Palmitoylation and membrane cholesterol stabilize μ-opioid receptor homodimerization and G protein coupling

<p>Abstract</p> <p>Background</p> <p>A cholesterol-palmitoyl interaction has been reported to occur in the dimeric interface of the β₂-adrenergic receptor crystal structure. We sought to investigate whether a similar phenomenon could be observed with μ-opioid receptor (OPRM1), and if so, to assess the role of cholesterol in this class of G protein-coupled receptor (GPCR) signaling.</p> <p>Results</p> <p>C3.55(170) was determined to be the palmitoylation site of OPRM1. Mutation of this Cys to Ala did not affect the binding of agonists, but attenuated receptor signaling and decreased cholesterol associated with the receptor signaling complex. In addition, both attenuation of receptor palmitoylation (by mutation of C3.55[170] to Ala) and inhibition of cholesterol synthesis (by treating the cells with simvastatin, a HMG-CoA reductase inhibitor) impaired receptor signaling, possibly by decreasing receptor homodimerization and Gαi2 coupling; this was demonstrated by co-immunoprecipitation, immunofluorescence colocalization and fluorescence resonance energy transfer (FRET) analyses. A computational model of the OPRM1 homodimer structure indicated that a specific cholesterol-palmitoyl interaction can facilitate OPRM1 homodimerization at the TMH4-TMH4 interface.</p> <p>Conclusions</p> <p>We demonstrate that C3.55(170) is the palmitoylation site of OPRM1 and identify a cholesterol-palmitoyl interaction in the OPRM1 complex. Our findings suggest that this interaction contributes to OPRM1 signaling by facilitating receptor homodimerization and G protein coupling. This conclusion is supported by computational modeling of the OPRM1 homodimer.</p

Predicting River Macroinvertebrate Communities Distributional Shifts under Future Global Change Scenarios in the Spanish Mediterranean Area

Several studies on global change over the next century predict increases in mean air temperatures of between 1°C to 5°C that would affect not only water temperature but also river flow. Climate is the predominant environmental driver of thermal and flow regimes of freshwater ecosystems, determining survival, growth, metabolism, phenology and behaviour as well as biotic interactions of aquatic fauna. Thus, these changes would also have consequences for species phenology, their distribution range, and the composition and dynamics of communities. These effects are expected to be especially severe in the Mediterranean basin due its particular climate conditions, seriously threatening Southern European ecosystems. In addition, species with restricted distributions and narrow ecological requirements, such as those living in the headwaters of rivers, will be severely affected. The study area corresponds to the Spanish Mediterranean and Balearic Islands, delimited by the Köppen climate boundary. With the application of the MEDPACS (MEDiterranean Prediction And Classification System) predictive approach, the macroinvertebrate community was predicted for current conditions and compared with three posible scenarios of watertemperature increase and its associated water flow reductions. The results indicate that the aquatic macroinvertebrate communities will undergo a drastic impact, with reductions in taxa richness for each scenario in relation to simulated current conditions, accompanied by changes in the taxa distribution pattern. Accordingly, the distribution area of most of the taxa (65.96%) inhabiting the mid-high elevations would contract and rise in altitude. Thus, families containing a great number of generalist species will move upstream to colonize new zones with lower water temperatures. By contrast, more vulnerable taxa will undergo reductions in their distribution area.This work was funded by GUADALMED-II (REN2001-3438-C07-06/HID), a project of excellence from “Junta de Andalucía” (RNM-02654/FEDER), the Spanish “Ministerio de Ciencia e Innovación” (CGL2007-61856/BOS), projects and a collaboration agreement between the “Spanish Ministerio de Medio Ambiente, Medio Rural y Marino” and the University of Granada (21.812-0062/8511)

Persistent left superior vena cava: Review of the literature, clinical implications, and relevance of alterations in thoracic central venous anatomy as pertaining to the general principles of central venous access device placement and venography in cancer patients

Persistent left superior vena cava (PLSVC) represents the most common congenital venous anomaly of the thoracic systemic venous return, occurring in 0.3% to 0.5% of individuals in the general population, and in up to 12% of individuals with other documented congential heart abnormalities. In this regard, there is very little in the literature that specifically addresses the potential importance of the incidental finding of PLSVC to surgeons, interventional radiologists, and other physicians actively involved in central venous access device placement in cancer patients. In the current review, we have attempted to comprehensively evaluate the available literature regarding PLSVC. Additionally, we have discussed the clinical implications and relevance of such congenital aberrancies, as well as of treatment-induced or disease-induced alterations in the anatomy of the thoracic central venous system, as they pertain to the general principles of successful placement of central venous access devices in cancer patients. Specifically regarding PLSVC, it is critical to recognize its presence during attempted central venous access device placement and to fully characterize the pattern of cardiac venous return (i.e., to the right atrium or to the left atrium) in any patient suspected of PLSVC prior to initiation of use of their central venous access device

A proposed structure for transmembrane segment 7 of G protein-coupled receptors incorporating an asn-Pro/Asp-Pro motif.

