Weighted geometric docking: incorporating external information in the rotation-translation scan

Weighted geometric docking is a prediction algorithm that matches weighted molecular surfaces. Each molecule is represented by a grid of complex numbers, storing information about the shape of the molecule in the real part and weight information in the imaginary part. The weights are based on experimental biochemical and biophysical data or on theoretical analyses of amino acid conservation or correlation patterns in multiple-sequence alignments of homologous proteins. Only a few surface residues on either one or both molecules are weighted. In contrast to methods that use postscan filtering based on biochemical information, our method incorporates the external data in the rotation-translation search, producing a different set of docking solutions biased toward solutions in which the up-weighted residues are at the interface. Similarly, interactions involving specified residues can be impeded. The weighted geometric algorithm was applied to five systems for which regular geometric docking of the unbound molecules gave poor results. We obtained much better ranking of the nearly correct prediction and higher statistical significance when weighted geometric docking was used. The method was successful even when the weighted portion of the surface corresponded only partially and approximately to the binding site.     

Seasonal differences in energy expenditure,flight characteristics and spatial utilization of Dalmatian Pelicans Pelecanus crispus in Greece

Animals typically adjust their behaviour to their changing environment throughout the annual cycle, modulating key processes such as the timing of breeding and the onset of migration. Such behavioural changes are commonly manifested in the movements and the energetic balance of individuals in relation to their species‐specific physiological characteristics, habitat attributes and the environmental properties of their distribution ranges. We used GPS and acceleration data collected using transmitters on free‐ranging birds to quantify annual movement patterns and estimate energy expenditure of the Dalmatian Pelican Pelecanus crispus, a large, soaring avian species which performs short‐distance migration and spends its entire annual cycle in mid‐latitudes. To assess the representativeness of our results, the transmitter effect was also tested. We found that daily trends in the overall dynamic body acceleration (ODBA; a proxy for energy expenditure) differed among seasons, with the highest values occurring during spring and the lowest during winter. Long inter‐lake flights were very rare in winter, and the number of flights and ODBA during spring was higher than during summer, suggesting greater motivation to move in spring. Although transmitters may have affected the birds, as none of the tagged birds bred, we found seasonal differences in behaviour and activity level. The observed patterns in differences in activity levels, long‐distance flights and flight characteristics between seasons suggest an annual rhythm of energy expenditure. These findings allow a better understanding of bird phenology, specifically regarding adaptations to wintering in a cold climate by reducing movement‐driven energy expenditure. Finally, the identification of periods with high and low energy expenditure may guide future conservation efforts by adjusting conservation plans in accordance with changing needs during the annual cycle.     

Purification and Identification of a Novel Leucine Aminopeptidase from <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>

A novel leucine aminopeptidase was purified from a Bacillus thuringiensis israelensis (Bti) culture. The purification stages included heating the concentrated supernatant to 65°C for 90 min, anion-exchange chromatography by DEAE cellulose, and hydrophobic chromatography by phenyl Sepharose. The specific activity of leucine aminopeptidase after the hydrophobic chromatography increased by 215.5-fold and the yield was 16%. The molecular weight of the active enzyme was 59 kDa. Mass spectrometry analysis of the 59-kDa leucine aminopeptidase revealed that this protein has at least 41% homology with the cytosol leucine aminopeptidase produced by Bacillus cereus. Maximal leucine aminopeptidase activity occurred at 65°C, pH 10 toward leucine as the amino acid terminus. The enzyme was strongly inhibited by bestatin, dithiothreitol, and 1,10-phenanthroline, indicating that the enzyme might be considered as a metallo-aminopeptidase that has disulfide bonds at the catalytic site or at a region that influences its configuration. Examination of the purified leucine aminopeptidase’s effect on the activation of the protoxin Cyt1Aa from Bti revealed that when it acts synergistically with Bti endogenous proteases, it has only a minor role in the processing of Cyt1Aa into an active toxin.     

Differential regulation of MMPs and matrix assembly in chicken and turkey growth-plate chondrocytes

Matrix metalloproteinases (MMPs) play a crucial role in growth-plate vascularization and ossification by processes involving proteolytic cleavage and remodeling of the extracellular matrix (ECM). Their regulation in the growth plate is crucial for normal vs. impaired matrix assembly. Tibial dyschondroplasia (TD), a prevalent skeletal abnormality in avian species, is characterized by the formation of a nonvascularized, nonmineralized plaque in the growth plate. Here, we show differential regulation of MMPs in cultured chondrocytes from chickens and turkeys; retinoic acid (RA) elevated MMP-2 activity in both species, but only in chicken did it induce MMP-9 activity. In contrast, phorbol 12-myristate 13-acetate (PMA) treatment induced MMP-9 activity in turkey chondrocytes but not in those of chicken. Moreover, we found different developmental patterns of TD in chickens and turkeys in-vivo as lower concentrations of, and shorter exposure to thiram were required in chicken than in turkey for TD induction. Growth-plate cartilage taken from thiram-induced lesions had lower gelatinolytic and caseinolytic activities compared with normal cartilage. Likewise, thiram reduced MMP-2 and MMP-13 activity in both chicken and turkey chondrocytes in vitro, although 10-fold higher concentrations were required for this effect in the latter. Finally, the combined treatments of RA or PMA with thiram induced MMP-9 activity in turkey but not in chicken chondrocytes. Furthermore, RA combined with thiram synergistically upregulated its activity in turkey but not chicken chondrocytes. Taken together, these results suggest that mechanisms of MMP regulation differ in the growth plates of these closely related avian species, resulting in altered matrix assembly as exemplified by TD development.     

