Direct observation of membrane insertion by enveloped virus matrix proteins by phosphate displacement

Enveloped virus release is driven by poorly understood proteins that are functional analogs of the coat protein assemblies that mediate intracellular vesicle trafficking. We used differential electron density mapping to detect membrane integration by membrane-bending proteins from five virus families. This demonstrates that virus matrix proteins replace an unexpectedly large portion of the lipid content of the inner membrane face, a generalized feature likely to play a role in reshaping cellular membranes.

The bile acid receptor TGR5 does not interact with β-arrestins or traffic to endosomes but transmits sustained signals from plasma membrane rafts.

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid, DCA, taurolithocholic acid, TLCA) and the selective agonists oleanolic acid (OA) and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide (CCDC) stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, assessed by confocal microscopy. DCA, TLCA and OA did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, determined by bioluminescence resonance energy transfer. CCDC stimulated a low level of TGR5 interaction with β-arrestin2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of extracellular signal regulated kinase (ERK1/2). BRET analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.

A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway

Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in an hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main precursor of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homestasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature.

The role of plasma membrane STIM1 and Ca(2+)entry in platelet aggregation. STIM1 binds to novel proteins in human platelets.

Ca(2+) elevation is essential to platelet activation. STIM1 senses Ca(2+) in the endoplasmic reticulum and activates Orai channels allowing store-operated Ca(2+) entry (SOCE). STIM1 has also been reported to be present in the plasma membrane (PM) with its N-terminal region exposed to the outside medium but its role is not fully understood. We have examined the effects of the antibody GOK/STIM1, which recognises the N-terminal region of STIM1, on SOCE, agonist-stimulated Ca(2+) entry, surface exposure, in vitro thrombus formation and aggregation in human platelets. We also determined novel binding partners of STIM1 using proteomics. The dialysed GOK/STIM1 antibody failed to reduced thapsigargin- and agonist-mediated Ca(2+) entry in Fura2-labelled cells. Using flow cytometry we detect a portion of STIM1 to be surface-exposed. The dialysed GOK/STIM1 antibody reduced thrombus formation by whole blood on collagen-coated capillaries under flow and platelet aggregation induced by collagen. In immunoprecipitation experiments followed by proteomic analysis, STIM1 was found to extract a number of proteins including myosin, DOCK10, thrombospondin-1 and actin. These studies suggest that PM STIM1 may facilitate platelet activation by collagen through novel interactions at the plasma membrane while the essential Ca(2+)-sensing role of STIM1 is served by the protein in the ER.

A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI-activated platelets: involvement of G6f, a novel platelet Grb2-binding membrane adapter.

Collagen-related peptide (CRP) stimulates powerful activation of platelets through the glycoprotein VI (GPVI)-FcR gamma-chain complex. We have combined proteomics and traditional biochemistry approaches to study the proteome of CRP-activated platelets, focusing in detail on tyrosine phosphorylation. In two separate approaches, phosphotyrosine immunoprecipitations followed by 1-D-PAGE, and 2-DE, were used for protein separation. Proteins were identified by MS. By following these approaches, 96 proteins were found to undergo PTM in response to CRP in human platelets, including 11 novel platelet proteins such as Dok-1, SPIN90, osteoclast stimulating factor 1, and beta-Pix. Interestingly, the type I transmembrane protein G6f was found to be specifically phosphorylated on Tyr-281 in response to platelet activation by CRP, providing a docking site for the adapter Grb2. G6f tyrosine phoshporylation was also found to take place in response to collagen, although not in response to the G protein-coupled receptor agonists, thrombin and ADP. Further, we also demonstrate for the first time that Grb2 and its homolog Gads are tyrosine-phosphorylated in CRP-stimulated platelets. This study provides new insights into the mechanism of platelet activation through the GPVI collagen receptor, helping to build the basis for the development of new drug targets for thrombotic disease.

Platelet actin nodules are podosome-like structures dependent on Wiskott-Aldrich syndrome protein and ARP2/3 complex

The actin nodule is a novel F-actin structure present in platelets during early spreading. However, only limited detail is known regarding nodule organization and function. Here we use electron microscopy, SIM and dSTORM super-resolution, and live-cell TIRF microscopy to characterize the structural organization and signalling pathways associated with nodule formation. Nodules are composed of up to four actin-rich structures linked together by actin bundles. They are enriched in the adhesion-related proteins talin and vinculin, have a central core of tyrosine phosphorylated proteins and are depleted of integrins at the plasma membrane. Nodule formation is dependent on Wiskott-Aldrich syndrome protein (WASp) and the ARP2/3 complex. WASp(-/-) mouse blood displays impaired platelet aggregate formation at arteriolar shear rates. We propose actin nodules are platelet podosome-related structures required for platelet-platelet interaction and their absence contributes to the bleeding diathesis of Wiskott-Aldrich syndrome.

