Sequence patterns associated with disordered regions in proteins

The relationship between amino acid sequence and intrinsic disorder in proteins is investigated. Two databases, one of disordered proteins and the other of globular proteins, are analyzed and compared in order to extract simple sequence patterns of a few amino acids or amino acid properties that characterize disordered segments. It is found that a number of reliable, nonrandom associations exists. In particular, two types of patterns appear to be recurrent: a proline-rich pattern and a (positively or negatively) charged pattern. These results indicate that local sequence information can determine disordered regions in proteins. The derived patterns provide some insights into the physical reasons for disordered structures. They should also be helpful in improving currently available prediction methods.     

Structures of vertebrate hyaluronidases and their unique enzymatic mechanism of hydrolysis

Human hyaluronidases (Hyals) are a group of five endo-beta-acetyl-hexosaminidase enzymes, Hyal-1, -2, -3, -4, and PH-20, which degrade hyaluronan using a hydrolytic mechanism of action. Catalysis by these Hyals has been shown to follow a double-displacement scheme. This involves a single Glu residue within the enzyme, the only catalytic residue, as the proton donor (acid). Also involved is a carbonyl group of the hyaluronan (HA) N-acetyl-D-glucosamine as a unique type of nucleophile. Thus the substrate participates in the mechanism of action of its own catalysis. An oxocarbonium ion transition state is postulated, but there is no formation of a covalent enzyme-glycan intermediate, as found in most such reactions. The major domain is catalytic and has a distorted (beta/alpha)8 triose phosphate isomerase (TIM) barrel fold. The C-terminal domain is separated by a peptide linker. Each Hyal has a different C-terminal sequence and structure, the function of which is unknown. These unique C-termini may participate in the additional function(s) associated with these multifunctional enzymes.     

Anchor profiles of HLA-specific peptides: analysis by a novel affinity scoring method and experimental validation

The study of intermolecular interactions is a fundamental research subject in biology. Here we report on the development of a quantitative structure-based affinity scoring method for peptide-protein complexes, named PepScope. The method operates on the basis of a highly specific force field function (CHARMM) that is applied to all-atom structural representations of peptide-receptor complexes. Peptide side-chain contributions to total affinity are scored after detailed rotameric sampling followed by controlled energy refinement. A de novo approach to estimate dehydration energies was developed, based on the simulation of individual amino acids in a solvent box filled with explicit water molecules. Transferability of the method was demonstrated by its application to the hydrophobic HLA-A2 and -A24 receptors, the polar HLA-A1, and the sterically ruled HLA-B7 receptor. A combined theoretical and experimental study on 39 anchor substitutions in FxSKQYMTx/HLA-A2 and -A24 complexes indicated a prediction accuracy of about two thirds of a log-unit in Kd. Analysis of free energy contributions identified a great role of desolvation and conformational strain effects in establishing a given specificity profile. Interestingly, the method rightly predicted that most anchor profiles are less specific than so far assumed. This suggests that many potential T-cell epitopes could be missed with current prediction methods. The results presented in this work may therefore significantly affect T-cell epitope discovery programs applied in the field of peptide vaccine development.     

A pharmacophore-based evolutionary approach for screening selective estrogen receptor modulators

We developed a pharmacophore-based evolutionary approach for virtual screening. This tool, termed the Generic Evolutionary Method for molecular DOCKing (GEMDOCK), combines an evolutionary approach with a new pharmacophore-based scoring function. The former integrates discrete and continuous global search strategies with local search strategies to expedite convergence. The latter, integrating an empirical-based energy function and pharmacological preferences (binding-site pharmacological interactions and ligand preferences), simultaneously serves as the scoring function for both molecular docking and postdocking analyses to improve screening accuracy. We apply pharmacological interaction preferences to select the ligands that form pharmacological interactions with target proteins, and use the ligand preferences to eliminate the ligands that violate the electrostatic or hydrophilic constraints. We assessed the accuracy of our approach using human estrogen receptor (ER) and a ligand database from the comparative studies of Bissantz et al. (J Med Chem 2000;43:4759-4767). Using GEMDOCK, the average goodness-of-hit (GH) score was 0.83 and the average false-positive rate was 0.13% for ER antagonists, and the average GH score was 0.48 and the average false-positive rate was 0.75% for ER agonists. The performance of GEMDOCK was superior to competing methods such as GOLD and DOCK. We found that our pharmacophore-based scoring function indeed was able to reduce the number of false positives; moreover, the resulting pharmacological interactions at the binding site, as well as ligand preferences, were important to the screening accuracy of our experiments. These results suggest that GEMDOCK constitutes a robust tool for virtual database screening.     

