Spontaneous formation of detergent micelles around the outer membrane protein OmpX

The structure and flexibility of the outer membrane protein X (OmpX) in a water-detergent solution and in pure water are investigated by molecular dynamics simulations on the 100-ns timescale and compared with NMR data. The simulations allow for an unbiased determination of the structure of detergent micelles and the protein-detergent mixed micelle. The short-chain lipid dihexanoylphosphatidylcholine, as a detergent, aggregates into pure micelles of approximately 18 molecules, or alternatively, it binds to the protein surface. The detergent binds in the form of a monolayer ring around the hydrophobic beta-barrel of OmpX rather than in a micellar-like oblate; approximately 40 dihexanoylphosphatidylcholine lipids are sufficient for an effective suppression of water from the surface of the beta-barrel region. The phospholipids bind also on the extracellular, protruding beta-sheet. Here, polar interactions between charged amino acids and phosphatidylcholine headgroups act as condensation seed for detergent micelle formation. The polar protein surface remains accessible to water molecules. In total, approximately 90-100 detergent molecules associate within the protein-detergent mixed micelle, in agreement with experimental estimates. The simulation results indicate that OmpX is not a water pore and support the proposed role of the protruding beta-sheet as a "fishing rod".     

Inhibition of interdomain motion in g-actin by the natural product latrunculin: a molecular dynamics study

As part of the cytoskeleton, actin is essential for the morphology, motility, and division of eukaryotic cells. Recent X-ray fiber diffraction studies have shown that the conformation of monomeric actin is flattened upon incorporation into the filament by a relative rotation of its two major domains. The antiproliferative activity of latrunculin, a macrolide toxin produced by sponges, seems to be related to its binding to monomeric actin and inhibition of polymerization. Yet, the mechanism of inhibition is not known in detail. Here, multiple explicit water molecular dynamics simulations show that latrunculin binding hinders the conformational transition related to actin polymerization. In particular, the presence of latrunculin at the interface of the two major domains of monomeric actin reduces the correlated displacement of Domain 2 with respect to Domain 1. Moreover, higher rotational flexibility between the two major domains is observed in the absence of ATP as compared to ATP-bound actin, offering a possible explanation as to why actin polymerizes more favorably in the absence of nucleotides.     

Issues in using whole slide imaging for diagnostic pathology: “routine” stains,immunohistochemistry and predictive markers

The traditional microscope, together with the “routine” hematoxylin and eosin (H & E) stain, remains the “gold standard” for diagnosis of cancer and other diseases; remarkably, it and the majority of associated biological stains are more than 150 years old. Immunohistochemistry has added to the repertoire of “stains” available. Because of the need for specific identification and even measurement of “biomarkers,” immunohistochemistry has increased the demand for consistency of performance and interpretation of staining results. Rapid advances in the capabilities of digital imaging hardware and software now offer a realistic route to improved reproducibility, accuracy and quantification by utilizing whole slide digital images for diagnosis, education and research. There also are potential efficiencies in work flow and the promise of powerful new analytical methods; however, there also are challenges with respect to validation of the quality and fidelity of digital images, including the standard H & E stain, so that diagnostic performance by pathologists is not compromised when they rely on whole slide images instead of traditional stained tissues on glass slides.     

A double-headed cathepsin B inhibitor devoid of warhead

Most synthetic inhibitors of peptidases have been targeted to the active site for inhibiting catalysis through reversible competition with the substrate or by covalent modification of catalytic groups. Cathepsin B is unique among the cysteine peptidase for the presence of a flexible segment, known as the occluding loop, which can block the primed subsites of the substrate binding cleft. With the occluding loop in the open conformation cathepsin B acts as an endopeptidase, and it acts as an exopeptidase when the loop is closed. We have targeted the occluding loop of human cathepsin B at its surface, outside the catalytic center, using a high-throughput docking procedure. The aim was to identify inhibitors that would interact with the occluding loop thereby modulating enzyme activity without the help of chemical warheads against catalytic residues. From a large library of compounds, the in silico approach identified [2-[2-(2,4-dioxo-1,3-thiazolidin-3-yl)ethylamino]-2-oxoethyl] 2-(furan-2-carbonylamino) acetate, which fulfills the working hypothesis. This molecule possesses two distinct binding moieties and behaves as a reversible, double-headed competitive inhibitor of cathepsin B by excluding synthetic and protein substrates from the active center. The kinetic mechanism of inhibition suggests that the occluding loop is stabilized in its closed conformation, mainly by hydrogen bonds with the inhibitor, thus decreasing endoproteolytic activity of the enzyme. Furthermore, the dioxothiazolidine head of the compound sterically hinders binding of the C-terminal residue of substrates resulting in inhibition of the exopeptidase activity of cathepsin B in a physiopathologically relevant pH range.     