Phenylephrine promotes phosphorylation of Bad in cardiac myocytes through the extracellular signal-regulated kinases 1/2 and protein kinase A.

Studies in non-cardiomyocytic cells have shown that phosphorylation of the Bcl-2 family protein Bad on Ser-112, Ser-136 and Ser-155 decreases its pro-apoptotic activity. Both phenylephrine (100 microM) and the cell membrane-permeating cAMP analog, 8-(4-chlorophenylthio)-cAMP (100 microM), protected against 2-deoxy-D-glucose-induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). In cardiac myocytes, phenylephrine primarily stimulates the alpha-adrenoceptor, but, at high concentrations (100 microM), it also increases the activity of the cAMP-dependent protein kinase, protein kinase A (PKA) through the beta-adrenoceptor. Phenylephrine (100 microM) promoted rapid phosphorylation of Bad(Ser-112) and Bad(Ser-155), though we were unable to detect phosphorylation of Bad(Ser-136). Phosphorylation of Bad(Ser-112) was antagonized by either prazosin or propranolol, indicating that this phosphorylation required stimulation of both alpha(1)- and beta-adrenoceptors. Phosphorylation of Bad(Ser-155) was antagonized only by propranolol and was thus mediated through the beta-adrenoceptor. Inhibitor studies and partial purification of candidate kinases by fast protein liquid chromatography showed that the p90 ribosomal S6 kinases, p90RSK2/3 [which are activated by the extracellular signal-regulated kinases 1 and 2 (ERK1/2)] directly phosphorylated Bad(Ser-112), whereas the PKA catalytic subunit directly phosphorylated Bad(Ser-155). However, efficient phosphorylation of Bad(Ser-112) also required PKA activity. These data suggest that, although p90RSK2/3 phosphorylate Bad(Ser-112) directly, phosphorylation of this site is enhanced by phosphorylation of Bad(Ser-155). These phosphorylations potentially diminish the pro-apoptotic activity of Bad and contribute to the cytoprotective effects of phenylephrine and 8-(4-chlorophenylthio)-cAMP.

The role of lipid composition on the interaction between a tryptophan-rich protein and model bacterial membranes

The interaction between tryptophan-rich puroindoline proteins and model bacterial membranes at the air-liquid interface has been investigated by FTIR spectroscopy, surface pressure measurements and Brewster angle microscopy. The role of different lipid constituents on the interactions between lipid membrane and protein was studied using wild type (Pin-b) and mutant (Trp44 to Arg44 mutant, Pin-bs) puroindoline proteins. The results show differences in the lipid selectivity of the two proteins in terms of preferential binding to specific lipid head groups in mixed lipid systems. Pin-b wild type was able to penetrate mixed layers of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) head groups more deeply compared to the mutant Pin-bs. Increasing saturation of the lipid tails increased penetration and adsorption of Pin-b wild type, but again the response of the mutant form differed. The results provide insight as to the role of membrane architecture, lipid composition and fluidity, on antimicrobial activity of proteins. Data show distinct differences in the lipid binding behavior of Pin-b as a result of a single residue mutation, highlighting the importance of hydrophobic and charged amino acids in antimicrobial protein and peptide activity.

Integration of steroid hormone initiated membrane action to genomic function in the brain

Estrogen is a ligand for the estrogen receptor (ER), which on binding 17beta-estradiol, functions as a ligand-activated transcription factor and regulates the transcription of target genes. This is the slow genomic mode of action. However, rapid non-genomic actions of estrogen also exist at the cell membrane. Using a novel two-pulse paradigm in which the first pulse rapidly initiates non-genomic actions using a membrane-limited estrogen conjugate (E-BSA), while the second pulse promotes genomic transcription from a consensus estrogen response element (ERE), we have demonstrated that rapid actions of estrogen potentiate the slower transcriptional response from an ERE-reporter in neuroblastoma cells. Since rapid actions of estrogen activate kinases, we used selective inhibitors in the two-pulse paradigm to determine the intracellular signaling cascades important in such potentiation. Inhibition of protein kinase A (PKA), PKC, mitogen activated protein kinase (MAPK) or phosphatidylinositol 3-OH kinase (PI-3K) in the first pulse decreases potentiation of transcription. Also, our data with both dominant negative and constitutive mutants of Galpha subunits show that Galpha(q) initiates the rapid signaling cascade at the membrane in SK-N-BE(2)C neuroblastoma cells. We discuss two models of multiple kinase activation at the membrane Pulses of estrogen induce lordosis behavior in female rats. Infusion of E-BSA into the ventromedial hypothalamus followed by 17beta-estradiol in the second pulse could induce lordosis behavior, demonstrating the applicability of this paradigm in vivo. A model where non-genomic actions of estrogen couple to genomic actions unites both aspects of hormone action.

