A specific interaction of small molecule entry inhibitors with the envelope glycoprotein complex of the Junín hemorrhagic fever arenavirus.

Arenaviruses are responsible for acute hemorrhagic fevers worldwide and are recognized to pose significant threats to public health and biodefense. Small molecule compounds have recently been discovered that inhibit arenavirus entry and protect against lethal infection in animal models. These chemically distinct inhibitors act on the tripartite envelope glycoprotein (GPC) through its unusual stable signal peptide subunit to stabilize the complex against pH-induced activation of membrane fusion in the endosome. Here, we report the production and characterization of the intact transmembrane GPC complex of Junín arenavirus and its interaction with these inhibitors. The solubilized GPC is antigenically indistinguishable from the native protein and forms a homogeneous trimer in solution. When reconstituted into a lipid bilayer, the purified complex interacts specifically with its cell-surface receptor transferrin receptor-1. We show that small molecule entry inhibitors specific to New World or Old World arenaviruses bind to the membrane-associated GPC complex in accordance with their respective species selectivities and with dissociation constants comparable with concentrations that inhibit GPC-mediated membrane fusion. Furthermore, competitive binding studies reveal that these chemically distinct inhibitors share a common binding pocket on GPC. In conjunction with previous genetic studies, these findings identify the pH-sensing interface of GPC as a highly vulnerable target for antiviral intervention. This work expands our mechanistic understanding of arenavirus entry and provides a foundation to guide the development of small molecule compounds for the treatment of arenavirus hemorrhagic fevers.

Clostridium difficile toxin B differentially affects GPCR-stimulated Ca2+ responses in macrophages: independent roles for Rho and PLA2.

Clostridium difficile toxins cause acute colitis by disrupting the enterocyte barrier and promoting inflammation. ToxB from C. difficile inactivates Rho family GTPases and causes release of cytokines and eicosanoids by macrophages. We studied the effects of ToxB on GPCR signaling in murine RAW264.7 macrophages and found that ToxB elevated Ca(2+) responses to Galphai-linked receptors, including the C5aR, but reduced responses to Galphaq-linked receptors, including the UDP receptors. Other Rho inhibitors also reduced UDP Ca(2+) responses, but they did not affect C5a responses, suggesting that ToxB inhibited UDP responses by inhibiting Rho but enhanced C5a responses by other mechanisms. By using PLCbeta isoform-deficient BMDM, we found that ToxB inhibited Ca(2+) signaling through PLCbeta4 but enhanced signaling through PLCbeta3. Effects of ToxB on GPCR Ca(2+) responses correlated with GPCR use of PLCbeta3 versus PLCbeta4. ToxB inhibited UDP Ca(2+) signaling without reducing InsP3 production or the sensitivity of cellular Ca(2+) stores to exogenous InsP3, suggesting that ToxB impairs UDP signaling at the level of InsP3/Ca(2+)coupling. In contrast, ToxB elevated InsP3 production by C5a, and the enhancement of Ca(2+) signaling by C5a was prevented by inhibition of PLA(2) or 5-LOX but not COX, implicating LTs but not prostanoids in the mechanism. In sum, ToxB has opposing, independently regulated effects on Ca(2+) signaling by different GPCR-linked PLCbeta isoforms in macrophages.

Modeling species-specific diacylglycerol dynamics in the RAW 264.7 macrophage.

A mathematical model of the G protein signaling pathway in RAW 264.7 macrophages downstream of P2Y(6) receptors activated by the ubiquitous signaling nucleotide uridine 5'-diphosphate is developed. The model, which is based on time-course measurements of inositol trisphosphate, cytosolic calcium, and diacylglycerol, focuses particularly on differential dynamics of multiple chemical species of diacylglycerol. When using the canonical pathway representation, the model predicted that key interactions were missing from the current network structure. Indeed, the model suggested that accurate depiction of experimental observations required an additional branch to the signaling pathway. An intracellular pool of diacylglycerol is immediately phosphorylated upon stimulation of an extracellular receptor for uridine 5'-diphosphate and subsequently used to aid replenishment of phosphatidylinositol. As a result of sensitivity analysis of the model parameters, key predictions can be made regarding which of these parameters are the most sensitive to perturbations and are therefore most responsible for output uncertainty.

Signaling and cross-talk by C5a and UDP in macrophages selectively use PLCbeta3 to regulate intracellular free calcium.