Activation of the West Nile virus NS3 protease: Molecular dynamics evidence for a conformational selection mechanism

The flaviviral nonstructural 3 protease (NS3pro) is essential for virus replication and is therefore a pharmaceutically relevant target to fight Dengue and West Nile virus (WNV). NS3pro is a chymotrypsin‐like serine protease which requires a polypeptide cofactor (NS2B) for activation. Recent X‐ray crystallography studies have led to the suggestion that the substrate binds to the two‐component NS2B‐NS3pro enzyme by an induced‐fit mechanism. Here, multiple explicit water molecular dynamics simulations of the WNV NS2B‐NS3pro enzyme show that the active conformation of the NS2B cofactor (in which its β‐loop is part of the substrate binding site) is stable over a 50‐ns time scale even in the absence of the inhibitor. The partial and reversible opening of the NSB2 β‐loop and its correlated motion with an adjacent NS3pro loop, both observed in the simulations started from the active conformation, are likely to facilitate substrate binding and product release. Moreover, in five of eight simulations without inhibitor (started from two X‐ray structures both with improperly formed oxyanion hole) the Thr132‐Gly133 peptide bond flips spontaneously thereby promoting the formation of the catalytically competent oxyanion hole, which then stays stable until the end of the runs. The simulation results provide evidence at atomic level of detail that the substrate binds to the NS2B‐NS3pro enzyme by conformational selection, rather than induced‐fit mechanism.     

Gene structure,transcripts and calciotropic effects of the PTH family of peptides in <Emphasis Type="Italic">Xenopus</Emphasis> and chicken

Background  

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken.     

Morphological and molecular characterization of populations of plains bristlegrass (<i>Setaria macrostachya</i> Kunth) in Chihuahua,México

Plains bristlegrass (Setaria macrostachya Kunth) is a native grass with forage value. However, due to the lack of grazing management practices, populations and thus genetic diversity, have been reduced. Morphological and genetic variability were analyzed on 44 populations of plains bristlegrass in the State of Chihuahua. Plants were transplanted in a common area under natural conditions. Two years later, morphological characterization was evaluated measuring nine variables, and genetic variability using AFLP molecular markers. The principal components analysis (PC) showed that the three first principal components explained 73.74% of the variation. The variables with the greatest contribution to the variance in PC1 were plant height and inflorescence length; in CP2, tiller number and leaf width; and in PC3, tiller thickness. Application of four pairs of primers, presented 186 total bands, from which 87.10% showed polymorphism and 12.90% monomorphism. The combination of EcoRI-AGG MseI-CAG primers detected the highest percentage (93%) of polymorphism with 40 polymorphic bands. The cluster analysis and Dice coefficient indicated that populations clump into two groups. The wide genetic variability and morphological characteristics detected among populations represent the basis for the selection of populations that could be used with different purposes in the rehabilitation of ecosystems. In addition, this study will allow establishment of in situ conservation strategies.     

Disordered binding of small molecules to Aβ(12-28)

In recent years, an increasing number of small molecules and short peptides have been identified that interfere with aggregation and/or oligomerization of the Alzheimer β-amyloid peptide (Aβ). Many of them possess aromatic moieties, suggesting a dominant role for those in interacting with Aβ along various stages of the aggregation process. In this study, we attempt to elucidate whether interactions of such aromatic inhibitors with monomeric Aβ(12-28) point to a common mechanism of action by performing atomistic molecular dynamics simulations at equilibrium. Our results suggest that, independently of the presence of inhibitors, monomeric Aβ(12-28) populates a partially collapsed ensemble that is largely devoid of canonical secondary structure at 300 K and neutral pH. The small molecules have different affinities for Aβ(12-28) that can be partially rationalized by the balance of aromatic and charged moieties constituting the molecules. There are no predominant binding modes, although aggregation inhibitors preferentially interact with the N-terminal portion of the fragment (residues 13-20). Analysis of the free energy landscape of Aβ(12-28) reveals differences highlighted by altered populations of a looplike conformer in the presence of inhibitors. We conclude that intrinsic disorder of Aβ persists at the level of binding small molecules and that inhibitors can significantly alter properties of monomeric Aβ via multiple routes of differing specificity.     

Structure-based tailoring of compound libraries for high-throughput screening: discovery of novel EphB4 kinase inhibitors

High-throughput docking is a computational tool frequently used to discover small-molecule inhibitors of enzymes or receptors of known three-dimensional structure. Because of the large number of molecules in chemical libraries, automatic procedures to prune multimillion compound collections are useful for high-throughput docking and necessary for in vitro screening. Here, we propose an anchor-based library tailoring approach (termed ALTA) to focus a chemical library by docking and prioritizing molecular fragments according to their binding energy which includes continuum electrostatics solvation. In principle, ALTA does not require prior knowledge of known inhibitors, but receptor-based pharmacophore information (hydrogen bonds with the hinge region) is additionally used here to identify molecules with optimal anchor fragments for the ATP-binding site of the EphB4 receptor tyrosine kinase. The 21,418 molecules of the focused library (from an initial collection of about 730,000) are docked into EphB4 and ranked by force-field-based energy including electrostatic solvation. Among the 43 compounds tested in vitro, eight molecules originating from two different anchors show low-micromolar activity in a fluorescence-based enzymatic assay. Four of them are active in a cell-based assay and are potential anti-angiogenic compounds.