a wider range of molecular types, for example nucleic acids, chemical entities, and molecular complexes. Extensive details about each supported molecular interaction can now be captured, including the biological role of each molecule within that interaction, detailed description of interacting domains, and the kinetic parameters of the interaction. The format is supported by data management and analysis tools and has been adopted by major interaction data providers. Additionally, a simpler, tab-delimited format MITAB2.5 has been developed for the benefit of users who require only minimal information in an easy to access configuration. CONCLUSION: The PSI-MI XML2.5 and MITAB2.5 formats have been jointly developed by interaction data producers and providers from both the academic and commercial sector, and are already widely implemented and well supported by an active development community. PSI-MI XML2.5 enables the description of highly detailed molecular interaction data and facilitates data exchange between databases and users without loss of information. MITAB2.5 is a simpler format appropriate for fast Perl parsing or loading into Microsoft Excel.

Partition coefficient and molecular flexibility: the concept of lipophilicity space.

The rationale of this study was to investigate molecular flexibility and its influence on physicochemical properties with a view to uncovering additional information on the fuzzy concept of dynamic molecular structure. Indeed, it is now known that computed molecular interaction fields (MIFs) such as molecular electrostatic potentials (MEPs) and lipophilicity potentials (MLPs) are conformation-dependent, as are dipole moments. A database of 125 compounds was used whose conformational space was explored, while conformation-dependent parameters were computed for each non-redundant conformer found in the conformational space of the compounds. These parameters were the virtual log P (log P(MLP), calculated by a MLP approach), the apolar surface area (ASA), polar surface area (PSA), and solvent-accessible surface (SAS). For each compound, the range taken by each parameter (its property space) was divided by the number of rotors taken as an index of flexibility, yielding a parameter termed 'molecular sensitivity'. This parameter was poorly correlated with others (i.e., it contains novel information) and showed the compounds to fall into two broad classes. 'Sensitive' molecules are those whose computed property ranges are markedly sensitive to conformational effects, whereas 'insensitive' (in fact, less sensitive) molecules have property ranges which are comparatively less affected by conformational fluctuations. A pharmacokinetic application is presented.

EADock : design of a new molecular docking algorithm and some of its applications