Studies in fibroblasts, neurons, and platelets have demonstrated the integration of signals from different G protein-coupled receptors (GPCRs) in raising intracellular free Ca(2+). To study signal integration in macrophages, we screened RAW264.7 cells and bone marrow-derived macrophages (BMDM) for their Ca(2+) response to GPCR ligands. We found a synergistic response to complement component 5a (C5a) in combination with uridine 5'-diphosphate (UDP), platelet activating factor (PAF), or lysophosphatidic acid (LPA). The C5a response was Galpha(i)-dependent, whereas the UDP, PAF, and LPA responses were Galpha(q)-dependent. Synergy between C5a and UDP, mediated by the C5a and P2Y6 receptors, required dual receptor occupancy, and affected the initial release of Ca(2+) from intracellular stores as well as sustained Ca(2+) levels. C5a and UDP synergized in generating inositol 1,4,5-trisphosphate, suggesting synergy in activating phospholipase C (PLC) beta. Macrophages expressed transcripts for three PLCbeta isoforms (PLCbeta2, PLCbeta3, and PLCbeta4), but GPCR ligands selectively used these isoforms in Ca(2+) signaling. C5a predominantly used PLCbeta3, whereas UDP used PLCbeta3 but also PLCbeta4. Neither ligand required PLCbeta2. Synergy between C5a and UDP likewise depended primarily on PLCbeta3. Importantly, the Ca(2+) signaling deficiency observed in PLCbeta3-deficient BMDM was reversed by re-constitution with PLCbeta3. Neither phosphatidylinositol (PI) 3-kinase nor protein kinase C was required for synergy. In contrast to Ca(2+), PI 3-kinase activation by C5a was inhibited by UDP, as was macropinocytosis, which depends on PI 3-kinase. PLCbeta3 may thus provide a selective target for inhibiting Ca(2+) responses to mediators of inflammation, including C5a, UDP, PAF, and LPA.

Use of a cAMP BRET sensor to characterize a novel regulation of cAMP by the sphingosine 1-phosphate/G13 pathway.

Regulation of intracellular cyclic adenosine 3 ',5 '-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed G(s)-dependent receptors for isoproterenol and prostaglandin E(2). Whereas two ligands, uridine 5 '-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via G(q)/calcium and G(i), the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for G(s)-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P(2) receptor and the heterotrimeric G(13) protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.

A targeted mass spectrometric analysis of phosphatidylinositol phosphate species.

The development of a new mass spectrometric lipid profiling methodology permits the identification of cellular phosphatidylinositol monophosphate/phosphatidylinositol bisphosphate/phosphatidylinositol trisphosphate (PIP/PIP2/PIP3) species that includes the fatty acyl composition. Using electrospray ionization mass spectrometry, we were able to resolve and identify 28 PIP and PIP2 compounds as well as 8 PIP3 compounds from RAW 264.7 or primary murine macrophage cell extracts. Analysis of PIP profiles after agonist stimulation of cells revealed the generation of differential PIP3 species and permitted us to propose a novel means for regulation and specificity in signaling through PIP3. This is the first reported identification of intact, cellular PIP3 by mass spectral analysis. The ability to analyze the fatty acyl chain composition of signaling lipids initiates new venues for investigation of the processes by which specific polyphosphoinositide species mediate.

Unravelling the signal-transduction network in B lymphocytes.

The Alliance for Cellular Signaling has chosen the mouse B lymphocyte as a model system to understand basic principles that govern cellular signalling. Progress to that end has focused initially on establishing a reproducible experimental cell system and characterizing essential signalling responses. Although unravelling this complex network will take years, findings revealed in the interim will prove immensely useful to the scientific community at large.

Overview of the Alliance for Cellular Signaling.

The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells--B lymphocytes (the cells of the immune system) and cardiac myocytes--to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.

2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers.

The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1'-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1'-methylindodi-carbocyanine halide (Cy5) N-hydroxysuccinimidyl ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in protein expression between laser capture microdissection-procured esophageal carcinoma cells and normal epithelial cells and to define cancer-specific and normal-specific protein markers. Analysis of the 2D images from protein lysates of approximately 250,000 cancer cells and normal cells identified 1038 protein spots in cancer cell lysates and 1088 protein spots in normal cell lysates. Of the detected proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in cancer cells. In addition to previously identified down-regulated protein annexin I, tumor rejection antigen (gp96) was found up-regulated in esophageal squamous cell cancer. Global quantification of protein expression between laser capture-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers.