An external site controls closing of the epithelial Na+ channel ENaC.

Members of the ENaC/degenerin family of ion channels include the epithelial sodium channel (ENaC), acid-sensing ion channels (ASICs) and the nematode Caenorhabditis elegans degenerins. These channels are activated by a variety of stimuli such as ligands (ASICs) and mechanical forces (degenerins), or otherwise are constitutively active (ENaC). Despite their functional heterogeneity, these channels might share common basic mechanisms for gating. Mutations of a conserved residue in the extracellular loop, namely the 'degenerin site' activate all members of the ENaC/degenerin family. Chemical modification of a cysteine introduced in the degenerin site of rat ENaC (betaS518C) by the sulfhydryl reagents MTSET or MTSEA, results in a approximately 3-fold increase in the open probability. This effect is due to an 8-fold shortening of channel closed times and an increase in the number of long openings. In contrast to the intracellular gating domain in the N-terminus which is critical for channel opening, the intact extracellular degenerin site is necessary for normal channel closing, as illustrated by our observation that modification of betaS518C destabilises the channel closed state. The modification by the sulfhydryl reagents is state- and size-dependent consistent with a conformational change of the degenerin site during channel opening and closing. We propose that the intracellular and extracellular modulatory sites act on a common channel gate and control the activity of ENaC at the cell surface.

Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays.

The SLC2 family of facilitated hexose and polyol transporters.

The SLC2 family of glucose and polyol transporters comprises 13 members, the glucose transporters (GLUT) 1-12 and the H(+)- myo-inositol cotransporter (HMIT). These proteins all contain 12 transmembrane domains with both the amino and carboxy-terminal ends located on the cytoplasmic side of the plasma membrane and a N-linked oligosaccharide side-chain located either on the first or fifth extracellular loop. Based on sequence comparison, the GLUT isoforms can be grouped into three classes: class I comprises GLUT1-4; class II, GLUT6, 8, 10, and 12 and class III, GLUT5, 7, 9, 11 and HMIT. Despite their sequence similarity and the presence of class-specific signature sequences, these transporters carry various hexoses and HMIT is a H(+)/ myo-inositol co-transporter. Furthermore, the substrate transported by some isoforms has not yet been identified. Tissue- and cell-specific expression of the well-characterized GLUT isoforms underlies their specific role in the control of whole-body glucose homeostasis. Numerous studies with transgenic or knockout mice indeed support an important role for these transporters in the control of glucose utilization, glucose storage and glucose sensing. Much remains to be learned about the transport functions of the recently discovered isoforms (GLUT6-13 and HMIT) and their physiological role in the metabolism of glucose, myo-inositol and perhaps other substrates.

Siglec-1 is a novel dendritic cell receptor that mediates HIV-1 trans-infection through recognition of viral membrane gangliosides.

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of immunity against pathogens. However, HIV-1 spread is strongly enhanced in clusters of DCs and CD4(+) T cells. Uninfected DCs capture HIV-1 and mediate viral transfer to bystander CD4(+) T cells through a process termed trans-infection. Initial studies identified the C-type lectin DC-SIGN as the HIV-1 binding factor on DCs, which interacts with the viral envelope glycoproteins. Upon DC maturation, however, DC-SIGN is down-regulated, while HIV-1 capture and trans-infection is strongly enhanced via a glycoprotein-independent capture pathway that recognizes sialyllactose-containing membrane gangliosides. Here we show that the sialic acid-binding Ig-like lectin 1 (Siglec-1, CD169), which is highly expressed on mature DCs, specifically binds HIV-1 and vesicles carrying sialyllactose. Furthermore, Siglec-1 is essential for trans-infection by mature DCs. These findings identify Siglec-1 as a key factor for HIV-1 spread via infectious DC/T-cell synapses, highlighting a novel mechanism that mediates HIV-1 dissemination in activated tissues.

Innate immunity: cytoplasmic DNA sensing by the AIM2 inflammasome.

Cytoplasmic double-stranded DNA triggers cell death and secretion of the pro-inflammatory cytokine IL-1beta in macrophages. Recent reports now describe the mechanism underlying this observation. Upon sensing of DNA, the HIN-200 family member AIM2 triggers the assembly of the inflammasome, culminating in caspase-1 activation, IL-1beta maturation and pyroptotic cell death.

Cloning of a cDNA encoding a plasma membrane-associated, uronide binding phosphoprotein with physical properties similar to viral movement proteins.