3 Summary 3. 1 English The pharmaceutical industry has been facing several challenges during the last years, and the optimization of their drug discovery pipeline is believed to be the only viable solution. High-throughput techniques do participate actively to this optimization, especially when complemented by computational approaches aiming at rationalizing the enormous amount of information that they can produce. In siiico techniques, such as virtual screening or rational drug design, are now routinely used to guide drug discovery. Both heavily rely on the prediction of the molecular interaction (docking) occurring between drug-like molecules and a therapeutically relevant target. Several softwares are available to this end, but despite the very promising picture drawn in most benchmarks, they still hold several hidden weaknesses. As pointed out in several recent reviews, the docking problem is far from being solved, and there is now a need for methods able to identify binding modes with a high accuracy, which is essential to reliably compute the binding free energy of the ligand. This quantity is directly linked to its affinity and can be related to its biological activity. Accurate docking algorithms are thus critical for both the discovery and the rational optimization of new drugs. In this thesis, a new docking software aiming at this goal is presented, EADock. It uses a hybrid evolutionary algorithm with two fitness functions, in combination with a sophisticated management of the diversity. EADock is interfaced with .the CHARMM package for energy calculations and coordinate handling. A validation was carried out on 37 crystallized protein-ligand complexes featuring 11 different proteins. The search space was defined as a sphere of 15 R around the center of mass of the ligand position in the crystal structure, and conversely to other benchmarks, our algorithms was fed with optimized ligand positions up to 10 A root mean square deviation 2MSD) from the crystal structure. This validation illustrates the efficiency of our sampling heuristic, as correct binding modes, defined by a RMSD to the crystal structure lower than 2 A, were identified and ranked first for 68% of the complexes. The success rate increases to 78% when considering the five best-ranked clusters, and 92% when all clusters present in the last generation are taken into account. Most failures in this benchmark could be explained by the presence of crystal contacts in the experimental structure. EADock has been used to understand molecular interactions involved in the regulation of the Na,K ATPase, and in the activation of the nuclear hormone peroxisome proliferatoractivated receptors a (PPARa). It also helped to understand the action of common pollutants (phthalates) on PPARy, and the impact of biotransformations of the anticancer drug Imatinib (Gleevec®) on its binding mode to the Bcr-Abl tyrosine kinase. Finally, a fragment-based rational drug design approach using EADock was developed, and led to the successful design of new peptidic ligands for the a5ß1 integrin, and for the human PPARa. In both cases, the designed peptides presented activities comparable to that of well-established ligands such as the anticancer drug Cilengitide and Wy14,643, respectively. 3.2 French Les récentes difficultés de l'industrie pharmaceutique ne semblent pouvoir se résoudre que par l'optimisation de leur processus de développement de médicaments. Cette dernière implique de plus en plus. de techniques dites "haut-débit", particulièrement efficaces lorsqu'elles sont couplées aux outils informatiques permettant de gérer la masse de données produite. Désormais, les approches in silico telles que le criblage virtuel ou la conception rationnelle de nouvelles molécules sont utilisées couramment. Toutes deux reposent sur la capacité à prédire les détails de l'interaction moléculaire entre une molécule ressemblant à un principe actif (PA) et une protéine cible ayant un intérêt thérapeutique. Les comparatifs de logiciels s'attaquant à cette prédiction sont flatteurs, mais plusieurs problèmes subsistent. La littérature récente tend à remettre en cause leur fiabilité, affirmant l'émergence .d'un besoin pour des approches plus précises du mode d'interaction. Cette précision est essentielle au calcul de l'énergie libre de liaison, qui est directement liée à l'affinité du PA potentiel pour la protéine cible, et indirectement liée à son activité biologique. Une prédiction précise est d'une importance toute particulière pour la découverte et l'optimisation de nouvelles molécules actives. Cette thèse présente un nouveau logiciel, EADock, mettant en avant une telle précision. Cet algorithme évolutionnaire hybride utilise deux pressions de sélections, combinées à une gestion de la diversité sophistiquée. EADock repose sur CHARMM pour les calculs d'énergie et la gestion des coordonnées atomiques. Sa validation a été effectuée sur 37 complexes protéine-ligand cristallisés, incluant 11 protéines différentes. L'espace de recherche a été étendu à une sphère de 151 de rayon autour du centre de masse du ligand cristallisé, et contrairement aux comparatifs habituels, l'algorithme est parti de solutions optimisées présentant un RMSD jusqu'à 10 R par rapport à la structure cristalline. Cette validation a permis de mettre en évidence l'efficacité de notre heuristique de recherche car des modes d'interactions présentant un RMSD inférieur à 2 R par rapport à la structure cristalline ont été classés premier pour 68% des complexes. Lorsque les cinq meilleures solutions sont prises en compte, le taux de succès grimpe à 78%, et 92% lorsque la totalité de la dernière génération est prise en compte. La plupart des erreurs de prédiction sont imputables à la présence de contacts cristallins. Depuis, EADock a été utilisé pour comprendre les mécanismes moléculaires impliqués dans la régulation de la Na,K ATPase et dans l'activation du peroxisome proliferatoractivated receptor a (PPARa). Il a également permis de décrire l'interaction de polluants couramment rencontrés sur PPARy, ainsi que l'influence de la métabolisation de l'Imatinib (PA anticancéreux) sur la fixation à la kinase Bcr-Abl. Une approche basée sur la prédiction des interactions de fragments moléculaires avec protéine cible est également proposée. Elle a permis la découverte de nouveaux ligands peptidiques de PPARa et de l'intégrine a5ß1. Dans les deux cas, l'activité de ces nouveaux peptides est comparable à celles de ligands bien établis, comme le Wy14,643 pour le premier, et le Cilengitide (PA anticancéreux) pour la seconde.

Envelope glycoprotein of arenaviruses.