Structural characterization of proteins using residue environments

A primary challenge for structural genomics is the automated functional characterization of protein structures. We have developed a sequence-independent method called S-BLEST (Structure-Based Local Environment Search Tool) for the annotation of previously uncharacterized protein structures. S-BLEST encodes the local environment of an amino acid as a vector of structural property values. It has been applied to all amino acids in a nonredundant database of protein structures to generate a searchable structural resource. Given a query amino acid from an experimentally determined or modeled structure, S-BLEST quickly identifies similar amino acid environments using a K-nearest neighbor search. In addition, the method gives an estimation of the statistical significance of each result. We validated S-BLEST on X-ray crystal structures from the ASTRAL 40 nonredundant dataset. We then applied it to 86 crystallographically determined proteins in the protein data bank (PDB) with unknown function and with no significant sequence neighbors in the PDB. S-BLEST was able to associate 20 proteins with at least one local structural neighbor and identify the amino acid environments that are most similar between those neighbors.     

Surprising Effects of Synaptic Excitation

Typically, excitatory synaptic coupling is thought of as an influence that accelerates and propagates firing in neuronal networks. This paper reviews recent results explaining how, contrary to these expectations, the presence of excitatory synaptic coupling can drastically slow oscillations in a network and how localized, sustained activity can arise in a network with purely excitatory coupling, without sustained inputs. These two effects stem from interactions of excitatory coupling with two different forms of intrinsic neuronal dynamics, and both serve to highlight the fact that the influence of synaptic coupling in a network depends strongly on the intrinsic properties of cells in the network.This work was partially supported by the National Science Foundation, under award DMS-0414023     

HIV protease inhibitor ritonavir increases endothelial monolayer permeability

HIV protease inhibitors (PIs) are often associated with metabolic and cardiovascular complications although they are effective anti-HIV drugs. In this study, we determined whether HIV PI ritonavir could increase endothelial permeability, one of the important mechanisms of vascular lesion formation. Human dermal microvascular endothelial cells (HMECs) treated with ritonavir showed a significant increase of endothelial permeability in a dose- and time-dependent manner assayed with a transwell system. Ritonavir significantly reduced the mRNA levels of tight junction proteins zonula occluden-1, occludin, and claudin-1 by 40-60% as compared to controls (P<0.05) by real-time PCR analysis. Protein levels of these tight junction molecules were also substantially reduced in the ritonavir-treated cells. In addition, HMECs treated with ritonavir (7.5, 15, and 30microM) showed a substantial increase of superoxide anion production by 10%, 32%, and 65%, respectively, as compared to controls. Antioxidants (EGCG and SeMet) effectively reduced ritonavir-induced endothelial permeability. Furthermore, ritonavir activated ERK1/2 (phosphorylation), but not P38 and JNK. Specific ERK1/2 inhibitor, PD89059, significantly abolished ritonavir-induced endothelial permeability by 92%. Thus, HIV PI ritonavir increases endothelial permeability, decreases levels of tight junction proteins, and increases superoxide anion production. ERK1/2 activation is involved in the signal transduction pathway of ritonavir-induced endothelial permeability.     