Activation of the G-protein coupled receptor 30 (GPR30) has different effects on anxiety in male and female mice

The GPR30, a former orphan GPCR, is a putative membrane estrogen receptor that can activate rapid signaling pathways such as extracellular regulated kinase (ERK) in a variety of cells and may contribute to estrogen's effects in the central nervous system. The distribution of GPR30 in the limbic system predicts a role for this receptor in the regulation of learning and memory and anxiety by estrogens. Though acute G-1 treatment is reported to be anxiogenic in ovariectomised female mice and in gonadally intact male mice, the effect of GPR30 activation is unknown in gonadectomised male mice. In this study, we show that an acute administration of G-1 to gonadectomised male mice, but not female mice, was anxiolytic on an elevated plus maze task, without affecting locomotor activity. In addition, though G-1 treatment did not regulate ERK, it was associated with increased estrogen receptor (ER)alpha phosphorylation in the ventral, but not dorsal, hippocampus of males. In the female, G-1 increased the ERK activation solely in the dorsal hippocampus, independent of state anxiety. This is the first study to report an anxiolytic effect of GPR30 activation in male mice, in a rapid time frame that is commensurate with non-genomic signaling by estrogen.

Regulation of Multidrug Resistance-Associated Protein 2 by Calcium Signaling in Mouse Liver

Mu hiding resistance associated protein 2 (Mrp2) is a canalicular transporter responsible for organic anion secretion into bile. Mrp2 activity is regulated by insertion into the plasma membrane; however, the factors that control this are not understood. Calcium (Ca(2+)) signaling regulates exocytosis of vesicles in most cell types, and the type II inositol 1,4,5-triphosphate receptor (InsP(3)R2) regulates Ca(2+) release in the canalicular region of hepatocytes. However, the role of InsP(3)R2 and of Ca(2+) signals in canalicular insertion and function of Mrp2 is not known. The aim of this study was to determine the role of InsP(3)R2-mediated Ca(2+) signals in targeting Mrp2 to the canalicular membrane. Livers, isolated hepatocytes, and hepatocytes in collagen sandwich culture from wild-type (WT) and InsP(3)R2 knockout (KO) mice were used for western blots, confocal immunofluorescence, and time-lapse imaging of Ca(2+) signals and of secretion of a fluorescent organic anion. Plasma membrane insertion of green fluorescent protein (GFP)-Mrp2 expressed in HepG2 cells was monitored by total internal reflection microscopy. InsP(3)R2 was concentrated in the canalicular region of WT mice but absent in InsP(3)R2 KO livers, whereas expression and localization of InsP(3)R1 was preserved, and InsP(3)R3 was absent from both WT and KO livers. Ca(2+) signals induced by either adenosine triphosphate (ATP) or vasopressin were impaired in hepatocytes lacking InsP(3)R2. Canalicular secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was reduced in KO hepatocytes, as well as in WT hepatocytes treated with 1,2-bis(o-aminophenoxy)ethane-N,N,N`,N`-tetra-acetic acid (BAPTA). Moreover, the choleretic effect of tauroursodeoxycholic acid (TUDCA) was impaired in InsP(3)R2 KO mice. Finally, ATP increased GFP-Mrp2 fluorescence in the plasma membrane of HepG2 cells, and this also was reduced by BAPTA. Conclusion: InsP(3)R2-mediated Ca(2+) signals enhance organic anion secretion into bile by targeting Mrp2 to the canalicular membrane. (HEPATOLOGY 2010;52:327-337)

Na plus -Glucose Cotransporter SGLT1 Protein in Salivary Glands: Potential Involvement in the Diabetes-Induced Decrease in Salivary Flow

