Prediction of indirect interactions in proteins

Background  

Both direct and indirect interactions determine molecular recognition of ligands by proteins. Indirect interactions can be defined as effects on recognition controlled from distant sites in the proteins, e.g. by changes in protein conformation and mobility, whereas direct interactions occur in close proximity of the protein's amino acids and the ligand. Molecular recognition is traditionally studied using three-dimensional methods, but with such techniques it is difficult to predict the effects caused by mutational changes of amino acids located far away from the ligand-binding site. We recently developed an approach, proteochemometrics, to the study of molecular recognition that models the chemical effects involved in the recognition of ligands by proteins using statistical sampling and mathematical modelling.     

Kinetics of proton release and uptake by channelrhodopsin-2

Electrophysiological experiments showed that the light-activated cation channel channelrhodopsin-2 (ChR2) pumps protons in the absence of a membrane potential. We determined here the kinetics of transient pH change using a water-soluble pH-indicator. It is shown that ChR2 released protons prior to uptake with a stoichiometry of 0.3 protons per ChR2. Comparison to the photocycle kinetics revealed that proton release and uptake match rise and decay of the P(3)(520) intermediate. As the P(3)(520) state also represents the conductive state of cation channeling, the concurrence of proton pumping and channel gating implies an intimate mechanistic link of the two functional modes. Studies on the E123T and S245E mutants show that these residues are not critically involved in proton translocation.     

Crystal structure of p44, a constitutively active splice variant of visual arrestin

Visual arrestin specifically binds to photoactivated and phosphorylated rhodopsin and inactivates phototransduction. In contrast, the p44 splice variant can terminate phototransduction by binding to nonphosphorylated light-activated rhodopsin. Here we report the crystal structure of bovine p44 at a resolution of 1.85 Å. Compared to native arrestin, the p44 structure reveals significant differences in regions crucial for receptor binding, namely flexible loop V–VI and polar core regions. Additionally, electrostatic potential is remarkably positive on the N-domain and the C-domain. The p44 structure represents an active conformation that serves as a model to explain the ‘constitutive activity’ found in arrestin variants.     

Synthesis and structure-activity relationships of 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-ones as novel series of potent β isoform selective phosphatidylinositol 3-kinase inhibitors

A series of 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-ones with excellent enzyme inhibition, improved isoform selectivity, and excellent inhibition of downstream phosphorylation of AKT has been identified. Several compounds in the series demonstrated potent (～ 0.100 μM IC(50)) growth inhibition in a PTEN deficient cancer cell line.     

Dendritic cell-mediated vaccination relies on interleukin-4 receptor signaling to avoid tissue damage after Leishmania major infection of BALB/c mice

Prevention of tissue damages at the site of Leishmania major inoculation can be achieved if the BALB/c mice are systemically given L. major antigen (LmAg)-loaded bone marrow-derived dendritic cells (DC) that had been exposed to CpG-containing oligodeoxynucleotides (CpG ODN). As previous studies allowed establishing that interleukin-4 (IL-4) is involved in the redirection of the immune response towards a type 1 profile, we were interested in further exploring the role of IL-4. Thus, wild-type (wt) BALB/c mice or DC-specific IL-4 receptor alpha (IL-4Rα)-deficient (CD11c^creIL-4Rα^−/lox) BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded bone marrow-derived DC exposed or not to CpG ODN prior to inoculation of 2×10⁵ stationary-phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4Rα-mediated signaling in the vaccinating DC is required to prevent tissue damage at the site of L. major inoculation, as properly conditioned wt DC but not IL-4Rα-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining lymph nodes of CD11c^creIL-4Rα^−/lox mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4Rα-mediated signaling in host DC to control parasite replication. In addition, no footpad damage occurred in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. We discuss these findings and suggest that the IL4/IL4Rα signaling pathway could be a key pathway to trigger when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.     

Evaluation of novel assays to assess the influence of different iron sources on the growth of Clostridium difficile

The ability of four Clostridium difficile strains to utilize various exogenous organic and inorganic iron sources for growth under iron-depleted (250 μM DPP) and iron-limited (75 μM DPP) conditions was analyzed in liquid broth cultures grown in tubes and in microtiter plates, and data compared with results from a bioassay developed on solid media. The growth profile of C. difficile varied depending on the iron source and availability. Addition of FeSO(4), FeCl(3), Fe citrate and ferritin allowed growth in an iron-depleted environment whereas glycoproteins (iron-saturated and low-iron lactoferrin, apo- and holo-transferrin) and heme proteins (hemoglobin, hematin and hemin) did not. All iron sources, except lactoferrin, were able to restore bacterial growth under iron-limited conditions to varying extents. The results demonstrated that the broth microtiter assay developed here was reproducible, reliable and convenient for high-throughput analysis of the growth of C. difficile compared to alternative traditional methods.     

Leaf essential oil composition of three species of Myrcianthes from Monteverde, Costa Rica

To examine the chemical diversity of essential oils from Myrcianthes species (Myrtaceae) as well as potential chemotaxonomic relationships between them, the leaf essential oils of M. fragrans (Sw.) McVaugh, M. rhopaloides (Kunth) McVaugh, and an undescribed species, Myrcianthes 'black fruit', from Monteverde, Costa Rica, were isolated by hydrodistillation and analyzed by GC/MS. The most abundant components of the essential oil of M. fragrans were 1,3,5-trimethoxybenzene (15.7%), (Z)-hex-3-en-1-ol (10.0%), alpha-cadinol (10.4%), eudesma-4(15),7-dien-1beta-ol (9.0%), caryophyllene oxide (7.8%), and spathulenol (7.5%). The leaf oils of two different samples of Myrcianthes rhopaloides were quantitatively different with one sample composed mostly of linalool (17.7%), alpha-cadinol (14.4%), spathulenol (11.1%), tau-cadinol (9.6%), and 1-epicubenol (6.9%), and the other was made up largely of (E)-hex-2-enal (46.1%), 1,8-cineole (12.5%), linalool (9.1%), alpha-cadinol (6.7%), and alpha-terpineol (4.4%). The major components in the leaf essential oil of Myrcianthes 'black fruit' were 1,8-cineole (38.3%), alpha-terpineol (21.2%), heptan-2-ol (15.5%), terpinen-4-ol (4.2%), and beta-pinene (3.8%). The leaf oil compositions of Myrcianthes in this study are very different from leaf oils from other members of Myrcianthes reported in the literature. A cluster analysis reveals large chemical variation not only between members of the genus, but also between samples of the same species.