Arenaviruses include lethal human pathogens which pose serious public health threats. So far, no FDA approved vaccines are available against arenavirus infections, and therapeutic options are limited, making the identification of novel drug targets for the development of efficacious therapeutics an urgent need. Arenaviruses are comprised of two RNA genome segments and four proteins, the polymerase L, the envelope glycoprotein GP, the matrix protein Z, and the nucleoprotein NP. A crucial step in the arenavirus life-cycle is the biosynthesis and maturation of the GP precursor (GPC) by cellular signal peptidases and the cellular enzyme Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P) yielding a tripartite mature GP complex formed by GP1/GP2 and a stable signal peptide (SSP). GPC cleavage by SKI-1/S1P is crucial for fusion competence and incorporation of mature GP into nascent budding virion particles. In a first part of our review, we cover basic aspects and newer developments in the biosynthesis of arenavirus GP and its molecular interaction with SKI-1/S1P. A second part will then highlight the potential of SKI-1/S1P-mediated processing of arenavirus GPC as a novel target for therapeutic intervention to combat human pathogenic arenaviruses.

Artificial zinc finger fusions targeting Sp1-binding sites and the trans-activator-responsive element potently repress transcription and replication of HIV-1.

Tat activates transcription by interacting with Sp1, NF-kappaB, positive transcription elongation factor b, and trans-activator-responsive element (TAR). Tat and Sp1 play major roles in transcription by protein-protein interactions at human immunodeficiency virus, type 1 (HIV-1) long terminal repeat. Sp1 activates transcription by interacting with cyclin T1 in the absence of Tat. To disrupt the transcription activation by Tat and Sp1, we fused Sp1-inhibiting polypeptides, zinc finger polypeptide, and the TAR-binding mutant Tat (TatdMt) together. A designed or natural zinc finger and Tat mutant fusion was used to target the fusion to the key regulatory sites (GC box and TAR) on the long terminal repeat and nascent short transcripts to disrupt the molecular interaction that normally result in robust transcription. The designed zinc finger and TatdMt fusions were targeted to the TAR, and they potently repressed both transcription and replication of HIV-1. The Sp1-inhibiting POZ domain, TatdMt, and zinc fingers are key functional domains important in repression of transcription and replication. The designed artificial zinc fingers were targeted to the high affinity Sp1-binding site, and by being fused with TatdMt and POZ domain, they strongly block both Sp1-cyclin T1-dependent transcription and Tat-dependent transcription, even in the presence of excess expressed Tat.

Protein interaction data curation: the International Molecular Exchange (IMEx) consortium.

The International Molecular Exchange (IMEx) consortium is an international collaboration between major public interaction data providers to share literature-curation efforts and make a nonredundant set of protein interactions available in a single search interface on a common website (http://www.imexconsortium.org/). Common curation rules have been developed, and a central registry is used to manage the selection of articles to enter into the dataset. We discuss the advantages of such a service to the user, our quality-control measures and our data-distribution practices.

Protein-protein interaction investigated by steered molecular dynamics: the TCR-pMHC complex.

We present a novel steered molecular dynamics scheme to induce the dissociation of large protein-protein complexes. We apply this scheme to study the interaction of a T cell receptor (TCR) with a major histocompatibility complex (MHC) presenting a peptide (p). Two TCR-pMHC complexes are considered, which only differ by the mutation of a single amino acid on the peptide; one is a strong agonist that produces T cell activation in vivo, while the other is an antagonist. We investigate the interaction mechanism from a large number of unbinding trajectories by analyzing van der Waals and electrostatic interactions and by computing energy changes in proteins and solvent. In addition, dissociation potentials of mean force are calculated with the Jarzynski identity, using an averaging method developed for our steering scheme. We analyze the convergence of the Jarzynski exponential average, which is hampered by the large amount of dissipative work involved and the complexity of the system. The resulting dissociation free energies largely underestimate experimental values, but the simulations are able to clearly differentiate between wild-type and mutated TCR-pMHC and give insights into the dissociation mechanism.

Molecular basis of interaction between Na,K-ATPase and FXYD proteins.