Oligogalacturonides are structural and regulatory homopolymers from the extracellular pectic matrix of plants. In vitro micromolar concentrations of oligogalacturonates and polygalacturonates were shown previously to stimulate the phosphorylation of a small plasma membrane-associated protein in potato. Immunologically cross-reactive proteins were detected in plasma membrane-enriched fractions from all angiosperm subclasses in the Cronquist system. Polygalacturonate-enhanced phosphorylation of the protein was observed in four of the six dicotyledon subclasses but not in any of the five monocotyledon subclasses. A cDNA for the protein was cloned from potato. The deduced protein is extremely hydrophilic and has a proline-rich N terminus. The C-terminal half of the protein was predicted to be a coiled coil, suggesting that the protein interacts with other macromolecules. The recombinant protein was found to bind both simple and complex galacturonides. The behavior of the protein suggests several parallels with viral proteins involved in intercellular communication.

Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii.

ABSTRACT: BACKGROUND: Sphingomonas wittichii strain RW1 can completely oxidize dibenzo-p-dioxins and dibenzofurans, which are persistent contaminants of soils and sediments. For successful application in soil bioremediation systems, strain RW1 must cope with fluctuations in water availability, or water potential. Thus far, however, little is known about the adaptive strategies used by Sphingomonas bacteria to respond to changes in water potential. To improve our understanding, strain RW1 was perturbed with either the cell-permeating solute sodium chloride or the non-permeating solute polyethylene glycol with a molecular weight of 8000 (PEG8000). These solutes are assumed to simulate the solute and matric components of the total water potential, respectively. The responses to these perturbations were then assessed and compared using a combination of growth assays, transcriptome profiling, and membrane fatty acid analyses. RESULTS: Under conditions producing a similar decrease in water potential but without effect on growth rate, there was only a limited shared response to perturbation with sodium chloride or PEG8000. This shared response included the increased expression of genes involved with trehalose and exopolysaccharide biosynthesis and the reduced expression of genes involved with flagella biosynthesis. Mostly, the responses to perturbation with sodium chloride or PEG8000 were very different. Only sodium chloride triggered the increased expression of two ECF-type RNA polymerase sigma factors and the differential expression of many genes involved with outer membrane and amino acid metabolism. In contrast, only PEG8000 triggered the increased expression of a heat shock-type RNA polymerase sigma factor along with many genes involved with protein turnover and repair. Membrane fatty acid analyses further corroborated these differences. The degree of saturation of membrane fatty acids increased after perturbation with sodium chloride but had the opposite effect and decreased after perturbation with PEG8000. CONCLUSIONS: A combination of growth assays, transcriptome profiling, and membrane fatty acid analyses revealed that permeating and non-permeating solutes trigger different adaptive responses in strain RW1, suggesting these solutes affect cells in fundamentally different ways. Future work is now needed that connects these responses with the responses observed in more realistic scenarios of soil desiccation.

Parallelizing remote sensing image geometric correction

Remote sensing spatial, spectral, and temporal resolutions of images, acquired over a reasonably sized image extent, result in imagery that can be processed to represent land cover over large areas with an amount of spatial detail that is very attractive for monitoring, management, and scienti c activities. With Moore's Law alive and well, more and more parallelism is introduced into all computing platforms, at all levels of integration and programming to achieve higher performance and energy e ciency. Being the geometric calibration process one of the most time consuming processes when using remote sensing images, the aim of this work is to accelerate this process by taking advantage of new computing architectures and technologies, specially focusing in exploiting computation over shared memory multi-threading hardware. A parallel implementation of the most time consuming process in the remote sensing geometric correction has been implemented using OpenMP directives. This work compares the performance of the original serial binary versus the parallelized implementation, using several multi-threaded modern CPU architectures, discussing about the approach to nd the optimum hardware for a cost-e ective execution.

Equine herpesvirus protein E10 induces membrane recruitment and phosphorylation of its cellular homologue, bcl-10.

v-E10, a caspase recruitment domain (CARD)-containing gene product of equine herpesvirus 2, is the viral homologue of the bcl-10 protein whose gene was found to be translocated in mucosa-associated lymphoid tissue (MALT) lymphomas. v-E10 efficiently activates the c-jun NH(2)-terminal kinase (JNK), p38 stress kinase, and the nuclear factor (NF)-kappaB transcriptional pathway and interacts with its cellular homologue, bcl-10, via a CARD-mediated interaction. Here we demonstrate that v-E10 contains a COOH-terminal geranylgeranylation consensus site which is responsible for its plasma membrane localization. Expression of v-E10 induces hyperphosphorylation and redistribution of bcl-10 from the cytoplasm to the plasma membrane, a process which is dependent on the intactness of the v-E10 CARD motif. Both membrane localization and a functional CARD motif are important for v-E10-mediated NF-kappaB induction, but not for JNK activation, which instead requires a functional v-E10 binding site for tumor necrosis factor receptor-associated factor (TRAF)6. Moreover, v-E10-induced NF-kappaB activation is inhibited by a dominant negative version of the bcl-10 binding protein TRAF1, suggesting that v-E10-induced membrane recruitment of cellular bcl-10 induces constitutive TRAF-mediated NF-kappaB activation.