Detection of nanometer-sized particles in living cells using modern fluorescence fluctuation methods

Nanosized materials are increasingly used in medicine and biotechnology but originate also from various aerosol sources. A detailed understanding of their interaction with cells is a prerequisite for specific applications and appraisal of hazardous effects. Fluorescence fluctuation methods are applied to follow the time-course of the translocation and distribution of fluorescent 20 nm polystyrene nanoparticles with negative surface charges in HeLa cells under almost physiological conditions. The experimental results demonstrate that singular particles enter the cell without significant contribution by endocytotic mechanisms and are distributed within the cytoplasm. Subsequently aggregation is observed, which can be blocked by cytotoxins, like Genistein and Cytochalasin B, interfering with cellular uptake processes. The observed non-active uptake is due to non-specific interactions with the cell surface and could be responsible for distribution of nanometer-sized materials in tissue.     

Differences in sensory projections between macro- and microchaetes in Drosophilid flies

From examination of the central axonal projections of sensory bristles on the notum of several species of Drosophilidae, we demonstrate different features that may indicate different functions for macro- and microchaetes. The large macrochaetes have conserved arborizations that correlate with their conserved position. Nevertheless, we find evidence for only two discrete projection patterns for bristles in the dorsocentral (DC) row, even when there may be four or five bristles present. We show that the small microchaetes of Drosophila melanogaster display regional specificity and subsets of contiguous bristles project to a common region in the thoracic ganglion. Interestingly, the axons of each of these subsets also form a specific fasciculation group on the scutum before joining the axon of a particular macrochaete. The positions of microchaetes on the scutum and the shape of the fasciculation groups vary between closely related species. There is no correlation between body size, bristle patterns, and fasciculation patterns. Furthermore, none of these traits correlate with the phylogenetic relationships between the species studied. We discuss the possibility that macro- and microchaetes may have different functions and that these have implications for evolutionary constraints on bristle patterns.     

Advertisement-call preferences in diploid-tetraploid treefrogs (Hyla chrysoscelis and Hyla versicolor): implications for mate choice and the evolution of communication systems

Signals used for mate choice and receiver preferences are often assumed to coevolve in a lock-step fashion. However, sender-receiver coevolution can also be nonparallel: even if species differences in signals are mainly quantitative, females of some closely related species have qualitatively different preferences and underlying mechanisms. Two-alternative playback experiments using synthetic calls that differed in fine-scale temporal properties identified the receiver criteria in females of the treefrog Hyla chrysoscelis for comparison with female criteria in a cryptic tetraploid species (H. versicolor); detailed preference functions were also generated for both species based on natural patterns of variation in temporal properties. The species were similar in three respects: (1) pulses of constant frequency were as attractive as the frequency-modulated pulses typical of conspecific calls; (2) changes in preferences with temperature paralleled temperature-dependent changes in male calls; and (3) preference functions were unimodal, with weakly defined peaks estimated at values slightly higher than the estimated means in conspecific calls. There were also species differences: (1) preference function slopes were steeper in H. chrysoscelis than in H. versicolor; (2) preferences were more intensity independent in H. chrysoscelis than in H. versicolor; (3) a synergistic effect of differences in pulse rate and shape on preference strength occurred in H. versicolor but not in H. chrysoscelis; and (4) a preference for the pulse shape typical of conspecific calls was expressed at the species-typical pulse duration in H. versicolor but not in H. chrysoscelis. However, females of H. chrysoscelis did express a preference based on pulse shape when tested with longer-than-average pulses, suggesting a hypothesis that could account for some examples of nonparallel coevolution. Namely, preferences can be hidden or revealed depending on the direction of quantitative change in a signal property relative to the threshold for resolving differences in that property. The results of the experiments reported here also predict patterns of mate choice within and between contemporary populations. First, intraspecific mate choice in both species is expected to be strongly influenced by variation in temperature among calling males. Second, simultaneous differences in pulse rate and pulse shape are required for effective species discrimination by females of H. versicolor but not by females of H. chrysoscelis. Third, there is greater potential for sexual selection within populations and for discrimination against calls produced by males in other geographically remote populations in H. chrysoscelis than in H. versicolor.