Résumé au large public Notre corps est constitué de différents types de cellules. La condition minimale ou primordiale pour la survie des cellules est d'avoir de l'énergie. Cette tâche est assumée en partie par une protéine qui se situe dans la membrane de chaque cellule. Nommé Na, K¬ATPase ou pompe à sodium, c'est une protéine pressente dans toutes les cellules chez les mammifères est composée de deux sous-unités, α et β. En transportant 3 ions de sodium hors de la cellule et 2 ions de potassium à l'intérieur de la cellule, elle transforme l'énergie chimique sous forme de l'ATP en énergie motrice, qui permet aux cellules par la suite d'échanger des matériaux entre l'espace intracellulaire et extracellulaire ainsi que d'ingérer des nutriments provenant de son environnement. Le manque de cette protéine chez la souris entraîne la mort de l'embryon. Des défauts fonctionnels de cette protéine sont responsables de plusieurs maladies humaines comme par exemple, un type de migraine. En dehors de sa fonction vitale, cette protéine est également engagée dans diverses activités physiologiques comme la contractilité musculaire, l'activité nerveuse et la régulation du volume sanguin. Vue l'importance de cette protéine, sa découverte par Jens C. Skou en 1957 a été honorée d'un Prix Noble de chimie quarante ans plus tard. Depuis lors, nous connaissons de mieux en mieux les mécanismes de fonctionnement de la Na, K-ATPase. Entre autre, sa régulation par une famille de protéines appelées protéines FXYD. Cette famille contient 7 membres (FXYD 1-7). L'un d'entre eux nommé FXYD 2 est lié à une maladie héréditaire connue sous le nom de hypomagnesemia. Nous disposons actuellement d'informations concernant les conséquences de la régulation par les protéines FXYD sur activité de la Na, K-ATPase, mais nous savons très peu sur le mode d'interaction entre les protéines FXYD et la Na, K-ATPase. Dans ce travail de thèse, nous avons réussi à localiser des zones d'interaction dans la sous- unité a de la Na, K-ATPase et dans FXYD 7. En même temps, nous avons déterminé un 3ème site de liaison spécifique au sodium de la Na, K-ATPase. Une partie de ce site se situe à l'intérieur d'un domaine protéique qui interagit avec les protéines FXYD. De plus, ce site a été démontré comme responsable d'un mécanisme de transport de la Na, K-ATPase caractérisé par un influx ionique. En conclusion, les résultats de ce travail de thèse fournissent de nouvelles preuves sur les régions d'interaction entre la Na, K-ATPase et les protéines FXYD. La détermination d'un 3ème site spécifique au sodium et sa relation avec un influx ionique offrent la possibilité 1) d'explorer les mécanismes avec lesquels les protéines FXYD régulent l'activité de la Na, ATPase et 2) de localiser un site à sodium qui est essentielle pour mieux comprendre l'organisation et le fonctionnement de la Na, K-ATPase. Résumé Les gradients de concentration de Na+ et de K+ à travers la membrane plasmatique des cellules animales sont cruciaux pour la survie et l'homéostasie de cellules. De plus, des fonctions cellulaires spécifiques telles que la reabsorption de Na dans le rein et le côlon, la contraction musculaire et l'excitabilité nerveuse dépendent de ces gradients. La Na, K¬ATPase ou pompe à sodium est une protéine membranaire ubiquitaire. Elle crée et maintient ces gradients en utilisant l'énergie obtenu par l'hydrolyse de l'adénosine triphosphate. L'unité fonctionnelle minimale de cette protéine se compose d'une sous-unité catalytique α et d'une sous-unité régulatrice β. Récemment, il a été montré que des membres de la famille FXYD, sont des régulateurs tissu-spécifiques de la Na, K-ATPase qui influencent