Deciphering structural stability and binding mechanisms of potential antagonists with smoothened protein

Identification of new potential inhibitors against Hedgehog pathway activator protein Smoothened (SMO) is considered to be of higher importance to improvise the future cancer therapeutics. Different SMO inhibitors/drugs (e.g. Cyclopamine, Vismodegib, Taladegib) used till date are found to be associated with several drug-related resistivity and toxicity. To explore the ability of new drug/inhibitor molecules, which can show better/similar binding and dynamic stability as compared to known inhibitors, virtual screening against SMO is performed followed by the comparative docking and molecular dynamic studies. ‘ZINC12368305’ is found to be the best molecule among the entire data-set, as it shows the highest binding affinity and stable conformations. Here, an integrative approach using Dynamic Graph Theory is introduced to gain the molecular insights of the structural integrity of these protein complexes at the residue level by analyzing the corresponding Protein Contact Networks along the Molecular Dynamics trajectories. The study further focuses to understand the detailed binding mechanisms of available inhibitor/drug molecules along with the newly predicted molecule. It is observed that a unique big cluster of low fluctuating residues at the vicinity of the drug binding pocket of the SMO in ZINC12368305-bound complex is present and driving it toward a more stable region. A close inspection on this site reveals the presence of a stable Pi–Pi interaction between the pyrazole group-associated phenanthrene ring of ZINC12368305 and aromatic ring of Phe484 of SMO, which could be the potential factor of ZINC12368305 to create a more stable complex with SMO as compared to the other inhibitors.     

Growth of Arabidopsis seedlings on high fungal doses of Piriformospora indica has little effect on plant performance,stress, and defense gene expression in spite of elevated jasmonic acid and jasmonic acid-isoleucine levels in the roots

The endophytic fungus Piriformospora indica colonizes the roots of many plant species including Arabidopsis and promotes their performance, biomass, and seed production as well as resistance against biotic and abiotic stress. Imbalances in the symbiotic interaction such as uncontrolled fungal growth result in the loss of benefits for the plants and activation of defense responses against the microbe. We exposed Arabidopsis seedlings to a dense hyphal lawn of P. indica. The seedlings continue to grow, accumulate normal amounts of chlorophyll, and the photosynthetic parameters demonstrate that they perform well. In spite of high fungal doses around the roots, the fungal material inside the roots was not significantly higher when compared with roots that live in a beneficial symbiosis with P. indica. Fifteen defense- and stress-related genes including PR2, PR3, PAL2, and ERF1 are only moderately upregulated in the roots on the fungal lawn, and the seedlings did not accumulate H₂O₂/radical oxygen species. However, accumulation of anthocyanin in P. indica-exposed seedlings indicates stress symptoms. Furthermore, the jasmonic acid (JA) and jasmonic acid-isoleucine (JA-Ile) levels were increased in the roots, and consequently PDF1.2 and a newly characterized gene for a 2-oxoglurate and Fe²⁺-dependent oxygenase were upregulated more than 7-fold on the dense fungal lawn, in a JAR1- and EIN3-dependent manner. We conclude that growth of A. thaliana seedlings on high fungal doses of P. indica has little effect on the overall performance of the plants although elevated JA and JA-Ile levels in the roots induce a mild stress or defense response.     

Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in plasma cholesterol regulation through modulation of low density lipoprotein receptor (LDLR) levels. Naturally occurring mutations can lead to hyper- or hypocholesterolemia in human. Recently, we reported that PCSK9 was also able to modulate CD81 in Huh7 cells. In the present study, several gain-of-function and loss-of-function mutants as well as engineered mutants of PCSK9 were compared for their ability to modulate the cell surface expression of LDLR and CD81. Although PCSK9 gain-of-function D374Y enhanced the degradation both receptors, D374H and D129N seemed to only reduce LDLR levels. In contrast, mutations in the C-terminal hinge-cysteine-histidine-rich domain segment primarily affected the PCSK9-induced CD81 degradation. Furthermore, when C-terminally fused to an ACE2 transmembrane anchor, the secretory N-terminal catalytic or hinge-cysteine-histidine-rich domain domains of PCSK9 were able to reduce CD81 and LDLR levels. These data confirm that PCSK9 reduces CD81 levels via an intracellular pathway as reported for LDLR. Using immunocytochemistry, a proximity ligation assay, and co-immunoprecipitation, we found that the cell surface level of PCSK9 was enhanced upon overexpression of CD81 and that both PCSK9 and LDLR interact with this tetraspanin protein. Interestingly, using CHO-A7 cells lacking LDLR expression, we revealed that LDLR was not required for the degradation of CD81 by PCSK9, but its presence strengthened the PCSK9 effect.     