Oral health complications in diabetes include decreased salivary secretion. The SLC5A1 gene encodes the Na(+)-glucose cotransporter SGLT1 protein, which not only transports glucose, but also acts as a water channel. Since SLC5A1 expression is altered in kidneys of diabetic subjects, we hypothesize that it could also be altered in salivary glands, contributing to diabetic dysfunction. The present study shows a diabetes-induced decrease (p < 0.001) in salivary secretion, which was accompanied by enhanced (p < 0.05) SGLT1 mRNA expression in parotid (50%) and submandibular (30%) glands. Immunohistochemical analysis of parotid gland of diabetic rats revealed that SGLT1 protein expression increased in the luminal membrane of ductal cells, which can stimulate water reabsorption from primary saliva. Furthermore, SGLT1 protein was reduced in myoepithelial cells of the parotid from diabetic animals, and that, by reducing cellular contractile activity, might also be related to reduced salivary flux. Six-day insulin-treated diabetic rats reversed all alterations. In conclusion, diabetes increases SLC5A1 gene expression in salivary glands, increasing the SGLT1 protein content in the luminal membrane of ductal cells, which, by increasing water reabsorption, might explain the diabetes-induced decrease in salivary secretion.

Soybean and sunflower oil-induced insulin resistance correlates with impaired GLUT4 protein expression and translocation specifically in white adipose tissue

Free fatty acids are known for playing a crucial role in the development of insulin resistance. High fat intake is known for impairing insulin sensitivity; however, the effect of vegetable-oil injections have never been investigated. The present study investigated the effects of daily subcutaneous injections (100 mu L) of soybean (SB) and sunflower (SF) oils, during 7 days. Both treated groups developed insulin resistance as assessed by insulin tolerance test. The mechanism underlying the SB- and SF-induced insulin resistance was shown to involve GLUT4. In SB- and SF-treated animals, the GLUT4 protein expression was reduced similar to 20% and 10 min after an acute it? vivo stimulus with insulin, the plasma membrane GLUT4 content was similar to 60% lower in white adipose tissue (WAT). No effects were observed in skeletal muscle. Additionally, both oil treatments increased mainly the content of palmitic acid (similar to 150%) in WAT, which can contribute to explain the GLUT4 regulations. Altogether, the present study collects evidence that those oil treatments might generate insulin resistance by targeting GLUT4 expression and translocation specifically in WAT. These alterations are likely to be caused due to the specific local increase in saturated fatty acids that occurred as a consequence of oil daily injections. Copyright (C) 2010 John Wiley & Sons, Ltd.

Alboserpin, a Factor Xa Inhibitor from the Mosquito Vector of Yellow Fever, Binds Heparin and Membrane Phospholipids and Exhibits Antithrombotic Activity

The molecular mechanism of factor Xa (FXa) inhibition by Alboserpin, the major salivary gland anticoagulant from the mosquito and yellow fever vector Aedes albopictus, has been characterized. cDNA of Alboserpin predicts a 45-kDa protein that belongs to the serpin family of protease inhibitors. Recombinant Alboserpin displays stoichiometric, competitive, reversible and tight binding to FXa (picomolar range). Binding is highly specific and is not detectable for FX, catalytic site-blocked FXa, thrombin, and 12 other enzymes. Alboserpin displays high affinity binding to heparin (K(D) similar to 20 nM), but no change in FXa inhibition was observed in the presence of the cofactor, implying that bridging mechanisms did not take place. Notably, Alboserpin was also found to interact with phosphatidylcholine and phosphatidylethanolamine but not with phosphatidylserine. Further, annexin V (in the absence of Ca(2+)) or heparin outcompetes Alboserpin for binding to phospholipid vesicles, suggesting a common binding site. Consistent with its activity, Alboserpin blocks prothrombinase activity and increases both prothrombin time and activated partial thromboplastin time in vitro or ex vivo. Furthermore, Alboserpin prevents thrombus formation provoked by ferric chloride injury of the carotid artery and increases bleeding in a dose-dependent manner. Alboserpin emerges as an atypical serpin that targets FXa and displays unique phospholipid specificity. It conceivably uses heparin and phosphatidylcholine/phosphatidylethanolamine as anchors to increase protein localization and effective concentration at sites of injury, cell activation, or inflammation.

Expression and purification of human respiratory syncytial virus recombinant fusion protein

The Human Respiratory Syncytial Virus (HRSV) fusion protein (F) was expressed in Escherichia call BL21A using the pET28a vector at 37 degrees C. The protein was purified from the soluble fraction using affinity resin. The structural quality of the recombinant fusion protein and the estimation of its secondary structure were obtained by circular dichroism. Structural models of the fusion protein presented 46% of the helices in agreement with the spectra by circular dichroism analysis. There are only few studies that succeeded in expressing the HRSV fusion protein in bacteria. This is a report on human fusion protein expression in E. call and structure analysis, representing a step forward in the development of fusion protein F inhibitors and the production of antibodies. (c) 2008 Elsevier Inc. All rights reserved.