ses propriétés de transport. Cependant, on connaît peu de chose au sujet de la nature moléculaire de l'interaction entre les protéines FXYD et la Na, K-ATPase. Dans cette étude, nous fournissons, pour la première fois, l'évidence directe que des résidus du domaine transmembranaire (TM) 9 de la sous-unité α de la Na, K-ATPase sont impliqués dans l'interaction fonctionnelle et structurale avec les protéines FXYD. De plus nous avons identifié des régions dans le domaine transmembranaire de FXYD 7 qui sont importantes pour l'association stable avec la Na, K-ATPase et une série de résidus responsables des régulations fonctionnelles. Nous avons aussi montré les contributions fonctionnelles du TM 9 de la Na, K-ATPase à la translocation de Na + en déterminant un 3ème site spécifique au Na+. Ce site se situe probablement dans un espace entre TM 9, TM 6 et TM 5 de la sous-unité α de la pompe à sodium. De plus, nous avons constaté que le 3ème site de Na + est fonctionnellement lié à un courant entrant de la pompe sensible à l'ouabaïne et activé par le pH acide. En conclusion, ce travail donne de nouvelles perspectives de l'interaction structurale et fonctionnelle entre les protéines FXYD et la Na, K-ATPase. En outre, les contributions fonctionnelles de TM 9 offrent de nouvelles possibilités pour explorer le mécanisme par lequel les protéines FXYD régulent les propriétés fonctionnelles de la Na, K-ATPase. La détermination du 3ème site au Na + fournit une compréhension avancée du site spécifique au Na + de la Na, K-ATPase et du mécanisme de transport de la Na, K-ATPase. Summary The Na+ and K+ gradients across the plasma membrane of animal cells are crucial for cell survival and homeostasis. Moreover, specific tissue functions such as Na+ reabsorption in kidney and colon, muscle contraction and nerve excitability depend on the maintenance of these gradients. Na, K-ATPase or sodium pump, an ubiquitous membrane protein, creates and maintains these gradients by using the energy from the hydrolysis of ATP. The minimal functional unit of this protein is composed of a catalytic α subunit and a regulatory β subunit. Recently, members of the FXYD family, have been reported to be tissue-specific regulators of Na, K-ATPase by influencing its transport properties. However, little is known about the molecular nature of the interaction between FXYD proteins and Na, K-ATPase. In this study, we provide, for the first time, direct evidence that residues from the transmembrane (TM) domain 9 of the α subunit of Na, K-ATPase are implicated in the functional and structural interaction with FXYD proteins. Moreover, we have identified regions in the TM domain of FXYD 7 important for the stable association with Na, K-ATPase and a stretch of residues responsible for the functional regulations. We have further revealed the functional contributions of TM 9 of the Na, K-ATPase α subunit to the Na+ translocation by determining a 3rd Na+-specific cation binding site. This site is likely in a space between TM 9, TM 6 and TM 5 of the a subunit of the sodium pump. Moreover, we have found that the 3rd Na+ binding site is functionally linked to an acidic pH- activated ouabain-sensitive inward pump current. In conclusion, this work gives new insights into the structural and functional interaction between FXYD proteins and Na, K-ATPase. Functional contributions of TM 9 offer new possibilities to explore the mechanism by which FXYD proteins regulate functional properties of Na, K-ATPase. The determination of the 3rd Na+ binding site provides an advanced understanding concerning the Na+ -specific binding site of Na, K-ATPase and the 3rd Na+ site related transport mechanism.