Protein instability during HIC: describing the effects of mobile phase conditions on instability and chromatographic retention

Hydrophobic interaction chromatography (HIC) is known to be potentially denaturing to proteins, but the effects of mobile phase conditions on chromatographic behavior are not well understood. In this study, we apply a model describing the effects of secondary protein unfolding equilibrium on chromatographic behavior, including the effects of salt concentration on both stability and adsorption. We use alpha-lactalbumin as a model protein that in the presence and absence of calcium, allows evaluation of adsorption parameters for folded and unfolded species independently. The HIC adsorption equilibrium under linear binding conditions and solution phase protein stability have been obtained from a combination of literature and new experiments. The effect of salt concentration on protein stability and the rate constant for unfolding on the chromatographic surface have been determined by fitting the model to isocratic chromatography data under marginally stable conditions. The model successfully describes the effects of added calcium and ammonium sulfate. The results demonstrate the importance of considering the effects on stability of mobile phase modifiers when applying HIC to marginally stable     

Proton-induced membrane fusion role of phospholipid composition and protein-mediated intermembrane contact

Glycolipid-phospholipid vesicles containing phosphatidate and phosphatidylethanolamine were found to undergo proton-induced fusion upon acidification of the suspending medium from pH 7.4 to pH 6.5 or lower, as determined by an assay for lipid intermixing based on fluorescence resonance energy transfer. Lectinmediated contact between the vesicles was required for fusion. Incorporation of phosphatidylcholine in the vesicles inhibited proton-induced fusion. Vesicles in which phosphatidate was replaced by phosphatidylserine underwent fusion only when pH was reduced below 4.5, while no significant fusion occured (pH ? 3.5) when the anionic phospholipid was phosphatidylinositol. It is suggested that partial protonation of the polar headgroup of phosphatidate and phosphatidylserine, respectively, causes a sufficient reduction in the polarity and hydration of the vesicle surface to trigger fusion at sites of intermembrane contact.     

Selection on QTL and complex traits in complex environments

Understanding genetic variation for complex traits in heterogeneous environments is a fundamental problem in biology. In this issue of Molecular Ecology, Fournier‐Level et al. ( 2013 ) analyse quantitative trait loci (QTL) influencing ecologically important phenotypes in mapping populations of Arabidopsis thaliana grown in four habitats across its native European range. They used causal modelling to quantify the selective consequences of life history and morphological traits and QTL on components of fitness. They found phenology QTL colocalizing with known flowering time genes as well as novel loci. Most QTL influenced fitness via life history and size traits, rather than QTL having direct effects on fitness. Comparison of phenotypes among environments found no evidence for genetic trade‐offs for phenology or growth traits, but genetic trade‐offs for fitness resulted because flowering time had opposite fitness effects in different environments. These changes in QTL effects and selective consequences may maintain genetic variation among populations.     

Computational design of protein-small molecule interfaces

The computational design of proteins that bind small molecule ligands is one of the unsolved challenges in protein engineering. It is complicated by the relatively small size of the ligand which limits the number of intermolecular interactions. Furthermore, near-perfect geometries between interacting partners are required to achieve high binding affinities. For apolar, rigid small molecules the interactions are dominated by short-range van der Waals forces. As the number of polar groups in the ligand increases, hydrogen bonds, salt bridges, cation–π, and π–π interactions gain importance. These partial covalent interactions are longer ranged, and additionally, their strength depends on the environment (e.g. solvent exposure). To assess the current state of protein-small molecule interface design, we benchmark the popular computer algorithm Rosetta on a diverse set of 43 protein–ligand complexes. On average, we achieve sequence recoveries in the binding site of 59% when the ligand is allowed limited reorientation, and 48% when the ligand is allowed full reorientation. When simulating the redesign of a protein binding site, sequence recovery among residues that contribute most to binding was 52% when slight ligand reorientation was allowed, and 27% when full ligand reorientation was allowed. As expected, sequence recovery correlates with ligand displacement.     

Developmentally regulated expression of a cell surface protein,neuropilin, in the mouse nervous system

Neuropilin (previously A5) is a cell surface glycoprotein that was originally identified in Xenopus tadpole nervous tissues. In Xenopus, neuropilin is expressed on both the presynaptic and postsynaptic elements in the visual and general somatic sensory systems, suggesting a role in neuronal cell recognition. In this study, we identified a mouse homologue of neuropilin and examined its expression in developing mouse nervous tissues. cDNA cloning and sequencing revealed that the primary structure of the mouse neuropilin was highly similar to that of Xenopus and that the extracellular segment of the molecule possessed several motifs that were expected to be involved in cell-cell interaction. Immunohistochemistry and in situ hybridization analyses in mice indicated that the expression of neuropilin was restricted to particular neuron circuits. Neuropilin protein was localized on axons but not on the somata of neurons. The expression of neuropilin persisted through the time when axons were actively growing to form neuronal connections. These observations suggest that neuropilin is involved in growth, fasciculation, and targeting for a particular groups of axons. © 1996 John Wiley & Sons, Inc.     

Limited chloroplast gene transfer via recombination overcomes plastomegenome incompatibility between Nicotiana tabacum and Solanum tuberosum

Green cybrids with a new nucleus-chloroplast combination cannot be selected after protoplast fusion in the intersubfamilial Nicotiana-Solanum combination. As an approach to overcome the supposed plastomegenome incompatibility, a partial plastome transfer by genetic recombination has been considered. After fusions of protoplasts of a light-sensitive Nicotiana tabacum (tobacco) plastome mutant and lethally irradiated protoplasts of wild-type Solanum tuberosum (potato), a single green colony was recovered among 2.5×10⁴ colonies. The regenerated plants had tobacco-like (although abnormal) morphology, but were normally green, and sensitive to tentoxin, demonstrating chloroplast markers of the potato parent. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed recombinant, nonparental patterns. A comparison with physical maps of the parental cpDNA demonstrated the presence of a considerable part of the potato plastome flanked by tobacco-specific regions. This potacco plastome proved to be stable in backcross and backfusion experiments, and normally functional in the presence solely of N. tabacum nucleus.