Molecular determinants of desensitization in an ENaC/degenerin channel.

Epithelial Na(+) channel (ENaC)/degenerin family members are involved in mechanosensation, blood pressure control, pain sensation, and the expression of fear. Several of these channel types display a form of desensitization that allows the channel to limit Na(+) influx during prolonged stimulation. We used site-directed mutagenesis and chemical modification, functional analysis, and molecular dynamics simulations to investigate the role of the lower palm domain of the acid-sensing ion channel 1, a member of the ENaC/degenerin family. The lower palm domains of this trimeric channel are arranged around a central vestibule, at ∼20 Å above the plasma membrane and are covalently linked to the transmembrane channel parts. We show that the lower palm domains approach one another during desensitization. Residues in the palm co-determine the pH dependence of desensitization, its kinetics, and the stability of the desensitized state. Mutations of palm residues impair desensitization by preventing the closing movement of the palm. Overexpression of desensitization-impaired channel mutants in central neurons allowed--in contrast to overexpression of wild type--a sustained signaling response to rapid pH fluctuations. We identify and describe here the function of an important regulatory domain that most likely has a conserved role in ENaC/degenerin channels.

Isolated integrin beta3 subunit cytoplasmic domains require membrane anchorage and the NPXY motif to recruit to adhesion complexes but do not discriminate between beta1- and beta3-positive complexes.

Integrin adhesion receptors consist of non-covalently linked alpha and beta subunits each of which contains a large extracellular domain, a single transmembrane domain and a short cytoplasmic tail. Engaged integrins recruit to focal structures globally termed adhesion complexes. The cytoplasmic domain of the beta subunit is essential for this clustering. beta1 and beta3 integrins can recruit at distinct cellular locations (i.e. fibrillar adhesions vs focal adhesions, respectively) but it is not clear whether individual beta subunit cytoplasmic and transmembrane domains are by themselves sufficient to drive orthotopic targeting to the cognate adhesion complex. To address this question, we expressed full-length beta3 transmembrane anchored cytoplasmic domains and truncated beta3 cytoplasmic domains as GFP-fusion constructs and monitored their localization in endothelial cells. Membrane-anchored full-length beta3 cytoplasmic domain and a beta3 mutant lacking the NXXY motif recruited to adhesion complexes, while beta3 mutants lacking the NPXY and NXXY motifs or the transmembrane domain did not. Replacing the natural beta subunit transmembrane domain with an unrelated (i.e. HLA-A2 alpha chain) transmembrane domain significantly reduced recruitment to adhesion complexes. Transmembrane anchored beta3 and cytoplasmic domain constructs, however, recruited without discrimination to beta1- and beta3-rich adhesions complexes. These findings demonstrate that membrane anchorage and the NPXY (but not the NXXY) motif are necessary for beta3 cytoplasmic domain recruitment to adhesion complexes and that the natural transmembrane domain actively contributes to this recruitment. The beta3 transmembrane and cytoplasmic domains alone are insufficient for orthotopic recruitment to cognate adhesion complexes.

hShroom1 links a membrane bound protein to the actin cytoskeleton.

hShroom1 (hShrm1) is a member of the Apx/Shroom (Shrm) protein family and was identified from a yeast two-hybrid screen as a protein that interacts with the cytoplasmic domain of melanoma cell adhesion molecule (MCAM). The characteristic signature of the Shrm family is the presence of a unique domain, ASD2 (Apx/Shroom domain 2). mRNA analysis suggests that hShrm1 is expressed in brain, heart, skeletal muscle, colon, small intestine, kidney, placenta and lung tissue, as well a variety of melanoma and other cell lines. Co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) experiments indicate that hShrm1 and MCAM interact in vivo and by immunofluorescence microscopy some co-localization of these proteins is observed. hShrm1 partly co-localises with beta-actin and is found in the Triton X-100 insoluble fraction of melanoma cell extracts. We propose that hShrm1 is involved in linking MCAM to the cytoskeleton.