Interaction of alpha-melanotropin with central dopamine systems: role of hormonal state and molecular structure

The tubero-infundibular and nigrostriatal DA neurone systems of rats respond to systemic (i.p.) injection of alpha-MSH (2-100 microgram/kg). The response of the tubero-infundibular (arcuate) DA neurones, an increase in cellular fluorescence intensity which can be interpreted as a sign of increased neuronal activity, is essentially the same in males, estrogen-progesterone-pretreated ovariectomized females and hypophysectomized males, whereas the type of response elicited by alpha-MSH in the nigral DA neurones depends upon the hormonal state of the animal. Differences between the two DA neurone groups exist also with regard to the effects of peptide fragments containing the two active sites of the alpha-MSH molecule. Results of lesion experiments in the lower brainstem (area postrema) and of blockade of muscarinic mechanisms by atropine further point to differences in the mechanisms underlying the peptide effects on the two neurone systems. The reaction of the tubero-infundibular DA system (which controls the pars intermedia of the pituitary) can be considered to reflect the activation of a feedback mechanism on MSH secretion, while the functional counterpart of the changes observed in the nigral system remains unknown at the present time.

Lack of evidence of the interaction of the Aß peptide with the Wnt signaling cascade in Drosophila models of Alzheimer's disease.

Background Alzheimer's disease (AD) is the leading form of dementia worldwide. The Aß-peptide is believed to be the major pathogenic compound of the disease. Since several years it is hypothesized that Aß impacts the Wnt signaling cascade and therefore activation of this signaling pathway is proposed to rescue the neurotoxic effect of Aß. Findings Expression of the human Aß42 in the Drosophila nervous system leads to a drastically shortened life span. We found that the action of Aß42 specifically in the glutamatergic motoneurons is responsible for the reduced survival. However, we find that the morphology of the glutamatergic larval neuromuscular junctions, which are widely used as the model for mammalian central nervous system synapses, is not affected by Aß42 expression. We furthermore demonstrate that genetic activation of the Wnt signal transduction pathway in the nervous system is not able to rescue the shortened life span or a rough eye phenotype in Drosophila. Conclusions Our data confirm that the life span is a useful readout of Aß42 induced neurotoxicity in Drosophila; the neuromuscular junction seems however not to be an appropriate model to study AD in flies. Additionally, our results challenge the hypothesis that Wnt signaling might be implicated in Aß42 toxicity and might serve as a drug target against AD.

CD28/CTLA4-B7 interaction is dispensable for T cell stimulation by mouse mammary tumor virus superantigen but not for B cell differentiation and virus dissemination.

B cells are the primary targets of infection for mouse mammary tumor virus (MMTV). However, for productive retroviral infection, T cell stimulation through the virally-encoded superantigen (SAG) is necessary. It activates B cells and leads to cell division and differentiation. To characterize the role of B cell differentiation for the MMTV life cycle, we studied the course of infection in transgenic mice deficient for CD28/CTLA4-B7 interactions (mCTLA4-H gamma 1 transgenic mice). B cell infection occurred in CTLA4-H gamma 1 transgenic mice as integrated proviral DNA could be detected in draining lymph node cells early after infection by polymerase chain reaction analysis. In mice expressing I-E, B cells were able to present the viral SAG efficiently to V beta 6+ T cells. These cells expanded specifically and were triggered to express the activation marker CD69. Further stages of progression of infection appeared to be defective. Kinetics experiments indicated that T and B cell stimulation stopped more rapidly than in control mice. B cells acquired an activated CD69+ phenotype, were induced to produce IgM but only partially switched to IgG secretion. Finally, the dissemination of infected cells to other lymph nodes and spleen was reduced and the peripheral deletion of V beta 6+ T cells was minimal. In contrast, in mice lacking I-E, T cell stimulation was also impaired and B cell activation undetectable. These data implicate B7-dependent cellular interactions for superantigenic T cell stimulation by low-affinity TCR ligands and suggest a role of B cell differentiation in viral dissemination and peripheral T cell deletion.

A molecular framework for light and gibberellin control of cell elongation.

Cell elongation during seedling development is antagonistically regulated by light and gibberellins (GAs). Light induces photomorphogenesis, leading to inhibition of hypocotyl growth, whereas GAs promote etiolated growth, characterized by increased hypocotyl elongation. The mechanism underlying this antagonistic interaction remains unclear. Here we report on the central role of the Arabidopsis thaliana nuclear transcription factor PIF4 (encoded by PHYTOCHROME INTERACTING FACTOR 4) in the positive control of genes mediating cell elongation and show that this factor is negatively regulated by the light photoreceptor phyB (ref. 4) and by DELLA proteins that have a key repressor function in GA signalling. Our results demonstrate that PIF4 is destabilized by phyB in the light and that DELLAs block PIF4 transcriptional activity by binding the DNA-recognition domain of this factor. We show that GAs abrogate such repression by promoting DELLA destabilization, and therefore cause a concomitant accumulation of free PIF4 in the nucleus. Consistent with this model, intermediate hypocotyl lengths were observed in transgenic plants over-accumulating both DELLAs and PIF4. Destabilization of this factor by phyB, together with its inactivation by DELLAs, constitutes a protein interaction framework that explains how plants integrate both light and GA signals to optimize growth and development in response to changing environments.