Src-mediated caveolin-1 phosphorylation affects the targeting of active Src to specific membrane sites

Src interactions with the plasma membrane are an important determinant of its activity. In turn, Src activity modulates its association with the membrane through binding of activated Src to phosphotyrosylated proteins. Caveolin-1 (Cav-1), a major component of caveolae, is a known Src phosphorylation target, and both were reported to regulate cell transformation. However, the nature of Src-Cav-1 interactions, a potential mechanism of their coregulation, remained unclear. Here we used fluorescence recovery after photobleaching beam-size analysis, coimmunoprecipitation, quantitative imaging, and far-Western studies with cells expressing wild type, as well as structural and activity mutants of Src–green fluorescent protein and Cav-1–monomeric red fluorescent protein, to measure their interactions with the membrane and with each other. We show dynamic Src–plasma membrane interactions, which are augmented and stabilized by Cav-1. The mechanism involves phosphorylation of Cav-1 at Tyr-14 by Src and subsequent binding of the Src SH2 domain to phospho–Cav-1, leading to accumulation of activated Src in focal adhesions. This novel Cav-1 function potentially modulates focal adhesion dynamics.     

Urokinase-type Plasminogen Activator Receptor (uPAR) Ligation Induces a Raft-localized Integrin Signaling Switch That Mediates the Hypermotile Phenotype of Fibrotic Fibroblasts

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.     

Water-Transfer Slows Aging in Saccharomyces cerevisiae

Transferring Saccharomyces cerevisiae cells to water is known to extend their lifespan. However, it is unclear whether this lifespan extension is due to slowing the aging process or merely keeping old yeast alive. Here we show that in water-transferred yeast, the toxicity of polyQ proteins is decreased and the aging biomarker 47Q aggregates at a reduced rate and to a lesser extent. These beneficial effects of water-transfer could not be reproduced by diluting the growth medium and depended on de novo protein synthesis and proteasomes levels. Interestingly, we found that upon water-transfer 27 proteins are downregulated, 4 proteins are upregulated and 81 proteins change their intracellular localization, hinting at an active genetic program enabling the lifespan extension. Furthermore, the aging-related deterioration of the heat shock response (HSR), the unfolded protein response (UPR) and the endoplasmic reticulum-associated protein degradation (ERAD), was largely prevented in water-transferred yeast, as the activities of these proteostatic network pathways remained nearly as robust as in young yeast. The characteristics of young yeast that are actively maintained upon water-transfer indicate that the extended lifespan is the outcome of slowing the rate of the aging process.     

Binding of Procollagen C-Proteinase Enhancer-1 (PCPE-1) to Heparin/Heparan Sulfate: PROPERTIES AND ROLE IN PCPE-1 INTERACTION WITH CELLS*

Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular matrix (ECM) glycoprotein that can stimulate procollagen processing by procollagen C-proteinases (PCPs) such as bone morphogenetic protein-1 (BMP-1). The PCPs can process additional extracellular protein precursors and play fundamental roles in developmental processes and assembly of the ECM. The stimulatory activity of PCPE-1 is restricted to the processing of fibrillar procollagens, suggesting PCPE-1 is a specific regulator of collagen deposition. PCPE-1 consists of two CUB domains that bind to the procollagen C-propeptides and are required for PCP enhancing activity, and one NTR domain that binds heparin. To understand the biological role of the NTR domain, we performed surface plasmon resonance (SPR) binding assays, cell attachment assays as well as immunofluorescence and activity assays, all indicating that the NTR domain can mediate PCPE-1 binding to cell surface heparan sulfate proteoglycans (HSPGs). The SPR data revealed binding affinities to heparin/HSPGs in the high nanomolar range and dependence on calcium. Both 3T3 mouse fibroblasts and human embryonic kidney cells (HEK-293) attached to PCPE-1, an interaction that was inhibited by heparin. Cell attachment was also inhibited by an NTR-specific antibody and the NTR fragment. Immunofluorescence analysis revealed that PCPE-Flag binds to mouse fibroblasts and heparin competes for this binding. Cell-associated PCPE-Flag stimulated procollagen processing by BMP-1 several fold. Our data suggest that through interaction with cell surface HSPGs, the NTR domain can anchor PCPE-1 to the cell membrane, permitting pericellular enhancement of PCP activity. This points to the cell surface as a physiological site of PCPE-1 action.