Transmembrane segment (TMS) 7 has been shown to play an important role in the signal transduction function of G-protein-coupled receptors (GPCRs). Although transmembrane segments are most likely to adopt a helical structure, results from a variety of experimental studies involving TMS 7 are inconsistent with it being an ideal alpha-helix. Using results from a search of the structure database and extensive simulated annealing Monte Carlo runs with the new Conformational Memories method, we have identified the conserved (N/D)PxxY region of TMS 7 as the major determinant for deviation of TMS 7 from ideal helicity. The perturbation consists of an Asx turn and a flexible "hinge" region. The Conformational Memories procedure yielded a model structure of TMS 7 which, unlike an ideal alpha-helix, is capable of accommodating all of the experimentally derived geometrical criteria for the interactions of TMS 7 in the transmembrane bundle of GPCRs. In the context of the entire structure of a transmembrane bundle model for the 5HT2a receptor, the specific perturbation of TMS 7 by the NP sequence suggests a structural hypothesis for the pattern of amino acid conservation observed in TMS 1, 2, and 7 of GPCRs. The structure resulting from the incorporation of the (N/D)P motif satisfies fully the H-bonding capabilities of the 100% conserved polar residues in these TMSs, in agreement with results from mutagenesis experiments. The flexibility introduced by the specific structural perturbation produced by the (NP/DP) motif in TMS 7 is proposed to have a significant role in receptor activation

Generation of a homology model of the human histamine H₃ receptor for ligand docking and pharmacophore-based screening

The human histamine H₃ receptor (hH₃R) is a G-protein coupled receptor (GPCR), which modulates the release of various neurotransmitters in the central and peripheral nervous system and therefore is a potential target in the therapy of numerous diseases. Although ligands addressing this receptor are already known, the discovery of alternative lead structures represents an important goal in drug design. The goal of this work was to study the hH3R and its antagonists by means of molecular modelling tools. For this purpose, a strategy was pursued in which a homology model of the hH₃R based on the crystal structure of bovine rhodopsin was generated and refined by molecular dynamics simulations in a dipalmitoylphosphatidylcholine (DPPC)/water membrane mimic before the resulting binding pocket was used for high-throughput docking using the program GOLD. Alternatively, a pharmacophore-based procedure was carried out where the alleged bioactive conformations of three different potent hH₃R antagonists were used as templates for the generation of pharmacophore models. A pharmacophore-based screening was then carried out using the program Catalyst. Based upon a database of 418 validated hH₃R antagonists both strategies could be validated in respect of their performance. Seven hits obtained during this screening procedure were commercially purchased, and experimentally tested in a [³H]Nα-methylhistamine binding assay. The compounds tested showed affinities at hH₃R with Ki values ranging from 0.079 to 6.3 lM

Management of semi-natural grasslands benefiting both plant and insect diversity: The importance of heterogeneity and tradition

Biodiversity of semi-natural grasslands depends on the management practices used. However, management systems suitable for one taxon, such as plants, can be detrimental to other taxa, such as insects, and vice versa. This study attempts to support conservation management planning by clarifying the effects of different grassland management practices on species richness and species composition of vascular plants, butterflies, moths, orthopterans and ground beetles, also taking into account the effects of climate and the landscape context. The study was performed in the White Carpathians Protected Landcape Area and UNESCO Biosphere Reserve (Czech Republic), which is famous for its grasslands with the globally highest fine-scale plant species richness. Different management practices (mowing, grazing, abandonment and mixed management; the latter including the previous three) were applied for at least five consecutive years at 34 sites, where plants and different insect groups were subsequently sampled. Effects of management on species richness of different taxonomic groups were assessed using generalised linear models, whereas the effects on species composition were assessed using redundancy analysis. Management influenced plant, butterfly and moth species richness, but the effects of particular management practices on all species and species of regional conservation importance differed between these taxonomic groups. Plant and moth species richness increased with mowing, but moth species richness decreased with grazing. Mixed management favoured plant and butterfly richness. Plant species composition was infuenced by mowing, grazing and mixed management while that of moths by mowing and grazing. Orthopterans and ground beetles did not respond significantly to management. Our results indicate that conservation management should comprise the traditional practices that have historically contributed to the formation of the biological diversity of the semi-natural grasslands in the study area. In particular, grazing may not be optimal for traditional hay-meadows and mowing should be carried out similarly as in pre-intensive farmland, creating spatio-temporal heterogeneity rather than uniformly cutting large grassland areas during a short period. In general, the optimal management should be heterogeneous, applying different practices in a mosaic or at different times during the season