Structural and functional interaction sites between Na,K-ATPase and FXYD proteins.

Several members of the FXYD protein family are tissue-specific regulators of Na,K-ATPase that produce distinct effects on its apparent K(+) and Na(+) affinity. Little is known about the interaction sites between the Na,K-ATPase alpha subunit and FXYD proteins that mediate the efficient association and/or the functional effects of FXYD proteins. In this study, we have analyzed the role of the transmembrane segment TM9 of the Na,K-ATPase alpha subunit in the structural and functional interaction with FXYD2, FXYD4, and FXYD7. Mutational analysis combined with expression in Xenopus oocytes reveals that Phe(956), Glu(960), Leu(964), and Phe(967) in TM9 of the Na,K-ATPase alpha subunit represent one face interacting with the three FXYD proteins. Leu(964) and Phe(967) contribute to the efficient association of FXYD proteins with the Na,K-ATPase alpha subunit, whereas Phe(956) and Glu(960) are essential for the transmission of the functional effect of FXYD proteins on the apparent K(+) affinity of Na,K-ATPase. The relative contribution of Phe(956) and Glu(960) to the K(+) effect differs for different FXYD proteins, probably reflecting the intrinsic differences of FXYD proteins on the apparent K(+) affinity of Na,K-ATPase. In contrast to the effect on the apparent K(+) affinity, Phe(956) and Glu(960) are not involved in the effect of FXYD2 and FXYD4 on the apparent Na(+) affinity of Na,K-ATPase. The mutational analysis is in good agreement with a docking model of the Na,K-ATPase/FXYD7 complex, which also predicts the importance of Phe(956), Glu(960), Leu(964), and Phe(967) in subunit interaction. In conclusion, by using mutational analysis and modeling, we show that TM9 of the Na,K-ATPase alpha subunit exposes one face of the helix that interacts with FXYD proteins and contributes to the stable interaction with FXYD proteins, as well as mediating the effect of FXYD proteins on the apparent K(+) affinity of Na,K-ATPase.

Three-dimensional models of 14α-sterol demethylase (Cyp51A) from Aspergillus lentulus and Aspergillus fumigatus: an insight into differences in voriconazole interaction.

Aspergillus lentulus, an Aspergillus fumigatus sibling species, is increasingly reported in corticosteroid-treated patients. Its clinical significance is unknown, but the fact that A. lentulus shows reduced antifungal susceptibility, mainly to voriconazole, is of serious concern. Heterologous expression of cyp51A from A. fumigatus and A. lentulus was performed in Saccharomyces cerevisiae to assess differences in the interaction of Cyp51A with the azole drugs. The absence of endogenous ERG11 was efficiently complemented in S. cerevisiae by the expression of either Aspergillus cyp51A allele. There was a marked difference between azole minimum inhibitory concentration (MIC) values of the clones expressing each Aspergillus spp. cyp51A. Saccharomyces cerevisiae clones expressing A. lentulus alleles showed higher MICs to all of the azoles tested, supporting the hypothesis that the intrinsic azole resistance of A. lentulus could be associated with Cyp51A. Homology models of A. fumigatus and A. lentulus Cyp51A protein based on the crystal structure of Cyp51p from Mycobacterium tuberculosis in complex with fluconazole were almost identical owing to their mutual high sequence identity. Molecular dynamics (MD) was applied to both three-dimensional protein models to refine the homology modelling and to explore possible differences in the Cyp51A-voriconazole interaction. After 20ns of MD modelling, some critical differences were observed in the putative closed form adopted by the protein upon voriconazole binding. A closer study of the A. fumigatus and A. lentulus voriconazole putative binding site in Cyp51A suggested that some major differences in the protein's BC loop could differentially affect the lock-up of voriconazole, which in turn could correlate with their different azole susceptibility profiles.